Analytical Chemistry Guide for M.Sc. Students

ANALYTICAL CHEMISTRY
For M.Sc. Students of Various Universities.
[COMPREHENSIVELY COVERING THE UGC SYLLABUS.]
DR. H. KAUR
READER
Postgraduate Department of Chemistry
N.A.S. (P.G.) College, Meerut, India.
~PRAGATI PRAKASHAN
PRAGATI PRAKASHAN First Edition : 2008
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Contents
1. INTRODUCTION................................... 1-39
Role of analytical chemistry 1
Types of analysis 2
Analytical methods 3
Classification of classical and instrumental methods of analysis 3
Selecting an analytical method 9
Steps involved in quantitative analysis 10
Neatness and cleanliness in laboratory 10
Selecting and handling of reagents 11
Organic reagents used in inorganic analysis 12
Safety in the analytical laboratory 14
Laboratory note book 15
Laboratory operations and practices 16
Sample preparation, dissolving the sample and sample decomposition 16
Stoichiometry 23
Volumetric glasswares 24
Gravimetric techniques 24
Analytical balance 33
Techniques of weighing using analytical balance 36
Weighing errors 36
Cleaning and calibration of glasswares 37
2. ERRORS AND EVALUATIONS . ...................... 40-61

Types and sources of errors 40
Systematic or determinate errors 40
Random or indeterminate errors 42
Effects of errors on analytical results 43
Accuracy. Absolute and relative errors 44
1?etermination of accuracy 45
Precision 45
Minimization of errors 46
Significant figures 48
Methods for reporting analytical data 48
Statistical evaluation of data 50
Statistical terms: Mean, mean deviation and median 51
Standard deviation 55
Reliability and rejection of results 58
The uses of statistics 60
Important relations 61
3. FOOD ANALYSIS. . . . . . .. . . . . .. . .. .. . . . . . . . . .. . ... 62-94
Introduction 62
Moisture analysis in foods 63
Ash analysis 66
Analysis of protein 70
Analysis of fat and crude fibre 74
Analysis of carbohydrates and starch 78
Determination of calcium 81
Analysis of phosphorus and potassium 82
Analysis of sodium by flame photometric method 84
Common adulterants in food 85
Contamination of food stuffs 87
Microscopic examination of food 89
Pesticide analysis of food products 90
Extraction, purification and analysis of organophosphates in food by
HnC 00 .
Analysis of insecticides in milk by HPLC 91
Gas chroma.tography for organophosphates in food 92
Thin layer chromatography for chlorinated pesticides in food products 93
4. TYPES OF WATER POLLUTION .................... 95-110

Water pollution 95
Complexing ligands in water 97
Origin of waste water 98
Ground and surface water pollution 99
Lake and river water pollution 103
Marine pollution. Effects of oil pollution i!1 marine water 105
Counter measures against oil spills 108
5. SOURCES OF WATER POLLUTION . ................ 111-116

Domestic and agricultural pollutants 111
Radioactive and thermal pollutants 112
Industrial eflluents 113
6. WATER POLLUTANTS AND THEIR EFFECTS . ........ 117-147

Inorganic pollutants, toxic metals and their detrimental effects 117
Organic pollutants, sediments and synthetic detergents 1'20
Oxygen demanding wastes and disease causing agents 125
Radioactive pollutants 128
Plant nutrients and eutrophication 130
Thermal pollutants in water 132
Pesticide pollutants 134
How pesticides endanger our life 142
Farm wastes and fertilizers 147
7. ANALYSIS OF WATER POLLUTANTS ... ............ 148-181
Objectives of water analysis 148
Chemical and physical examination of water 148
Parameters for water analysis :
Colour, turbidity and conductivity 151
Total solids, acidity and alkalinity 153
Hardness, chloride, sulphate and fluoride 156
Silica and phosphate 160
Different forms of nitrogen 162
Measurement of dissolved oxygen (DO) 165
Measurement of chemical oxygen demand (COD) 168
Measurement of biochemical oxygen demand (BOD) 169
Total organic carbon 172
Pesticide analysis 172
Analysis of pesticides by TLC and GC 173
Analysis of insecticides by HPLC 174
Water pollution laws and water quality standards 176
8. HEAVY METAL POLLUTION . ...................... 182-198

Metal toxicity 182
Public health significanctl of heavy metals :
. Cadmium, chromium, copper 183
Lead, zinc, manganese, mercury, arsenic 186
Instrumental techniques for the analysis of heavy metals in water 193
Analysis of heavy metals using atomic absorption spectrophotometry 193
Analysis of copper 195
DPP for the determination of copper and zinc in tap water 195
Analysis of lead in water 197
9. SOIL ANALYSIS ................................. 199-223

Components of soil 199
Micro and macro plant nutrients 202
Analysis of soil : Soil moisture measurement 206
Determination of soil pH 207
Determination of total nitrogen and nitrate nitrogen in soil 209
Determination of total phosphorus and PO!- ion 212
Determination of silica and lime 214
Determination of magnesium and manganese 216
Determination of sulphur 218
Determination of salts in soil 220
Determination of sodium and potassium in soil by flame photometry 221
10. FUEL ANALYSIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 224-246

Fuels 224
Classification of fuels 224
Solid fuels 224
Grading of coal 225
Liquid fuels 226
Gaseous fuels 227
Producer gas, water gas and natural gas 227
Calorific value of fuel 231
Proximate and ultimate analysis of coal 235
Determination of calorific value of coal using proximate and ultimate
analysis 238
Flash and fire point of liquid fuels 240
Aniline point of liquid fuels 243
Carbon residue of liquid fuels 244
Octane and cetane numbers 245
11. CLINICAL CHEMISTRY . . . . . . . . . . . . . . . . . . . . . . . . . .. 247-278

Composition of blood 247
Functions of blood 249
Collection and preservation of samples 249
Clinical analysis : Serum electrolyte 251
Estimation of blood chloride, sodium and potassium 251
Estimation of serum calcium and bicarbonate 253
Estimation of blood glucose 255
Estimation of blood urea 257
Estimation of blood urea nitrogen 259
Estimation of uric acid in serum 260
Estimation of total serum protein 261
Estimation of serum albumin and globulin 262
Estimation of serum barbiturates 263
Estimation of serum acid and alkaline phosphatase 264
Immunoassay. Principles of radio immunoassay 266
Applications of radio immunoassay 269
Blood gas analysis 270
Trace elements in the body: 273
Calcium, Magnesium, Sodium, Potassium
Copper, Zinc, Manganese, Iron, Iodine
12. DRUG ANALYSIS . .............................. . 279-292

Narcotics 279
Dangerous drugs 280
Classification of drugs 280
Screening of drugs by gas chromatography 283
Screening of drugs by GC-MS 285
Screening of drugs by thin layer chromatography and HPLC 288
Analysis of drugs by fluorimetric method 290
Analysis of drugs by UV and IR spectrophotometric methods 290
INDEX G" • • • • • • • • • • • • • • • • " • • • • • • • • • • • • • • • • • • • • • • • • • • • (iHiv)

o
1
INTRODUCTION
ANALYTICAL CHEMISTRY
Analytical chemistry is a scientific discipline that develops and applies
methods, instruments and strategies to gain infonnation about the
composition and nature of matter. It is an interdisciplinary branch of science
which plays an important role in nearly all streams of chemistry such as
inorganic, organic, physical, industrial and biochemistry. It also finds
extensive applications in environmental science, agricultural science,
oceanography, clinical chemistry, solid state research and electronics. The
scope of analytical chemistry is very broad and embraces a wide range of
manual, chemical and instrumental techniques.
Role of Analytical Chemistry.
Analytical chemistry seeks ever imposed means of determining the
chemical composition, purity and quality of substances by qualitative and
quantitative analysis.
• It is the science of chemical characterisation of matter. It provides the
techniques and tools needed for insight into our material world.
• Manufacturing industries rely upon analytical chemistry for
testing raw materials and for assuring the quality of finished products
whose composition is critical. The quality of manufactured products
often depends on proper chemical proportions and measurement of
the constituent is a necessary part of quality control. The
semiconductor industry is an example of an industry whose very
existence depends on accurate determination of substances present in
extremely minute quantities.
• Several industrial and household products, alloys, polymers, fuels,
paints, perfumes, and pharmaceuticals are analysed by the
procedures developed by analytical chemists before being sold to the
consumer.
• Many industrial processes give rise to pollutants which can create a
health problem. Quantitative analysis of air, water and soil samples
must be carried out to determine the levels of pollution and to
establish safe limits for pollutants.
• Environmental quality is often evaluated by testing for suspected
contaminants using the techniques of analytical chemistry.
• In medicine, analytical chemistry is widely used for clinical laboratory
tests to assist in diagnosis of illness and in monitoring the conditions
of patients. The composition and purity of drugs detennine their
efficacy.
(1)
2 ANALYTICAL CHEMISTRY
• The nutritional value of food is determined by chemical analysis for

major components such as proteins and carbohydrates and trace
components like vitamins and minerals. Even the calories in a food
are often calculated from its chemical analysis. Also the food can be
analysed for contaminants (e.g., pesticide residues) and for essential
nutrients such as vitamin content.
• In farming, the nature and level of fertiliser application is based on
information obtained by analysing the soil to determine its content of
the essential plant nutrients, NPK and trace elements required for
healthy plant growth.
• Geological surveys require the services of analytical chemists to
determine the composition of numerous rocks and soil samples
collected in the field. One example is the qualitative and quantitative
examination of moon rock brought back to Earth in 1969 by the first
American astronauts to land on the moon.
• Much government legislation can only be enforced by the work of
analytical chemists, e.g., national and international agreements on
atmospheric and water pollution, food safety measures, regulations on
substances injurious to health and laws governing the misuse of
drugs.
• Analytical chemistry also make important contributions to fields as
diverse as forensics, archaeology and space science.
TYPES OF ANALYSIS
Analysis involves the resolution of a chemical compound into its
proximate or ultimate parts, the determination of its elements or of foreign
substances it may contain. The first requirement in a completely unknown
sample is to ascertain what substances are present in it and which type of
impurities are contained in the sample. The solution of such problems lies
within the province of qUalitative analysis. Having ascertained the nature
of the constituents of a given sample, the next step is to determine how much
of each (or specified) component is present. Such determinations lie within the
realm of quantitative analysis and a variety of techniques are available to
supply the required information. Depending upon the nature of information
sought, chemical analysis may be classified into four types.
1. Proximate analysis, in which the amount of each element in a
sample is determined with no concern as to the actual compounds
present.
2. Partial analysis deals with the determination of selected
constituents in the sample.
3. Trace constituent analysis, a specialised instance of partial
analysis, deals with the determination of specified components
present in very minute quantity.
4. Complete analysis, when the proportion of each component of the
sample is to be determined.
On the basis of sample size, analytical methods are often classified as
follows:
INTRODUCTION 3
1. Macro involves the analysis of quantities of 0·1 g or more.

2. Meso (Semimicro) measures quantities ranging from 10-2 g to
10-1 g.
3. Micro deals with quantities in the range 10-3 g to 10-2 g.
4. Submicro is concerned with samples in the range 10-4 g to 10-3 g.
5. Ultramicro measures quantities below 10-4 g.
A major constituent is one accounting for 1 to 100% of the sample
under investigation. A minor constituent is present in the range 0·01 to
1%; a trace constituent is present at a concentration of less than 0·01%.
Further subdivisions include : trace corresponds to 102 - 104 J..lg g-1 or
102 - 104 ppm, microtrace to 10-:-1 - 102 pg g-1 or 10-7 - 10-4 ppm,
nanotrace to 10-1 -102 fg g-1 or 10-10 _10-7 ppm.
When the sample weight is small (0·1-1·0 mg), the determination of a
trace component at the 0·01% level may be referred to as subtrace analysis.
If the trace component is at the microtrace level, the analysis is termed
submicro trace. Determination of a component at the trace level (0·1 mg) is
called ultra trace analysis while with a component at the microtrace
level, the analysis is termed as ultra-microtrace.
CLASSIFICATION OF ANALYTICAL METHODS
Analytical techniques employed in quantitative analysis are based
upon:
(a) The quantitative performance of suitable chemical reactions.
(b) Measuring the amount of reagent needed to complete the reaction or
ascertaining the amount of reaction product obtained.
(c) Appropriate electrical measurements (e.g. potentiometry).
(d) Measuring certain optical properties (e.g. absorption spectra). In
some cases, a combination of optical or electrical measurements and
quantitative chemical reaction (e.g. amperometric titration) may 'be
used.
Common techniques indispensable to analytical chemistry (Table 1) are
categorised as follows :
1. Classical methods 2. Instrumental methods
3. Non-destructive methods 4. Radioactive methods
5. Special methods
1. Classical Methods.
The quantitative execution of chemical reactions is the basis of classical
methods of analysis.
(i) Gravimetric Analysis. In gravimetric analysis, the substance
being determined is converted into an insoluble precipitate which is collected,
dried and weighed. It involves several steps such as preparation of the
sample, precipitation, digestion, filtration, washing, drying and weighing. In
case of electrogravimetry, electrolysis is carried out and the material
deposited on one of the electrodes is weighed. Weight measurement or changes
of energy are also important in thermal methods of analysis, where these
features are recorded as a function of temperature.
•
Thermogravimetry records the change in weight. DTA records the
difference in temperature between a test substance and an inert reference
material. DSC records the energy required to establish a zero temperature
difference between a test substance and a reference material.
(ii) Titrimetric Analysis. Titrimetric analysis depends on the
measurement of volumes of solution of the interacting substances. The
substance to be determined is allowed to react with an appropriate reagent as
a standard solution and the volume of solution needed for complete
stoichiometric reaction is determined. The common titrimetric reactions are
neutralisation (acid-base) reactions, oxidation-reduction reactions, iodometry
or iodimetry reactions, precipitation reactions and complex-forming reactions.
Volumetry also measures the volume of gas evolved or absorbed in a
chemical reaction.
Advantages of Classical Methods.
• Classical methods are based on absolute measurements.
• Procedure is simple and accurate.
• There is no need to calibrate a sample of known composition.
• The method is suitable for a non-routine and on occasional analysis.
• Equipment required is cheap and readily available.
Limitations of Classical Methods.
• Procedure is time consuming.
• Accuracy decreases with decreasing concentration.
• Lack of specificity and versatility.
2. Instrumental Methods.
The methods which measure an electrical property, absorption of
radiation or the intensity of an emission, require the use of a suitable
instrument, polarograph, spectrophotometer etc. and in consequence are
referred to as instrumental methods.
(A) Electroanalytical Methods. These methods involve the
measurement of current, voltage or resistance in relation to the concentration
of a certain species in solution. Techniques are:
(i) Coulometry (measurement of current and time needed to
complete an electrochemical reaction).
(ii) Voltametry (measurement of current at a micro-electrode at a
specified voltage).
(iii) Potentiometry (measurement of potential of an electrode in
equilibrium with an ion to be determined).
(iv) Conductimetry (measurement of the electrical conductivity of a
solution).
(B) Spectroscopic Methods. Spectroscopic methods of analysis
depend on:
• Measurement of the amount of radiant energy of a particular
wavelength absorbed or emitted by the sample.
• Bending, scattering or delayed emission of radiant energy.
INTRODUCTION 5
Table 1. Classification of instrumental methods of analysis.

S.No. Method Principle Property measured
1. Electroanalytical Change in the electrical Mass of deposited
methods properties of the system. substance.
(A) Electrogravimetry Electrolysis is carried out. Material deposited on
one of the electrode is
weighed.
(B) Coulometry Deposition of matter on an Quantity of electricity
electrode during electrolysis. and time.
(C) Conductimetry Change in electrical conduc- Electrical conductivity,
tivity of a solution during Electrical resistance.
chemical reaction.
(D) Amperometry Potential applied between the Current through the
indicator electrode and electrolytic cell IS
depolarised reference electrode measured.
is kept constant.
(E) Potentiometry Change in electrode potentials Electrode potential.
of a system during chemical
reaction.
(F) Polarography Electrode polarisation. Voltage, current.
2. Spectroscopic Interaction of matter with Radiant energy of a

methods electromagnetic radiation. particular wavelength.
(A) Atomic Atomising the specimen. Absorption of
Absorption radiation.
Spectroscopy
(B) Absorption Absorption of poly-and mono- Optical density of the
Spectrophotometry chromatic radiant energy by solution.
(colorimetry, molecules and ions in solution.
photoelectro-
colorimetry).
(C) Emission Sample is subjected to an Position and intensity
Spectroscopy electric arc or spark plasma. of spectral lines.
Emission of radiation.
(D) X-ray Emission of X-ray spectrum by Position and intensity
Spectroscopy atoms. of spectral lines.
(E) Raman Absorption of monochromatic Same.
Spectroscopy radiation by matter and
emission of new radiation
differing from that absorbed
by the wavelength.
(F) Turbidimetry Absorption and scattering of a Amount of light
light beam by turbid media. stopped or scattered
by a suspension.
Continued ..... .
S.No. Method Principle Property measured

(G) Nephelometry Reflection and scattering of a Same.
light beam by colloidal
solution.
(H) Refractometry Refraction of light by matter. Refractive index.
3. Mass Spectroscopy Ionisation of atoms, ions and Position and intensity

molecules by a combined of signals in mass
action of electric and magnetic spectrum. Mass to
fields and appearance of mass charge ratio.
spectra.
4. Nuclear Magnetic Nuclear magnetism (resonance Position and intensity
Resonance absorption of electromagnetic of lines of NMR
radiation by matter in spectrum.
magnetic field).
5. Radiometric Conversion of stable isotopes Intensity of radiation,
Methods of an element to radio isotopes. Induced radioactivity.
(A) Isotope Dilution Change in specific activity of Radioactivity.
the compounds labelled with a
radioisotope.
6. Kinetic Methods Speed of chemical reaction Concomitant change
may be increased by the in the absorbance of
addition of catalyst. solution for visible or
UV radiation.
7. Thermal Methods Recording as a function of Change in weight or
temperature and time. energy.
(A) Thermogravimetry, Weighing of the substance Change in weight.
TG while it is being heated.
(B) Differential Heat effects associated with Difference in
Thermal physical and chemical changes temperature.
Analysis, DTA of a substance are recorded
when it is heated.
(C) Differential Energy necessary to establish Change in energy.
Scanning a zero temperature difference
Colorimetry, DSC between a test substance and a
reference material.
(i) Absorption Methods. These methods are usually classified
according to the wavelength involved as visible, ultraviolet or infrared
spectrophotometry.
Atomic absorption spectroscopy (AAS) involves atomising the
specimen, often by spraying a solution of the sample into a flame and then
studying the absorption of radiation from an electric lamp producing the
spectrum of the element to be determined.
(ii) Emission Methods. Emission methods subject the sample to heat
or electrical treatment so that atoms are raised to excited states causing
INTRODUCTION 7
them to emit energy. The intensity of this energy is then measured. Here are
some of the common excitation techniques.
• Flame photometry uses a solution of the sample injected into a flame.
• Emission spectroscopy subjects the sample to an inductively
coupled plasma and then examining the emitted light (which may
extend into the ultraviolet region).
• Fluorimetry takes a substance in a fluorescent reagent and excites
it using visible or ultraviolet radiation.
(iii) Magnetic Resonance Spectroscopy. Nuclear magnetic
resonance (NMR) spectroscopy is concerned with the study of interaction of
energy with spin-active nuclei which have permanent magnetic moments and
quanti sed nuclear spin states.
• Electron spin resonance (ESR) spectroscopy. Electrons in the
free radicals, atoms, ions or molecules (having unpaired electrons)
change their spin under the influence of applied magnetic field and
spectra arising is called ESR or EPR.
(iv) Photoelectron Spectroscopy (PES). In PES, a beam of photons
of known energy is allowed to fall on the sample and kinetic energy of the
ejected electrons is measured. PES can be studied either using X-ray photons
(XPES) or UV photons (UVPES).
(v) Scattering Methods. Nephelometric and turbidimetric methods
measure the amount of light stopped or s<:attered by a suspension.
C. Chromatographic and Electrophoretic Methods. These are
essentially separative processes for mixtures of substances but equipped with
modern detector systems, they are also adapted to identifY components of
mixtures.
3. Non-Destructive Methods (X-ray Methods).
(i) Electron Probe Micro Analysis. Primary X-rays are produced
when high speed electrons collide with a solid target. It is possible to identify
certain emission peaks which are characteristic of elements contained in the
target. The wavelengths of the peaks can be related to the atomic number of
the elements producing them, so they provide a means of identifYing elements
present in the target sample. Further, under controlled Gonditions, the
intensity of peaks may be used to determine the amounts of the various
elements present. This forms the basis of electron probe micro analysis,
in which a small target area of the sample is selected for identification.
(ii) X-ray Fluorescence. When a beam of short wavelength primary
X-rays strikes a solid target, it emits X-rays at wavelengths characteristic of
the atoms involved. This is referred to as secondary or fluorescence
radiation. It is possible to get an idea of sample composition by examining
the peak heights of the .fluorescence radiation. X-ray fluorescence analysis
is a rapid process which finds applicatlons in metallurgical research, in
processing of metallic ores and in cement industry. Crystalline material will
diffract a beam of X-rays. X-ray powder diffractometry can be used to
identifY components of mixtures.
4. Radioactive Methods.
Radioactive methods involve measuring the intensity of radiation from
a naturally radioactive material and also measuring radioactivity induced by
exposing the sample to a neutron source (activation analysis), or isotope
dilution and radio immunoassay. Typical applications include
determination of trace elements and quality control in semiconductor
manufacturing.
5. Special Methods.
(i) Optical Methods. Refractometry can be used to measure the
refractive index of liquids and to check the purity of the compound. In
conjunction with a calibration curve, it can be employed to analyse a mixture
of two liquids. Optical rotatory dispersion and circular dichroism
coupled to polarimetry correlate measurement of optical rotation and
absorption.
(ii) Kinetic Methods. Kinetic methods are based on the fact that the
speed of a given chemical reaction may be increased by the addition of a small
amount of catalyst, and within limits, the rate of catalysed reaction will be
governed by the amount of catalyst present. If a calibration curve is prepared
showing variation of reaction rate with the amount of catalyst used, then
measurement of reaction rate will make it possible to determine how much
catalyst has been added in certain instance. This provides a sensitive method
for determining sub-microgram amounts of appropriate substances.
Non-destructive methods, radioactive methods, optical and kinetic
methods also require the use of an instrument, hence may be considered as
instrumental methods.
Advantages of Instrumental Methods.
• Instrumental methods are general1y more sensitive and selective than
the classical methods.
• These techniques are normally applicable at concentrations far too
small to be amenable to determination by chemical methods.
• Determination is much faster than purely chemical procedures.
• Measurements obtained are reliable. Accuracy is obtainlild.
• Even complex samples can be handled easily.
• Finds wide application in industry.
• In most cases a microcomputer can be interfaced to the instrument so
that absorption curves, polarograms, titration curves etc. can be
plotted automatically. In fact, by the incorporation of appropriate
servo-mechanisms, the whole analytical process may be completely
automated.
Limitations of Instrumental Methods.
Despite the advantages possessed by instrumental methods in many
directions, their wide-spread adoption has not rendered the purely chemical
or classical methods obsolete, the situation is influenced by the following
factors:
INTRODUCTION 9
• With instrumental methods it is necessary to carry out a calibration

operation using a sample of material of known composition as
reference substance.
• Many instruments are expensive and their use will only be justified if
numerous samples have to be analysed, or when dealt with the
determination of substances present in minute quantities (trace,
subtrace or ultratrace analysis).
• Whilst an instrumental method is ideally suited to the performance of
a large number of routine determinations, for an occasional,
non-routine analysis it is often simpler to use a chemical method than
to go to the trouble of preparing requisite standards and carrying out
the calibration of an instrument.
• The sensitivity and accuracy depends on the instrument.
• Sizable space is required.
• Specialised training is needed.
SELECTING AN ANALYTICAL METHOD
To select the most appropriate method, analysts must be familiar with
the practical details of the various techniques and the theoretical principles
on which they are based. They should consider the conditions under which
each method is reliable, know possible interferences and, finds ways to
circumvent any problem. Analysts will be concerned with accuracy, precision,
speed, sensitivity, selectivity, availability of the instrument, level of analysis,
time and cost of the procedure involved.
Factors Affecting the Selection of an Analytical Method.
Different analytical techniques have differing degrees of sophistication,
sensitivity, selectivity, cost and time requirements. So an important task for
the analyst is the selection of the best procedure for a given determination.
This will require careful consideration of the following criteria.
• The type of analysis required; elemental or molecular, routine or
occasional.
• Nature of the information which is sought.
• Size of the sample available.
• Problems arising from the nature of the material to be investigated,
e.g., radioactive substances, corrosive substances, substances affected
by water.
• Possible interference from components of the material.
• The proportion of the constituent to be determined.
• The purpose for which the analytical data are required.
• Complexity of the material to be analysed.
• The concentration range which needs to be investigated.
• The accuracy required.
• The time required to complete the analysis.
• The number of analysis of similar type which have to be performed.
• Does the nature of the specimen or the magnitude of the sample
available indicate the use of non-destructive methods of analysis as
opposed to the more commonly applied destructive methods involving

dissolution of the sample prior to the application of normal analytical
techniques?
STEPS INVOLVED IN QUANTITATIVE ANALYSIS
Following steps must be considered carefully and assessed when
confronted with an unfamiliar quantitative determination in order to
minimise errors and to maintain accuracy and reproducibility.
1. Sampling. Procedure employed for sampling depends on the size
and physical nature of the sample.
2. Preparation of Analytical Sample. Analytical sample can be
prepared by
(i) reduction in particle size,
(ii) mixing for homogeneity,
(iii) drying,
(iv) determination of sample weight or volume.
3. Dissolution of the Sample. It can be achieved by heating,
ignition, fusion, using solvents or dilution.
4. Removal of Interferences. Interferences from the desired
constituents can be removed by filtration, selective precipitation,
masking, selective oxidation or reduction, solvent extraction, ion
exchange, chromatographic separation (GLC, TLC, HPLC) etc.
Note that samples of liquids and gases are frequently amenable to
automatic treatment; most readi1y automated are the instrumental
procedures for measurement, calibration, optimisation, statistical
treatments and data presentation.
5. Sample Measurement and Control of Instrumental Factors.
It can be achieved by standardisation, calibration, optimisation,
measurement of response, absorbance, emission signal, potential and
current etc.
6. Employing spectroscopic investigations.
7. Calculation of analytical results and for the sample, statistical
or chemometric evaluation of data.
8. Presentation of Data. It can be done by print out, data plotting
or storage (archiving).
NEATNESS AND CLEANLINESS IN LABORATORY

An analyst must be aware of basic scientific techniques, analytical
procedures and handling of simple equipments to obtain accurate,
reproducible and reliable results. The importance of set routines for
cleanliness and orderly working should be emphasised. The following points
should be kept in mind while working in a laboratory.
1. Benches must be kept clean and tidy and all spillages of liquids or
solids cleaned away immediately.
2. All glassware should be clean and rinsed with distilled or deionised
water before use. The outsides <if the vessels may be dried with a
lint-free glass cloth which is reserved exclusively for this purpose
INTRODUCTION 11
and laundered frequently. The cloth should not be used on the inside
of the vessels.
3. The working surface of the .bench must not be cluttered with
apparatus. All the apparatus associated with some particular
operation should be assembled together on the bench. This is
extremely essential to avoid confusion when duplicate determina-
tions are performed. The apparatus which are not in use should be
returned to the locker.
4. The container must be labelled so that the solution, precipitate or
filtrate can be readily identified. The vessel must be covered to
prevent contamination of the contents by dust.
5. For temporary labelling, a chinagraph pencil or a felt-tip pen (glass
marker pen) is preferred to wax pencils or adhesive labels. Adhesive
labels are employed for long term use.
6. Reagent bottles should never be allowed to accumulate on the bench,
these must be replaced on the reagent shelves immediately after use.
7. Reagent solutions should be poured in the testing vessel.
8. Never insert spoon, spatula or knives into the reagent bottle. Use
clean porcelain spoon for taking solid chemicals.
9. All determinations should be performed in duplicate.
10. A stiff covered note book should be used for recording experimental
observations.
SELECTING AND HANDLING OF REAGENTS
The purest reagents should be used for quantitative analysis, wherever
possible use reagents of analytical quality. Analar chemicals from BDH Ltd.
(Merck) conform to ACS specifications. Certain manufacturers market
chemicals of high purity and each package of these analysed chemicals has a
label giving the manufacturer's limits of certain impurities. Following points
should be considered while handling the reagents.
• Liquid reagents should be poured from the bottle; a pipette should
never be inserted into the reagent bottle. Avoid contamination of the
stopper of reagent bottle.
• When the liquid reagent is poured from a bottle, the stopper should
never be placed on the working bench or shelf.
• The stopper should be placed upon a clean watch glass.
• The stopper should be returned to the bottle immediately after the
reagent has been removed.
• The reagent bottles must be kept scrupulously clean, particularly
round the neck or mouth of the bottle.
• If a reagent of adequate purity for a particular determination is not
available then the purest available product must be purified. Liquids
can be purified by fractional distillation and solids by
recrystallisation.
• The reagents should be tested by standard methods for the impurities
that might cause errors in the determination.
• Use specially purified reagents such as the BDH Aristar chemicals to
detect lower limits in various types of instrumental analysis.
• Solid reagents must be sufficiently dry.

• Standard solutions of accurately known concentrations need to be
stored correctly.
• Unless specifically directed, never return any excess reagent to a
bottle to avoid contamination of the entire solution.
• Reagent bottle should not be opened for a longer time.
• Observe regulations for the safe disposal of surplus reagents.
ORGANIC REAGENTS USED IN INORGANIC ANALYSIS

1. Dimethyl glyoxime.
CH3-C=NOH
I
CH 3-C=NOH
Dimethyl glyoxime solution reacts with Ni 2+ ion in ammonical medium
and forms scarlet red neutral red chelate of bis-Cdimethyl glyoximato) nickel
CII). The formation of this chelate can be used for the identification,
estimation of Ni2+ and its separation from Co 2+ ions.
2. a-Nitroso !3-naphthol.
~OH
l2JSJ
a-Nitro so !3-naphthol solution reacts with slightly acidic solution of
Co2+ ion to form its red brown chelate of octahedral geometry. This test is not
shown by Ni 2+ ion.
3. S-Hydroxy quinoline (Oxine).
~
yN~
OH
Oxine is employed for the separation of AI from Be in acidic medium, Mg
from Ca in alkaline medium and for gravimetric estimations of Cu, Ni, Co,
Zn, Pb, Cd and Mg etc.
4. Cupferron.
/NO
~N'ONH4
Cupferron, the salt of nitrosophenyl hydroxylamine reacts with Fe(ITI)
to form bidentate inner complex of iron in which a labile hydrogen (or NH;t
ion) is replaced and oxygen of the nitroso group co-ordinates. The reagent is
highly selective for Nb, Ta, W, Hg(I), Fe(III) and Zn in acid solution.
5. Dithizone or Diphenyl thiocarbazone.
N=N-C-NH-NH
@ll@
INTRODUCTION 13
Dithizone is specially used for the estimation of Pb which produces a

brick red complex with the reagent. It is also used for the spectrophotometric,
complexometric and quantitative determination of Cu, Zn, Cd, Pb, Hg, TI
and Bi.
6. Dithio-oxamide (Rubeanic acid), NH2 CS.CSNH2 • Dithio-
oxamide quantitatively precipitates Cu, Co, Ni in strongly ammonical
solutions. Traces of Cu, Ni, Co can be determined spectrophotometric ally with
this reagent. It is also employed as a delicate spot test reagent for Cu, Co, Ni.
The reagent forms coloured complexes with Ru, Os, Pd and Pt in acidic
medium.
7. a-Benzoin oxime.
C6H5-CH-OH
I
C6Ho-C=NOH
The reagent yields a green precipitate Cu(C 14H n 0 2N) with Cu in
ammonical solution. Cu may thus be separated from Cd, Ph, Ni, Co, Zn, AI
and Fe. a-Benzoin oxime quantitatively precipitates Mo as an addition
compound in mineral acid solution.
s. SaIicylaIdoxime.
~OH
&CH=NOH
Salicylaldoxime has been employed for the determination of Cu. Tartaric
acid is used as the masking agent.
9. Magneson (p-Nitrobenzene azo resorcinol).
02N--@-N~N-(jj)-OH
HO .
Magneson forms a blue lake with Mg(OH)2 m alkaline medium.
Ammonium salts reduce the sensitivity of the test.
10. Benzidine.
H2N-@-@-NH2
Benzidine is used as the dihydrochloride for the precipitation of
sulphate in acidic solution.
11. Anthranilic acid.
~NH
&COOH
Sodium salt of anthranilic acid is useful for quantitative determination
of Cu, Cd, Ni, Zn and Co at controlled pH.
12. 1, 10-Phenanthroline.
r0n
t:N">-<"NJ
The reagent yields extremely stable red colour with Fe2+ ion
[Fe(C 12H s N 2)s]2+. The test is highly sensitive over a wide pH range (2 to 9)
and conform to Beer's law. The ferrous complex of 1, 10-phenanthroline
(ferroin) is a valuable redox indicator.
13. Acetyl acetone.
o °
II
CHs-C-CH2-C-C " HS
Acetyl acetone reacts with Cu2+ ions to form neutral inner chelate which
can be separated from Fes+ ions by solvent extraction. Trifluoroacetyl acetone
has been used in the separation of Zr and H£
14. Ethylene diamine tetra-acetic acid.
HOOCH2C " /CH2COOH
N-CH2-CH2-N
HOOCH2C / '-.....CH2COOH
Disodium salt of EDTA has been used for the estimation of Mi+ and
Ca2+ ions by complexometric titrations at pH 10 using Eriochrome Black T as
an indicator.
15. Alizarin red.
cQ5:::iN
o
a
To the Als+ salt solution add NH 40H, alizarin red and acetic acid in
excess. Red colouration is obtained which is destroyed by F- ions. Hence the
reagent is very sensitive for the detection of V- ions.
SAFETY IN THE ANALYTICAL LABORATORY
There is always a potential hazard in a chemistry laboratory and
regrettably, accidents do occur on occasions. However, they can be minimised
by adhering to the laboratory safety code, by taking all necessary safety
precautions and by carrying out a hazard assessment before any
experimentation. Following points should be borne in mind.
• Many chemicals encountered in analysis are poisonous and must be
handled carefully.
• It is necessary to carry out a safety assessment of the poisonous
chemicals, concentrated acids, PARs, PCBs, KCN and halogenated
solvents.
• Many operations involving chemical reactions are potentially
dangerous, so recommended procedures must be carefully followed
and obeyed.
• Always clean up spilled chemicals. Do not leave broken or chipped
glassware lying around working bench.
INTRODUCTION 15
• Reagent bottles and apparatus should be placed properly after use.

• Neutralise acid spills with sodium bicarbonate and alkali spills with
boric acid.
• Mercury spills must be vacuumed up with a suction flask or dusted with
sulphur powder. Protect yourself from highly toxic mercury vapours.
• Use fume hood while heating acids or using volatile organic
solvents. Use a safety shield when working with fuming reactions.
• Perform only the authorized experiments and never work alone.
Special care should be taken when working with flammable organic
solvents since many have been identified as acute or chronic toxic
substances, frequently carcinogenic.
• Avoid breathing fumes.
• An analyst working in the laboratory should locate fire extinguishers,
safety showers, exits, fire blankets, first aid equipments and eye
fountains.
• All workers should familiarise themselves with local safety
requirements which may include compulsory wearing of lab coats,
rubber gloves and safety spectacles.
• Do not eat or drink or use laboratory equipment for storing or holding
food.
• Safety standards in laboratories have been tightened now and
monitored by government officials fraquently.
• Avoid getting chemicals on your skin, even solids, and wash off any
contamination with large volumes of water. Immediately remove any
clothing contaminated with corrosive substances.
LABORATORY NOTEBOOK
• A stiff covered notebook should be used for recording experimental
observations as they are made.
• Never use loose leafs. Number pages consecutively.
• Never tear out pages. If not used, put a line through the page.
• Date each page and record the name of the project.
• Devote a double page to each determination. Use one page for the
experimental observations and the other for brief description of the
procedure followed with special features associated with the
determination.
• The record must conclude with the relevant calculations, so the
equations for the principal chemical reactions should be shown
together with a clear exposition of the procedure for calculating the
result.
• Finally, appropriate comments should be made about the degree of
accuracy and the level of precision.
• Now the modern laboratory instruments are frequently interfaced or
they possess built-in computers to produce printed records of the
analytical results in the form of spectra or chromatogram. These
equipments process the data and compare it automatically with
standards and previous results stored in computer memory.
• The printed data or any chart should be permanently attached within

the laboratory notebook.
• The results should be roughly checked to confirm their consistency
with the printed data.
LABORATORY OPERATIONS AND PRACTICES

Quantitative analysis of a compound involves several laboratory
operations, apparatus and techniques such as sample preparation, weighing
and dissolving the sample, sample decomposition, stoichiometry and complete
analysis by volumetric or gravimetric techniques.
SAMPLE PREPARATION FOR ANALYSIS

Preparing the substances for analysis represents the first, and often the
most difficult step in the analytical protocol. At first sight, an analyst must
know: What is in the sample? What concentrations are present? What health
risks do these materials present? One cannot analyse accurately unless a
representative sample can be obtained from a complex matrix.
Sampling.
Sampling is the process of extracting from a large quantity of material
a small portion which is truly representative of the composition of the whole
material. The purpose of analysis is to determine the quality or composition
of a material. It is essential to adopt adequate sampling procedures to know
the validity of the analytical results.
Sampling Procedure.
The reliable method for sampling is one in which portions of the material
are selected based upon statistical probabilities. The result obtained from the
given sample may form the basis of assessing the value of a large
consignment of the commodity from which the sample was drawn.
Sampling in Different Physical States.
During sampling it is the number of analyte particles which is important
rather than the amount (mass) of the sample. The relationship n oc lJR 2
applies in all cases where R is the relative standard deviation and n is the
number of particles. The consequence ofthis relationship means that different
approaches are used for each of the three phases. For gases and liquids the
particle size is very small, e.g., 1 em3 _.of a pure gas at STP contains
2·5 x 1019 particles and 1 cm3 of liquid, - 2·5 x 1016, but for solids the
requirement of homogeneity depend upon taking sufficient analyte particles
to be representative. Since modem methods of analysis often require
considerably less than 1 mg of material in order to perform a number of
analyses, this small amount is obtained by first collecting a gross sample
and then reducing it down to the analytical sample.
Sampling of Gases.
Sampling techniques for gases and vapours involve gas sampling
vessels, static sensors, entrapment and real-time analysis. A number of
INTRODUCTION 17
instruments (electrochemical sensors) and techniques (IR, GC-MS) are

available for instant analysis.
Sampling of Liquids.
Several approaches are used to obtain a gross liquid sample viz., small
static systems, flowing contained volumes, open flowing systems. The protocol
depends upon whether it is desirable to obtain a number of discrete samples
which will be analysed separately or to bulk the individual samples taken to
give a composite sample. Automatic collectors can either produce a single
composite sample or can collect up to 24 separate samples in individual
containers. The separate aliquots ofliquids can be analysed individually and
the results combined or the aliquots can be combined into one gross sample
and replicated analysis performed.
Sampling of Solids.
For gases and liquids, although both spatial and chronological variations
may occur, at a particular location and time most gas and liquid samples are
homogeneous and contain a large number of particles. This is not true in solid
sampling where inhomogeneity of the material, small variations in positions
and particle size can be highly significant. Generally sampling of solids
requires an even more rigorous and systematic approach than for gases and
liquids. It is found that sampling errors are inversely proportional to the
number of particles taken. The easiest way to sample a homogeneous material
is the grab sample. The whole of the material to be analysed is known as
the population or the lot. This may be a single, more or less homogeneo~s
mass or it may contain a number of individual units. From this is drawn the
gross sample or bulk sample. This is obtained by taking increments from
the lot in such a way that, when combined, they should be representative of
the lot. The analytical sample is taken from this prior to analysis.
Note that:
• Correct sampling of materials is important to
(i) obtain a representative portion of the material for analysis and
(ii) prevent the occurrence of accidents with sampling hazardous
materials.
• Sampling variance, V is inversely proportional to the number of
sampling increments (n), i.e., V = kIn where k is a constant dependent
on the size of the increment and variation within the bulk material.
• Sample types are:
(i) Bulk samples,
(ii) stratified (segregated) samples and
(iii) discrete units (packets, bottles etc.).
Bulk samples are those which may contain several components and
particle sizes but which do not have an observable boundary between
one part of the sample and another.
Sampling Statistics.
Errors in measurement are classified as determinate (systematic) errors,
which give a measure of accuracy and indeterminate (random) errors, which
are a measure of precision. For normal statistical manipulations it is often

assumed that the determinate errors are reduced at a low value and the
remaip.ing uncertainty in the result is due to random error that are quantified
by calculating the standard deviation. However, the standard deviation of a
method is composed of the standard deviation of each step in the analysis.
Hence while analysing real samples, the overall standard deviation So is
given by
so=--.jsl+s~ or vo=sl+s~ or Vo=(VA+VS)
where SA is the analytical standard deviation and Ss the sampling standard
deviation.
SA can be separated into two discrete components :
(i) Same-sample variation i.e., variation in instrument or signal
response for repetitive measurements of the same sample.
(m Different-sample variation is the within-lot variance and it
depends on the relative composition of the sample and the
homogeneity of the particles.
The sampling variation S S or between-sample variation measures
the variability between different parts of the population.
Note that it is better to take a large number of samples (to improve
sampling precision) and to analyse them with a method of moderate precision
than to use a high-precision technique on a small number of samples.
Composite samples can also greatly increase the overall precision of the
experiment under suitable circumstances.
Sampling plan should be prepared by considering
(i) size of the sample,
(ii) number of samples to be taken and
(iii) individual or composite sample.
Before analysis most samples are dried at 383 K to remove moisture.
WEIGHING THE SAMPLE

Chemical balance is used for weighing the sample. The material is
usually weighed in a weighing bottle which is stoppered and stored 'in a
desiccator. The bottle is weighed before and after the withdrawl, so the weight
of substance is obtained by difference. Weight burettes are used for work
demanding highest accuracy in transferring various quantities of liquids,
non-aqueous solutions or viscous liquids.
DISSOLVING THE SAMPLE

• Many inorganic substances can be dissolved directly in water or acids
(HCI, HNO g, H 2S04, HF, HCI04 ) but materials such as minerals,
refractories and alloys must be tested with a variety of reagents to
select a suitable solvent.
• Almost all acetates, arsenates, borates, carbonates, bicarbonates,
oxalates, chromates, phosphates and sulphides of cations are soluble
while sulphates and some halides are insoluble in water.
INTRODUCTION 19
• Some inorganic substances will not dissolve in acids, so fusion with

an acidic or basic flux in the molten state may be employed to render
them soluble.
• Most organic substances can be readily dissolved in a suitable organic
solvent and some are soluble in water or can be dissolved in aqueous
solutions of acids or alkalis.
SAMPLE DECOMPOSITION
Methods generally adopted for decomposing and dissolving an analytical
sample include:
1. Heating with strong acids or bases. 2. Fusion reagents (fluxes).
3. Microwave heating. 4. Combustion methods.
1. Decomposing Samples with Acids in Open Vessels.
Mineral acids are generally employed to decompose inorganic samples in
open vessel. The suspension is heated by flame, hot plate or heating mantle.
Following reagents may be used to dissolve difficult materials.
(i) Concentrated acids. Concentrated HCI dissolves many metals
(above H2 in the electrochemical series) and metallic oxides. Hot conc-
entrated HN03 dissolves most metals except Sb, Sn and W which are
converted to slightly soluble acids. Hot concentrated H 2S04 dissolves several
substances, many organic materials are charred and then oxidised by this
treatment.
(ii) Aqua regia (75 vol % HCI and 25 vol % HN0 3 • Due to its strong
oxidising character, it is a very potent solvent. The effectiveness of the acid is
increased by adding other oxidants such as Br2 and H20 2 .
(iii) Hydrofluoric acid. HF is used mainly to decompose silicates,
minerals and steel. Excess of HF is removed by evaporation with H 2S0 4
leaving a residue of metallic sulphates. Use HF with great care since it
causes painful skin burns.
(iv) Perchloric acid. HCI0 4 attacks stainless steels and a number of
iron alloys that do not dissolve in other acids. Hot concentrated HCI0 4 gives
explosive reactions with organic substances or easily oxidised inorganic
compound. Evaporation involving perchloric acid should be performed in a
fume cupboard free from combustible organic materials. For safety, the
compound should be treated first with concentrated nitric acid, the mixture
heated and then add small quantities of HCI0 4 until the oxidation is
complete. When 60 vol % HN0 3, 20 vol % HCI04 and 20 vol % H 2S04 is used,
the perchloric acid is also evaporated along with HN03 leaving a sulphuric
acid solution of the components for analysis. The organic part of the
material under investigation is destroyed and the process is called
wet ashing. Use perchloric acid with great care since it is dangerously
explosive.
2. Decomposing Inorganic Substances by Fusion Reagents (Fluxes).
Fusion reagents (alkali metal salts), commonly known as fluxes are used
to decompose sub!;!tances (e.g., mineral oxides, silicates and some iron alloys)
that are not soluble in normal solvehts or acids. Typical fluxes include
anhydrous Na2C03' either alone or mixed with KNOa or Na202, NaOH, KOH,
K 2S 20 7 (potassium pyrosulphate).
Procedure of Fusion of Fluxes.
To carry out the fusion, a layer of flux is placed at the bottom of the
crucible, then an intimate mixture of the flux and finely powdered substance
added. The crucible should be about half-filled and kept covered during the
whole process. The crucible is heated gradually at first and the temperature
slowly raised to the required temperature. The final temperature should not
be higher than is actually necessary.
When the fusion has been completed (30 to 60 minutes), the crucible is
gently rotated and tilted by tongs so that molten material distributes itself
around the walls of the container and solidifies as a thin layer. When cold,
the crucible is placed in a pyrex beaker, porcelain dish or platinum basin and
covered with water. Acid is added, if necessary. The vessel is covered with
clock glass and temperature is maintained to 368-373 K until solution is
achieved. Typical fluxes used to decompose inorganic materials are illustrated
in Table 2.
Types of Fluxes.
The flux employed will depend on the nature of the insoluble substance.
(i) Acidic fluxes such as pyroborates, pyrosulphates and acid fluorides
are used to decompose basic materials.
(ii) Basic fluxes (carbonates, hydroxides, peroxides, metaborates)
attack acidic materials.
Table 2. Typical fluxes.

Flux Melting point, K Crucible used Substance decomposed
Na2C03 1124 Pt Silicates, alumina, phosphates
and sulphates
N~C03+KN03 - Pt or Ni Samples containing S, Sb, As,
or Na202 Cr etc.
NaOH or 591 Au, Ni, Ag Silicates, SiC, minerals
KOH 653
LiB0 2 1123 Pt,Au Ceramics, silicates, slags
~S207 or 573 Pt HF and Th phosphates,
Na2S 207 slightly soluble oxides.
B20 3 850 Pt Silicates, oxides
Na202 Decomposes Fe, Ni Acid insoluble alloys of Fe, Ni,
Cr, Mo, W, Li, Pt alloys, Sn,
Cr, Zr minerals
CaC03 + NH4CI - Ni Silicates
INTRODUCTION 21
(iii) Oxidising fluxes (Na202 or Na2C03 mixed with KN0 3) are used
in oxidising medium.
Characteristics of some fluxes.
(i) Sodium carbonate. Sample containing silicates or refractory
material can be decomposed by Na2C03 at 1273 K. The cationic constituent of
the sample is cunverted to acid soluble carbonate and non-metallic constituent
to soluble sodium salts.
(ii) Lithium metaborate. Anhydrous LiB0 2 is especially suitable for
materials containing silica, when the resulting mass is dissolved in dilute
acids.
Advantages claimed for LiB02 •
• Fusion with lithium metaborate are usually quicker (15 min) and can
be performed at a lower temperatures than with other fluxes.
• The loss of Pt from the crucible is less with LiB02 fusion than with a
Na2C03 fusion.
• No gases are evolved during the fusion or dissolution of the melt. So
there is no loss of material due to spitting.
• Many elements can be determined directly in the acid solution of the
melt without the need for tedious separations.
3. Microwave Decomposition of the Sample.
Microwave digestion of the sample can be carried out by several devices.
(i) Atmospheric pressure vessels.
Atmospheric pressure vessel utilises open vessel system, has a focussed
microwave cavity and a tubing for the insertion and removal of reagents.
Since the reagents operates at atmospheric pressure so there is evolution of
gases during the digestion process.
(ii) Moderate pressure microwave vessels.
Microwave digestion vessels are constructed from teflon (m.p. 573 K) or
quartz or borosilicate glass vessels. A closed digestion vessel of teflon body
consists of a cap and a safety valve. It can operate at 120 ± 10 Psi pressure
to decompose silicates.
(iii) High pressure microwave vessels (Microwave bombs).
Many of the substances which require fusion treatment to render them
soluble will in fact dissolve in mineral acids if the digestion with acid is
carried out under high pressure and high temperature. Such drastic
treatment requires a container capable of withstanding requisite pressure
and also resistant to chemical attack. Such conditions are met in acid
digestion vessels (microwave bombs). Digestion vessels comprise a stainless
steel pressure vessel (50 cm3) with a screw-on lid and fitted with a teflon
liner. They can withstand pressures of 8-9 MPa (80-90 atm) and may be
heated to 453 K. Under these conditions, the refractory material can be easily
decomposed in about 40 minutes.
Advantages.
• Decomposition of the sample is time and cost saving.
• There is no need for expensive platinum ware.
• No losses can occur during the treatment.
• The resulting solution is free from the heavy loading of alkali metals.
(iv) Microwave ovens.
Microwave ovens are now being used extensively for decomposing
several compounds. In the oven, 12 moderate pressure vessels can be heated
at a time. The vessels can rotate through 360· so the average energy received
by each vessel is nearly the same. Decomposition is much rapid and drying
times for precipitates is greatly reduced. Further, the loss of volatile
components of the sample is virtually eliminated. Microwave decomposition
is often easy to automate thereby reducing operation times required to
prepare samples for analysis.
(v) Microwave furnaces.
Microwave furnaces consist of a small chamber to accomodate only a
crucible. The chamber is made up of SiC which is surrounded by quartz
insulation. The advantage of microwave furnace over muffle furnace is the
speed, at which the high temperature (1273 K) is reached within two minutes.
4. Decomposing Organic Compounds by Combustion Methods.
(i) Combustion in an open crucible (Dry ashing).
The organic sample is heated in an open crucible until all carbonaceous
matter has been oxidised, leaving a residue of inorganic components as oxide.
The residue is dissolved in dilute acid and the solution can be analysed by
suitable procedure. The technique is inapplicable when the inorganic
component is volatile.
(ii) Combustion-tube method.
In automated combustion tube analysers, the sample is ignited in a
stream of helium. Oxygen is passed over an oxidation catalyst consisting of a
mixture of silver vanadate and silver tungstate. Halogens and sulphur are
removed with a packing of silver salts. A packing of hot copper is placed at
the end of combustion train to remove 02 and to convert NOx to N 2. In this
method C, H, N can be determined quickly. The exit gases consisting of a
mixture of H 2 0, CO2 , N and helium is collected in a glass bulb.
(iii) Combustion with O2 in a sealed container (Schoniger method).
Analysis of organic compounds for elements (S, P or halogens) is
performed by combustion of organic material in 02; the inorganic constituents
are thus converted to forms which can be determined by titrimetric or
spectrophotometric methods.
Procedure. Schoniger flask, containing few em3 of absorbing solution
(e.g. aqueous NaOH), is filled with oxygen and sealed using the stopper with
the platinum basket attached (Fig. 1a). About 5-10 mg of sample is weighed
iNTRODUCTION 23
on to a shaped piece of paper (Fig. Ib)

which is folded in such a way that the
tail (wick) is free. This is then placed
in a platinum basket suspended from
the ground glass stopper of a 1 dm 3
flask. I
I
I
I
The wick of the sample paper ---}----:---

I I
I I
can be ignited by remote electrical ---t----~--
I I
I I
control or an infrared lamp. After
combustion, the flask is shaken for 2
minutes to ensure complete (a) (b)
absorption. Organic S present in Fig. 1. (a) Safety oxygen flask.
solution is converted to S03 and S02 (b) Paper shape for wrapping sample.
by the combustion, absorbed in H 20 2
and determined as sulphate.
The combustion products of organic halides are absorbed in NaOH
containing some H 20 2. Chlorides can be determined by argentimetric
potentiometric titration while bromides by mercurimetric titration.
Phosphorus from orthophosphate can be absorbed by either H 2S04 or
RN0 3 and determined spectrophotometric ally by molybdenum blue method.
STOICHIOMETRY
Analytical procedures may either be stoichiometric or non-stoichio-
metric.
1. Stoichiometric Methods.
Here the constituent whose amount is being measured (R A ) undergoes a
reaction with another substance (R B ).
RA +RB ---t PC+PD
The amount of desired constituent, R A can be calculated by applying the
law of definite and combining proportions, provided the amount of any of the
products (Pc or PD ) or of the reagent is known. Wet chemical methods
(gravimetric and volumetric), coulometric methods, gas analytical methods
and certain separation techniques are stoichiometric.
2. Non-stoichiometric Methods.
They can not be written as exact well-defined reactions. These methods
are based on the accurate measurement of a physical property which changes
in a proportion to the concentration of the desired constituent. Here it is only
necessary to calibrate the procedure. Most instrumental methods are
non-stoichiometric. Some examples are :
• Optical methods based on
(a) absorption of energy (UV and IR spectrometry) and
(b) emission of energy (flame photometry, fluorescence,
chemiluminescence ).
• Electrical methods, e.g., potentiometry, polarography, conductivity
etc.
• Other non-stoichiometric methods include mass spectrometry,

refractometry, polarimetry and activation analysis etc.
VOLUMETRIC GLASSWARES
Volumetric glasswares (flasks, burettes, pipettes, syringes) are used for
the measurement of volumes.
Volumetric Flasks. They are employed in making up standard
solutions to a given volume and for obtaining (with the aid of pipette), aliquot
portions of a sample solution to be analysed. The mark extends around the
neck. In order to avoid errors due to parallax, the lower edge of the
meniscus of the liquid should be tangential to the graduation mark and both
the front and back of the mark should be seen as a single line.
Pipettes.
(i) Transfer pipettes have one mark and deliver a constant volume
of liquid under certain specified conditions.
(ii) Graduated or measuring pipettes are employed to deliver various
small volumes as required.
(iii) Syring pipettes or micro pipettes have fixed or variable volume
and are used for dispensing large numbers of identical volumes
very quickly.
Use of transfer pipette. A suitable pipette filler is attached to the
upper end of the pipette (filling should never be carried out by mouth suction).
Rinse the pipette with a solution and discard it. It should then be filled with
the liquid to 2 cm above the mark. Any adhering liquid is removed by wiping
with a filter paper, the liquid is allowed to run out slowly until the bottom of
the meniscus just reaches the mark. The pipette must be held vertically and
with the mark at eye level. The liquid is then allowed to run into the receiving
vessel with the tip touching the wall of the vessel.
When the discharge has ceased, the jet is held in contact with the vessel
for a draining time of 15 s. The liquid in the jet of pipette must not be
removed by blowing.
Burettes.
Burettes are used to deliver fractional volumes of a solution of known
concentration which reacts stoichiometrically with another reactant. Burettes
should be clamped vertically in the holder. To deliver liquid from a burette in
a conical flask during titration, one hand is used to swirl the vessel and the
other is used to control the stopcock. To read the position of the meniscus,
the eye must be at the same level as the meniscus in order to avoid errors
due to parallax.
Piston burettes can provide automatic plotting of titration curves.
They also allow a variable rate of delivery as the end point is approached. So
there is no risk of over shooting.
GRAVIMETRIC TECHNIQUES
Gravimetric analysis employs preparation of the sample (described
earlier), precipitation, digestion, filtration, washing, drying, incineration and
weighing of the product.
INTRODUCTION 25
PRECIPITATION "'
Many methods exist for the precipitation of compounds. When the ionic
product exceeds the solubility product, the solution is supersaturated and
precipitation occurs.
Factors which Determine Successful Analysis by Precipitation.
1. The precipitate should be so insoluble that no appreciable loss occurs
when it is collected by filtration.
2. The precipitate must be coarse, dense, granular and can be readily
separated from the solution by filtration.
3. The precipitate must be convertible into a pure substance of definite
chemical composition.
Procedure for Efficient Precipitation.
1. Precipitation should be carried out in dilute solution, due regard
being paid to the solubility of the precipitate, time required for
filtration and the subsequent operations to be carried out with the
filtrate. This will minimise errors due to co-precipitation.
2. Precipitation is effected in hot solutions, provided the solubility and
stability of the precipitate permits. At higher temperature
(i) solubility is increased with a consequent reduction in the degree of
supersaturation. (ii) Coagulation is assisted' and sol formation
decreased. (iii) The velocity of crystallisation is increased thus
forming better crystals.
3. The precipitant is added slowly with constant stirring (without
splashing). A slight excess of the reagent should be added to take
advantage of common ion effect. Large excess may lead to complex
formation.
4. Mter the precipitate has settled, a few drops of the precipitant should
be added to determine whether further precipitation occurs.
5. Crystalline precipitates should be digested over night, except in those
cases where post-precipitation may occur. As a rule, digestion on the
steam bath is desirable. This process decreases the effect of
co-precipitation and yields readily filterable precipitates. Digestion
has little effect upon gelatinous or amorphous precipitates.
6. The precipitate should be washed with the appropriate dilute solution
of electrolyte. Pure water causes peptisation.
7. If the precipitate is contaminated by co-precipitated impurities, it can
be purified by reprecipitation.
8. Precipitation from a homogeneous solution is generally employed to
prevent supersaturation.
9. Precipitates so formed may be crystalline (e.g., BaS04), curdy (AgCl),
gelatinous [(Fe(OHhl or flocculent.
Co-precipitation.
The contamination of precipitate by substances that are normally
soluble under the conditions of precipitation is called co-precipitation. Thus
the soluble impurities become incorporated into the crystal lattice of the
precipitate during its formation. Co-precipitation may be due to isomorphous

replacement or adsorption followed by entrapment or occlusion (that is,
trapping of foreign ions and solvent within the crystal).
Co-precipitation occurs by :
(i) Adsorption at the surface of precipitate. Surface adsorption will
be maximum for gelatinous precipitates and minimum for those of
pronounced macrocrystalline character. Precipitates with ionic lattices appear
to conform to the Paneth-Fajans-Hahn adsorption rule, which states that
the ion that is most strongly adsorbed by an ionic substance (crystal lattice) is
that ion which forms the least soluble salt. AgI adsorbs silver acetate much
more strongly that it does AgN0 3 since the former is less soluble. The
deformability of the adsorbed ions and electrolytic dissociation of the adsorbed
compound have a considerable influence; the smaller the dissociation of the
compound, the greater is the adsorption. Thus H 2S, a weak electrolyte, is
strongly adsorbed by metallic sulphides.
(ii) Occlusion of foreign substance during the process of crystal
growth from the primary particles. The extent of occlusion by adsorption
and entrapment is affected by the solubility of the entrapped substance. Here
the impurities fit in the crystal lattice of the precipitate and it is occluded in
the form of mixed crystal or a solid solution (BaS04 + PbS04; BaS04 + BaCr04).
If the impurity is isomorphous or forms a solid solution with the precipitate,
the amount of co-precipitation may be very large, since there will be no
tendency for elimination during the aging process.
Fractional Precipitation.
Fractional precipitation occurs when a precipitating agent is added to a
solution containing two anions, both of which form slightly soluble salts with
the same cation.
Post-precipitation.
Post-precipitation occurs on the surface of the first precipitate after its
formation. It occurs with sparingly soluble substances which form
supersaturated solution; they have an ion in common with the primary
precipitate. In the precipitation of calcium as oxalate in the presence of
magnesium, the magnesium oxalate separates out gradually upon the calcium
oxalate. The longer the precipitate is allowed to stand in contact with the
solution, the greater is the error due to this cause.
DIGESTION (AGEING)
The precipitate is allowed to stand at room temperature or at a flame,
hot plate or on a water bath. Digestion has little effect upon amorphous or
gelatinous precipitates. The net result of digestion is to reduce the extent of
co-precipitation and to increase the size of particle, rendering filtration easier.
FILTRATION
Filtration involves the isolation of the precipitate from the mother liquor.
The object is to get the precipitate and the filtering medium quantitatively
free from the solution. The systems used for filtration include
INTRODUCTION 27
(i) filter paper (ii) crucibles (iii) filter pulp mats

(iv) asbestos (v) filter membranes etc.
The choice of filtering medium will be controlled by the nature of the
precipitate.
(i) Filter Papers.
Filter papers are available in various degrees of porosity. The texture of
the filter paper must allow rapid filtration while retaining the smallest
particles of precipitate. Whatman filter paper (ash less) is available in three
grades.
Qualitative grade. Whatman filter paper No.1, known as qualitative
grade filter paper, is used to filter precipitates of average fineness. Whatman
No.3 and No.4 filter papers are used to filter very fine and gelatinous
precipitates respectively.
Semi-quantitative grade. Whatman No. 30 and No. 31 filter papers
are used to separate gelatinous and fine precipitates from the solution. The
ash of an 11 cm circle of these papers is 0·00003 g.
Quantitative grade. Quantitative filter papers (Whatman Nos. 40, 41,
42) have small ash content; this is achieved during manufacture by washing
with HCI and HF acids. The sizes generally chosen are circles of 7·0, g·O, 11·0
and 12·5 cm diameter. The ash of an 11 cm circle should not exceed 0·0001 g.
Three textures of filter papers are generally used, one for very fine
precipitates, one for medium size precipitates and one for gelatinous
precipitates and coarse particles.
Hardened filter papers. These papers are made by further treatment
of quantitative filter papers with acid. They have an extremely small ash,
much greater mechanical strength when wet and are more resistant to acids
and alkalis.
Table 3. Characteristics for Whatman series of hardened

filter papers (ash 0.008%).
Filter paper Hardened ashless
Number 540 541 542
Speed Medium Fast Slow
Particle size retention Medium Coarse Fine
The size of the filter paper selected for a particular operation is

determined by the bulk of precipitate and not by the volume of the liquid to
be filtered. Other types of filter papers are Munktels (Swedish paper), Carl
Schiecher and Schull (German paper).
Procedure.
To carry out rapid filtration, the funnel containing the properly fitted
(making 60· angle) fluted filter paper is placed in a funnel. The paper is
moistened with water and pressed down tightly to the sides. A clean beaker
is placed so that the stem of the funnel just touches the side; this will prevent
splashing. The liquid to be filtered is then poured down a glass rod into the
filter paper. The lower end of the stirring rod should be very close to the filter
paper but not quite touching on the side. The paper is never filled completely
with the solution. The last traces of the precipitate in the beaker should be
transferred to the funnel by holding the glass rod across the beaker, tilting it
and directing a jet of water from a wash bottle.
Filtration by suction is rarely necessary with gelatinous and fine
precipitates. The suction will draw the particles into the pores of the paper
and the speed of filtration is reduced.
(ii) Crucibles.
The precipitate may react with the cellulose of the filter paper so it is
convenient to collect the precipitate in a crucible for direct weighing.
Following types of crucibles are employed.
Gooch crucibles. A mat of asbestos (filtering medium) is placed on a
perforated porcelain plate (witt plate) in Gooch crucible.
Sintered glass crucibles. These crucibles are made of resistance glass
and have a porous disc of sintered ground glass fused into the body of the
crucible. The filter d~sc is made in varying porosities, zero is the coarset and
five the finest. The range of pore diameter for various grades is as follows.
Porosity 0 1 2 3 4 5
Pore diameter (J.UU) 200-250 100-120 40-50 20-30 5-10 1-2
Depending upon the pore size these crucibles are graded as G-l, G-2, G-3
etc. Porosity 4 (or G-4, G is a kind of glass) is suitable for very fine
precipitates (such as BaS04), porosity 3 for precipitates of medium particle
size (such as AgCl).
Advantages.
• Sintered glass crucibles are resistant to most reagents except HF and
hot concentrated acids.
• These crucibles can be heated to 773 K with gradual heating. They
can be readily cleaned and dried at 373 K
Porcelain crucibles. Porcelain crucibles are often used for igniting
precipitates and heating small quantities of solids because of their cheapness
and ability to withstand high temperatures. Some reactions, such as fusion
with Na2C03 and evaporation with HF cannot be carried out in porcelain
crucibles due to resultant chemical attack. A slight attack also occurs with
pyrosulphate fusions.
Fused-silica crucible is resistant to heat shocks because of its very
small coefficient of expansion. It is not attacked by acids at a high
temperature except HF and H 3P04.
However, fused-silica crucible is attacked by fused alkalis and
carbonates. It is more brittle than ordinary glass and requires longer time for
heating and cooling than platinum crucible.
Platinum crucible. Crucibles made of 95% Pt and 5% Au (non-wetting
alloy) are used in preparing samples for X-ray fluorescence investigation.
INTRODUCTION 29
Fusion samples are readily removed from these crucibles. At high

temperatures platinum permits the flame gases to diffuse through it and this
may cause reduction of some substances. Hence if a covered crucible is heated
by a gas flame, there is reducing atmosphere in the crucible. But with an open
crucible, diffusion into the air is so rapid that this effect is not appreciable.
Platinum is attacked by heating with aqua regia, HCl, H3P04 and liquid
mixtures which evolve Br2 or 12 as well as fusion with oxides, peroxide,
nitrates, sulphides, cyanide, molten Pb, Ag, Cu, Zn, Bi, Sn, Au, Sb, Si and
volatile halides.
Solid carbon produced creates hazard. It may be burnt off at low
temperatures with free access to air, without harm to the crucible. Ashing in
presence of carbonaceous matter should riot be conducted in platinum
crucible, since metallic elements which may be present will attack platinum
under reducing conditions. Beside above crucibles, other crucibles of Ni, Ag
and AI may also be employed.
How to Use Crucible for Filtration.
A new crucible should be washed Venledca P
with concentrated HCl and then with
distilled water. A hot 0.1 M solution of
the tetrasodium salt of EDTA is an
excellent solvent for dissolving
precipitates (except sulphides) from the
porous plate fitted in the crucible. Then
S
the dried and weighed crucible is
supported in an adoptor. The crucible is
half-filled with the solution, suction is
applied and the precipitate is transferred Chamber
slowly for filtration. With both sintered
glass and porous-base crucibles, avoid
filtering materials that may clog the
filter plate.
Buchner funnel. Large
quantities of material are filtered
through a Buchner funnel. One of the Removable
disadvantages of porcelain Buchner ~25mmfilter
funnel is that, the filter plate cannot be
removed for thorough cleaning. In a
modern polythene version, the funnel Outlet
can be unscrewed for cleaning both sides
of the plate. Special disposable filter
Collar
funnels (Fig. 2) are particularly useful in
filtering small quantities of radioactive
materials and proteins. Fig. 2. Disposable filter funnel.
(iii) Filter Pulp.

Filter pulp is available in moist form as small pellets. The colloidal
hydrated oxides, gelatinous silica and finely divided barium sulphate can be
filtered using filter pulp with the precipitate.
(iv) Synthetic Filter Membrane.

These membranes have uniform microporous screens. For many aqueous
solutions, a cellulose acetate membrane (CAM) or glass fibre-polypropylene
membrane can be used but for non-aqueous or aggressive solutions, the
membrane and its housing can be of PTFE or polysulphone plastics.
Centrifuge. Separation of mother liquor from recrystallised salts,
collection of precipitates (that are difficult to filter) and decantation is better
achieved by using a centrifuge.
Factors on which filtration depends.
(i) Size of the particles to be filtered.
(ii) Chemical reactivity of the particles.
(iii) Purpose of filtration.
(iv) Settled precipitate should be filtered to accelerate filtration.
WASHING
The object of washing is to free the precipitate from the dissolved
substances or absorbed ions with the minimum amount of washing liquid and
to reduce the loss of precipitate by solution. The washing solution should have
a minimum solvent effect on the precipitate and should not help in peptizing
it even after electrolytic ions have been washed off.
It is better to wash with a number of small portions of the washing
liquid, which are well drained between each washing, than with one or two
large portions or by adding fresh portions of the washing liquid while solution'
still remains on the filter paper. An ideal washing liquid should possess
the following qualities.
• It should have no solvent action on the precipitate but dissolve foreign
substances easily.
• It should have no dispersive action on the precipitate.
• It should form no insoluble product with the precipitate.
• It should be easily volatile at the drying temperature of the precipitate.
• It should not contain any substance which may interfere with
subsequent determination in the filtrate.
In general, pure water should not be used unless it is certain that it will
not dissolve appreciable amounts of the precipitate. If the precipitate is
soluble in water, a common ion is usually added, since any electrolyte is less
soluble in a dilute solution containing one of its ions than it is in pure water.
Categories of Wash Solutions.
(i) Solutions which prevent the precipitate from becoming
colloidal and passing through the filter..Ammonium nitrate solution is used
for washing iron (III) hydroxide and 1% HN03 for washing AgCl.
(ii) Solutions which reduce the solubility of the precipitate. The
wash solution containing a common ion with the precipitate may reduce the
solubility of the precipitate.
(iii) Solutions which prevent the hydrolysis of salts of weak acids
and bases.The addition of an acid to the wash solution prevents the
INTRODUCTION 31
hydrolysis of ferric or similar salts. Some precipitates tend to oxidise during

washing, then the precipitate cannot be allowed to run dry. A special solution
which reconverts the oxidised compound into original condition must be
employed, e.g., acidified H 2S water is used for CuS. Gelatinous precipitates
like Al(OH)3 require more washing than crystalline precipitates such as
CaC 2 0 4·
• If the precipitate settles rapidly, washing by decantation may be
carried out.
• The main bulk. of the precipitate is first transferred by mixing with
the wash solution and pouring off the suspension. Precipitate
adhering to the sides of the beaker is transferred to the filter paper
with the aid of a wash bottle using a policeman.
• Completeness of the washing can be tested by applying an appropriate
qualitative test.
DRYING AND IGNITING PRECIPITATES

The choice of drying or igniting depends on the temperature to which the
precipitate is heated. Drying applies when the temperature is below 523 K
and ignition applies from 523 K to 1473 K. Precipitates for drying should be
collected on filter paper or in sintered glass or porcelain filtering crucibles.
Precipitates for ignition are collected on filter paper, porcelain or silica
crucibles.
• Ignition is simply effected by placing in a special ignition dish and
heating with the appropriate burner.
• Crucibles may be placed in a thermostatically controlled electric
drying oven or muffle furnace equipped with a pyrometer.
• Thermogravimetry gives information on the temperature range to
which a precipitate should be heated for a particular composition.
INCINERATION
The well drained filter paper and precipitate are carefully detached from
the funnel, folded and ignited in the weighed crucible to a constant weight.
In case the precipitate (e.g., Filter
BaS04, PbS04, Bi20 3, CuO) is
reduced by the burning paper
or decomposed on strong
heating, the filter paper is
separately incinerated. The
precipitate from the filter
paper is carefully transferred
to the watch glass placed over
a glazed paper and covered
with an inverted funnel Fig. 3. Incineration of the filter paper.
(Fig. 3).
The filter paper is folded several times to make it like a cone and is held
just above the crucible by a clean tongs. It is burnt with a non luminous
bunsen flame. The ash is collected in a weighed crucible on a glazed paper.
Particles falling on the crucible are collected with the help of a feather and
transferred to crucible. The crucible is heated strongly so as to burn all carbon
to a white ash. It is cooled in the desiccator. The main part of the precipitate
is transferred to the crucible. Finally, the precipitate is brought to constant
weight of heating to the necessary temperature.
COOLING AND DRYING IN DESICCATOR

The crucible is allowed to cool in desiccator. A desiccator is a covered
glass container containing a drying agent such as anhydrous CaCl2 or
CaS04, P20S, silica gel, activated alumina etc. The relative efficiencies of
various drying agents (and residual water per dm 3 of air in mg) is :
CaCl2 (1.5), NaOH sticks (0.8), H 2S04 (0.3), silica gel (0.03), KOH sticks
(0.014), A1 20 3 (0.005), CaS04 (0.005), molecular sieve (0.004).
Mg(CI0 4)2 (0.002), P 2 0 5 (0.00002).
Action of desiccants. It can be considered from two view points.
(i) The amount of moisture that remains in the closed space of
desiccator is related to the vapour pressure of inexhausted
desiccant that is, the vapour pressure measures the extent to which
the desiccant can remove moisture.
(ii) A second factor is the weight of water that can be removed per unit
weight of desiccant, i.e., the drying capacity.
Note that:
• A substance cannot be dried by a desiccant which has a vapour
pressure greater than the vapour pressure of the substance itself. In
general, substances that form hydrates have higher vapour pressures
but they have also greater drying capacities.
• When a hot crucible is placed in a desiccator, about 5s should elapse
for the air to become heated and expand before putting the cover.
When reopening, the cover should be slided gradually in order to
prevent any sudden inrush of air, which might blow material out of
the crucible. Air rushes in to fill the partial vacuum created when the
expanded gas content of the desiccator cools down.
• A vacuum desiccator (Fig. 4) is used for drying large quantities of
solids. These desiccators, made
of glass, plastics or metal, are
designed to withstand reduced
pressure. The desiccator may be
evacuated, so drying is much
more rapid than the ordinary
type. Evacuate the desiccator
when it is surrounded by an
adequate guard in the form of Fig. 4. Vacuum desiccators.
a stout wire cage.
WEIGHING OF THE PRODUCT

The operation of heating, cooling and weighing the crucible is repeated
till a constant weight of the product is obtained.
INTRODUCTION 33
ANALYTICAL BALANCE
Weighing is an essential part of chemical analysis, both for measuring
the sample and for preparing standard solutions. Standard laboratory
weighings are typically made to three or four significant figures so the
weighing device must be accurate and sensitive. There are various
sophisticated ways of achieving this, but the most accurate and versatile
device for measuring mass is analytical balance.
Analyst actually deals with masses rather than weights. The weight (w)
of an object is the force of attraction due to gravity that is exerted upon the
object, i.e.,
w=mg
where m is the mass and g is the acceleration due to gravity. Since the
gravitational attraction varies with altitude, the weight of the object is
variable. Mass is the quantity of matter of which the object is composed and
is invariant.
Recently the design of analytical balance has been fundamentally
altered and the traditional free-swinging, equal arm, two pan chemical
balance together with its box of weights is uncommon.
TYPES OF ANALYTICAL BALANCES

1. Single-Pan Mechanical Balance. ~ L1 ----l-*141---L2- - . J
Principle. The mechanical balance 8 C
is a first class lever that compares two
masses Ml (unknown mass) and M2
(known mass). The fulcrum A lies between
Fig. 5. Principle of
the points of application of forces B and C
analytical balance.
(Fig. 5).
The principle of operation is based on the fact that at balance
Ml Ll = M2 L2· If Ll and L2 are constructed to nearly equal, then, at balance
M 1 =M2· A pointer is placed on the beam to indicate the state of balance.
When the balance is unloaded, adjust the value of M2 until the pointer
returns to its original position on the scale. Since the ratio of masses is the
same as the ratio of weights in equal arm balance so the known masses are
called standard weights.
Operation of Single-Pan Balance. A first class lever
(unsymmetrical) is pivoted on a knife edge. A single pan is situated at one end
on which the object is placed. When the balance is not in use, a series of
weights (160 g to 200 g) are counter balanced by a single weight on the other
end of the beam which also acts as a part of damping piston (Fig. 6).
When an object is placed on the pan, individual weights are removed
from the end of the beam to restore it to equilibrium. This is achieved by
means of knobs in front of the balance that lift weights from the beam. These
weights will be equal to the weight of the objects on the pan. Actually, the
beam is not brought completely to balance but weights (whole gram or 0.1 g)
are removed.
Weight control
knobs
Damping
counter weight
Pan arrest
Pan
release
Fig. 6. Schematic diagram for a typical single pan balance.

The imbalance of the beam is registered optically and
automatically on an illuminated vernier scale by a light ray reflected from
an engraved optical scale on the beam. The imbalance may be read on a
digital counter. The last digit (0.1 mg) are read from the scale. Sensitivity of
the balance varies with the load because it is governed by the centre of
gravity of the beam. Calibration of the vernier or digital readout to read the
amount of imbalance is done at a given
sensitivity, that is, at a given load. So the load
must remain constant.
The original no-load reading is known as
the zero point and the position under load is
called the rest point. In operation, the rest point
is made to coincide with the zero point. The zero
point (to read zero) IS adjusted by vernier by
means of a knob.
All weights of a single-pan balance are
removed by means of control knobs on the front
of the balance. The weights removed are
registered on a counter on the front of the
balance. The beam is brought to rest rapidly with
the help of air piston damper. Fig. 7. Typical single pan
balance.
INTRODUCTION 35
A three-position beam arrest knob is used to protect the knife edges and
beam. The centre position arrests the pan and beam, a second position
releases the pan for use and third position completely releases the pan to
allow the balance to come to rest. From a typical single-pan balance (Fig. 7).
weighings can be made in less than a minute. Mechanical balances are being
replaced by electronic balances but many are still in use.
2. Electronic Balances.
Modern electronic balances provide convenience in weighing and are free
from mechanical failure and sensitivity to vibration. It eliminates the
operation of dailing weights, smooth release of balance beam and pan support,
turning and reading micrometers etc.
Operating Principle of an Electronic Balance.
Electronic balances operate by applying an electromagnetic restoring
force to the support for the balance pan. The pan rests on the arm of a movable
hanger whose position is monitored by an electrical position scanner (Fig. 8).
Position
scanner
Movable ha:ng~e:r3~U~[i~;~~~L___
Coil Temperature sensor
Fig. 8. Operating principle of electronic balance.
When the sample is placed on the pan, the resultant displacement of the
support is cancelled out. The magnitude of restoring force is governed by the
value of current flowing in the coils of the electromagnetic compensation
system. This compensation current is proportional to the weight placed on the
pan. A microprocessor converts the value of
current to the corresponding weight value (in
grams) which appears as a digital display.
Electronic balances incorporate a tary facility
where the weight of the container can be
automatically subtracted.
Working. A single control bar is used to
switch the balance on and off, to set the display
to zero and to tare a container automatically on
the pan. The majority of balances incorporate a
self-testing system and a built-in weight
calibration system. These balances can be
coupled to a printer which gives a printed record
ofthe weight. A digital-display electronic balance
is shown in Fig. 9. Fig. 9. Electronic balance.
Since results are available as an electrical signal, they can be processed

by a computer and stored. Weighing statistics can be automatically calculated.
Weight Ranges of Electronic Balances.
Electronic balances cover the four weight ranges.
(i) Macrobalance weighs up to 160 g and reading to 0.1 mg.
(ii) Semimicrobalance weighs up to 30 g and reading to 0.01 mg.
(iii) Microbalance is sensitive up to 20 g and reading to 1 Ilg.
(iv) Ultramicrobalance is sensitive up to 5 g and reading to 0.1Ilg.
3. Electrochemical Quartz Balance.
Electrochemical balance utilizes a thin quartz crystal disk oscillating at
10 MHz. The frequency of oscillation changes with any change in mass and
the frequency change measured by the instrument is converted to mass units.
A film of gold is evaporated on the quartz and the gold substrate can be coated
with the sample of interest.
These highly sensitive balances are available with 100 Ilg range that
can detect 1 Ilg (10-9 g) changes.
TECHNIQUES OF WEIGHING USING ANALYTICAL BALANCE

• Keep the balance clean. Remove dust from the pan and pan floor with
a camel hair brush.
• Never exceed the stated maximum load of the balance.
• Objects to be weighed should always be handled with tongs or a loop
of clean paper.
• Objects to be weighed should be allowed to attain the temperature of
the balance before weighing. If the object has been heated, sufficient
time (30 to 40 min) must be given for cooling.
• Substances must be weighed in suitable containers such as weighing
bottles, crucibles, watch glasses or beakers.
• Liquids and volatile or hygroscopic solids should be weighed in
stoppered weighing bottles. Avoid exposing the balance to corrosive
atmosphere.
• Addition of chemicals to the receptacle must be done outside the
balance case.
• Place the object on the pan when the balance is at rest and close the
pan compartment.
• Set the on-off control of the balance to the on position, observe the
value shown on the digital display and record it.
• If the balance is linked to the printer, confirm that the printed result
agrees with the digital display. Return the control to the off position.
• When all weighings have been completed, remove the object, clear up
any accidental spillage and close the pan compartment.
WEIGHING ERRORS
Inspite of careful weighing, some errors may persist due to following
causes.
1. Defective balance.
INTRODUCTION 37
2. Changes in the weight of weighing vessel.

3. Buoyancy effects.
4. Errors in recording the weights.
1. Defective Balance.
Weighing errors may arise due to defective balance construction and
weights, chipped knife edges, magnetic dumping and dust etc.
2. Changes in the Weight of Weighing Vessel.
Changes in the weight of weighing vessel may be due to
(i) absorption or loss of moisture,
(ii) electrification of the surface caused by rubbing,
(iii) difference in temperature between the weighing vessel and the
balance case.
• These errors may be removed by wiping the vessel gently with a
linen cloth and allowing it to stand for 30 minutes in proximity to the
balance before weighing.
• The electrification is slowly dissipated on standing. It can also be
eliminated by subjecting the vessel to the discharge from an antistatic
gun.
• Substances which have been heated in an oven or ignited in a crucible
are allowed to cool in desiccator. Volatile, hygroscopic and efforescent
substances must be weighed in tightly closed vessels.
3. Buoyancy Effects on the Object Container and Weights.
• Buoyancy occurs when an object is immersed in a liquid and its actual
weight is diminished by the liquid it displaces.
• If the object and the weights have the same density, hence the same
volume, then no errors will be introduced.
• If the substance has a lower density than the weights, the substance
will displace a greater volume of air than the weights. It will therefore
weigh less in air than in vacuum. Conversely, if a heavier material
(Pt) is weighed, the weight in a vacuum will be less than the apparent
weight in air.
4. Errors in, Recording the Weights.
Many weighing errors are due to mistakes made in checking the weights
on the balance pan or in reading the digital display on electronic balances.
The accurate reading of weights is best carried out by checking the weights
as they are added and then removed from the pan. Any digital display should
be carefully read at least twice.
CLEANING OF GLASS WARES

Glass wares must be perfectly clean and free from grease to obtain the
reliable results. To test the cleanliness the glass ware is filled with distilled
water and then water withdrawn, only an unbroken film of water remains
there. If the water collects in drops, the apparatus is dirty and must be
cleaned.
Methods for Cleaning Glass Wares.

1. Detergent Teepol. Teepol, a mild and inexpensive detergent, may
be used for cleaning glass apparatus. For cleaning burette, 2 cm3 of 10% stock
solution diluted with 50 cm3 of distilled water is poured into the burette and
allowed to stand for 30 s. The detergent is then run off and burette is rinsed
three times with distilled water. A pipette may be similarly cleaned using
1 cm 3 of the stock solution dHuted with 30 cm3 of deionised water.
2. Decon 90. Decon 90 is claimed to be especially effective in removing
contaminations due to radioactive nuclides.
3. Chromic acid Cleaning Mixture. Fill the apparatus carefully
with chromic acid (a saturated solution of sodium or potassium dichromate in
concentrated H2S04) and allow to stand overnight. Sodium dichromate is
usually preferred because it is much cheaper and more soluble (70 g per
dm 3) than potassium dichromate (5 g per dm\
Chromic acid is poured off, the apparatus is rinsed with deionised water
and allowed to dry. Sodium dichromate and sulphuric acid mixture should be
filtered through glass wool to remove sludge which may block the tips of
burettes and pipettes.
4. Mixture of H 2S04 and HNOa. This fuming mixture is used when
the vessel is very greasy. Handle this mixture with great care.
5. Mixture of KOH and Spirit. This extremely effective degreasing
agent, acting much quicker than the cleaning mixture, is prepared by
dissolving 100 g of KOH in 50 cm3 of water and after cooling making up to
1 dm 3 with methylated spirit. Use it with great care.
CALIBRATION OF GLASS WARES
Procedure. The calibration procedure involves determining the weight
of water contained in or delivered by a particular apparatus. The temperature
of water is observed and from the known density of water at that
temperature, the volume of water can be calculated. Buoyancy of air affects
the observed weight of low density objects. Table 4 lists calculated volumes
for 1 g of water in air, corrected for buoyancy with stainless steel weights of
density 7.8 g/cm3 ,
Table 4. Volume of 1 g of water at various temperatures.

Temp. Volume Temp. Volume Temp. Volume Temp. Volume
(K) (em3) (K) (em3 ) (K) (em3 ) (K) (em3 )
283 1·0013 289 1·0021 295 1·0033 301 1·0047
285 1·0015 291 1·0023 297 1·0037 302 1·0051
287 1·0017 293 1·0027 299 1·0044 303 1·0053
In all calibration operations the apparatus to be calibrated must be

carefully cleaned and placed adjacent to the balance which is to be employed,
together with a supply of distilled water, so that they assume the temperature
of the room. Flasks should be dried by rinsing with a little acetone and then
blowing a current of air to remove acetone.
INTRODUCTION 39
Volumetric Flask Calibration.

Weigh the clean, dry stoppered flask. Fill it to the mark with distilled
water. There should be no droplets on the neck. The flask and water should
be equilibrated to room temperature. Weigh the filled flask and record the
temperature of water to 273.01 K The increase in weight represents the
weight of water contained by the flask.
Pipette Calibration.
The pipette is filled with the distilled water (placed in the balance room
for an hour) slightly above the mark. Water is run out until the meniscus is
exactly on the mark. The drop adhering to the jet is removed by bringing the
surface of water contained in a beaker in contact with the jet. Deliver the
water from the pipette into a clean, weighed stoppered flask holding the jet
of pipette in contact with the side of the vessel. The pipette is allowed to drain
for 15 s after the outflow has ceased. Reweigh the flask, note the temperature
of water and calculate the capacity of pipette with the aid of Table 4.
Burette Calibration.
It is essential to check leakage and delivery time before calibration.
Test for leakage. The stopcock is cleaned and assembled in the
burette. The burette is placed in the holder, filled with deionised water,
adjusted to zero mark and any drop of water adhering to the jet removed by
filter paper. The burette is allowed to stand for 20 minutes. If the meniscus
has not fallen by more than one scale division, the burette may be considered
satisfactory.
Test for delivery time. Separate the components of the stopcock, dry
grease and reassemble. Fill the burette to zero mark with deionised water and
place in the holder. Open the stopcock fully and note the time taken for the
meniscus to reach the lowest graduation mark of the burette. It should agree
with the time marked on the burette.
For burette calibration, fill the burette with distilled water. Weigh a
clean, dry stoppered flask. Adjust the burette to zero mark and remove any
drop adhering to the jet. Place the flask under the jet, open the stopcock fully
and allow water to flow into the flask.
As the meniscus approaches the desired calibration point on the burette,
reduce the rate of flow and adjust the meniscus to the required mark.
Restopper and reweigh the flask. Repeat this procedure for each graduation
(every 5 cm3 ) to be tested. Note the temperature ofthe water. Using Table 4,
the volume delivered at each point is calculated from the weight of water
collected.
o
2
ERRORS AND EVALUATION
INTRODUCTION
The function of analyst is to obtain a result as near to the true value as
possible by the correct application of the analytical procedure employed.
Quantitative analysis is not simply a case of taking a sample, carrying out a
single determination and then claiming that the value obtained is irrefutable.
It requires a sound knowledge of the chemistry involved, of the possibilities
of interferences from other ions, elements and compounds as well as of the
statistical distribution of values. In chemical analysis, when numerical data
and numerical results are measured with the maximum accuracy that the
instrument, method and analyst are capable of, it has been observed that the
results of successive determination differ among themselves to some extent.
The values obtained may be correct within the possible limits of
measurements. The average value of a series of measurements is accepted as
the most probable value. The reliability of the result depends upon the
magnitude of the difference between the average value and the true value.
TYPES AND SOURCES OF ERRORS

The errors, which affect an experimental result may conveniently be
divided into following categories :
1. Systematic, Determinate or Constant Errors.
These are the errors which can be avoided, or whose magnitude can be
determined and the measurements rectified. A determinate error is
characterised by the fact that it affects to the same degree the results of a
series of determination. Systematic errors can be further classified depending
upon the system measured, observer and the instrument.
(A) Personal Errors.
These errors are due to factors for which the individual analyst is
responsible' and are not connected with the method or procedure. Personal
errors may arise from the constitutional inability of an individual to make
certain observations accurately. Some persons are unable to judge colour
changes sharply in visual titrations, which may result in a slight overstepping
of the end point. Other examples of personal errors are :
(i) Mechanical loss of material in various steps of an analysis.
(ii) Errors in reading a burette.
(iii) Improper washing of a precipitate.
(iv) Insufficient cooling of crucible before weighing.
(v) Using impure reagents.
(40)
ERRORS AND EVALUATION 41
(vi) Ignition of precipitate at incorrect temperatures.

(vii) Allowing hygroscopic materials to absorb moisture before or during
weighing.
(viii) Failure to apply buoyancy corrections when required.
(ix) Errors in calculations.
(8) Operational Errors.
These errors are mostly physical III nature and occur when sound
analytical technique is not followed, e.g., non-representative sampling,
introduction of foreign material in a sample. At times distinction between
methodic and an operative error will be only arbitrary, as with errors
resulting from the solubility of precipitates, in which the difference is of
degree and not of kind.
(e) Instrumental and Reagent Errors.
These arise :
(i) From the faulty construction of balances.
(ii) Use of improperly calibrated weights.
(iii) Graduated glassware and other instruments.
(iv) Attack of reagents upon glasswares resulting in the introduction of
foreign materials.
(v) Volatilisation of platinum at very high temperatures.
(vi) Use of reagents containing impurities.
(D) Methodic Errors.
These errors originate from incorrect sampling and incompleteness of a
reaction. These are the most serious errors because often they are difficult to
detect. Examples include wrong standardisation of a pH meter, background
absorption in atomic absorption spectroscopy, faulty detector response in
chromatographic and spectroscopic methods. In gravimetric analysis errors
may arise owing to co-precipitation and post precipitation, decomposition or
volatilisation of weighing forms on ignition, weighing of the hygroscopic
substances, weighing imperfectly cooled substances, solubility of the
precipitate in presence of the excess of the precipitant or washing liquid and
precipitation of substances other than the intended ones or the formation of
colloidal precipitates.
In titrimetric analysis errors may occur owing to :
(i) Failure of reactions to proceed to completion.
(ii) Occurrence of 'induced and side reactions.
(iii) Reaction of substances other than the constituent being determined.
(iv) Difference between the observed end point and the stoichiometric
equivalence point of a reaction.
Errors in redox titration may be due to redox potential, rate of
establishment of equilibrium, pH, presence of certain catalysts and
non-reversibility of redox indicators. Errors in complexometric titrations
depend upon the process used to detect the end point. Here the relative error
is obtained from the formula, e = (p - 1) x 100% where p is the ratio of metal
concentration. Possible causes of errors in electrometric titrations are the
slow potential stabilization, undesirable electrode polarisation, side reactions

occurring at electrode surface. In precipitation titrations, the main cause
of error is the solubility of the precipitate. The excess indicator used as a
precipitant also introduces error. For example, in Mohr's method, a large
excess of K2Cr04 forms Ag2Cr04 before the equivalence point has been
reached.
(E) Additive and Proportional Errors.
The absolute value of an additive error is independent of the amount of
the constituent present in the determination. Loss in weight of a crucible in
which a precipitate is ignited and errors in weight are the well known
examples of additive errors. The presence of this error is revealed by taking
samples of different weights. The absolute value of a proportional error
depends upon the amount of the constituent. Proportional error may arise
from an impurity in a standard substance which leads to an incorrect value
for the molarity of a standard solution. Other proportional errors may not
vary linearly with the amount of the constituent but exhibit an increase with
the amount of constituent present. When Na2S04 is coprecipitated with
BaS04 its amount increases with the amount of BaS04 in direct ratio. One
example is the ignition of Al 20 3. At 1473 K the aluminium oxide is anhydrous
and non- hygroscopic. Ignition of various weights at an appreciably lower
temperature will show a proportional type of error.
2. Random or Indeterminate Errors.
These errors are accidental and so intangible over which the analyst has
no control. These errors are revealed by the small differences in the
successive values of a measured quantity when the measurements are made
by the same analyst. Random errors can be divided into two classes.
(A) Variations within Determinate Errors. These errors can not be
prevented from variation, e.g., in igniting a precipitate of AI(OH)3 to constant
weight, an analyst may obtain successive values which vary without a definite
trend. This variation could be due to varying amounts of water in the weighed
residue of Al20 3. There are appreciable weight changes in hygroscopic
Al 20 3 due to slightly different periods of ignition, exposure to the atmosphere
and cooling of desiccator.
(B) Erratic Errors. The analyst has no control over erratic errors.
Weighing with a sensitive balance subjected to vibrations will show erratic
errors. The mathematical model that best satisfies the magnitude of random
error and the frequency of its occurrence is called the Normal (or Gaussian)
distribution (Fig. 1). An inspection of this error curve shows :
(i) Small errors occur more frequently than large ones.
(ii) Positive and negative errors of the same numerical magnitude are
equally likely to occur.
(iii) Narrow peaked curve with steep slopes indicates a relatively high
degree of precision.
(iv) A broad curve indicates a relatively low degree of precision.
- -
432101234
-va Error +ve Error
Fig. 1. Magnitude of errors.
3. Errors in Measurements.
(A) Errors in weighing may be due to insensitivity of the balance, wrong
suspension of the ring-riders, placing the weights at edge of the pan and use
of non-calibrated weights etc. Difference in temperature between the object
weighed and the balance also causes error in weighing. It has been estimated
that a difference of 1K causes an error of about 0·15 mg.
(B) Errors in measuring solutions may be due to incorrect use of
glasswares.
4. Gross Errors.
Common gross errors due to carelessness of the analyst are :
(i) Use of numerically incorrect conversion factors.
(ii) Wrong selection of the method.
(iii) Unsuitable storage of samples.
5. Other Errors.
(i) Errors in radiometric analysis.
(ii) Errors in chromatography.
(iii) Photometric errors.
EFFECTS OF ERRORS ON ANALYTICAL RESULTS

• Systematic error in a series of replicate measurements causes all the
results to be too high or too low. It may lead to bias in measurement
results affecting all the data. An example constitutes unsuspected loss
of a volatile substance while heating a sample.
• The effect of constant (systematic) error becomes more serious as the
size of the quantity measured decreases.
• Solubility losses lower the results of a gravimetric analysis.
• With constant errors, the absolute error is constant with sample size
but the relative error varies when the sample size is changed.
• Proportional errors increase or decrease according to the size of the
sample taken for analysis.
• One way of reducing the effect of constant error is to increase the size
of sample. Consider the following example.
A 0.50 mg of precipitate is lost when it is washed with 200 em3 of wash
liquid. If the precipitate weighs 500 mg, the relative error due to
solubility loss is (0.50/500) x 100% = 0.1%. Loss of same quantity
from 50 mg of precipitate results in a relative error of 0.1%.
• The presence of interference contaminants in the sample affect the
results. High results are observed for the percentage of Cu (in
presence of Fe) because the 12 produced will be a measure of Cu and
Fe in the sample.
• Gross errors affect results that differ markedly from all other data in
a set of replicate measurements.
ACCURACY
Accuracy has been defined as the concordance between a measured value
and the accepted true or most probable value. It follows, therefore, that
systematic errors cause a constant error (either too high or too low) and thus
affect the accuracy of a result.
Measure of Accuracy.
Accuracy can be expressed in terms of absolute and relative error.
Absolute Error. The absolute error (d) of a measured value is a
numerical difference between the true value ()1) and the measured or observed
value (x), i.e., d = )1- x. Absolute error can be positive if)1 > x and negative
if)1 < x. Sometimes absolute error is expressed by the absolute value, without
respect to the sign.
Relative Error. The relative error(e) is the absolute error divided by
the true value. Thus,
Relative error is expressed in terms of percent or in parts per thousand

(ppt).
e = ~ x 1000 ppt.
)1
Observed value and relative error give true or correct value,
x
)1 =-1- (for e:;:.1)
-e
The relative maximum error of the final result Ep can be defined as
the sum of all relative errors of all values experimentally obtained, i.e.,
f
Ep=Ex +Ex +Ex ±I.xj
1 2 3 1
The magnitude of relative error depends upon the magnitude of the
absolute error and the absolute value ofthe quantity determined. Smaller the
absolute error and greater the magnitude of the absolute value, smaller will
be the relative error. Relative errors can be minimised by precise
measurements and eliminating the error of procedure. It is a measure of the
accuracy of the result and permits a comparison ofthe accuracy of the results
expressed in different units.
Exercise 1. The result of an analysis was determined as 15·752 g while the

accepted value was 15·872 g. Calculate the absolute and relative error.
Solution. Absolute error = True value - Observed value
= 15·872 -15·752 = 0·120
·
R eIa t lve Absolute error 1000
error = Tru 1 x
eva ue
= 0·120 x 1000/15·872 = 7·56
Determination of Accuracy.
1. Absolute Method.
A synthetic sample containing known amounts of the constituents in
question is used. Known amounts of the constituent can be obtained by
weighing out pure element or compounds of known stoichiometric
composition. The test of the accuracy of the method is carried out by taking
varying amounts of the constituents and proceeding according to specified
instructions. The difference between the mean of an adequate number of
results and the amount of the constituent actually present (usually expressed
as parts per thousand, ppt) is a measure of the accuracy of the method in the
absence of foreign substances. The effect of foreign elements should also' be
studied. Accuracy of the method is likely to be largely controlled by the
separation of the pure sample from foreign elements before its determination.
2. Comparative Method.
If several fundamentally different methods of analysis for a given
constituent are available, e.g., gravimetric, titrimetric, spectrophotometric or
spectrographic etc., the agreement between at least two methods of
essentially different character can usually be accepted as indicating the
absence of an appreciable systematic error (a systematic error is one which
can be evaluated experimentally or theoretically).
PRECISION
Precision may be defined as the concordance of a series of measurements
of the same quantity. It is expressed in terms of numerical difference between
a given experimental value and the mean value of all the experimental
results. Accuracy expresses the correctness of a measurement and
precision the reproducibility of a measurement. Precision always accompanies
accuracy but a high degree of precision does not imply accuracy. Precision
may be expressed as the standard deviation, the coefficient of variation, the
range of the data or as a confidence interval about the mean value.
Example. A substance was known to contain 49·10 ± 0·02% of a constituent
X. The results obtained by two analysts using the same substance and the
same analytical method were as follows :
Analyst 1. % X: 49·01; 49·25; 49·08; 49·14.
The arithmetic mean is 49·12% and the results range from 49·01% to
49·25%.
Analyst 2. % X: 49·40; 49·44; 49·42; 49·42.

The arithmetic mean is 49·42% and the results range from 49·40% to
49·44%. The results of the analysis can be summarised as follows :
A. The values obtained by Analyst 1 are accurate (very close to the
correct result), but the precision is inferior to the results given by
Analyst 2. The value obtained by Analyst 2 are very precise but are
not accurate.
B. The results of Analyst 1 face on both sides of the mean value and
could be attributed to random errors. It is apparent that there is a
constant (systematic) error present in the results of Analyst 2.
MINIMISATION OF ERRORS
Systematic errors can often be materially reduced by one of the following
methods:
1. Calibration of Apparatus and Application of Corrections.
All instruments such as burettes, pipettes, measuring flasks and weights
etc., should be calibrated, and the appropriate corrections applied to the
original measurements. In cases, where an error cannot be eliminated, it is
possible to apply a correction for the effect that it produces. Thus an impurity
in a weighed precipitate may be determined and its weight deduced. Also
recalibrate instruments frequently.
2. Running a Blank Determination.
This consists in carrying out a separate determinatipn, the sample being
omitted, under exactly the same experimental conditions as are employed in
the actual analysis of the sample. The object is to find out the effect of the
impurities introduced through the reagents and vessels and to determine the
excess of standard solution necessary to establish the end point under the
conditions met within the titration of the unknown sample. A large blank
correction is undesirable because the exact value then becomes uncertain and
the precision of the analysis is reduced.
3. Running a Control Determination.
The method consists in carrying out a determination under (as nearly as
possible) identical experimental conditions upon a quantity of a standard
substance which contains the same weight of the constituent as is contained
in the unknown sample. The weight of the constituent x in the unknown can
then be calculated from the relation :
Result found for standard = Weight of constituent in standard
Result found for unknown x
Here it must be pointed out that National Bureau of Standards in
USA supply standard samples of irons, steels, non-ferrous alloys and primary
standards e.g., sodium oxalate, potassium hydrogen phthalate, benzoic acid
and arsenic (III) oxide.
4. Running Parallel Determination.

These serve as a check on the result of a single determination and
indicate only the precision of the analysis. The values obtained in parallel
determinations should agree well among themselves. These values should not
vary by more than three parts per thousand. If larger variations are shown,
the determinations must be repeated until satisfactory concordance is
obtained. A good agreement between duplicate and triplicate determinations
does not justify the conclusion that the result is correct; a constant error may
be present. The agreement merely shows that the accidental errors, or
variations of the determinate errors are same in parallel determinations.
5. Internal Standards.
It involves adding a fixed amount of a reference material (the internal
standard) to a series of known concentrations of the sample to be determined.
The ratios of the physical value (absorption or peak size) of the internal
standard and the series of known concentrations are plotted against the
concentration values. It should give a straight line. Any unknown
concentration can then be determined by adding the same quantity of internal
standard and finding where the ratio obtained falls on the concentration
scale. The method is of particular interest in spectroscopic and
chromatographic determinations.
6. Standard Addition.
A known amount of the constituent being determined is added to the
sample, which is then analysed for the total amount of constituent present.
The difference between the analytical results for samples with and without
the added constituent gives the recovery of the amount of added constituent.
Accuracy of the procedure is enhanced if the recovery is satisfactory. The
procedure is specially applied to physico-chemical processes such as
polarography and spectrophotometry.
7. Isotopic Dilution.
A known amount of an element being determined (containing a
radioactive isotope), is mixed with the sample and the element is isolated in
a pure form (as a compound), which is weighed. The radioactivity of the
isolated material is measured and compared with that of the added element.
The weight of the element in the sample can then be calculated.
8. Amplification Methods.
When very small amount of the material to be measured is beyond the
limits of the apparatus, then the material can be reacted in such a way that
every molecule produces two or more molecules of some other measurable
material, the resultant amplification may then bring the quantity to be
determined within the scope of the apparatus or method available.
9. Use of Independent Method of AnalYSis.
In some cases the accuracy of a result may be established by carrying
out the analysis in an entirely different manner. Thus iron can first be
determined gravimetrically by precipitation as iron (III) hydroxide after

removing the interfering elements, followed by ignition of the precipitate to
iron (III) oxide. It may then be determined titrimetrically by reduction to iron
(II) state, and titration with a standard solution of an oxidising agent, such
as cerium (IV) sulphate or potassium dichromate. Similarly the strength of
hydrochloric acid can be determined both by titration with a standard
solution of a strong base and by precipitation and weighing as AgCl. If the
results obtained by the two different methods are concordant, it is highly
probable that the values are correct within small limits of error.
SIGNIFICANT FIGURES
The term digit denotes anyone of the ten numerals, including zero. A
significant figure is a digit which denotes the amount of the quantity in the
place in which it stands. The digit zero is a significant figure except when it
is the first figure in a number. For example, in the quantities 1·3680 and
1·0082 g the zero is significant but in the quantity 0·0035 kg the zeros are not
significant figures. They serve only to locate the decimal point and can be
omitted by suitable choice of units, i.e., 3·5 g. The first two numbers contain
five significant figures but 0·0035 contains only two significant figures.
Observed quantities should be recorded with one uncertain figure retained.
Thus the weights are determined to the nearest tenth of a milligram.
Consider the weight 2·2756 g. This means that the weight is less than
2·2757 g and more than 2·2755 g. A weight of 2·270 g would signify that the
weight is nearer to 2·270 g than it is to either 2·271 g or 2·269 g.
The digits of a number which are needed to express the precision of the
measurement from which the number was derived are known as significant
figures. The significant figurt'ls in addition and subtraction are based on
absolute uncertainty which is equal to the largest absolute uncertainty of any
individual number. The concept of significant figures in multiplication and
division operations is based on relative uncertainty.
Exercise 2. How many significant figures does each of the following numbers
have?
(a) 2500 (b) 0·004 (c) 5000·0 (d) 0·02765 (e) 4·20 x 1010.
Solution. (a) 4 (b) 1 (c) 5 (d) 4 (e) 3.
RULES FOR COMPUTATION

METHODS FOR REPORTING ANALYTICAL DATA
Rule 1. In expressing an experimental analytical data, never retain
more than one doubtful digit. Eliminate all digits that are not significant.
Rule 2. Retain as many significant figures in a result or in any data as
will give only one uncertain figure. Thus a volume which is known to be
between 25·5 cm3 and 25·7 cm3 should be written as 25·6 cm3 , but not as
25·60 cm3 , since the latter would indicate that the value lies between 25·59
and 25·61 cm3 . Also, if a weight, to the nearest 0·1 mg is 5·2600 g, it should
never be written as 5·260 g or 5·26 g, since in the latter case an accuracy of a
centigram is indicated and in the former a milligram.
Rule 3. (a) In rounding off quantities to the correct number of

significant figures, add one to the last figure retained if the following figure
is 5 or ove~. This is known as rounding up. Thus the number 8·856 has been
rounded upto the digit 8·86.
(b) If the last digit discarded is less than 5, leave the next digit
unchanged. This is called rounding down. For example, the number 7·64 is
rounded to 7·6. It must be noted that rounding never changes the power of
10. Therefore it is better to express numbers in exponential notation before
rounding. For instance, in rounding 67832 to four figures, the value is
6·783 x 104 •
Exercise 3. Round each of the following numbers to four, three and two
significant figures.
(a) 1·00727 (b) 28755 (c) 0·002045 (d) 169992.
Solution. (a) 1·007, 1·01, 1·0 (b) 2·876 x 104 , 2·88 X 104 , 2·9 X 104
(c) 2·045 x 10-3, 2·05 x 10-3, 2·0 x 10-3
(d) 17·00, 17·0, 17.
Exercise 4. Round 5174·55 to five, three and two figures.
Solution. 5·1746 x 103 , 5·17x 103 , 5·2x 103 •
Rule 4. In addition or subtraction, there should be in each number only
as many significant figures as there are in the least accurately known
number. Thus the addition 168·11 + 7·045 + 0·6832 should be written as
168·11 + 7·05 + 0·68 = 175·84.
The sum or difference of two or more quantities can not be more precise
than the quantity having the largest uncertainty.
Rule 5. In multiplication or division, retain in each factor one more
significant figure than is contained in the factor having the largest
uncertainty. The percentage precision of a product or quotient can not be
greater than the percentage precision of the least precise factor entering into
the calculation. Thus the multiplication of 1·26 x 1·336 x 0·5834 x 25·8652
should be done using the values 1·26 x 1·336 x 0·583 x 25·87 and the result
expressed to three significant figures.
When a large number of multiplications and divisions are to be made then
. . (0·46308) (0·0808)
logarIthm table should be used. ConSIder an example, (2.809) (0.052) . The
factor with least number of significant figute is 0·052. It has two significant
figures (5·2 x 10-2 ). Hence the answer should be expressed in two significant
figures.
When a calculation involves both addition (or subtraction) and
multiplication (or division) do the addition first so as to determine the number
of significant figures in the answer.
. 5 E
E xerClse "fi fi
. xpress slgm cant 19ures m
. 27·736 -27·140
23.024 .
Solution. Subtraction in the numerator gives
27·736 - 27·140 = 0·596. Now division of 0·596/23·024 provides the answer in
three significant figures, i.e.,
27·736 - 27·140 0·596 -2

23.024 = 23.024 = 2·58 x 10 •
Exercise 6. Calculate significant figures in 21·697 - 20·802.

Solution. Three significant figures (21·697 - 20·802 = 0·895).
Exercise 7. How many significant figures are there in
0·0625 + 0·0612 + 0·0531.
Solution. Four significant figures (each added term has only three
significant figures) i.e., 0·1768.
Rule 6. Computation involving a precision not greater than one fourth
of 1% should be made with a 10 inch slide rule. Slide rule is a good method
for checking the calculations made by logarithms. Use of logarithms has
been recommended where a large number of multiplications and divisions are
to be made.
USE OF CALCULATORS AND COMPUTERS
In addition to the normal arithmetic functions, a suitable calculator for
statistical work should enable the user to evaluate the mean and standard
deviation, correlation coefficient and linear regression. The results obtained
by the use of calculator must be carefully scrutinised to ascertain the number
of significant figures to be retained and must be checked against a rough
arithmetical calculation to ensure that there are no gross computational
errors.
Micro-computers are used for processing large amounts of data. These
facilitate the collection and processing of the data, which may be I:?tored on
floppy or hard discs for later use. There is a large number of commercial
software available for performing advanced statistical calculations. Also
standard programs now exist in BASIC.
STATISTICAL EVALUATION OF DATA

Statistical evaluation of analytical data comprises of several phases
viz. decision about some preliminary matters, collection of data, security of
data, treatment, summarisation and analysis of data as well as statistical
inference. Some important applications of statistical methods for the
evaluation of analytical results are illustrated below.
• Mathematical statistical calculations offer the method for the
determination of criteria of accuracy and precision as well as
occurrence of errors. Statistical methods are, therefore, employed in
interpreting the average of results obtained in replicate
determinations.
• Estimating the probability that an experimental mean and a true
accepted value are different that is, whether the difference is real or
it is the result of random error. This test is particularly useful for
investigating systematic errors in a method and determining whether
the two samples belong to the same source or not.
• Deciding with a certain probability whether an apparent scheme in a
set of replicate measurements is the result of gross error and can thus
be rejected or whether it is a legitimate part of the population that

must be retained while calculating the mean of the set.
• When a small number of observations are made the value of standard
deviation does not give a measure of how close the sample mean
might be to the true mean. But it is possible to calculate a
confidence interval to estimate the range within which the true
mean may be found. While comparing more than two means, the
possible sources of errors may be : the random error associated with
replicate measurements and the variation that may arise between the
individual analysis. These variations may be calculated and their
effects estimated by a statistical method known as the analysis of
variance (ANOVA).
• Using Pearson's correlation coefficient to establish whether there
is a linear relationship between two variables.
• Employing Chemometrices which may be defined as the application
of mathematical and statistical methods to design and/or optimise
measurement procedures and to provide chemical information by
analysing relevant data.
STATISTICAL TERMS
MEAN.
The mean is the numerical value obtained by dividing the sum of a set
of measurements by the number of individual results in the set. It is also
known as arithmetic mean or average. (Average is the mathematical mean
of the replicate determinations). If we consider a series of n observations
arranged in ascending order of magnitude : x1> x2' Xg ... xn _ 1> x n ' the
arithmetic mean (or simply the mean) is given by:
_ Xl + x2 + Xg ... + ... + xn _ 1 + xn
X= ... (1)
n
Mean, m is also given by m = "fMn In
where M = individual measurement, n = total number of measurements.
Exercise 8. Calculate the results given by the replicate determinations of
chloride in a metal chloride :
Xl =32·22, x2 = 32-64, x3 =32·52, x4 =32·46.
Solution. Sum of four determinations is 129·84, n = 4.
The mean will, therefore, be x = 129·8414 = 32·46%.
Exercise 9. Find the arithmetic mean of first five natural numbers.
Solution. x= 1 + 2 + 3 + 4 + 5 = 15 = 3.0
5 5
Exercise 10. Calculate the value of A if the mean of 6, 4, 7, A and 10 is 8.
Solution. 8 = 6 + 4 + 7 + A + 10
5
40 = 27 + A or A =40 - 27 :;:: 13
Exercise 11. The mean of 40 observations was 160. On rechecking, it was

found that the value of 165 was wrongly copied as 125 for computation of
mean. Calculate the correct mean.
Solution. Mean, m = :r. Mn1n
:r.Mn
160 = ----:w-
6400 =:r.Mn
Incorrect value of :r.Mn = 6400
Correct value of :r.Mn = Incorrect value of :r.Mn - Incorrect
reading + Correct reading
= 6400 - 125 + 165 = 6440
:r.Mn
Correct mean = Correct value of--
n
= 6440 = 161
40
Arithmetic Mean in a Discrete Frequency Distribution.
Arithmetic mean in a discrete frequency distribution may be computed by
direct, indirect and step deviation method.
Direct Method. If a variate x takes values xl, x2 ... Xn with
corresponding frequencies flo f2 ... fn respectively, then mean of these values
is given by
or
Exercise 12. Calculate the mean of following distribution :

x=4 6 9 10 15
f= 5 10 10 7 8
Solution. Calculation of mean.
Xi 4 6 9 10 15
fi 5 10 10 7 8 n=l:fi=40
fi Xi 20 60 90 70 120 l:fixi = 360
:r.fi Xi 360
Arithmetic mean = x = - - = - =9
'if;, 40
Exercise 13. If the mean of distribution is 1·46, find the missing frequencies
in the following frequency distribution.
x=0 1 2 3 4 5 Total
f = 46? ? 25 10 5 200
Solution. Suppose the missing frequencies are 11 and f2.
Calculation of mean.
Xi 0 1 2 3 4 5
Ii 46 fl f2 25 10 5 n =86 +fl +f2
'-
fixi 0 fl 2f2 75 40 25 r.. Ii xi = 140 + fl + 2f2
Since n=200, So 200=86+/\ +/z
or 114 =f1 +f2 ... (2)
"Lfi xi
Also Mean = 1·46 = - -
n
2f2
= 1·46= 140 +f1 + -
200
or 292 = 140 + f1 + 2 f2
152 =f1 +2f2 ... (3)
Solving equations (2) and (3) we get '1 = 76 and '2 = 38.
Mean Deviation.
The mean deviation of a single measurement is the mean of the
deviations of all the individual measurements. It can be calculated by :
(i) Determining the arithmetical mean of the results.
(ii) Calculating the deviation of each measurement from the mean.
(iii) Dividing the sum of the deviations (regardless of sign) by the
number of measurements. The mean or average deviation d is calculated by
- "LIMn-ml
d = -'--~-":'"
n
where, IMn -ml = absolute value of the deviation of the Mnth number from
the mean.
Exercise 14. Analysis of a given quantity gave the following nine values.
Assuming the errors to be random ones calculate mean. 46·62, 46·47, 46·64,
46·76, 46·53, 46·60, 46·71, 46·60, 46·71.
"LMn
Solution. The mean m =-n-
= 46·62 + 46·47 + 46·64 + ... 46·71 = 46.627
9
The difference between anyone of the values and the mean (46·627) is
the deviation Xi of that value from the mean. These deviations regardless of
the sign are: 0·007, 0·157, 0·013, 0·133, 0.:997, 0·027, 0·083, 0·027, 0·083
respectively. The mean or average deviation d is
- "L IMn - m I "LXi 0·007 + 0·157 + 0·013 + ...
d= =-= =0·070
n n 9
where "LXi is the sum of the individual deviation from the mean.
Relative Mean Deviation.

It is the mean deviation divided by the mean. It is expressed in terms of
percentage or parts per thousand. In the above numerical
. d .. 0·070 x 100 0 15
R eIatIve mean eVlatlOn = 46.627 =. nt -/0
MEDIAN
Median is the middle result when replicate data are arranged according
to increasing or decreasing values. Or median is a value about which all the
other values are' equally distributed.
Median of an ungrouped data. For an ungrouped data xl> x2' ... x n '
the median is computed as follows :
1. Arrange the data in ascending or descending order of magnitude.
2. Determine the total number of observations (n).
3. If the data, n is odd, then
Median = Value of [ n ; 1
4. If the data, n is even, then
r observation.
.
MedIan =
Value of
(2'n]th observation + Value
2
of (n
2' + 1]th observation
... (4)
Exercise 15. Calculate the median of the following data :
37, 31, 42, 43, 46, 25, 39, 45, 32
Solution. Arrange the data in ascending order.
25, 31, 32, 37, 39, 42, 43, 45, 46
No. of observations = 9 (odd).
..
9
Medi= Value of ( ; 1
= Value of 5
r observation
th observation
=39
Note. If the 'data is an odd numbered set, the median is the middle
value.
Exercise 16. Calculate the median of the data given below,'
15, 24, 21, 13, 12, 16, 25, 18, 10, 22
Solution. Arrange the data in ascending order
10, 12, 13, 15, 16, 18, 21, 22, 24, 25
Here n = 10 (even). Applying equation (4), we get
Value of 5th observation + Value of 6th observation
Me di an=
2
= 16 + 1812 = 17
Note. If the data is an even numbered set, median is the average of

middle two values.
Determination of Median in case of a Discrete Frequency
Distribution.
1. Arrange the data in ascending or descending order of magnitude.
2. Obtain the cumulative frequencies.
th
3. Find the size of (N ; 1) item, where N is the total frequency.
4. Median is located at the value of the variable in whose cumulative
frequency the value Of[N; 1Jh item falls.
Exercise 17. Calculate the median size of the following data:

Size (X) 4 5 6 7 8 9 10
Frequency (F) 17 12 15 18 11 13 14
Solution. Compute cumulative frequencies (CF).
Size'(X) 4 5 6 7 8 9 10
Frequency (F) 11 12 13 14 15 17 18 [N= 100]
C.F. 11 23 36 50 65 82 100
. = Value of [N
MedIan + 1J = 1002+ 1 = 50· 5th Item.
-2- .
Median =7
Characteristics of Median.
• Median can be calculated graphically, while mean cannot be.
• Median is not affected by absolute value.
• Median is the only average which is used while dealing with the
qualitative data which cannot be measured quantitatively.
STANDARD DEVIATION
Standard deviation (also called root mean square deviation) cr
measures how closely the data are clustered about the mean. The smaller the
standard deviation, the more closely the data are present about the mean.
S.D. of a single measurement can be obtained by extracting the square root
of the quotient obtained by dividing the sum of the square of the individual
deviations of the number of measurements made.
1 ]1/2 .... I[~ (Xi]

cr=;,~(Mn-m)2
[ ='1-;;-
When the number of values is small the denominator is (n -1) rather
than n. The above equation may also be written as
s =[~ (Mn - m)2]1/2 =~[~ (Xi]

(n -1) n-1
Considering the above exercise (14), the standard deviation of a single

measurement
(0-007)2 + (0-157)2 + ___ ]1/2
s =[ 9_ 1 = 0-091
While the standard deviation of the mean s = 0-091/...[9 =0-030.

This value gives an indication of the reliability of the mean_
If we consider equation (1) then the standard deviation may be defined
by:
s= ~ (Xl - X )2 + (x2 - X )2 + ___ (xn - X )2
___ (5)
n-1
In this equation the denominator is n - 1 rather than n when the
number of measurement is small. Equation 5 may be written as
s = ~ r. (x - x )2
n-1
The square of the standard deviation is called the variance. Another
measure of precision, known as Relative standard deviation (RSD) is given
by:
RSD=slx
This measure is often expressed as a percentage, known as the
coefficient of variance ( CV)_ It is given as CV = s x 1001 x .
Exercise 18. Analysis of a sample of haematite gave the following percentage
values for the iron content: 7-08, 7-21, 7-12, 7-09, 7-16, 7-14, 7-07, 7-14, 7-18
and 7-11.
Find out the mean, standard deviation and coefficient of variation for the
values.
Solution. Mean x =7-13%,
x x-x (x-x)2
r. (x _x)2 = 0-0182
7-08 -0-05 0-0025 s = "",0-0182/9 = "",0-0020
7-21 0-08 0-0064 =±0-045%
C_V. = 0-045 x 100/7-13 = 0-63%.
7-12 -0-01 0-0001
The mean of several readings (x)
7-09 -0-04 0-0016 will make a more reliable estimate of the
7-16 0-03 0-0009 true mean 01) than is given by one
measurement. The greater the number of
7-14 0-01 0-0001 measurements (n), the closer will the
7-07 -0-06 0-0036 sample average approach to the true
value. The standard error of the mean Sx
7-14 0-01 0-0001 is given by:
s
s =--
7-18 0-05 0-0025 x..Jn
In the above exercise (18)
7-11 -0-02 0-0004
= + 0-045 = + 0.014
Lx = 71-30 0-0182 Sx - ill -
If 100 measurements were made,

_ + 0·045 _ + 0~0045
sx-- vlOO --
Thus the precision of a measurement may be improved by increasing the
number of measurements.
Exercise 19. Two analysts gave the following observations.
Analyst 1. (a) 49·01 (b) 49·21 (c) 49·08
Analyst 2. (a) 49·40 (b) 49·42 (c) 49·44
Calculate precision and accuracy of both the analysts. Which analyst is
more precise and which one is more acourate ?
Solution. Analyst 1.
- 49·01 + 49·21 + 49·08 _ 147·30 - 4910
Mean m- 3 - 3 - ..
149.01 - 49·10 I = 0·09

149.21- 49·10 1 = 0·11
L (Mn - m) 0.22
149.08 - 49·10 1 = 0·02 Mean Deviation = = - - = 0·07
n 3
J
= 0·22
Analyst 2. Mean m = 49·40 + 49~42 + 49·44 = 14~26 = 49.42.
149.40 - 49.421 = 0·02

149.42 - 49.421 = 0·00
L(Mn -m) 0.04
149.44 - 49. 42 1 = 0·02 Mean Deviation = n ='-3- = 0·01.
0·04
Since mean deviation is a measure of precision and absolute difference
is a measure of accuracy. So analyst 2 is more precise and more accurate than
the analyst 1.
Exercise 20. The percentage of constituent A in a compound AB are
22·6~, 22·64, 22·54 and 22·53%. Calculate mean deviation and relative mean
deviation.
Solution. Mean m = 22·61 + 22·64 + 22·54 + 22·53 = 90·32 = 22.58
4 4
Deviation, 22·61 - 22·58 = + 0·03
22·64 - 22·58 = + 0·06
22·54 - 22·58 = - 0·04
22·53 - 22·58 = - 0·05 Mean Deviation = 0'i8= 0·045

0·18
Relative mean deviation= 0.0~~.~8100 = 0·2% = 2·0 parts per thousand.

Exercise 21. Calculate the standard deviation for an element whose
percentage in a sample were found to be 20-8, 21·6, 22·1, 22·0, 23·3, 21·9 and
22-8%.
Solution. Mn (Mn _m)2
20·8 1·6129
LMn
21·6 0·2209 m = - - = 154·5/7 = 22·07
n
22·1 0·0009
22·0 0·0049 s= [ L(Mn -m)2] 11'

n-1
23·3 1·5129
21·9 0·0289
[ r
/2
22·8 0·5329 = 3.9~43 = 0.8077.
154·5 3·9143
RELIABILITY OF RESULTS
Statistical figures obtained from a set of measurements are of limited
value by themselves. Analysis of the results can be considered in two main
categories.
(1) The reliability ofthe results; and
(2) Comparison of the results with other set of data or true value.
The values should be rejected only when a suitable statistical test has
been applied or when there is an obvious chemical or instru.mental reason
that could justify exclusion of a result. Consider the following example.
Exercise 22. Lead was determined in a sample of dust. Following values
were obtained 4·3, 4·1, 4·0, 3·2 fJg g-l. Should the last value, 3·21lg g-l be
rejected?
Solution. Here Q test may be applied to solve this problem.
1Questionable value - Nearest value 1
Q= Largest value - Smallest value
13 .2 - 4 .0 1 0·8
Q= 4.3-3.2 =TI=0.727
Thus Q calculated is O· 727 but the critical value of Q for a sample size
of four is 0·831. Hence the result 3·21lg g-l should be retained. If three more
measurements were made i.e., 4·3, 4·1,4·0,3·2,4·2, 3·9,4·0 Ilg g-l then,
13 .2 - 3·91 0.7
Q = 4.3 - 3·2 = 1.1 = 0·636.
The value of Q critical for a sample size of seven is 0·570, so rejection of

the value 3·21lg g-l is justified. (Note. Q value has no regard to algebraic
sign).
REJECTION OF RESULTS
When a series of replicate analysis are performed, one of the results
appears to differ markedly from the rest. It is now to be decided whether to
reject or retain such a result. There is no uniform criteria to do so. Experience
is the best basis for judging the validity of a particular observation as a
statistical test would be. However, following tests are available for
determining whether or not a rejection is justified.
1. Average Deviation.
After the average deviation for a series of measurements have been
obtained, the data may be tested as follows. Reject the doubtful value and
determine the mean and average deviation of the retained value. If x ;;:: 4d
i.e., if the deviation ofthe suspected value from the mean is atleast four times
the average deviation, then the rejection is justified. Consider exercise 14.
Suppose that a tenth value is 46·34. Deviation of this suspected value from
the mean is 46·627 - 46·34 =0·287. Average deviation is 0·070. Since o· 287 is
more than 4 times the average deviation, the rejection is justified.
2. Standard Deviation.
A normal distribution curve is plotted (Fig. 2) for 100 measurements of
a sample. Here frequency of occurrence of a measurement is plotted against
the value of the measurement. The curve shows that upon taking another
measurement there would be 68·26% chance and it would fall between ± 10"
of the mean. The chance would be 95·46% if ± 20" were taken, while ± 30" would
give a 99· 7% chance that the result would fall within this range. Thus a value
of 30" is used as a criteria for rejecting a measurement.
-30 -20 -10 Mean 10 20 30

Measurement value
Fig. 2. Normal distribution curve.
.60 ANALYTICAL CHEMISTRY
COMPARISON OF THE MEANS OF TWO SAMPLES

When a new analytical method is being developed it is usual practice to
compare the values of the mean and precision of the new (test) method with
those of an established (reference) procedure. The significance, t when
comparing two sample means Xl and x2 is given by the expression:
xl -X2
t =-~=======:;==
8 "';l/nl + 1/n2
p
where 8p the pooled standard deviation is calculated from the two sample
standard deviations 81 and 82 as given below:
8 = -V (nl - 1) 81 + (n2 - 1) 8~
P nl +n2 - 2
THE USES OF STATISTICS

• Statistics is the science of collection, presentation, analysis and
interpretation of numerical data. It is an extremely essential tool for
the analyst.
• Statistical laws like correlation, regression, dispersion,
approximation, probability, test of significance are required for
evaluating the analytical data.
• Statistical dimensions can be extended by applying purely
mathematical methods of differentiation, integration, algebra and
trignometry.
• Mathematical statistical calculations offer the method for the
determination of criteria of accuracy and precision.
• Statistical methods permit the measurement of imperfection and
uncertainty.
• Quick statistical tests are now available that are called non-para-
metric methods. Calculations involving non-parametric methods are
very simple, so they are amenable to a quick evaluation. Instead of
the mean, the median is used as a measure of central tendency.
• Statistics Software Packages. In statistics, a spreadsheet is a
powerful software program that can be used for data analysis, doing
repetitive calculations and displaying the calculations graphically or
in chart form. They have built in functions, for example, standard
deviation and other statistical functions, for carrying out
computations on data that are input by the analyst. Popular
spreadsheet programs include Microsoft Excel, Lotus and Quattro
Pro. All operate basically the same but differ in specific commands
and syntax.
• LlNEST for Additional Statistics. LINEST program of excel
allows us to quickly obtain several statistical functions for a set of
data, in particular the slope and its standard deviation, the coefficient
of determination and the standard error of the estimate. Excel has a
large number of mathematical and statistical functions for
automatically calculating the mean.
IMPORTANT RELATIONS
• Average deviation of a single measurement is the mean of the
deviations of all the individual measurements, i.e., d = L Bin where
L B is the sum of all deviations from the mean, n is the total number
of values.
• Average deviation of mean CD) is equal to the average deviation of a
single measurement divided by the square root of the number of
measurements made. D = d/--!n where D = average deviation of
mean, n = no. of readings, d = average deviation of a single
measurement.
• Standard deviation is a precise and reliable measure of deviation.
~fU,2
Standard deviation of a single measurement(8) is 8 = -" ~--1 where
n-
L B2 is the sum of squares of all deviations from mean and n is the
total number of measurements.
• Standard deviation of mean(S) is obtained from the standard
deviation (8) by dividing it by the square root of the number of
measurements. S = 81--!n. Usually (28) is taken as a reasonable limit
within which the true value is likely to lie.
o
3
FOOD ANALYSIS
INTRODUCTION
Chemical analysis of food is done to determine the acceptability,
nutritive value, quality, composition and authencity of the food products.
Major steps in the analysis include
(i) to select and prepare samples,
(ii) to perform the assay
(iii) to calculate and interpret the data.
The choice of the analytical method is usually based on the nature of
sample, the specific reason for the analysis and characteristic of the method
itself, such as specificity, speed, accuracy, precision, cost of equipment and
training of personnel.
Sampling and Sample Preparation.
Sampling is the most variable step in the overall analysis of food. It
involves following steps :
1. Selection of Sampling Procedures.
The first requirement in sampling is to clearly define the population to
be sampled. The sampling procedure selected depends on the purpose of
inspection, nature of the product, nature of the test method and the nature of
the population being investigated. Increasing the sample size will increase the
reliability of the final results.
2. Sampling for Attributes or Variables.
Attributes are those characteristics that are present or not present.
Variables are those characteristics that are measured on a continuous scale.
The actual value obtained is compared to the expected value and the deviation
determined.
Sampling plans. These may be single, double or multiple. Selection of
appropriate sampling plan depends on the overall quality of the lot and the
cost of sampling. Multiple sampling plans reduce sampling costs by rejecting
low-quality lots or accepting high-quality lots. Every sampling plan has
inherent risks associated with it. Sampling plans depend on whether the
population is homogeneous or heterogeneous.
Preparation of Food Samples.
Sample preparation depends on the nature of the food and the type of
analysis. Very small samples should not be used, as this leads to moisture loss
during preparation and subsequent handling. Sample preparation involves :
(62)
FOOD ANALYSIS 63
(i) Grinding. Mills, bowl cutters, meat mincers, tissue grinders,

mortars and pestles or blenders are most useful for moist samples.
(ii) Enzymatic Inactivation. Food materials are rich in enzymes.
Enzymes can be inactivated by inorganic compounds, by a shift in
pH or by salting out. Oxidative enzymes may be controlled by
reducing agents.
(iii) Lipid Protection. Lipids create a particulate problem in sample
preparation. Foods high in fat are difficult to grind at room
temperature. These foods may be ground in a frozen state.
Low-temperature storage under nitrogen is usually recommended
to protect moist foods.
(iv) Microbial Growth and Contamination. Micro-organisms are
present in nearly all foods and if not controlled can alter the
composition of the sample. Freezing, drying and chemical
preservatives can control microbial growth. The method of
preservation depends on the nature of the food, the expected
contamination, the storage period, conditions and the analyses that
are to be performed.
Evaluation of Analytical Data.
Computers and graphics softwares are employed for the calculations of
analytical result.
MOISTURE ANALYSIS IN FOODS

Need for Moisture Assay.
The moisture content of foods is important to food processors and
consumers for a variety of reasons.
1. Moisture is used as a quality factor for jams, jellies, sugar syrups.
2. Moisture is a quality factor in the pre!}ervation of food products. It
affects stability in dehydrated vegetables and fruits, dried milk,
spices and herbs.
3. Reduced moisture is used for convenience in packaging of
concentrated milks and fruit juiees, liquid cane sugar and corn
sweetner
4. Moisture is an inexpensive filler.
Forms of Water in Foods.
1. Free water. Free water acts as the dispersing agent for colloids
and the solvent for salts.
2. Adsorbed water. This water is held lightly to proteins or is
occluded in cell walls or protoplasm.
3. Water of hydration. This water is bound chemically, e.g., lactose
monohydrate. Depending on the form of the water present in a food,
the method used for determining moisture may measure the water
content in food.
Table 1. Moisture content (%) of some foods.

Moisture Moisture
Food Food
content content
Potatoes, white 79.8 Oranges 86.0
Cucumbers 95.1 Watermelons 92.6
Green beans 90.1 Apples 84.5
Milk (3.5 % fat) 87.4 Grapes 81.5
Cheese (4.2% fat) 78.3 Peanuts 1.8
Yogurt 89.0 Walnuts 3.1
Ice cream 63.2 Ground beef (10% fat) 68.3
Dry legumes, peas 10.5 Chicken, fryer 57.5
Bread 35.0 Chicken eggs 73.7
Flour, wheat 12.0 Butter 15.5
Honey 17.2 Oils, salad 0.0
PROCEDURES FOR MOISTURE ANALYSIS

1. Drying Methods.
The dry matter that remains after moisture removal is referred to as
total solids. The food sample can be dried in forced draft oven, vacuum oven
or microwave oven etc.
Oven drying methods. The sample is heated under specified
conditions and the loss of weight is used to calculate the moisture content of
the sample. The moisture content value obtained is highly dependent on the
type of oven used, conditions in the oven, time and temperature of drying.
Using oven drying procedures, the moisture and total solid contents of foods
can be calculated as follows :
M
-;0
M OIS
. t ure (tIwt)
w Weight
= . of water in sample x 100
WeIght of wet sample
% Moisture (wtiwt) = Wt. of wet sample - Wt. of dry sample x 100
Wt. of wet sample
% Total solids (wtlwt)= Wt. of dry sample x 100
Wt. of wet sample
2. Distillation Procedures for Spices and Condiments.
Direct and reflux distillation techniques involve co-distilling the water
in a food sample with a high boiling point solvent that is immiscible in water,
collecting the mixture that distils off and then measuring the volume of water.
Distillation methods cause less thermal decomposition of some foods than
oven drying at high temperature. Water is measured directly in the
distillation procedure (rather than by weight loss) but reading the meniscus
of a receiving tube to determine the volume of water is less accurate than a
weight measurement.
FOOD ANALYSIS 65
3. Chemical Method for Low-Moisture Foods.

Karl Fischer Titration Method. Principle. The method is used for
the determination of water in low-moisture foods like dried fruits and
vegetables, roasted coffee, oils, fats, sugar or proteins. The method involves
reduction of 12 by S02 in presence of water.
2H20 + S02 + 12 ~ H 2S0 4 + 2HI
This was modified to include methanol and pyridine to dissolve 12 and S02'
C5H 5N.I2 + C5H 5N.S02 + C5H 5N + H 20 ~ 2C 5H 5N.HI + C5H 5N.S03
C5 H 5N.S03 + CH30H~ C 5H 5NHS04.CH3
These reactions show that for each mol of water, 1 mol of each 12, S02
and methanol and 3 mols of pyridine are used. For general work, a methanol
solution is used that contains 12, S02 and pyridine in the ratio of 1 : 3 : 10
and at a concentration so that 3·5 mg water is equal to 1 mL of reagent.
Titration Procedure.
Iodine and S02 are added to the sample in a closed chamber protected
from atmospheric moisture. The excess of iodine that cannot react with H 20
can be determined visually. The colour is red brown at the end point. The
method is suitable for samples with a moisture content greater than 0·03%.
Karl Fischer reagent (KFR) is added directly as the titrant if the
water in the sample is accessible. If water in the solid sample is inaccessible
to the reagent, the moisture is extracted from the food with methanol. The
methanol extract is then titrated with KFR.
Determination of KFR Water Equivalence (KFR eq).
The KFR eq value represents the equivalent amount of water that reacts
with 1 mL of KFR. The KFR eq can be established with pure water, a
water-in-methanol standard or sodium tartrate dihydrate.
36 g H 20/mol Na2C4H406.2H20 x S x 1000
KFR eq (mg H20/mL) = 230.08 g/mol x A
KFR eq = KFR water equivalence, S =Wt. of sodium tartrate dihydrate
(g), A = mL of KFR required for titration of sodium tartrate dihydrate.
KFReqxKg
% Moisture content = S x 100
where, Kg = mL of KFR used to titrate sample, S =wt. of sample (mg).
Sources of Error in the Karl Fischer Titration Method.
(i) Atmospheric moisture must not be allowed to infiltrate the
reaction chamber.
(ii) Moisture adhered to the walls of glasswares must be dried.
(iii) Owing to incomplete water extraction, cereal grains must be
ground to a fine powder.
(iv) Certain food constituents may interfere. For example,
carbonyl compounds react with methanol to form acetals and
release water, to overestimate moisture content. Ascorbic acid is

oxidised by KFR to dehydroascorbic acid to overestimate moisture
content. Unsaturated fatty acids will react with iodine, so moisture
content is overestimated.
4. Coulometric Titration.
It is ideal for samples with very low levels of moisture, from 0.03%
to ppm levels. In this method, 12 is electrolytically generated to titrate the
water. The amount of 12 required to titrate the water is determined by the
current needed to generate the iodine.
5. Physical Methods.
(i) Electrical Method.
(a) Dielectric Method. Moisture content in certain foods can be
determined by measuring the change in capacitance or resistance
to an electric current passed through a sample
(b) Conductivity Method. The conductivity of an electric current
increases with the percentage of water in the sample.
(H) Hydrometry. The moisture content in salt brines, beverages and
sugar solutions can be calculated by measuring specific gravity or density by
pycnometer or hydrometers.
(iii) Refractometry. Moisture in liquid sugar products and condensed
milks can be determined using a Baume hydrometer (solids), a Brix
hydrometer (sugar content), refractometer, or by gravimetric means. The
refractometer has been valuable in determining the soluble solids in fruit
products.
The refractive index of an oil, syrup or other liquid is a dimensionless
constant that can bft used to determine the nature of the food. When a beam
of light is passed from one medium to another and the density of the two
differs, then the be.am of light is bent. Bending of the light beam is a function
of the media and the sines of the angles of incidence and refraction at any
given temperature and pressure, and is thus a constant.
_ Sine of incident ray angle
Refractive index, 11
- Sine of refracted ray angle
whenever refractive indices of standard fluids are given these are prefaced
with 11IP = a value from 1·3000 to 1·7000. Here 20 refers to the temperature
in ·C, D is the wavelength of the light beam, the D line of the sodium
spectrum (or 589 nm from white light).
ASH ANALYSIS'
Ash refers to the inorganic residue remaining after complete oxidation
of organic matter in a food stuff. Determination of ash content is a part of the
proximate analysis for nutritional evaluation. Ashing is the first step in the
preparation of a food sample for specific elemental analysis.
Ash Content in Foods. The ash content of most fresh food rarely
exceed 5% while dried beef may contain 11·6% ash (wet weight basis).
FOOD ANALYSIS 67
Table 2. Ash content of selected foods on wet weight basis.

Percent Percent Percent
Food Food Food
ash ash ash
Apples 0.3 Whole wheat flour 1.7 Butter 2.5
Bananas 0.8 Brown rice 1.0 Cream 2.9
Dried fruits 2.3 Corn meal 1.3 Milk 0.7
Potato 1.0 White rice 0.7 Yogurt 0.8
Eggs, roast beef, fish fillet, hamburger contain 1·0, 3·0, 1·3 and 1·1
percent ash content respectively. Fats, oils and shortenings have 0·0 to 4·09%
ash, starch contains 0·3% and wheat germ 4·3% ash. Meat, poultry and sea
foods contain 0·7 to 1·3% ash.
METHODS FOR ASH ANALYSIS

Sample Preparation.
(i) Fat and Sugar Products. Animal products, spices and syrups
require treatments prior to ashing because of high fat and moisture
(spattering, swelling) or high sugar content (foaming) that may result in loss
of sample. Meats, sugars and syrups need to be evaporated to dryness. One
drop of olive oil (ashless) is added to allow steam to escape as a crust is
formed on the product. Cheese, sea food, spices may cause smoking and
burning upon ashing. Allow this to finish by keeping the muftle door open.
Ashing of the same sample may follow drying and fat extraction.
(ii) Plant Materials. Plant materials are dried prior to grinding. The
sample may be used for multiple determinations (i.e., protein, fibre). Fresh
stem and leaf tissues should be dried in two stages (i.e., first at a lower
temperature of 55·C, then at a higher temperature) to prevent artifact lignin.
Plant material with 15% moisture may be ashed before drying.
1. Dry Ashing.
Principle. Dry ashing is incineration at high temperature (550·C) in
muftle furnace. Ashing time is reduced with microwaving. Water and volatiles
are v~pourised and organic substances are burned in air to CO2 and oxides
of N 2 .• Most of the minerals are converted to oxides, chlorides, sulphates,
phosphates and silicates. Elements such as Pb, Fe, Se, Hg may partially
volatilise with this method so other methods must be used if ashing is a
preliminary step for elemental analysis.
Procedw:e.
• Weigh 5 to 10 g sample in a tared crucible.
• Place crucible in muftle furnace. Ignite at 550·C for 12 to 18 hours.
• Open the door of muffle furnace carefully to avoid losing ash that may
be fluffy.
• Quickly transfer the crucible to a desiccator for cooling and weigh it.
Calculations.
nt A h (d
-to S ry b aSIS
.) = Wt. after ashing - tare wt. of crucible
.
Wt. of sample x dry matter coefficIent
where dry matter coefficient = % solids/100.
For example, if corn meal is 87% dry matter, the dry matter coefficient
would be 0·87. If ash is calculated in wet-weight basis, delete the dry matter
coefficient. If moisture was determined in the same crucible prior to ashing,
the denominator becomes (dry sample wt. - tared crucible wt.).
However, if carbon is still present after the initial incineration, several
drops of HN03 or H 20 should be added and the sample re-ashed. If the carbon
persists, such as with high-sugar samples then
• Suspend the ash in water.
• Filter through ashless filter paper because this residue tends to form a
glaze.
• Dry the filtrate.
• Re-ash the paper and dried filtrate in muffle furnace.
Note that:
• Glycerin, alcohol and hydrogen will accelerate ashing.
• Samples such as jellies will spatter and can be mixed with cotton
wool.
• Ashing of cereals can be accelerated by adding alcoholic solution of
magnesium acetate. An appropriate blank determination is necessary.
• High fat samples should be extracted either by using the crude fat
determination procedure or by burning off prior to closing the muflle
furnace .
• Use crucible cover to prevent spattering of salt-rich foods.
2. Wet Ashing.
Wet ashing or wet digestion is a procedure for dissolving minerals and
oxidising substances with high fat content (meats etc.) using oxidising
agents.
Procedure.
• A 1 g dried, powdered sample is placed in 150 mL Griffin beaker.
• Add 10 mL HN03 and allow it to soak overnight.
• Add 3 mL of 60% HCI04 (Place a beaker under pipette tip during
transport) and heat on a hot plate upto 350°C until frothing stops and
RN0 3 is almost evaporated.
• Continue boiling until perchloric reaction occurs. Place watch glass on
beaker. Sample should be colourless. Do not evaporate the liquid to
dryness.
• Cool the beaker. Wash watch glass with minimum. deionised water.
Add 10 mL 50% HCI.
• Transfer to 50 mL volumetric flask and dilute with deionised water.
• Wash the hood pre cautiously after last sample.
Preparation for iron analysis in a meat. Boil 2 g sample in 30 mL
HN0 3 at 350·C on hot plate until 10 mL remain. Add 10 mL of 60% HCI04
FOOD ANALYSIS 69
and continue boiling until copious fumes occurs. Place watch glass in beaker.
Dilute to 100 mL in a volumetric flask following oxidation.
Advantages of Wet Ashing.
• Minerals usually remain in solution.
• There is little or no loss from volatilization because of lower
temperature.
• The oxidation time is short and requires a hot plate, hood, tongs and
safety equipments.
Disadvantages.
• Wet ashing requires constant operation attention.
• Corrosive reagents are necessary.
• Only a few samples can be handled at one time.
• Perchloric acid reacts with iron (in the assay for iron) to form ferrous
perchlorate. It forms an insoluble complex with o-phenanthrolene in
the procedure. It should not interfere with atomic absorption
spectrophotometry.
3. Modified Dry-wet Ash Oxidation.
• Evaporate moist samples (25 to 50 mL) at 100°C overnight or in a
microwave oven.
• Heat on a hot plate until smoking ceases.
• Ash at 525°C for 3 hours.
• Cool and wet with deionised distilled water and 3 mL HN03 .
• Dry and incinerate at 525°C for 1 to 2 hours.
• Weigh sample after cooling in desiccator.
4. Low Temperature Plasma Ashing.
Principle. Low temperature plasma ashing refers to a specific type of
dry ashing method whereby foods are oxidised in a partial vacuum by nascent
oxygen formed by radiofrequency electromagnetic field generator. Highly
volatile elements are preserved by this method.
Instrumentation. The equipment consists of a glass system with a
variable number of chambers for samples that may be evacuated by a vacuum
pump.
Procedure.
The ground material is inserted into individual glass boats which are
inserted into separate glass chamber. The chambers are sealed and a vacuum
is applied. A small flow of 02 or air is introduced into the system maintaining
minimum vacuum. The frequency generator is then activated at a frequency
less than 14 mHz and adjusted by the amount of wattage applied (50 to 200
watts) to control incineration. The progress of ashing may be observed
through the chambers.
Advantages.
(i) There is less chance of losing trace elements by volatilization.
(ii) The low temperature (I50°C) used with plasma ashers keeps the
microscopic and crystalline structures unaltered.
Disadvantages. The major disadvantages are small sample capacity,

expense of the equipment and operator's time.
OTHER ASH MEASUREMENTS

1. Soluble and Insoluble Ash in Water.
These measurements are an index of the fruit content of preserves and
jellies.
• Weigh the total ash.
.• Add 10 mL distilled water, cover the crucible and heat to boil.
• Filter on ashless filter paper and rinse with hot distilled water five to
six times.
• Dry and re-ash filter paper for 30 minutes.
• Weigh and calculate as percent water-insoluble ash.
• Calculate soluble ash by subtracting insoluble ash from total ash or
dry the filtrate, re-ash and weigh.
2. Ash Insoluble in Acid.
This ash determination is used to measure surface contaminations
(silicates) of fruits, vegetables, wheat and rice coatings.
• Add 25 mL of 10% HCI to total ash or water-insoluble ash.
• Boil for 5 minutes. Filter on ashless filter paper and wash with hot
distilled water.
• Re-ash dried filter paper and residue for 30 minutes.
• Weigh and calculate as a percentage.
3. Alkalinity of Ash.
The ash of fruits and vegetables is alkaline (Na, K, Ca, Mg) while that
of meats and some cereals is acidic (CI, S, P). Alkalinity of ash is used to
determine the acid-base balance of foods and to detect adulteration of food
with minerals. The salts of citric, malic and tartaric acids yield carbonates
upon combustion. Phosphates may interfere. The alkalinity of ash has be~n
used as a quality index of fruit and, fruit juices.
Procedure.
• Place ash (total or water-insoluble ash) in platinum dish and
accurately add 10 mL 0·1 N HCI.
• Heat on a steam bath.
• Cool and transfer to a volumetric flask.
• Titrate the excess HCI with 0·1 N NaOH using methyl orange as an
indicator.
• Express in terms of mL 1 N acid/100 g sample.
In case of insoluble ash, its alkalinity can be determined by titrating
directly with 0·1 N HCI using methyl orange. Express in terms of mL
1 N acid/100 g sample.
ANALYSIS OF PROTEIN
Protein is a common ingredient of all food materials. Analysis of protein
is required to know
FOOD ANALYSIS 71
• Total protein content,

• Amino acid composition,
• Protein content during isolation and purification,
• Nonprotein nitrogen and
• Nutritive value (digestibility, protein efficiency ratio or nitrogen
balance) of a protein.
Table 3. Protein content of selected foods (on wet weight basis).

Percent Percent Percent
Food item Food item Food item
protein protein protein
Milk 3.5 Wheat flour 13.3 Potato 1.6
Non fat dry milk 35.9 Rice 7.5 Soybean 34.1
Egg, raw 12.9 Starch 0.3 Beef, dried 34.3
Almonds 18.6 Cod fish 28.5 Apples 0.2
1. Protein (Casein) Content in Milk.

Milk protein (casein) can be separated from milk and analysed by the
following method.
Procedure.
• Dilute 200 mL milk to one litre with distilled water in a 2 L beaker.
• Add 1 g glacial acetic acid when a white precipitate settles down.
• Decant off the aqueous layer and wash the precipitate with water.
• Grind the precipitate with a little 0·1% NaOH solution to neutralise
the acid.
• Filter the resultant suspension through muslin cloth by pressing it
hard until the liquid coming out is faintly turbid.
• Acidify the filtrate by adding glacial acetic acid so that the solution
contains 0·1% of it.
• Wash the precipitate obtained from decanted water, neutralise with
0·1% NaOH solution and filter.
• Repeat the process of precipitation and washing.
• Finally drain off all the water from the precipitate and make its paste
with rectified spirit.
• Again filter the precipitate with alcohol and then with ether to remove
fats.
• Dry it in air oven when protein (casein) is obtained as a white
amorphous powder.
• Weigh the yield and find out the amount of protein in grams.
2. Protein (Casein) Content in Butter. It can be calculated as,
Percentage of protein = (Percentage of moisture + lactic acid + fat - 100.
3. Analysis of Crude Protein Content of Food.
Kjeldahl Method.
Sample Preparation. Solid foods are ground to pass a 20-mesh
screen. Sample should be homogeneous.
Digestion.
Place accurately weighed sample in Kjeldahl flask. Add H 2S04 and
catalyst (Hg, Cu or Se or Se02 : CuS04 in 3: 1 ratio) for complete breakdown
of organic matter. During digestion, protein N is liberated to form NH! ions.
H 2S0 4 oxidises organic matter and combines with ammonium formed. Carbon
and hydrogen are converted to CO 2 and H20.
H 2S04 ,d
Protein(N) ) (NH4)2S04
catalyst
Neutralisation and Distillation.

Digest is diluted with water. Alkali containing sodium thiosulphate i!)
added to neutralise H 2S04, Sodium thiosulphate helps to release N from Hg
which tends to bind NH!- The ammonia so formed is distilled into boric acid
solution containing indicators methylene blue and methyl red.
(NH4)2S04 +2NaOH ~ 2NHa + Na2S04 + 2H20
NHa + HaBOa ~ NH! + H 2B03" (Borate ion)
Titration. Borate anion (proportional to the amount of N) is titrated
with standardised HCI.
H 2B03" + H+ ~ HaBOa
Calculations. Moles HCI = Moles NHa = Moles N in the sample.
A reagent blank should be run to subtract reagent N from the sample N.
% N = N HCI x Corrected acid volume x 14 g N x 100
g of sample mole
where corrected acid vol. = mL std. acid for sample-mL std. acid for blank.
A factor is used to convert percent N to percent crude protein. Most
~" "
~~. '''; ... protein contain 16% N, so the conversion factor is 6·25 (1~~ = 6.25).
~~ 'f~
~ 1~ = % Protein or % N x 6·25 = % Protein.

Table 4. Nitrogen to protein conversion factors for various foods.
%Nin %Nin %Nin
Food Factor Food Factor Food Factor
protein protein protein
Egg or 16.0 6.25 Milk 15.7 6.38 Wheat 18.0 5.70
meat
Corn 16.0 6.25 Oat 17.2 5.83 Soybean 17.5 5.71
Alternate Procedures.
In place of distillation and titration with acid, NHa or N can be
quantitated by :
(i) Nesslerisation.
NH40H + 2HgI2 + 2KI + 3KOH ~ NH4Hg2I + 7KI + 4H20
Red-orange, 440nm
FOOD ANALYSIS 73
The method is' rapid and sensitive but ammonium dimercuric

iodide is colloidal and colour is not stable.
(ii) NHa + Phenol + Hypochlorite ~ Indophenol (Blue, 630 nm)
(iii) Direct measurement of NHa using ion, chromatographic method.
(iv) Micro Kjeldahl method is used to measure microgram quantities of
proteins.
Kjeldahl method is accurate and applicable to all types of
foods but it measures total organic nitrogen and not just protein
nitrogen.
4. Ultra Violet Absorption Method.
Principle. Proteins show strong absorption at UV 280 nm du~ to
tryptophan and tyrosine residues in proteins. Since the content of tryptophan
and tyrosine in each protein is constant, the absorbance at 280 nm could be
used to estimate the concentration of proteins, using Beer's law.
Since each protein has a unique amino acid composition, the extinction
coefficient (E280) or molar absorptivity (Em) must be determined for
individual protein for protein content estimation.
Procedure.
Proteins are solubilised in alkali or buffer. Absorbance of protein solution
is read at 280 nm against a reagent blank. Protein content is calculated by :
Absorbance, A =abc, where a = absorptivity,
b = cell or cuvette path length, c = concentration.
Advantages.
• UV method is used to determine protein contents of milk and meat
products.
• The method is rapid, sensitive, nondestructive and used widely in
post-column detection of proteins.
Disadvantages.
• UV has not been used widely in food systems.
• Nucleic acid also absorb at 280 nm. The absorption 280 nm/260 nm
ratios for pure protein and nucleic acids are 1·75 and 0·5. One can
correct the absorption of nucleic acids at 280 nm ifthe ratio of 2801260
nm in known.
5. Biuret Method.
A violet-purple colour is obtained when cupric ions are complexed with
peptide bonds (present in proteins) under alkaline conditions. Absorbance of
colour occurs at 540 nm. Colour intensity (absorbance) is proportional to the
protein content of the sample.
Other Methods. Protein can also be analysed by measuring the
physico-chemical properties of proteins by Lowry method, ninhydrin
method, turbidimetric method, dye binding method and Bradford method.
ANALYSIS OF FAT
Fats are esters of fatty acids with triacylglycerols. To analyse food for fat
contents accurately, it is necessary to know the general compositions of the
lipids in the foods and the physical and chemical properties of lipids and
foods. An accurate analysis of lipids in foods is important for nutritional
labelling, to determine whether the food meets the standard of identity and
is uniform.
Percent lipids (wet weight basis) of some foods are : Lard, oils
(-100), butter and margarine (80), salad (40-70), almonds (54), walnuts (64),
soybeans (18), milk (4·3), grains (3-5), germ (10), bacon (65), eggs (12), apples
(0·4), avocados (26·4) and asparagus (0·2).
Methods.
Methods used for fat analysis are :
1. Solvent extraction methods (continuous solvent extraction,
semicontinuous solvent extraction and discontinuous solvent
extraction methods).
2. Non-solvent wet extraction methods (Babcock method, Gerber
method, detergent method).
3. Refractive index method.
4. Instrumental methods.
5. Calorimetric method.
MOJONNIER METHOD FOR THE ANALYSIS OF MILK FAT
1. Discontinuous Solvent Extraction Method.
Principle. Fat is extracted with a mixture of ethyl ether and petroleum
ether. Extracted fat is dried to a constant weight and expressed as percent fat
by weight.
Preparation of Sample. Weigh or measure the test portion of a
homogeneous milk sample. If lumps of cream do not disperse, warm the
sample to 38°C and cool the warmed sample to 20°.
First Extraction.
• Weigh 10 g milk into a Mojonnier fat extraction flask.
• Add 1·5 mL NH4 0H and shake vigorously. NH40H neutralises acidic
sample and dissolves protein.
• Add 10 mL of 95% ethanol to prevent gel formation and shake for 1
minute ..
• Add 25 mL ethyl ether to dissolve the lipid and shake well.
• Add 25 mL petroleum ether and shake. It removes moisture from the
ethyl ether extract and dissolves more nonpolar lipid.
• Centrifuge for 30 s at 600 rpm.
• Decant ether solution from the flask into the previously weighed
Mojonnier fat dish.
Second Extraction.
• Add 5 mL of 95% ethanol and shake vigorously for 15 s.
• Add 15 mL ethyl ether and shake for 60 s.
FOOD ANALYSIS 75
• Add 15 mL petroleum ether and shake for 60 s.

• Centrifuge for 30 s at 600 rpm.
• Decant solution into the same Mojonnier dish.
Third Extraction.
• Add 15 mL ethyl ether and 15 mL petroleum ether. Shake for 60 s.
• Centrifuge for 30 s at 600 rpm and decant solution into the same
Mojonnier dish.
• Evaporate the solvent in the dish on hot plate at 100·C in a hood.
• Dry the dish and fat to a constant weight in a forced air oven at
100·C.
• Cool the dish to room temperature and weigh.
Calculations. % Fat = 100 x {[(wt. of dish + fat) - (wt. of dish)] - (av.
wt. of blank residue)}lwt. of sample.
A pair of reagent blanks must be prepared. For reagent blank
determination, use 10 mL distilled water instead of milk sample. Reagent
blank should be < 0·002 g. Duplicate analyses should be < 0·03% fat.
MOjonnier Method for Fat in Flour.
• Mix 2 g sample and 2 mL ethanol in a 50 mL beaker.
• Add 10 mL HCl. Heat the beaker at 80°C in water bath with stirring
for 30 minutes for hydrolysis.
• Add 10 mL alcohol and cool. The acid hydrolysed flour is extracted by
a mixture of ethyl ether and petroleum ether as described in the
Mojonnier method for milk fat.
2. Non Solvent Wet Extraction Methods.
Gerber Method for Milk Fat.
Principle.
Sulphuric acid and amyl alcohol are added to a known volume of milk.
H 2S04 digests proteins and carbohydrates, releases fat and generates heat.
Procedure.
• Add 10 mL of H 2S04 at 20·C into a Gerber milk bottle.
• Accurately measure milk sample (11 mL) into a Gerber bottle using a
Gerber pipette. Add 1 mL isoamyl alcohol. Tighten the stopper and
shake. Centrifuge the bottle for 4 minutes.
• Heat the bottle in a water bath at 60·C for 5 minutes. Read the fat
content from the graduations on the bottle neck.
Applications. Gerber method is simpler, faster and has wider
application to a variety of dairy products. The isoamyl alcohol generally
prevents charring of sugar.
3. Detergent Method for Milk Fat.
Milk is pipetted into a Babcock test bottle. An anionic detergent, dioctyl
sodium phosphate, is added to disperse the protein layer to liberate fat.
In case of other food products, a strong hydrophilic nonionic polyoxye-
thylene detergent, sorbitan monolaurate is added to separate fat. The
percent fat is measured volumetrically and expressed as percent fat.
76 ANALYTICAL CH~MISTRY
4. Refractive Index Method for Processed Meat.

The refractive index is characteristic of each kind of fat and the values
vary with degree and type of unsaturation, oxidation, heat treatment,
temperature and fat content. Fat is extracted with a solvent and the
refractive index of the solvent is compared to the refractive indices of the
extracted fat solution and fat.
5. Instrumental Methods.
Infrared Method for Milk Fat. Infrared method is based on
absorption of infrared energy by fat at a wavelength of 5·73 ~. The more the
energy absorption at 5·73 ~, the higher the fat content of the sample. This
method was used to determine the fat content of milk using a standard curve
of the infrared absorptions and fat content determined by a standard
analytical method.
ANALYSIS OF CRUDE FIBRE
Dietary fibre is defined as lignin plus plant polysaccharides that
cannot be digested by human enzymes. Major components of fibre are
cellulose, hemicellulose, proteins, lignin and hydrocolloids.
1. Gravimetric Methods.
(i) Crude Fibre.
Crude fibre is analysed by sequential extraction of the sample with
1·25% H2S04 and 1·25 % NaOH. The insoluble residue is dried, weighed and
ashed to correct for mineral contamination of the fibre residue. Crude fibre
measures cellulose and lignin in the sample but hemicellulose, pectins and
hydrocolloids are solubilised and not detected.
(ii) Total, Insoluble and Soluble Fibre.
Duplicate samples of dry, fat extracted ground foods are enzymatically
digested with a-amylase, amyloglucosidase and protease to remove starch
and protein. Insoluble fibre is collected by filtration. Soluble fibre is
precipitated by adding 78% ethanol to filtrate. The filtered fibre residues are
washed with ethanol, acetone, oven dried and weighed. One duplicate is
analysed for protein and the other is incinerated to determine ash content.
Fibre = I{esidue weight - (Weight of protein + Ash).
This (AOAC) method can be used to determine fibre content of all foods.
2. Chemical Methods for the Analysis of Fibre.
Chemical procedures collect macromolecules in the amylase-
amyloglucosidase digest by filtration with or without ethanol precipitation.
The polysaccharides in the precipitate are hydrolysed with H 2S04 and
quantitated colorimetrically or chromatographically (GC or HPLC).
The Southgate's method, Englyst-Cummings method and Theander-
Marlett methods are most widely used for the analysis of fibre.
Theander-Marlett Method.
Principle. Free sugars and lipids are extracted with ethanol and
hexane. Starch is removed by enzymatic digestion and insoluble fibre is
separated from soluble fibre. Fibre fractions are hydrolysed with H 2S04 and
FOOD ANALYSIS 77
sugar content of the acid hydrolysates is determined. Lignin is determined

gravimetrically. Fibre =:= Monosaccharides + Lignin.
Procedure. A flow diagram for this approach is illustrated in Fig. 1.
A dry, ground sample of food (lOg).
+
Sonicate and extract with ethanol and then hexane (2 times).
+
Filter between extractions with Whatman No. 65 paper.
+
Extracted residues are dried and weighed to determine sugar and lipid loss.
+
Dry sample, 4 to 5 g.
+
Digest starch with Termamyl in 75 niL of 0'1 M"acetate buffer, pH 5'0,
with 70 ppm Ca2+ at 96°C for 1/2 an hour.
+
Digest starch with amyloglucosidase for 16 hours.
+
Centrifuge and filter to separate soluble and insoluble fibre.
i
+ +
Filtrate. Pellet and filter retentate.
+
Collect soluble polysaccharides ppt.
+
Wash with ethanol, acetone and
with ethanol or by dialysing dry overnight under vacuum (40°C).
filtrate and freeze-drying the
dialysate. ~
Hydrolyse cellulose with
+
Hydrolyse soluble fibre with
12N H 2S04 (lhr).
IN H 2S04 (3hrs. 100°C). ~

Dilute acid to IN and hydrolyse
+
Soluble fibre, analyse sugars. Uronic
non-cellulose, insoluble
polysaccharides for 3 hrs.
acids are analysed colorimetrically.
Neutral sugars are measured by ~
HPLCorGC. Centrifuge and vacuum filter.
Wash filter retentate with water.
Dried filter retentate is Klason Supernatant and filtrate.

lignin. Insoluble fibre (sugars +
lignin). +
Insoluble fibre. It is sum of
sugars + lignin weight.
Total fibre =Soluble fibre + Insoluble fibre.
Fig. 1. Theander-Marlett scheme for the analysis of fibre.
This method for measuring fibre provides the most accurate estimate
of fibre over a wide range of foods.
ANALYSIS OF CARBOHYDRATES
Carbohydrates play an important role in human nutritions as energy
reserves. These are classified as monosaccharides (simple sugars),
oligo saccharides and polysaccharides.
Table 5. Carbohydrate content (wet weight basis) of selected foods.

% Carbohy- % Carbohy- % Carbohy-
Food Food Food
drate drate drate
Honey 75.10 Starch (potato) 83.10 Potato 15.40
Broccoli 2.30 Carrot 3.59 Apple 12.40
Milk (2%) 4.78 Orange 9.19 Grape 16.11
Importance of Carbohydrate Analysis. Carbohydrate analysis of raw

materials and processed foods can be used to provide a wealth of informations.
The fingerprint oligosaccharide patterns can be used to detect food
adulteration In addition, carbohydrate breakdown products can be used to
determine if a food has been irradiated.
METHODS OF ANALYSIS
1. Chemical Methods for the Analysis of Monosaccharides and
Oligosaccharides.
(i) Munson and Walker method.
(ii) Lane Eynon method.
(iii) Nelson-SomogyI method.
(iv) Alkaline ferricyanide method.
(v) Phenol-sulphuric acid method.
(vi) Anthrone method.
Most of the chemical techniques are based on the reaction of reducing
sugars with chemical reagents to yield precipitates or coloured complexes,
which are quantitated by solubilization, then titration or by spectro-
photometric determination.
Munson and Walker Method for the Analysis of Reducing Sugars.
Carbohydrates are oxidised on heating with an excess of cupric
sulphate and alkaline tartrate in basic medium to keep copper as copper
(Cu"l hydroxide.
• Upon heating, water is driven off and copper oxide is converted to
cuprous oxide.
• Cuprous oxide precipitates as the carbohydrates are oxidised and
can be determined by following methods :
(i) Gravimetric method.
(ii) Electrolytic deposition from HN0 3 where the copper oxide is
dissolved in HN03 and then deposited on Pt electrodes. The weight gain of
the electrode is related to the reducing sugar content.
FOOD ANALYSIS 79
(iii) By titration with sodium thiosulphate. Cuprous oxide is

dissolved in nitric acid. It undergoes oxidation to cupric nitrate. KI is added
and the iodide is oxidised to 12 which is titrated with thiosulphate using
starch indicator.
(iv) By titration with KMn04 • Cuprous oxide is reacted with ferric
sulphate. Fe 3+ is reduced to Fe2+. Ferrous ion is then titrated with KMn04
resulting in a colour change.
CU20 + Fe2(S04)3 ~ 2FeS04 + CuS04 + CuO
10FeS04 + 2KMn04 + 8H2S04 ~ 5Fe2(S04)3 + ~S04 + 2MnS04 + 8H20
However, the mechanism of the reaction is quite complex as
carbohydrates in basic solutions undergo tautomerisation, unsaturation and
base elimination.
Reducing sugar + Cu2+ + Base ~ Oxidised sugar + CU20
Modified Munson and Walker Method for the Analysis of Glucose,
Fructose and Invert Sugar.
Modified method involves the use of an excess of alkaline copper citrate
(in place of tartrate) and N~C03' Following the reduction, the excess copper
citrate is reacted with excess KI and the liberated 12 is titrated with sodium
thiosulphate to analyse carbohydrates.
2. Analysis of Reducing Sugars (Before Inversion).
• Take 1% reducing sugar solution (e.g., honey) in a rinsed burette.
Pipette out accurately 5 mL of Fehling solution A and 5 mL of Fehling
solution B into a 100 mL conical flask and dilute it with 40 mL of
distilled water.
• Fehling solution A. Dissolve 69·278 g CuS04 in distilled water and
dilute to 1000 mL.
• Fehling solution B. Dissolve 346 g of Rochelle salt (sodium
potassium tartrate) and 100 g NaOH in distilled water and dilute to
1000 mL.
• Heat the mixture of conical flask on hot plate.
• Run down 1% honey solution from the burette into the mixture of
conical flask till the solution turns brick-red in colour. .
• Add 1 mL of 0·2% methylene blue indicator solution.
• Again add honey solution till the end point is indicated by the colour
change from blue to red.
• Note the reading of the burette.
Calculation.
'l1 t 1 d . Strength of Fehling solution x 50 x 0·98
o a re ucmg sugar = Titre value of the honey solution
Calculation of Strength of Fehling Solution.
Strength of Fehling solution is obtained by titration against 0·5% glucose
(0·5 g in 100 mL of water) solution. If x mL of 0·5% glucose solution is
required for complete reduction of 10 mL of Fehling solutions A and B then,
Strength of Fehling solution = ~~~ x :0
Conversion factor from glucose (mol. wt. 180) to starch (mol. wt.
162).
10 parts of glucose are equal to 9 parts of starch.
. 162 9
·. ConverSIOn from glucose to starch = 180 = 10 = 0·9
3. Analysis of Reducing Sugars (After Inversion).

• Pipette out 1 mL of 10% honey (reducing sugar) in 100 mL conical
flask.
• Add 2 mL glacial acetic acid and heat to boil. Keep it for 2 hours.
• Neutralise it with Na2C03'
• Make up the solution to 100 mL with water in a measuring flask.
• Titrate this solution against 10 mL of Fehling solution A and B as
done in previous experiment.
Calculation.
Reducing sugar (after inversion)
= Strength of Fehling solution x 50 x 0·98
Titre value of the honey solution
4. Physical Methods for the Analysis of Carbohydrate Syrups.
Physical methods like polarimetry, refractometry and specific
gravity are useful as rapid quality control techniques for pure carbohydrate
syrups (honey, dextrose syrups) and juices.
5. Enzymatic Methods.
(i) Total Change Method. Sufficient enzyme is added to convert all of
the substrate in the food sample to product. The amount of the substrate in
the sample is then determined from the total change in the sample either as
substrate disappearance or as product formation.
(ii) Rate Assay or Kinetic Method. First the initial rate of the
enzyme substrate reaction is determined. From this relation the
concentration of enzyme, substrate, activator or inhibitor may be determined.
6. Modern Analytical Methods.
Modem methods afford accurate analysis of structurally similar
carbohydrates at trace concentrations and are :
(i) HPLC.
(ii) Microscale HPLC.
(iii) High performance capillary electrophoresis.
(iv) Micellar electrokinetic capillary chromatography (MECC).
(v) Capillary gas chromatography.
(vi) Supercritical fluid chromatography.
(vii) Mass and NMR spectroscopy.
ANALYSIS OF STARCH
• All natural food starch contains two types of homopolysaccharide
materials that is, amyloses and amylopectins.
• Lower molecular weight carbohydrates and lipids can be removed
from starch by extraction with 80% ethanol. Further treatment with
FOOD ANALYSIS 81
hot water removes most of the amylose and dextrins leaving

amylopectin as residue.
• Starch can be extracted from cereals by its treatment with hot
CaCl2 solution, treatment of a gelatinised sample with perchloric acid
or extraction with dimethyl sulphoxide.
• Samples with high protein levels can be treated with Carrez reagent
which results in protein precipitation.
• Following extraction, starch can be quantitatively determined
(i) by polarimetry (specific optical rotation for all cereal starches is
+203),
(H) by acid hydrolysis using H 2S04 or HCI04,
(iii) by enzymatic hydrolysis employing glucoamylase followed by
reducing sugar analysis.
• Recently glucose produced from starch hydrolysis has been
determined using cerium oxidation. The reaction involves
colorimetric conversion of Ce(IV) to Ce(III) which can be monitored at
445 nm. This rapid analysis is used to determine starch content of a
number of co:r.p.mercial starch hydrolyzates.
• Glucose produced from starch hydrolysis can also be determined by
HPLC or GC methods. Protein and C I3 NMR has been used to analyse
modified starches.
DETERMINATION OF CALCIUM
Calcium is a common constituent of food and in spices it can be analysed
as follows:
Reagents Required.
• Dilute HCI. 2 Volumes of conc. HCI (relative density 1·19) diluted
with 5 volumes of water.
• NH40H (relative density 0·90) and saturated ammonium oxalate
solution.
• Standard 0·1 KMn04 solution, standardised against sodium
oxalate.
• Dilute ~S04' 1 Volume of conc. H 2S04 (relative density 1·84) diluted
with 4 volumes of water.
• Acetic acid. 1 Volume of glacial acetic acid diluted with 2 volumes of
water.
• Bromocresol green indieator solution (0·04 %, m/v). Grind 0·1 g
bromocresol green with 14·3 mL of 0·01 N NaOH in an agate mortar.
Transfer the contents to a 250 mL flask and make up the volume with
water.
Procedure.
• Weigh 2 to 4 g of the material and obtain its total ash (as described
earlier).
• Digest the ash in dish with dilute HCl. Evaporate to dryness. Treat
the residue with dilute Hel and again evaporate to dryness on a water
bath. Treat the residue with 10 mL conc. HCI. Add 50 mL of water
and filter in a 250 mL beaker.
• Wash the residue with hot water and collect the washing in the same
beaker.
• Add to the solution in the beaker 0·5 mL of bromocresol green
indicator and then NH 40H till the colour changes to blue. Adjust the
pH at 4·5 by adding acetic acid until the colour changes to distinct
green.
• Filter the solution. Wash with hot water. Collect the washing in the
same beaker and heat to boil.
• Add saturated ammonium oxalate solution dropwise till the
precipitate appears and then add excess solution. Heat to boil.
• Digest for 3 hours. Decant the solution through ashless filter paper.
• Pour 20 mL of hot water on the precipitate and again decant the clear
solution.
• Dissolve any precipitate remaining on the filter paper by washing
with hot dilute HCI into the original beaker. Wash the filter paper
with hot water.
• Reprecipitate by adding NH40H and a little ammonium oxalate
solution.
• Digest for 3 hours. Filter through the same filter paper. Wash with
hot water until it is chloride free.
• Perforate the apex of the filter cone. Wash precipitate into the beaker.
Wash the filter paper with hot dilute H 2S04 and titrate with standard
KMn04 solution at 70·C.
Calculation.
2·8 NY
Calcium (as CaO) percent by mass = M where
N = Normality of standard KM:n04 solution.
V = Volume (mL) of the standard KM:n04 solution used for titration.
M = mass (g) of the material taken for the test analysis.
Calcium can also be calculated as follows :
Ca mg/100 g
Titre x 0·2 x Total volume of ash solution x 100
=~~--~~~~~~~~~~~==~~==~~~~~-
Volume taken for estimation x wt. of sample taken for ashing
ANALYSIS OF PHOSPHORUS
Principle.
Phosphorus reacts with molybdic acid to form a phosphomolybdate
complex. It is then reduced with aminonaphthol sulphonic acid to the complex
molybdenum blue which is measured colorimetrically.
Reagents. 1. Molybdate solution. Dissolve 25g of ammonium
molybdate in 400 mL of water. Add 500 mL of 10 N H 2S04 and make up the
volume to 1 L with water.
2. Aminonaphthol sulphonic acid solution. Dissolve in water 0·5 g
1-amino-2-naphthol-4-sulphonic acid, 30 g NaHSO g and 6 g Na2S0g. Make up
the volume to 250 mL.
FOOD ANALYSIS 83
3. Standard phosphate solution. Dissolve 0·4389 g KH2P04 in

water, add 10 mL of 10 N H 2S04 and make up to 1 L with water
(1 mL = 0·1 mg P). Add 1 mL of chloroform as preservative.
Procedure.
To 5 mL of ash solution obtained by dry ashing, add 5 mL of molybdate
reagent and mix. Add 2 mL of aminonaphthol sulphonic acid solution, mix and
make up the volume to 50 mL. Prepare similarly a blank using water in place
of sample. Allow to stand for 10 minutes and measure the colour at 650 run
setting the blank at 100% transmittance.
Standard Curve.
Dilute 10 mL standard potassium phosphate solution to 50 mL with
water (1 mL = 0·02 mg P). Pipette aliquots of this solution from 5 to 40 mL
into 50 mL volumetric flasks. Add 5 mL of molybdate reagent and mix. Add
2 mL of amino naphthol sulphonic acid reagent, mix, make up the volume to
50 mL and measure the colour as in sample. Plot concentration against
absorbance.
Calculation. Read phosphorus content from the calibration curve.
Pmg/lOOg=
= mg ofP in the aliquot of ash solution x Total volume of ash solution x 100
mL of ash solution x Wt. of sample taken for ashing
ANALYSIS OF POTASSIUM BY FLAME PHOTOMETRIC METHOD

Prinoiple.
Potassium in solution is atomised into an oxyhydrogen flame. The flame
excitE)s atoms of potassium causing them to emit radiations at specific
wavelengths. The amount of radiation emitted is measured on a
spectrophotometer. Under standard conditions it is proportional to the
concentration of potassium in solution.
Reag!ents.
1. KCI stock solution. Dissolve 1·909 g KCI in distilled water and
make up the volume to 1 litre (1·0 mg KlmL or 1000 ppm).
2. Standard solution. Measure 150 mL stock standard solution
(containing 150 ppm of K) and 5 mL HCI into a flask and make solution to 1
litre. In order to compensate for minute interferences caused by other ions
in the determination of potassium, it is necessary that the standard solution
be augmented with equivalent concentrations of those ions that occur in
highest proportions in the sample being analysed. A background of emission
spectra similar to that of plant extract is obtained when the standard contains
150 ppm Ca, 75 ppm Mg and 15 ppm P.
Standard Curve.
Dilute aliquots of standard solution from 0 to 150 mL making each
aliquot to a volume of 150 mL with 0·5 % HCI. Atomise setting the top
standard at 100% transmittance. Note the luminosity of the flame for each
concentration. Draw a standard curve by plotting concentration on abscissa

and the percentage luminosity on the ordinate.
Procedure.
Dilute an aliquot of ash solution so that it contains less than 150
ppm potassium. Add HCI so that the concentration of acid is same as that in
the standard solution. Atomise the diluted extract in a calibrated flame
photometer with the wavelength dial set at 768 nm and transmittance set at
100% for the top standard solution of potassium. From the standard curve
note the concentration.
Calculation.
u = ppm found from standard curve x Volume made up x Dilution x 100
"'''mg/lOO g Weight of sample x 1000
ANALYSIS OF SODIUM BY FLAME PHOTOMETRIC METHOD
Principle.
Sodium in solution is atomised into an oxyhydrogen flame. Excited
sodium atoms emit radiations at specific wavelengths. The amount of
radiation emitted is proportional to the concentration of sodium in solution.
Reagents.
1. NaCI stock solution. Dissolve 2·5418 g of NaCI in 1 L of distilled
water in volumetric flask (1 mL = 1 mg Na).
. 2. Standard solution. Measure 10 mL of stock standard solution
(containing 10 mg of Na) and 5 mL of HCI into alL volumetric flask and
make up the volume with water. This solution contains 10 ppm of Na.
In order to compensate for minute interference produced by other ions
in the det~rmination of sodium, precautions identical to the case in potassium
should be taken.
Standard Curve. Draw standard curve between concentration and
percent luminosity of sodium as in potassium.
Procedure.
Dilute an aliquot of plant extract so that it contains less than 10 ppm of
Na. Add sufficient HCI so that the concentration of acid is the same as that
in the standard solution. Atomise the diluted extract in a calibrated flame
photometer with the wavelength dial set at 589 nm and the transmittance at
100% for the top standard solution of sodium.
Calculation.
N~/100g=
_ ppm found from the standard curve x Volume made up x Dilution x 100
- Weight of sample x 1000
FOOD ADULTERATION
Food adulteration implies the addition of foreign matter or removal of
certain valuable ingradient from it. A food article shall be deemed to be
adulterated if
FOOD ANALYSIS 85
(i) it is not of the superior nature,

(ii) something has been abstracted from it,
(iii) it is kept under unsanitary conditions,
(iv) it consists of rotten, decomposed or filth substances and
(v) the quality of the article is deteriorated.
The adulterants used always mix well with the food articles in respect
of colour, shape, size and appearance. The various factors that lead to
unscruplous people to adulterated food are higher demand, quick gain,
cheapness, easy availability of adulterants and leniancy in the enforcement of
food laws etc.
COMMON ADULTERANTS IN FOOD
Adulterants used in specific foods are listed below:
• Milk. The addition of starch, skimmed milk, water or removal of fat.
• Milk Powder. Starch, moisture, fat deficiency.
• Pure Ghee. Vanaspati ghee, animal fat, rancid stuff, excess
moisture.
• Vanaspati Ghee. Animal body fat, rancid fat, argemone oil, sesame
oil, prohibited colours as well as flavour.
• Vegetable Oils. Mineral oils, rancid oil, argemone oil, rubber seed
oil, tea seed oil, oil soluble dyes, watermelon seed oil and other cheap
oils.
• Butter. Animal fat, starch, excess moisture, rancid stuff, vanaspati
ghee, prohibited colours.
• Pulses (Mansoor, gram). Khesari dal, sand, dirt, coal tar dyes.
• Dal (Arhar). Khesari dal coloured with metanil yellow.
• Wheat rice. Stones, dirt, sand, grit, heavy insect infestation.
• Wheat flour (Atta, suji, maida). Sand, dirt, soapstone, excess
bram, chalk powder, foreign starch.
• Mustard. Argemone seed.
• Gram flour (Besan). Pea flour, maize flour, khesari pulse flour,
sand, dirt, coal tar dyes.
• Turmeric powder. Lead chromate, yellow earth, foreign starch,
powder of rice and maize, talc, sand, grit.
• Turmeric whole. Lead chromate, metanil yellow.
• Common salt. Fine white sand, excess moisture, excessive salt
other than N aCI.
• Vinegar. Mineral acids, coal tar dye, synthetic vinegar sold as malt
or wine, less amount of acetic acid.
• Cane sugar. Fine white sand, dirt, invert sugar, urea, iron fillings,
suji (semolina).
• .Ajwain. Fine sand and foreign seeds.
• Asafetida (Bing). Sand, grit, resins, gums, chalk, coal tar dyes,
foreign resins.
• Coriander (Dhania). Cow dung, saw dust, horse dung, powdered
bran, foreign starch.
• Cumin. Synthetic zeera, mud, foreign seeds.
• Chillies. Brick powder, coloured saw dust, talcum powder, foreign

starch, powdered bran.
• Cardamom (choti elaichD. Exhaused spices.
• Cinnamon (Dalchini). Cassia bark.
• Garam masala (powdered mixture ofv.;;rrious spices and condiments).
Sand, grit, coal tar, dyes, starch etc.
• Tea leaves. Saw dust, mash husk, cashew husk, tamarind seed,
powder, colour.
• Coffee powder. Used coffee chicory, roasted date seeds, tamarind
husk, starch.
• Black pepper. Dried peels of papaya, foreign barriers, peelings of
urd dal.
• Amchur. Fine sand, heavy insect infestation.
• Sweets. Metanil yellow.
• Ice cream. Artificial sweetner, starch and non-permitted colours.
• Soft drinks. Prohibited colours, flavours, sweetners, saccharine,
mineral acids, bacterial and metal contaminations.
• Processed foods. Prohibited food additive, solvent residues and
microbial contamination.
• Cinnamon. Cassia bark.
HARMFUL EFFECTS OF FOOD ADULTERANTS

1. The consumption of adulterated foods has a slow poisoning effect. The
victims of edible oil adulterated with argemone oil show epidemic
dropsy.
Argemone poisoning leads to gastrointestinal disturbances, swelling
of limbs, hypertension, hyper pigmentation and death owing to
cardiac arrest.
2. Mixing of turmeric powder with lead chromate causes lead
poisoning, stiffness of the limbs, brain damage, anaemia and even
abortion.
3. An excessive intake of khesari dal in pulses causes permanent
paralysis of limbs.
4. The use of coal tar and non-permitted colours in dals, sweets or tea
leaves and that of mineral oils in edible oils can prove to be potential
carcinogens.
5. The consumption of meat from antibiotic fed animals causes multiple
drug resistance, hardening of arteries and coronary heart diseases.
6. The excessive intake of nitrate and nitrite in drinking water and meat
products cause methaemoglobinaemia, cancer and tumours in kidney,
trachea, oesophagus and lungs.
7. Gossypol in cotton seed flour and phallidine in mushrooms causes
cancer in man.
However, the weakest link in the prevention of adulteration is the lack
of awareness among people. They must be aquainted with the type of
adulterants used in food and punishment should be given to persons involved
in adulteration.
FOOD ANALYSIS 87
CONTAMINATION OF FOOD STUFFS

Food stuffs like canned fruits, vegetables and milk products contaminate
with microbes due to underprocessing or leakage. Underprocessing is the
failure to destroy all bacteria capable of subsequent growth in the product
during the heat process. Leakage is due to the contamination of the product
after III adequate heat process either due to faulty seam or damage to the can
after sealing.
Organisms Causing Food Contamination.
Food spoilage organisms are mainly of two types.
1. Thermophilic group such as
(i) Flat sour.
(ii) Thermophilic anaerobes.
(iii) Sulphide spoilage microbes.
2. Mesophilic group include
(i) Putrefactive anaerobes.
(ii) Butyric anaerobes.
(iii) Aciduric flat sours.
In each group, organisms have been found to produce spores
highly resistant to heat.
(iv) Lactobacilli
(v) ;Yeasts
(vi) Moulds.
The organism involved in food spoilage have definite relationship to the
acidity of the food stuff.
Food spoilage above pH 4·5.
• Bacillus stearothermophilus cause flat sour spoilage in low acid
foods (pH 5·3) like peas, corn, potatoes.
• Bacillus coagulants contaminate in medium acid (pH 4·6) canned
products such as com, spinach, asparagus.
Food spoilage is due to underprocessing or leakage.
• Thermophilic anaerobes, e.g., clostridium thermosaccharolyticum
group spoil vegetables like green beans, spinach, asparagus at pH 4·5
to 5·0. The food product may swell, burst, produce CO2 and H 2 .
• Clostridium nigrificans group cause sulphide spoilage in peas, lima
bean, cauliflower, beet, potato, french bean (pH 5.3) and produce
H 2S. Bacillus subtilis, B. mesentericus, B. polymyxa, B. macerans
(aerobic spore formers) contaminate low acid (pH 5·3 and above) foods
like peas, com, beet etc.
• Mesophilic anaerobes including clostridium botulinum disintegrate
solid food particles, produce putrid odour, CO2, H2 in cabbage, turnip,
pumpkin, carrot, okra etc. at pH 5·3 to 4·5.
• Butyric acid anaerobes (Cl. butyricum) deteriorate vegetables, corns,
soups and sauces.
• Cocci, moulds and yeasts cause frothy fermentation in liquors and
brines due to leakage.
. 88 ANALYTICAL CHEMISTRY
• Clostridium nigrificans cause sulphur stinkers in food stuffs.

• Mesophilic spore bearers, obligate or facultative anaerobes putrefy
vegetables, corns, wheat and fruits.
• Micrococci, Leuconostoc, aerobic, anaerobic spore formers and
gram-negative nonsporing rods cause leakage spoilage in juices, soups
and canned foods.
Food Spoilage below pH 4·5.
• Bacillus coagulants (B. thermoacidurans) cause occasional spoilage
in tomato juice due to under processing.
• Moulds cause softening or disintegration in canned fruits due to
leakage of containers.
• Cl. pasteurianum and Cl. butyricum, mesophilic, obligate anaerobes
spoil pears, figs, pine apples due to spore forming anaerobes.
• B. polymyxa group (B. macerans) contaminate citrus juices, prunes,
jack fruit and peach etc.
• Leuconostoc pleofructi, Leuconostoc mesenteroides cause gaseous
fermentation in canned pine apple and ropiness in peaches.
• Micrococci spoil prunes, rhubarb, pickles, chutneys at pH 3·7 or lower.
Note that: Frozen fruits and vegetables may spoil due to enzymes or
psychrophiles.
• LeafY vegetables grown in polluted streams or sewage farms may
contaminate with Escherichia coli.
• Deterioration in pickles may be due to low salt content of brine,
incomplete curing, poor quality vinegar, or underprocessing.
• Fermented pickles of sauerkraut type contain lactobacillus,
leuconostoc and halophiles.
• Ketchups and sauces may contaminate gas-producing yeasts.
• Sugars, molasses and syrups may contain thermophiles
(B. stearothermophilus, Cl. nigrificans). Osmophilic yeasts,
Aspergillus, penicillium may cause inversion.
Miscellaneous Micro-organisms Causing Food Contamination.
• Zearalenone, an oestrogenic mycotoxin produced by Fusarium,
readily colonise wheat, barley, rice, maize, cereals and bread.
• Fumonisins mycotoxins (Bl> B 2 , Bs) are produced by Fusarium
moniliforme and F. prediferatum. Fumonisins are found in maize and
rice.
• Listeria contaminate with dairy products (cheese, yoghurt), meat and
vegetables.
• Moulds of the genera Aspergillus and Penicillium produce
Ochratoxins which may contaminate coffee, cocoa, wine, beer, barley
and spices.
• Aspergillus flavus and A. parasiticus produce alfatoxins that
damage a variety of foods including nuts, dry fruits, cereals and
herbs.
• Deoxynivalenol (DON) or Vomitoxin. DON is related to
compounds known as Trichothecenes formed by Fusarium
FOOD ANALYSIS 89
graminearum and F. culmorum. DON contaminate mainly cereals,

grains, wheat, rice, maize, oats, barley, bear, bread and infant food.
• Trichothecenes are the largest group of mycotoxins produced by
fusarium moulds. There are over 40 different Trichothecenes
including 2, 3-acetyl DON, nivalenol and diacetoxyscirpenol (DAS).
Trichothecenes contaminate wheat, barley, oats and rice etc.
MICROSCOPIC EXAMINATION OF FOOD

1. Plate Method.
Microscopic examinations of food are performed by adding enriched food
samples to a microtitre plate. The plate is washed and a highly specific
monoclonal antibody conjugate is added to it which binds to microbe
antigens forming an immune complex. The plate is washed again to remove
any unbound conjugate. The presence or absence of microbe is determined by
the addition of a colourless substrate which produces a coloured product in
presence of a particular micro-organism.
Result can be read visually on a microplate reader at 450 nm.
2. Coliform Count Method for Examining Fresh Fruits and Vegetables.
Reagent required is Ringer solution. It is used at one-quarter the
original strength, and is prepared by dissolving 2·15 g NaCI, 0·075 g KCI,
0·12 g CaC12 (dry) and 0·5 g Na2S203.5H20 in 1000 mL of distilled water.
Method. Shake 100 g of the vegetable with 200 mL of Ringer solution.
Allow it to stand for 20 minutes and decant. Test positive tubes for the
presence of E coli. Report the results as the number of organisms per 100 g
vegetable. Good quality vegetables should show less than 500 E. coli per
100 g.
3. Total Bacteria Count for Examining Frozen Fruits and Vegetables.
Preparation of Dextrose-Tryptone Broth or Agar Culture
Medium. The ingredients are 10 g tryptone, 5 g dextrose, 0·04 g bromocresol
purple and 1 L water. Steam the ingredients until dissolved. Adjust pH to 7.0.
Filter, distribute 20 mL each to large culture tubes (22 x 175 mm), plug with
cotton wool and sterilise.
Procedure.
For plating, add 1 g of inoculum to the sterilised petridish and pour 10
mL of melted agar medium. Mix the inoculum in the agar and allow to
incubate. The growth of organism in the broth is indicated by the change of
colour from purple to yellow. The colonies appear on the surface with a
typical spot in the centre. Count total bacteria growing at TC in 5 days
and also count lactic acid bacteria, moulds and yeasts. Frozen peas rapidly
deteriorate on thawing. Slime usually contains large number of
Leuconostocs which gives them a yellow appearance but sometimes heavy
growth of coryneforms produces ammonia and this neutralises the acid
formed by Leuconostoc. Counts on frozen vegetables are low (l06/g).
4. Microbial Count in Vegetables and Fruits.

Culture. Inoculate dextrose-tryptone agar, tomato juice and incubate
at 30°C aerobically and anaerobically.
Counts. Count for lactic acid bacteria, flat sour thermophiles
(B. stearothermophilus), moulds, yeast and H 2S producing anaerobes.
• In both carbonated and non-carbonated drinks, the microbial count
diminished in time.
• In fruit juices, lactic acid and acetic acid bacteria may grow at pH 4.
Fruit juices of orange, lemon and grape fruit used for soft drinks and
beverages must be sterile and give a negative result, when checked
for yeasts by mixing one part of juice with nine parts of sterile 10%
(w/v) sucrose solution and incubated at 26·5°C for 14 days. The
absence of gas bubbles and a white deposit of yeast cells indicates a
yeast-free juice.
5. Direct Microscopic Examination of Food.
Take the food content in a sterile container for subculturing. Make
smears with a sterile inoculating loop. Stain with methylene blue and
Gram-stain. The presence of Gram positive rods suggests underprocessing
while cocci, yeasts etc. suggest leakage spoilage.
PESTICIDE ANALYSIS OF FOOD PRODUCTS

Chromatographic techniques used for the analysis of pesticides in food
stuffs are HPLC, GLC and TLC.
General Extraction and Purification of the Food Samples.
Extract 20 g of finely ground food (cereal) sample with dichloromethane
in the Soxhlet extractor for 3 hours. Transfer it to a Kudema Danish flask. If
the cereal extract is cloudy, pass it through a column containing 20 g of
anhydrous Na2S04' Wash the column with 40 mL of hexane or isopropyl
alcohol and mix the washing to the extractor before transferring it to the
evaporator. Add 3 drops of liquid paraffin. Heat the contents on a steam bath
until the volume becomes 3 mL. Finally evaporate to dryness in the
dechlorination flask in a current of dry air at 35°C.
ANALYSIS OF ORGANOPHOSPHATES IN FOOD BY HPLC

Organophosphate insecticides present in food products may be
parathion, malathion, methyl parathion, diazenon, trithion and EPN etc.
Reagents.
(i) H 2S0 4
(ii) Acetone
(iii) Hexane
(iv) Dichloromethane
(v) Methanol as eluting solvent
(vi) Magnesia. It is prepared by making slurry of 500 g MgO with
water. Heat on steam bath for 30 min, filter, dry at 120°C and
pulverise to pass through sieve No. 60. Store in a closed container.
FOOD ANALYSIS 91
Experimental Technique.
Extraction of the food sample. Extract the sample (1 L) in a
separatory funnel and acidify to pH 2 with H 2S04 . Add 60 mL of acetone and
shake. Then extract with 60 mL dichloromethane and hexane (1 : 1) by
shaking for 2 minutes. Water layer is transferred in the original sample
container and organic phase is collected in Kudema Danish flask. Extraction
is repeated twice with 50 mL each of dichloromethane and hexane and the
solvent is treated as above. Solvent extract volume is then reduced to 0·5 mL
under reduced pressure. Few microlitre of this concentrate is injected to
HPLC for the analysis of organophosphates.
Standard Conditions for Operating HPLC.
• The column of the HPLC is packed with 5 )lm C-8 bonded-phase
particles.
• The width and height dimensions of column are 4·5 x 250 mm.
• Flow rate of eluting solvent methanol (67 mL CH30H + 33 mL H 20)
is maintained at 2 mL per minute.
• A UV detector is attached to the instrument.
Process. The organophosphates are separated by using the gradient
elution system of mobile phase and
the elute monitored by UV detector Peak identification
at 254 nm. Chromatograms showing 1. Methyl parathion
2. Ciodrin
the peaks of various
3. Parathion
organophosphates in food sample so 4. Dyfonate
obtained are shown in Fig. 2. 5. Diazinon
Calculations are based on 6.EPN
peak height measurement of the 3 7. Ronnel
sample and the standard for 8. Trithion
determining the concentration of 1 8
4
species. 2 5 6
Analysis of Chlorinated
Insecticides in Milk by HPLC. 7
Automated HPLC pesticide analyser
can be used for the analysis of
chlorinated insecticides in milk by
injecting raw milk onto a short silica
precolumn where the fat was
retained and the pesticide eluted
with hexane. The less polar fat
eluting organochlorines such as DDT, Time,min
DDE and a-BHC were stored at the Fig. 2. Chromatograms of various
organophosphates in food sample.
top of a longer analytical column.
Pesticides such as f3-BHC, y-BHC and dieldrin which eluted later were
resolved from each other by the precolumn and passed directly into an
electron capture detector by means of a computer controlled
pneumatically-operated switching value for determining their concentration
in the milk.
GAS CHROMATOGRAPHY FOR ORGANOPHOSPHATES IN FOOD

Organophosphates such as malathion, parathion, diazenon, chlorthion,
meta-systox in food stuffs can be analysed by gas chromatography.
Analytical Steps.
Sample Preparation.
It involves chopping, grinding or macerating the food sample containing
organophosphate.
Extraction and Purification.
Extraction is carried to recover as much of the pesticides as possible by
solubilising them.
• A 250 g of the chopper or macerated composite sample is blended with
acetonitrile or acetone.
• Anhydrous salt (NaCI or Na2S04) can be added to absorb water. Or
water can be added so that the crude extract can be purified with a
subsequent partitioning step with an organic solvent. The solvent is
separated from insoluble solids by filtration.
• Sometimes emulsions are formed. Emulsion formation can be
minimised by adding a small quantity of saturated salt solution or
with a few drops of alcohol or an antifoaming agent or by centrifuging
the mixture.
• For samples of fruits, vegetables, wine, milk (that contain more
than 70% water), 100 g aliquot is taken. For dry samples, 10 to 50 g
are presoaked with water to bring the water content to 100 g.
• For matrices like butter, fats and animal tissues sufficient water is
added to the sample (10 to 30 g) to obtain a total of 100 g of water.
• Acetonitrile (200 mL) and dichloromethane (150 mL) or acetone (200
mL) and petroleum ether (150 mL) are added to water-amended
sample together with NaCI (30 g).
• The mixture is blended at high speed for 2 minutes.
• The organic phase is dried over Na2S04, reduced in volume to 3 mL,
diluted with 5 mL of an appropriate solvent and reconcentrated.
• The dilution-reconcentration steps are repeated to ensure the
complete removal of those extraction solvents that can affect the
operation of the detector.
• The resulting concentrate can be used for the analysis by gas
chromatography. For fruits and vegetables (100 g), the
co-extractives amount to a small fraction of 1 g.
Standard Conditions for Operating Gas Chromatograph.
• Capillary column, packed column or megabore columns may be used.
The column of GC is packed with chromosorb roHP. Capillary GC
columns are fabricated from fused silica and are jacketed with a
polyamide coating. These are 0·25 mm in diameter and can range in
length up to 25 m.
FOOD ANALYSIS 93
• Active stationary phase is usually permanently bonded to the inner

surface of the column.
• The temperature of the gas chromatograph is kept to 200'C for 10
minutes. The temperature of the injector a,nd detector is maintained
at 225'C.
• N2 is used as carrier gas and its flow rate is maintained at 25 mL per,
minute.
Procedure for the Analysis.
The purified extract dissolved in a volatile solvent is added to the head
of a glass column containing an adsorbing material (stationary phase), which
is maintained at elevated temperature.
• An autoinjector can deliver a preset volume of sample to the
chromatograph.
• Components of the sample mixture are volatilised at the operating
temperature of the instrument.
• Components partition between the stationary phase and a carrier gas
(mobile phase) according to their relative affinities for the two phases.
• Now ancillary devices are used in automated gas chromatographs
to increase the degree of automation of the separation, detection and
quantitation of chromatograms of pesticides in foods.
• A recording integrator can determine the retention time of each
organophosphate in the food sample as well as peak area/peak height
and the corresponding area/height ratio. The accurate measurement
of peak area or peak height is achieved using sophisticated
mathematical algorithms.
• Microcomputer based GC software packages can be used to compare
test chromatograms with a control (pesticide-free) chromatogram of
the same food matrix.
THIN LAYER CHROMATOGRAPHY FOR CHLORINATED

PESTICIDES IN FOOD PRODUCTS
Chlorinated pesticides, the economic poisons employed to regulate the
impact of various pests in agriculture, contaminate in crops and food stuffs.
These are DDT, BHC, aldrin, dieldrin, chlordane, lindane, toxaphene,
heptachlor, methoxychlor etc.
Extraction and Purification of the Food Sample.
Follow the same procedure as employed in HPLC.
Procedure for the Analysis.
Prepare the glass plate by coating silica gel slurry or alumina layer. If
adsorption TLC is to be performed, the layer of sorbent (stationary phase) is
activated by heating to 200'C for 4 hours. The food sample containing
chlorinated pesticides is applied as a spot near one end of the plate. The plate
is placed in a closed chamber saturated with hexane. The solvent migrates up
the plate by capillary action and the sample components are separated.
The chromatogram is developed with chromogenic reagent 0·2%

AgNO g or 0·5% solution of p-dimethylamino hydrochloride made in sodium
ethoxide. Moisten the plates by spraying distilled water. Heat the plate in
front of ultraviolet lamp for 1 minute. Violet to green spots are produced
by chlorinated pesticides in food sample.
RF values of some pesticides are: Aldrin 0·79, DDT 0·60, BHC 0·4,
dieldrin 0·17 and methoxychlor 0·12.
Table 6. Chlorinated pesticides contamination in food samples.

%
% Sample Sample
Insecticide Insecticide
with with
Source detected and Source detected and
insecticide insecti-
range (ppm) range (ppm)
residue cide
residue
Milk 80 DDT 0.25 to 0.50 Fruits 60 DDT 0.1
Dieldrin 0.01
Eggs 60 DDT 0.025 - 1.0 Meat 20 DDT 0.2
Lindane 0.2 Dieldrin 0.01
Vegeta- 70 DDT 0.2 Cereals 38 DDT 0.25-1.0

bles Lindane 2.0 Lindane 0.2
Heptachlor 2.0 Endrin 1.0
chlordane 2.0 BHC 0.1
Endrin 2.0
HPTLC.
High-performance thin layer chromatography is a new technique in
which TLC plates are coated with smaller, more uniform particles of
controlled porosity. This permits better detection of organochlorines in food
stuffs.
o
4
TYPES OF WATER POLLUTION
WATER POLLUTION
Water is a vital natural resource which is essential for multiplicity of
purposes. About 80% of the earth surface (i.e., 80% of the total 50,000 million
hectares area) is covered by water. Out of the estimated 1011 million km 3 of
the total water present on earth, only 33400 m 3 of water is available for
drinking, agriculture, domestic, power generation, industrial consumption,
transportation and waste disposal. In the chemical process industrial water
is used as a reaction medium, a solvent, a scrubbing medium and a heat
transfer agent. As a source of life for man, plants and other forms of life, it
cannot be replaced by any other solvent. But today water resources (rivers,
lakes, ponds, streams) are founding it more and more difficult to escape from
pollution.
The signs of water pollution are obvious to all such as bad taste of
drinking water, offensive odours from water bodies, unchecked growth of
aquatic weeds in water, decrease in number of fish in fresh water, oil and
grease floating on water surfaces. These factors disturb the normal uses of
water for public water supply, recreation and aesthetics, aquatic organisms
and wild life, agriculture and industry.
The problem is-what would happen when the water pollutants
gathered in the sea water reach the threshold levels of toxicity at which
phytoplanktons cease to function? Also what would happen when dissolved
oxygen would be depleted by biological oxygen demand? The plant and animal
life would disappear, because of death. Bacterial decomposition shifts from
aerobic to anaerobic conditions. The products of metabolism will change.
Under aerobic conditions, the products so formed are not so toxic.
C ~ CO2 , S ~ H 2S0 4, N ~ NH3 + HN03, P ~ H3P04
Under anaerobic conditions, the products tend to be odouriferous,
turbid and likely to be more toxic.
The main worry is-How is this situation to be faced? What arguments
should be proposed to the concerned pullution problems? Industrial societies
do indulge a lot of water pollutants making it foul, unpleasant, turbid and
unfit for drinking purposes.
DEFINITIONS OF WATER POLLUTION

Water pollution"'may be defined in a number of ways :
• Alteration in physical, chemical and biological characteristics of water
which may cause harmful effects on ~an and aquatic biota.
(95)
• Addition of excess of undesirable substances to water that make it

harmful to man, animal and aquatic life, or otherwise cause
significant departures from the normal activities of various living
communities in or around water.
• Any adverse change in conditions or composition of the water so that
it becomes less suitable for the purpose for which it would be suitable
in its natural state.
• Water pollution altogether nullifies or decreases the suitability of
water resources for human consumption or for the support of man's
natural life process. (Felfoldy 1982)
• Foreign substances, either from natural or anthropogenic sources,
contaminated with toxicants may be harmful to life because of their
toxicity, reduction of normal oxygen level of water, aesthetically
unsuitable and spread epidemic diseases. (WHO 1986)
• Water gets polluted when its normal functions and properties are
altered. It indicates the state of deviation from the quality and purity
of water sample.
• Pollution means the presence of any toxic substance in water that
degrades the quality to constitute a hazard or impair its usefulness.
(USPHS)
• Pollution reduces the number of species and destroys the balance of
life in streams. It is evidenced by the biological indices of community
diversity.
• Water pollution is caused due to harmful solids, liquids or gases which
are non-permissible, undesirable, unpleasant and objectionable.
• Discharge of any trade effluent, sewage or solid, liquid, gaseous
substances into water. This contaminant in water by foreign
substances may be direct or indirect and likely to cause nuisance. It
renders such water injurious to public health, domestic, commercial,
industrial, agricultural and other uses.
• Water pollution is considered not only in terms of public health but
also in terms of conservation, aesthetics, preservation of natural
beauty and resources.
• Water pollution actually represents the state of deviation from the
pure condition whereby its normal function and properties are
affected. Any shift in the naturally dynamic equilibrium existing
among environmental segments i.e., hydrosphere, lithosphere,
atmosphere or sediments give rise to the state of water pollution.
CLASSIFICATION OF WATER POLLUTION
Water pollution can be classified into four categories viz., physical,
chemical, biological and physiological pollution of water.
Physical Pollution of Water. It brings about changes in water with
regard to its colour, odour, taste, turbidity, density and thermal properties etc.
Chemical Pollution of Water. It causes changes in acidity, alkalinity
and dissolved oxygen. It may be caused either by inorganic pollutants or
organic pollutants which may be biodegradable (domestic waste) or
non-biodegradable (pesticides).
TYPES OF WATER POLLUTION 97
Biological Pollution of Water. Bacterial pollution in water is caused

by the excretory products of man, animals and birds. The main pollutants
belong to coliform group and certain subgroups, faecal streptococci and
miscellaneous organisms. Biological pollution is also brought about by
bacteria, viruses, algae, diatoms like protozoa, rotifers, crustaceans and plant
toxins.
Physiological Pollution of Water. Physiological pollution of water is
caused by several chemical agents such as chlorine, sulphur dioxide, hydrogen
sulphide, ketones, phenols, amines, mercaptans and hydroxy benzene.
USPHS has laid down the limits of 0·002 ppm. for these substances.
COMPLEXING LIGANDS IN WATER

Naturally occurring chelating ligands such as amino acids and humic
acids exist in natural water and soil. Synthetic chelating agents like sodium
ethylene diamine tetra acetate (EDTA), sodium nitrilo triacetate (NTA),
sodium tripolyphosphate and sodium citrate are discharged in small amounts
into aquatic systems. The abundance of chloride in sea water leads to the
formation of chloro complexes. Various ligands found in natural water and
waste water contain a variety of functional groups which can coordinate to
metal ions viz., Fe2 +, Cu2+, Zn2+, Co2+, Ni2+, Cd2+, Ba2+, Ca2+ and Mn2+.
o o
II /H .. II
R-C-O- R-N:"H/N~ R-O-P-O-
I
Carboxylate Phenoxide Aliphatic and aromatic o
group group amino group Phosphate group
Humic compounds. Humic substances occur naturally during the
decomposition of vegetation. They occur as deposits in soil, marsh sediments
and decayed vegetables. These non-degradable materials are classified into
humin, humic acid and fulvic acid on the basis of solubility.
The properties of water are influenced by the humic substances due
to their acid-base, adsorptive and complexing properties. The soluble fulvic
acid has an effect on the properties of water, while the insoluble humin and
humic acids affect water quality through exchanges of cations, organic
materials etc., with water.
Humic substances are high molecular weight polyelectrolytic macro-
molecules. They consist of a carbon skeleton with a high degree of aromatic
character and bulky functional groups containing oxygen. Humic substances
have elementary composition: C 45 to 55%, H 3 to 6%, 0 30 to 45%, N 1 to
5%, S 0 to 1%. They may also consist of protein-like materials and carbo-
hydrate fraction. The decomposition products of humic acid are :
OH H-C=O COOH
@yoH
H3CO~OCH3 OH
HoJ§lOH
Catechol Syringaldehyde 3, 5-Dihydroxy benzoic acid
Humic substance chelate metal ions with a carboxylic and a phenolic

hydroxyl group.
o
II
lQr
C'-...o
o O./M
I
Phenolic hydroxyl group Two carboxyl group Carboxyl group

Insoluble humic substances, the humin and humic acids exchange
cations with water and have the capacity to accumulate large quantities of
metals. Fulvic acid has a chemical formula, C lOH 15 (COOH)6(OH)5(CO)2 and
formula weight 666.
ORIGIN OF WASTE WATER

Water after its use is disposed off into the sea or a stream. Such
disposable water which cannot be used for domestic or industrial purposes is
termed as waste water. The water after use emerging out of industry is
termed as waste water or industrial effluent while water that emerges out
after domestic consumption is called domestic effluent or sewage.
Waste water usually contains soluble organics, suspended solids, traces
of chemical and heavy metals.
Main Origin or Sources of Waste Water.
1. Domestic sewage.
2. Industrial waste water.
3. Agricultural run off and storm water.
4. Urban run off waste water.
Origin of Industrial Waste Water.
(i) Textile Industry. Waste water emerges from textile industry at
different stages of processing such as
• Desizing • Bleaching • Mercerising
• Dyeing • Printing • Finishing
(ii) Wool Industry. Waste water originates during
• Scouring • Oiling • Sizing
• Filling • Dyeing
The waste water is highly coloured and contains greases, soaps,
alkalies, olive oil, starch, soda ash, detergents, chromium salts etc.
(iii) Pulp and Paper Industry. Waste water is generated during
cellulosic raw material preparation, pulping with chemicals,
washing, bleaching, chemical'recovery, screening of pulp and paper.
The effluent is dark brown, has high content of suspended and
dissolved solids (1350 mgIL and 1650 mgIL), high COD (1600 mgIL)
etc.
(iv) Electroplating Industry. Surface cleaning, stripping or pickling
and electroplating contribute to waste water stream. The effluents
contain NaOH, Na2C03, H 2S0 4 , toxic metals, aldehydes, oil,

grease, TDS and saccharin etc.
(v) Leather Tanning Industry. Tannery waste water originates
during soaking, liming, dehairing, deflushing, deliming, vegetable
tanning, bating, pickling, chrome tanning, dying, fat liquoring etc.
(vi) Fertilizer and Pesticide Industries. Waste water originates
from spill over of the final fertilizer products, boiler blow down,
cooling processes etc.
(vii) Cane Sugar Industry. Effluents emerge during spillage from
sugarcane juice extraction, clarification, wash waters from filter
press, spray pond overflow, floor washings, spillage from pan
boiling. Waste water has high BOD, obnoxious odour, TDS and high
carbonaceous matter.
(viii) Edible Oil Refinery. Sources of waste waters are spill-overs,
filter washings, neutralisation of excess fatty acids.
(ix) Oil Fields and Oil Refinery. Effluents originate during oil
pumping, desalting, demulsification, refining operations.
(x) Soap and Detergent Industry. Origins of waste water are spill
over of chemicals, floor washings, processing waste. Water contains
alkyl benzene sulphonate, builders, borax, surfactants etc.
(xi) Food Industry. Food industry encompasses canning, dairies,
breweries, distilleries and some pharmaceutical industries. Origins
of waste water are spillage, washing of cans, butter, cheese
equipments, screening, precipitation etc.
(xii) Energy Industries. Waste hot water emerges from thermal and
nuclear power plants.
TYPES OF WATER POLLUTION

Water pollution may be divided into five categories on the basis of
spurces and storage of water viz. ground water pollution, surface water
pollution, lake water pollution, river water pollution and sea water pollution.
GROUND WATER POLLUTION

Today the accelerated pace of development, rapid industrialisation and
population density have increased the demand of water resources. Ground
water, a gift of nature, is about 210 billion m 3 including recharge through
infiltration, seepage and evapotranspiration. Out of thi~ nearly one-third is
extracted for irrigation, industrial and domestic use, while most of the water
is regenerated into rivers. Over 98% of the fresh water on the earth lies below
its surface. The remaining 2% is what we see in lakes, rivers, streams and
reservoirs. Of the fresh water below the surface about 90% satisfies the
description of ground water i.e., water which occurs below the water table.
About 2% water occurs as soil moisture in the unsaturated zone above the
water table and is essential for plant growth.
Ground water acts as a reservoir by virtue of large pore space in earth
materials, as a conduit which can transport water over long distances and
as a mechanical filter which-improves water quality by removing suspended
solids and bacterial contamination. It is the source of water for wells and
springs i.e., the recommended source of rural domestic use. It is replenished
by precipitation through rain, snow, sleet and haiL
The major portion of water (about 1650 cubic kms) which goes to earth
crust is retained by its upper layer as soil moisture. Only 500 cubic kms
percolate down to the ground water deposits. About 120 cubic kms of water
applied to agricultural fields moves down to ground water table and about 50
cubic kms of surface flow also end up as ground water. Thus a total of 670
cubic kms of fresh water enters the ground annually.
Factors Affecting Ground Water Pollution.
Today human activities are constantly adding numerous wastes to
ground water reservoirs at an alarming rate. Ground water contamination is
generally irreversible i.e., once it is contaminated, it is difficult to restore the
original water quality of the aquifer. The extent of water pollution depends
on the following factors :
1. Rain fall pattern.
2. Depth of water table.
3. Distance from the source of contamination.
4. Soil properties such as texture, structure and filtration rate.
Sources of Contamination in Ground Water.
Underground sources of drinking water, especially in outskirts of larger
cities are highly polluted. Ground water is threatened with pollution from the
following sources.
1. Domestic wastes.
2. Industrial effluents.
3. Agricultural discharges.
4. Run off from urban areas.
5. Soluble effluents.
6. Waste water treatment lagoons.
7. Mine spills.
8. Seepage pits.
9. Earthen septic tanks.
10. Urban and rural garbages.
11. Refuse dumps.
12. Barnyard manures.
13. Transport accidents.
14. Leaching of pollutants.
Protecting Ground Water from Pollution.
Following steps are suggested for the protection of ground water :
1. The contaminant sources should be carefully surveyed.
2. Location of industrial and municipal disposal sites should be decided
keeping in view the ground water levels and flow pattern in the area.
3. In case of toxic industrial effluents, steps should be taken for
predisposal treatment by the industry itself.
4. Location of wells for drinking water supplies should be decided with

utmost caution.
5. Surrounding contaminant's sources and flow directions should be
considered.
SURFACE WATER POLLUTION

Surface Waters.
The emergence of industrial revolution encouraged the growth of
factories which grossly polluted the surface waters of rivers, oceans, lakes and
ponds etc. To 1150 cubic kms of surface water may be added 200 cubic kms
of surface flow which comes from outside India. The surface flow is enlarged
by addition of about 450 cubic kms of fresh water from ground water flow
while 50 cubic kms are added as runoff from irrigated areas. The surface loses
almost 50 cubic kms of its water which percolates down to the ground water
deposits. The total surface flow per year is 1800 cubic kms which are
distributed among a number of river basins.
Surface Water Should be Free from the Following Contaminants.
• Compounds which impart colour, odour and turbidity, e.g., oils,
greases, phenols, toxic metals and organics.
• Substances which may precipitate to form objectionable deposits or
float on the surface as oil, scum and debris.
• Toxic radio nuclides which actuely affect the physiology of men,
animals and aquatic organisms.
• Substances which are likely to result in enhancing the growth of
undesirable aquatic life.
• Heavy materials that check the growth of aquatic flora and fauna.
• Chlorinated compounds, chloroform and chloramines used during
chlorination of surface water should not exceed the permissible dose
(1000 ppb) fixed by EPA.
• Ozonization instead of chlorination of water should be applied.
Factors Affecting Surface Water.
• The nature and extent of surface water pollution depends on the
following factors :
• Hydrological characteristics of diluting biocides and the extent of self
purification.
• Vegetation, soil type and degree of weathering rocks.
• Waste water disposal systems and techniques for treatment of
domestic and urban sewage including pretreatment of industrial
waste waters.
• Physical, chemical and biological characteristics of waste water
entering the surface waters.
• Hygienic and health situation of the communities residing near
surface waters.
Sources of Surface Water Pollution.

Surface water comes in direct contact with the atmosphere, seasonal
streams, rivulets and surface drains. So there occurs a continuous exchange
of dissolved and atmospheric gases while the wastes are added through water
conveyance. Recently, US Department of Health, Education and Welfare
(HEW) has classified surface water pollutants into following categories.
1. Sewage and waste.
2. Industrial effluents.
3. Particulates and atmospheric gases.
4. Infectious agents.
5. Mineral and chemical compounds.
6. Dissolved toxic pollutants and surface run off.
7. Thermal pollutants.
8. Radioactive nuclides.
9. Organic chemical exotics.
In polluted surface waters, the ions like Na+, K+, M~+, SO~-, H 2P04 and
H4P20~- interact forming a variety of complexes, thereby deteriorating the
quality of surface water. Chemical processes like ion exchange, chelation,
precipitation, coagulation, aggregation, oxidation, reduction and dissolution
are operating simultaneously making the surface water an extremely complex
system.
In South, pollution problem in surface water is caused by the setting of
coconut husks. Chemical industries, textiles, paper and pulp industries
discharge maximum amount of polluted water. An estimate indicates that
about 140 m 3/tonne water is required per 1000 metre of cotton cloth. Paper
mills and News print at Nepanagar MP alone discharge 18 million gallons of
industrial effluents per day into water.
Factors Affecting Nutrient Loss in Surface Water.
Polluted surface water is highly degraded and contains much less
nutrient content. Main factors affecting nutrient loss in water are as follows:
• Irrigation and soil conservation. • Rainfall pattern and topography.
• Temperature and evaporation. • Soil erosion and sedimentation.
• Nature of vegetation cover. • Hydrological features of the sea.
Vora estimated that in India alone nearly 5·37 million metric tonnes of
nutrients (NPK) are washed away into the rivers and seas annually.
Processes Aggravating the Pollution Problems in Water.
The surface water pollution is complex due to numerous processes which
occur simultaneously in nature.
• Naturally occurring radionuclides in water.
• Chemical chain of reactions occurring in water.
• Variation in the rate of flow of water.
• Toxic resulting products in water.
• Biological activities of aquatic flora and fauna.
• Extent of water quality modification with time and space.
LAKE WATER POLLUTION

In India, coastal lakes and estuaries cover about 2·6 million hectares of
water area. The rapid pace of industrialisation and urbanisation have posed
a serious threat to these vast varieties of water resources. The world's largest
lake Salty Caspian sea in USSR and Iran covers about 1,70,000 square km
area. The largest fresh water lake Superier in North America covers 31,820
square km and the deepest lake Baikal in Siberia is 1940 m deep. Lakes like
Dal and Nagin in Kashmir, Loktak in Manipur and Hussain Sagar in
Hyderabad have seriously chocked by aquatic weeds due to eutrophication.
Sukha Tal (dry lake) and Saria Tal (rotten lake) represent high pollution
level in Kumaun region. South India's biggest natural fresh water lake,
Kolleru is fastly disappearing. Asia's largest Manchar lake is leading
towards doom. Its sweet water lake J aisamand has continue to languish,
throttled by pollution, encroachment and heavy siltage. The lake receives
high percentage of organic matter and nutrients as a result of which water
hyacinth increases by 50 tonnes per day. Lonar lake which is 1830 kms in
diameter and 150m deep is highly polluted by numerous wastes. Nagin lake
have reduced from 27m to 18m during the last 80 years due to siltation. In
1991, a red layer of algae surfaced all along the bank in Dal lake turning it
to a marsh.
Sources of Pollutants in Lake Water.
• Discharge of organic wastes from hills. Industrial toxic effluents.
• Waste sludges and dumping of tailings. Sedimentation and siltation.
• Telbal Nullah adds about 800 tonnes of silt every year in Dal lake.
• Phytoplanktons and eutrophication.
• The deterioration of the aesthetic and life supporting qualities of
natural lakes and estuaries is caused by excessive fertilization due to
eftluents rich in N, P, K. Thus water contains several dissolved gases
(N2, CO2, H 2, C1 2, NH 3, S02, NO x , H 2S), dissolved mineral salts (Na,
Ca, Mg, K, Fe, Mn, Co), suspended matters (sand, clay, silt) and
microbes. Also some of the dumped chemicals that were safely flushed
away (e.g., Hg, Cd, As, Pb, PCBs etc.) are now coming to haunt us.
How Excess Nutrients Kill a Lake.
Lakes are dying because of asphyxiation and becoming oligotrophic
(unproductive). Excess of nutrients accelerate eutrophication. In the bottom
layer biological decomposition of organic materials consume all the 02
resulting in O2 depletion. So anaerobic population dominate causing the
following reduction reactions. NOs ~ N02' ~ NH3 ~ N 2, SO~- ~ H 2S. Metal
ions precipitate as metal sulphides and settle as sediments.
RIVER WATER POLLUTION

Rivers are the most dynamic water resource of the earth's ecosystem.
Their major function being the transportation of water. They also carry to the
sea dissolved and particulate matter from crystal weathering and erosion
from land. Good quality water is inadequate even for the normal living and
is getting polluted due to numerous discharges. Major Indian rivers such as
Ganga, Yamuna, Tapti, Narmada, Sone, Chambal, Daha, Damodar,
Krishna, Cauvery, Brahmaputra, Mahi are severely polluted. Table 1
gives an idea of the quantum of discharge in major rivers of the world.
Table 1. Discharge, drainage area and particulate load of the

world's major rivers.
Discharge Drainage area Total suspended
River m 2lYr. x 1011 m 2 x 1012 solids glYr. x 1012
Amazon 66·9 6·0 800
Brahmaputra 6·3 1·2 800
Ganga 5·9 1·3 1600
Hoang Ho 1.03 - 2083
Indus 2·11 - 616
Yangtze 60 2·0 552
Mississipi 5·8 3·5 309
Nile 0·9 - 122
Ganga and Mississipi are comparable in their discharge quantities, the

total suspended solid carried by Ganga is five times that of the Mississipi. The
Missiouri river in the US, a 2470 km sewer is used for removing industrial
wastes. The lake Baikal, renowned for its pristine pure water and hailed as
an ecological paradise is being increasingly threatened due to excessive
pollutants.
The following conclusions are drawn from the river water pollution:
• Water quality is deteriorated every where.
• Damodar river (River of sorrow) is extremely polluted. Chambal river
is much polluted up to 32 kms.
• Water resources contain toxic metals such as As, Hg and other
chemicals such as ammonia, cyanide, organic matter etc., in different
concentrations.
• Coliform content also increases up to 100 fold in the river water
during monsoon.
In Mumbai and Kalyan belt, millions of litres of untreated sewage and
industrial effluents are discharged into the river water. Water is highly acidic
(pH = 1·5) in the Kallu river near Kalyan town. Acute water pollution
problems prevail in Hooghly near Kolkata, Gomti near Kanpur,
Damodar near Durgapur and Yamuna near Agra and Delhi.
Effects of Pollutants on River Water.
(i) Salt and alkali levels in water increase. Nutrient load due to
residual fertilisers increase. Pesticides accumulate in the
environment.
(ii) The major adverse impacts of sewage pollution are deoxygenation,

high BOD load, rapid eutrophication and accumulation of heavy
metals in water.
(iii) Industrial effluent, though less than one-third of bulk of sewage,
has the most dangerous impact on the biosphere due to toxic
pollutants. It causes fish kill, destruction of aquatic habitat for
micro-organisms and contamination of human food chain. Rivers of
the world are nothing but open sewers fit only to carry urban
wastes, half burnt bodies, pesticides and industrial effluents etc.
MARINE POLLUTION
Marine pollution is defined as the discharge of waste substances into the
sea resulting in harm to living resources, hazards to human health, hindrance
to fishery and impairment of quality for use of sea water. Marine pollution is
associated with the changes in physical, chemical and biological conditions of
the sea water. This water is also unfit for human consumption and industrial
purposes because of high salt content. Chemically it is a solution of 0·5 m NaCI
and 0·005 m MgS0 4 containing traces of all conceivable matter in the universe.
Like the land, the air, the rivers, the lakes, our seas and oceans also suffer from
pollution. The oceans are deemed to be our last and endless dust bin.
Sources of Oil Pollutants in Marine Water.
At present annual consumption of refined petroleum products is more
than 25 billion barrels (800 billion gallons). Such large scale consumption is
associated with some losses in water. The total annual influx of petroleum
hydrocarbons into the ocean is about 10 million metric tonnes by tankers. Oil
tankers are also responsible for oil spill in seas.
• Cargo tanker washings at sea. About 3 million tonnes of oil are
added annually to the sea by using sea water as ballast for empty
tankers. It is mixed into water when the ballast gets dumped and
carries residual oil from the tanker. It can coat 20 feet wide and half
an inch deep oil layer for 8700 miles in a beach.
• Import oil losses. Collisions in port contribute one million tonne of
oil in sea water annually.
• Bilge pumping at sea. The dumping of bilge contents by ships adds
nearly 5,00,000 tonnes of oil per year in sea water, while total influx
of oil into ocean has been 5 to 10 million tonnes annually.
• Recent oil based technology and vessel accidents near sea shore add
to extensive marine oil pollution. It is estimated that two million
tonnes of used lubricating oil are added every year in coastal waters.
Maritime accidents due to collision, fire, explosion or grounding
also result in oil release in water.
• International discharge of oily wastes from tank washings and
accidental spillages pollute the sea water severely. From Indra Dock
basin alone, more than 90,000 litres of waste oil was collected in 1984.
A recent report shows that about 20 billion tonnes of wastes per year
from industries, homes, farms and municipalities end up in sea.
• Oil leakage from 20,000 miles of pipe lines which cross water ways
may undergo corrosion, cracks or punctures and would lead to oil
pollution in sea water.
• The blowout of wells, disposal of drilling muds, accidental damages
to offshore, drilling rigs add to oil pollution in water.
• Oily wastes from oil fields or refineries near the coast produce
problems in coastal water. Shipping operations at the coastal belt add
light diesel oil and crude petroleum to sea water.
• Today refined petroleum products meet more than 60% of the world's
energy requirement. Annually about 25 billion barrels (800 billion
gallons) of petroleum products are consumed on a global basis. Such
large consumption results in some losses which severely pollute the
marine environment.
• The hazardous crude petroleum is a complex mixture of paraffins
(25%), cyclo paraffins (20%), aromatics (5%) and several toxic organic
compounds. The total influx of petroleum hydrocarbons into oceanic
water is 10 million metric tonnes per year generated due to
transportation activities which result in acute oil pollution in sea.
• Oil spills mixed with urban sewage, silt, plastics, pesticides and
insiduous toxic compounds are pervasive and complex the pollution
problems in sea. Toxic substances abound oil spilling into the oceans
at 10 times the rate of natural seeps, while lead being deposited on
soil and in waterways 100 times. Cadmium being released 40 times,
radionuclides many times and acidity of precipitation over millions of
square kilometres increasing 10-fold, are ultimately added to oceans.
Transportation of Oil-Spill in Marine Environment.
Oil is transported mainly by wind currents, waves and tides in marine
ecosystem. The dispersal of oil and its persistence in sea water are affected
by the kind of oil, chemical composition, specific gravity, temperature and the
state in which it is discharged into the sea. The distributed oil is then
subjected to natural processes like evaporation, dissolution, emulsification,
oxidation, sedimentation and uptake by marine organisms.
Due to these interactions, the volatile components of oil, such as, low
boiling aromatics (benzene, phenanthrene), paraffins (n-hexane, 2, 3-dimethyl
hexane), cycloparaffins (cyclohexane, 2, 3-bicyclo octane) readily evaporate.
Highly soluble aromatics can be removed by dissolution. Less resistant
paraffins get readily degraded by bacteria. Heavy oil residues disintegrate as
tar lumps or tar bills washed into the beaches.
Major Accidents.
World's ocean system has been subjected to 9,151 cases of oil spills
between 1967 and 2003. A total of 5,432,000 tonnes of oil has been split into
the ocean.
EFFECTS OF OIL POLLUTION IN MARINE WATER

Oil pollution in water has been an inevitable consequence of the
dependence of rapidly growing population on oil based technology. The
detrimental effects of oil pollution in sea water are as follows :
1. Physical Effects of Oil in Water.

• Reduction in dissolved oxygen. Oil films retard significantly the
rate of oxygen uptake by water.
• Reduction in light penetration. Oil slick decreases the light
intensity up to 90%. Such diffused light may hamper the photo-
synthesis in aquatic life.
• Smothering. Smothering coats of oil have killed lichens and algae
along the shore lines.
2. Effects of Oil Pollution on Marine Ecosystem.
• Extensive spreading of oil affects the floating plantation and marine
life severely. In areas of oil exploration, fishing gear and craft
operations get critically affected by crude oil and lumps of oily tar.
• Waste from oil refineries and discharged petroleum from ships cause
heavy damage to fishery. Recently heaps of dead fish were washed
ashore between Dabolim and Velcao coast in Goa. It created a big
scare especially among fisherman. Fish catches have to be discarded
because of tainting. It was believed that fish mortality occurred due
to dangerous effluent discharge from the Zuari.Agro Chemical
Fertilizer factory.
• Oil spilling causes lethal toxicity on aquatic flora.
• Direct oil coating unable the fishes to respire and clog their gill slits.
Soluble aromatics present in oil acutely affect the aquatic organisms
by disrupting their biological, physiological and behavioural activities.
• Adult marine organisms can not survive on exposure of 1 to 100 ppm.
of soluble aromatics contained in oil, while even 0·1 ppm acts as a
lethal dose for fish larvae.
• Sub-lethal dose, i.e., 10 to 100 ppb of oil component disturbs chemical
sensing and communication system of marine organisms.
• Aromatic hydrocarbons in oil may accumulate in aquatic plant
tissues even at low concentration of 10 ppb. These are carcinogenic
and adversely affect plant metabolism. Naphthalene and
phenanthrene are extremely toxic to marine biota. Benzpyrene
accumulates in food chain in fish which may cause cancer in man.
• Inhalation of aromatics result in acute intoxication. Benzene, even in
minute traces is particularly toxic. Its long exposure may cause
anaemia and leUkopenia in man.
• Hydrocarbons in oil get incorporated in body tissues of marine
animals. These are quite stable like heavy metals and pesticides.
• Hydrocarbons cause anaesthesia and necrosis in a wide variety of
lower animals while their higher doses result in cell damage and
death. Studies conducted on oil spills close to sea shore revealed the
immediate massive destruction of marine life like fish, worms, crabs,
invertebrates and lobsters etc.
• Oil pollutants may block the taste receptors of organisms and may
mimic the natural stimulation which gives rise to false responses to
organisms.
• Emulsified oil may reach the bottom of sea damaging aquatic animals
and plants. Oil may serve as a concentration medium for fat soluble
poisons like pesticides. These poisons may seriously accumulate in
aquatic biota posing deleterious effects.
3. Effects of Oil Pollution on Man.
Oil pollution in marine water also affects man critically in the following
ways:
• Paraffins, like methane and ethane are asphyxiants, i.e., they cause
acute suffocation. Some paraffins are central nervous system
depressants. Liquid paraffins can remove oil from exposed skin
causing dermatitis and pneumonia in lung tissues.
• Breathing higher concentrations of unsaturated cycloparaffins can
result in irritation and anaesthesia. Aromatic thiophenes,
benzothiophenes and mercaptans damage liver and kidneys.
• Crude oil contains sulphur compounds, small amount of nitrogen,
little olefins, metals like iron, nickel and vanadium. These are
extremely lethal. Carbonyl sulphide is dangerously poisonous. It is
also toxic to rats at 2900 ppm. Actually it dissociates to hydrogen
sulphide which acts on central nervous system resulting in death
mainly from respiratory paralysis.
4. Effects of Oil Pollution on Birds.
Ironically the oil that drives millions of vehicles around the world,
sometimes drives countless birds and animals to a most cruel death.
• Birds are specially vulnerable to damage from oil coating. The spilled
oil break down their natural insulating oils and waxes which shield
the birds from water. Ultimately they lose insulation, start shivering
and may freeze to death in winter. About 25,000 birds died in Torry
Canyon incident.
• Oil spilling in sea water causes abnormally low body temperature in
birds resulting in hypothermia. Nearly 150 rare species of·bald
eagles also became victims, when they ingested oil during Exxon
Valdez accident, scavenging an oily sea bird c~rasses.
• About 1000 sea otters died when their fur became saturated with oil
by losing insulation. Several birds developed respiratory ailments
because volatile components of oil weakened membranes in their
lungs. Others suffered from liver and kidney damage caused by
ingesting oil while cleaning their coats.
In addition to these severe effects, oil and tar coated beaches are
anaesthetic.
COUNTER MEASURES AGAINST OIL SPILLS

Several methods have been devised to deal with oil floating on the sea.
Physical Methods.
• The simplest method is to skim the oil off the surface with a suction
device.
• The floating oil can be absorbed using a suitable absorbing material

like polyurethane foam. Chopped straw and saw dust can also be
used to absorb oil from the sea water.
• Chemicals can be used to coagulate the oil.
• By spreading a powder of high density over the oil patch by which oil
can be sunk to the bottom.
• Chalk treated with stearate and 10% sand in water slurry removes the
oil considerably.
Chemical Methods.
1. Dispersion. Dispersion of oil is most satisfactory method for
removing oil from the sea surface. Natural dispersion also removes some oil
from the water. During this process small droplets of oil which are bigger than
dissolved molecules get incorporated into water in the form of a dilute oil-in
water suspension. Crude oil consists of little nitrogen, sulphur and oxygen
bearing compounds which can act as natural surfactants. The surfactant
decreases the oil water interfacial tension dissociating the oil into tiny
droplets.
A dispersant contains a surfactant, a solvent and a stabilizer which
cause the oil to spread farther and disperse. The solvent enables the surface
active agents to mix with it and penetrate into the oil slick forming emulsions.
The stabilizer fixes the emulsion and prevents it from coalescing once it is
formed. Dispersion increases the slick surface area increasing microbial
decomposition. Although dispersants are effective in diffusing a oil slick, these
chemicals are toxic to aquatic biota.
2. Evaporation. Evaporation removes about 50% of oil during an oil
slick's life time. Low boiling hydrocarbons, such as, benzene, toluene and
xylenes are lost rapidly from the water surface. Much of the oil that
evaporates is photo-oxidized in air while some of it may return to the seas as
atmospheric fall out. Photolysis and physico-chemical changes in oil
cause it to coalesce forming tar balls which would sink in water decreasing
its toxicity.
3. Emulsification. It is an effective weathering process in which water
is incorporated into the floating oil forming a water-in-oil emulsion. Such
emulsions, containing 20 to 80% water are often viscous and are called
mousse. Once mousse formation starts, clean up operation becomes difficult,
as it is almost impossible to pump.
However, mousse can be disintegrated by wind and wave action into tar
balls that wash ashore. Since emulsified oil droplets sink in water body, oil
stays in water with its toxic components like benzene, toluene or xylene
entering the food chain with disastrous long term effects.
4. Absorbents. An oil spill can be cleaned up by using absorbents, such
as, peat, moss, saw dust, straw and pine bark etc. Synthetic absorbents
include polystyrene, polyethylene, polypropylene and polyurethane which are
quite promising. On applying to the oil slick, they absorb the oil and prevent
it from spreading. When the absorbent material is removed from the water,
the oil also gets removed.
5. Burning the oil slick. Burning oil slicks. on the open seas is
comparatively less successful because the more volatile light fractions
evaporate quickly from the oil slick. The water also removes heat much faster
and the process leads to extensive air pollution.
6. Using chemical additives. Chemical additives can be used to
solidify oil from water surface. Mechanical methods involving the use of
additives and skimmers have been satisfactorily used to remove oil slicks.
7. Floating booms. Now floating booms are in common use in
harbours and areas where transfer of petroleum products occur.
S. Improved navigation aids. Hazard warning instrumentation and
offshore drilling operations can effectively protect the water from oil pollution.
Development of submerged pyramid shaped canopies to cover the drill hole
area, use of mechanical or pneumatic (air curtain) walls around the drill site
and physical encapsulation of drill and its hole can be suggested to escape
from the pollution hazards of sea water.
9. Using micro-organism in oil clean up. Microbes can be deployed
as voracious scavengers removing all kinds of oil pollutants. Various varieties
of Pseudomonas can consume esteric compounds and hydrocarbons from the
oil. The gene secreting enzymes are found on plasmids, small and
semi-autonomous rings of DNA. Some microbes can ingest dispersed oil
droplets and subsequently deposit them as faecal pellets.
o
5
SOURCES OF WATER POLLUTION
INTRODUCTION
Water is essential for the survival of any form of life. Today water
resources have been the most exploited natural system since man strode the
earth. Pollution of water bodies is increasing tremendously due to rapid
population growth, industrial proliferations, urbanisation, increasing living
standards and wide spheres of human activities. Time is, perhaps not too far
when pure and clean water, particularly in densely populated and
industrialised water scarce areas may be inadequate for maintaining the
normal living standards. Water pollution is mainly caused by natural
processes (volcanic eruptions, decomposed vegetable and animal matter) and
anthropogenic sources such as domestic, agricultural, mining and industrial
etc.
SOURCES OF WATER POLLUTION

1. Domestic Effluents.
About 75% of water pollution is caused by domestic sewage, sullage, food
processing plants, waste water and sludge from cess pools etc. Sewage
contains 99·9% water and 0·1% solids (30% inorganic matter and 70% organic
matter). Inorganic solid consists of grit, metals and salts while a major
portion of organic solids contain proteins, fats and carbohydrates. Sullage
comprises of detergents, soap, fats, grease used for washing utensils, clothes
and milk products.
Average composition of sewage in mg/L is :
Total dissolved solids 2100, BOD (5 days) 500, dissolved solids 1000,
ammonical nitrogen 25, oil and grease 100, sulphate 1000, chlorides 600,
phenolic compounds 5·0, sodium 60, zinc 15 and Cu, Cr, Pb, Ni - 2 mgIL.
Domestic sewage composed of spent water containing wine, faeces and
soapy wastes makes the water extremely anaesthetic. Accumulation of
domestic wastes in water bodies retards the self regulatory capabilities of
aquatic organisms. Self purifYing ability of water is lost and it becomes unfit
for domestic purposes. Moreover, the decomposition of wastes by aerobic
microbes decreases due to extreme water pollution.
2. Agricultural Pollutants.
Agricultural pesticides, fertilizers, farm wastes, manure slurry, plants
and animal's debris, drainage from sullage cause excessive pollution in water
resources. Modern agricultural practices uses a large number of
agrochemicals, growth hormones, nutrient solutions etc. All these chemical
(111)
residues along with organic debris from the remains of harvested crops are
trapped by run-off water causing pollution problems in receiving waters. The
agricultural run-off is considerably rich in NPK nutrients, organic matter and
pesticides. While NO) and PO~- cause eutrophication, pesticides have been
reported to get bioaccumulated and biomagnified through food chains
resulting in secondary poisoning to man, animals and predatory birds.
3. Radioactive Pollutants.
Living organisms are continuously exposed to a variety of radiation
sources illustrated below :
Natural sources of radiation are:
• Solar rays, gamma rays, cosmic rays.
• Electromagnetic radiations
• Environmental and particulate radiations.
• Radionuclides in the earth crust, and
• Internal radiations.
Anthropogenic sources. Recently man made sources have begun to
add large doses of radiation to the existing natural radioactive pollution to
which our bodies have got accustomed with several ill effects. Major sources
of radioactive pollutants are :
• Nuclear power plants and nuclear reactors.
• Radioactive materials used in nuclear weapons.
• Mining and processing of ores to produce radio-isotopes.
• Radioactive fall out from nuclear bombs.
• Emission from the industrial use of nuclear energy.
• Leakage from underground nuclear detonations.
• Use of radio-isotopes in medicine, industry, agriculture and research
operations.
• Major radio-pollution in water is caused by uranium, radium and
plutonium.
Once the radionuclides find access into the aquatic environment, they
enter the ecocycling processes and entirely disrupt the metabolic pathways.
As compared to organic poisons, injurious effects of radionuclides are
exceedingly high.
4. Thermal Pollutants.
Thermal pollution involves warming up of an aquatic ecosystem to the
point where desirable organisms are adversely affected. Following sources
contribute to thermal pollution.
• The operation of nuclear power plants.
• Nuclear fuel processing units.
• Coal-fired thermal power plants.
• Industrial effluents.
• Domestic sewage etc.
Release of heat from vario~s sources is :
Thermal power plants 75%, effluents 6%, sewage 9% and biological
activities 4 %. Hydroelectric power stations have negative effects of thermal
loading. Discharge of unutilised heat adversely affect the aquatic biota.
SOURCES OF WATER POLLUTION 113
5. Industrial Effluents.
Industrial activities generate a variety of waste products which are
generally discharged into water streams. The nature of industrial wastes
depends upon the industrial processes in which these are generated. The
pollutants usually associated with industrial effiuents are organic matter,
inorganic dissolved salts, suspended solids, fertilizing materials, thermal
constituents in the form of heat, micro-organisms and pathogens (Table 1).
Table 1. Nature of industrial effluents.

S.No. Industry Nature of effluents
1. Thermal power Heat, heavy metals, dissolved solids and inorgnic
plants compounds.
2. Pulp and paper Suspended solids, high or low pH, colour, fibres, BOD, COD,
high temperature, fibres, dissolved substances.
3. Rubber industry Chlorides, suspended and dissolved solids, variable pH and
high BOD.
4. Steel mills Acids, phenols, cyanogen, low pH, alkali, lime stone,
oils,fine suspended solids, cyanides, cyanates, iron salts,
ores and coke.
5. Cotton industry Sodium, organic matter, colour, high pH and fibres.
6. Oil refineries Acids, alkalies, phenols, tarry or resinous materials and
petroleum oils.
7. Metal plating Metallics, toxic cyanides, cadmium, chromium, zinc, copper,
aluminium and low pH.
8. Iron foundry Coal, clay, suspended solids and iron.
9. Pesticides Aromatic compounds, acidity and high organic matter.
10. Acids Low pH and organic content.
11. Antibiotics Toxic organics and high acidity or alkalinity.
12. Synthetic drugs High suspended and dissolved organic matter including
vitamins, high acidity or alkalinity.
13. Tanneries Calcium, chromium, more salt content, colour, dissolved and
suspended matter.
14. Distilleries Very high COD, low pH, high organic matter, high
suspended and dissolved solids containing nitrogen,
potassium.
15. Organic Toxic compounds, phenols, high acidity, alkalinity.
chemical
industry
16. Explosive Alcohol, metals, TNT and organic acids.
Contd. ...
S.No. Industry Nature of effluents

17. Fertilizers High pH, high ammonia, high fluoride content, acidity or
alkalinity, organic matter, nitrogen, phosphorus and
potasGium.
18. Photographic Organic and inorganic reducing agents, silver and alkalies.
products
19. Synthetic mills Zinc, toxic substances, sulphides and high pH.
20. Dairy products High BOD, alkalies, acids, greases, fats, lactose, colloidal
solids mainly proteins.
2l. Beet sugar High BOD, high suspended and dissolved organic matter.
22. Fruits and Dissolved and colloidal organic matter.
vegetables
23. Beer High dissolved solids containing nitrogen and fermented
starches and allied products.
24. Soft drinks High BOD, high pH and suspended solids.
25. Yeast High BOD and high organic matter.
26. Pickles High suspended solids, colour and organic matter.
27. Slaughter High suspended solids, dissolved organic matter and
houses proteins.
EFFLUENTS FROM SOME TYPICAL INDUSTRIES

Effluents from Chemical Industries.
The waste water emerging from heavy chemicals, fertilizer plants and
pesticide factories fall under this category. The waste water is acidic and
usually coloured with strong offensive odour. Waste water from pesticide
industry contains abnormally high concentration of organic phosphorus
compounds which is extremely toxic to flora and fauna. Phosphatic industry
waste contains tallow and slimes, suspended matter with acidic character and
high BOD. The non-degradable matter increases BOD due to the presence of
soaps, detergents and fatty acids.
Effluents from Food, Beverage and Drug Industries.
The effluents from food, pharmaceutical and drug industries have a very
high BOD, dissolved organic matter and suspended organic solvents. Waste
water from canned food, dairy products, meat poultry preparation contains
high level of proteins, carbohydrates, amino acids, alkali 'metals and
phosphates. Rice processing industry waste contains a high level of starch.
Effluents from Distillery.
The distillery waste which is fermented contains a high level of nitrogen,
total solids, suspended solids, Na, K, Ca and Fe (Table 2).
SOURCES OF WATER POLLUTION 115
Table 2. Nature of distillery waste water.

Parameter Level Parameter Level Parameter Level
Colour Brown-black odour Offensive Temperature 90-96°C
BOD 3500-4500 COD 3500-9500 Total solids 9200 mg/L
mg/L mg/L
Total N 1000-1500 pH 4·0-5·0 Sodium 120-280
ppm ppm
Potassium 800-12000 Calcium 300-900 Iron 30-80 ppm
ppm ppm
Sulphate 1500-4500 Chloride 4500-8000 Suspended 2000-14000
ppm ppm solids mg/L
Effluents from Material Industries.

Such industries compose of steel, electroplating, foundry, smity, paper
pulp, rubber and glass industries. Their wastes have colloidal colouring
matter, sulphate, non degradable lignin sulphonates, waxes, dyes and
bleaching agents. Steel industry waste is acidic due to high mineral contents,
cyanides and alkalies. The pickel waste contains Zn, Cr, Fe and rubber
industries waste contains latex coagulated rubber, suspended solids and has
a high pH value (Table 3).
Table 3. Characteristics of industrial effluent and sewage in India.

Suspended Other
BOD
(mg/L) COD solids Consti- Pollution
Industry pH (mg/L) (SS) tuents Aspects
(20°C)
(mg/L) (mg/L)
Paper industry 9·0 200 1600 1350 SOc8OO High pH, SS
Sugar factory 8·0 650-820 60-100 1500:...1800 - High pH,
SS, colour
toxicity
Tannery 9·5 7000 - 3200 Cr-15 to 20 High pH
and BOD,
SS, colour
Straw board 7·0-13·0 2000 5000 3000 - High pH,
SS, BOD
Cotton textiles 8·0-11-0 200-600 - 30-50 Dyes, Alkali,
detergents BOD, dyes
Cr-5
Steel mill - 630 - - Oil, grease, Highly
NH3/N-1000 toxic, NH3 ,
Phenols CN, Phenols
1300
Steel (finished) - 280 - 310 Phenol 98, Phenols,
NH3/N-440 NH3 ,CN
Contd. ...
Suspended Other
BOD
COD solids Consti- Pollution
Industry pH (mgIL)
(mgIL) (SS) tuents Aspects
(20°C)
(mgIL) (mgIL)
Refinery - 200 - - Sulphides, Phenols

oil-30 and
phenol-30 mineral oil
Fertilizers 8·0 30 330 3700 NH 3/N-510, Toxic due to
As NH3
Dairy 8·0 820 1350 690 Oil, grease High BOD,
SS, Grease,
high pH,
COD
Sewage 7·8 350 500 200 NH3, High SS,
Albuminoid BOD
Effluents from Thermal and Nuclear Power Plants.

The effluents of these industries contain Pb, Cu, Hg, V, Fe, Al, Ca, Mn,
In, F, S04 and silica etc. The heated discharges from thermal power plants
raises the temperature of water by IO°C. Nuclear waste contains hazardous
radioactive isotopes of several elements.
Service Industries Effluents.
These include laundry and photographic process industries. The waste
from laundry contains soap, detergents and is alkaline in nature.
Photographic industry is composed of developers, hypo, silver bromide spent
solutions, fixing inorganic reducing agents. They contain large amount of
suspended solids with turbidity of 300 to 400 JU. The wastes contain
nondegradable organic matter.
o
6
WATER POLLUTANTS AND
THEIR EFFECTS
INTRODUCTION
Water, the most abundant and natural resource, is extremely essential
for the survival of all organisms. But today clean water has become a precious
commodity and its quality is threatened by numerous water pollutants.
WATER POLLUTANTS AND THEIR EFFECTS

1. Inorganic Pollutants and Toxic Metals.
This category of water pollutants consists of inorganic salts, metal
compounds, mineral acids, metals and detergents. These pollutants enter the
water system by the activities in chemical industries, metallurgical processes
and various natural sources. Inorganic pollutants may be categorized as
follows:
(i) Acids and alkalies. Industries manufacturing hydrochloric, nitric,
sulphuric and phosphoric acids, and bases of sodium, potassium and calcium
etc., discharge enormous amount of acids and alkalies in water system.
(ii) Soluble and insoluble salts. A variety of salts when present
beyond the threshold limit, pose chronic water pollution problems. Some
metalloids of iron, cadmium, sodium, lithium and silica in water are
extremely toxic to life.
(iii) Polyphosphates. Polyphosphates in detergents are the major
source of phosphorus in water. They result in extreme eutrophic situation and
serve as algal nutrient.
(iv) Acid mine drainage. Coal mines discharge considerable
quantities of sulphuric acid and also ferric hydroxide into local streams
through seepage. These chemicals are formed by the reaction occurring
between air, water and iron pyrites (FeS2) present in coal seams. FeS2 is
stable in absence of air but when coal seams are exposed to air in mining
operations, oxidation reactions occur producing huge quantities of acid.
Micro-organisms also play an important role in the overall process involving
a series of reactions. FeS2 undergoes oxidation in the presence of moisture.
2FeS2 + 4H20 + 602 ~ 2Fe 3+ + 4H2S04 ... (1)
... (2)
Reaction (2) proceeds slowly at pH below 3·5 but can be catalysed by iron
bacterium Thiobacillus ferroxidant, while at pH 4·5 bacterium Metallogenium
accelerates the reaction. Ferrobacillus ferro-oxidants also favour the reaction.
(117)
It has been estimated that 0·5 x 10-3 M H 2S04 decreases the pH of stream
water upto 3·0. An estimate showed that 4 x 109 kg. of H 2S04 enters into
United States streams every year and its 60% originates in abandoned mines
causing massive fish kill. The water beds contaminated with acid mine
eftluents get coated with yellow deposit of amorphous semigelatinous ferric
hydroxide. Large fluxes of strong acids have been able to overwhelm the
buffering capacity of water causing drastic drop in pH. However, carbonate
rocks and bicarbonate ions can maintain the natural buffer system in water
as follows:
CaC0 3 + H 2S04 ~ Ca2+ + SO~- + H 20 + CO2
But as the pH increases, the ferric hydroxide in water covers the
particles of carbonate rock with impermeable membrane which inhibits
further neutralization of sulphuric acid.
(v) Other chemicals. The inorganic metals and organic species
interact with each other forming toxic organo-metallic compounds. The
interaction of these compounds play a major role in redox equilibria,
neutralization, colloid formation and micro-organism's mediated reactions in
aquatic ecosystem. Various industrial products, plastics and synthetic fibres
when consigned to incinerators emit highly toxic vapours and particulates in
air. They ultimately enter the water systems with rain fall. Similarly,
discarded plastic materials, poly vinyl chloride (PVC) produce toxic
hydrochloric acid. These chemicals are highly irritating even in low
concentrations and extremely lethal in higher doses to aquatic organisms.
TOXIC METALS
Toxic metals are added in aquatic system from industrial processes,
domestic sewage discharge, street dust, land run off and fossil fuel burning.
Traces of heavy metals such as Hg, Cd, Pb, As, Co, Mn, Fe and Cr have been
identified as deleterious to aquatic ecosystem and human health. Hard water
contains carbonate and sulphate ions which combine with lead forming
insoluble protective coating of lead carbonate and lead sulphate.
The major sources of lead poisoning have' been the steel and paint
industries. About 80% of lead retained in the body enters the bone affecting
the metabolic activities. Another major source of lead pollution is the burning
of gasoline containing the anti knock additive lead tetra ethyl, Pb(C2H 5)4' In
fish, Hg is present as dimethyl mercury, (CH3)2Hg, a toxic substance which
is known to concentrate in the food chain. This compound is synthesised from
elementary mercury by certain anaerobic bacteria living at the bottom of
lakes and rivers. Dimethyl mercury appears to concentrate in brain tissues
and remain their for long periods of time. Symptoms of Hg-poisoning arise
when concentration of (C2H5)2Hg in the brain reaches 5 ppm. 12 ppm is
highly fatal. Manganese also enters the water bodies through domestic
wastes, industrial eftluents and dry cell batteries. Its chronic exposure leads
to neurological disorders. Selenium content of most drinking waters is found
as 10 ppb. Its excessive amount creates' carcinogenic effect on man and
animals.
WATER POLLUTANTS AND THEIR EFFECTS 119
Table 1. Tolerance limits for trace metals in drinking water.

Tolerance Limits Tolerance Limits
Element Element
(in ppm.) (in ppm.)
Al 1·0 Be 0·50
B 0·75 As 0·001
Cd 0·001 Cr 5·0
Co 0·2 Cu 0·2
Ph 5·0 Li 5·0
Mn 2·0 Mo 0·005
Ni 0·5 Se 0·005
Zn 5·0 V 10·0
The enhanced level of heavy metals is concerned because ofthe following

reasons:
• They accumulate in human body.
• Metals create sublethal and chronic effects to organisms even in
minute concentrations.
• Due to carcinogenic and teratogenic effects in man.
• Their trace amounts cause phytotoxic and synergistic effects in living
organisms.
Detrimental Effects of Inorganic Pollutants.
• Acidic pollutants destroy most invertebrates and micro-organisms at
pH below 4·0 which prevent self-purification of the stream.
• Acids are reported to be lethal to fish. Acid mine drainage is the major
cause of fish kill. Excessive precipitation increases the mine-drainage
out put and deteriorate water quality.
• .Fe(OH)3H2fi04 of acid mine water is most injurious to aquatic biota
in water bodies.
• Alkalies wastes coming from chemical manufacturing plants, kier
liquors, wool scouring wastes, tannery and cotton mercerizing wastes,
raise the pH of the water upto 12. Thus alkalies also destroy bacteria
and micro-organisms in water.
• Strong alkalies like NaOH and KOH are known to produce
asphyxiation by the coagulation of gill secretions in fish.
• Water containing excessive salts become brackish and inhibit
penetration of sunlight in water.
• Acids mostly produce hydrogen sulphide gas when they come in
contact with sludge and mud of ponds and rivers at low pH. The gas
is highly toxic.
• Water at low pH (-6·0) can bring about excessive corrosion of plumbing
systems, piers, boats and metals.
• Acidic water having pH 4·0, increases the solubility of Fe, Al and Mg
salts. Excess of dissolved metals pose deleterious effect on aquatic life.
• High acidity and alkalinity of water damage agricultural fields.

Irrigated water contains higher concentration of salts in the range of
25 to 8000 mg / L. Today 30% of the irrigated land in the world is
severely affected by water salinity.
• Excess of inorganic pollutants like carbonates, sulphates, bicarbonates
of calcium and magnesium make the water hard and unsuitable for
boilers. Iron and manganese salts make water turbid and unsuitable
for drinking purposes.
• Not only the soluble salts affect aquatic life, but they cause serious
diseases in man. Nitrate coming from nitrogenous organic matter
causes mathemoglobinemia in children in the range of 20 to 40 ppm.
Detrimental Effects of Toxic Metals.
• Trace metals in water act as cumulative poisons and accumulate in
living organisms causing chronic diseases. Metallic contaminants
destroy bacteria and hinder purification system of rivers.
• Heavy metals have a great affinity to attack sulphur bonds, protein,
carboxylic acid and amino group thereby disrupting the cell
metabolism.
• Metals in water bind the cell membrane in aquatic organisms affecting
transport processes through the cell wall. They also tend to precipitate
phosphatic compounds and catalyze their decomposition.
• Toxic materials cause chromosome damage and interfere with the
process of heredity in man. Ag, Cd, Pb and Sb make the water
dangerous to drink.
• In adults, a concentration of 80 micrograms of arsenic per 100 g of
blood causes brain damage in children.
• WHO pointed out that shell fish can concentrate mercury to the level
of 10 mg per kg. Mercury poisoning caused Minamata disease in
Japan in 1953.
• Copper in 2 parts per hundred million of water is fatal to sticklebacks.
• Heavy metal ions precipitate the mucous secretion of the gills in fish.
These precipitates occupy the interlameller spaces arresting the
movement of gill filaments and block their respiratory' tract.
• Metallic contaminants from the industrial wastes get precipitated and
settle down with sewage sludge. It prevents the further use of water for
industrial and domestic purposes.
• Metals are considered to be indestructible poisons. Their dispersion
into seas, rivers and streams for a long duration may be highly
dangerous because they may affect the production of atmospheric
oxygen, contaminate the water and affect aquatic life.
2. ORGANIC POLLUTANTS
Organic pollutants enter into water system through domestic sewage,
industrial wastes from paper mills and tanneries, waste from slaughter
house, meat packing plants, food processing plants, plant nutrients,
detergents, biocides, run off from crop lands and decomposition products of
organic compounds. The addition of carbohydrates, fatty acids, proteins,

soaps, detergents and oils cause organic pollution of water. A recent estimate
showed that more than 70 million tonnes of organic chemicals are synthesized
every year and have multiplied 10 times since 1950.
Organic pollutants in water may be categorized as follows :
(i) Carbohydrates and proteins. Household wastes in sewage and
faecal matter are the principal contributors of carbohydrates and proteins in
water. Sugar, starch, cellulose, sucrose, glycogen, dextrin and alginic acid are
added to water.
Main components of protein deteriorating water bodies consist of amino
acids, albumin, gelatin, casein and keratin etc. These compounds undergo
putrefaction by bacterial action to release sulphur and phosphorus
compounds. They produce sulphuretted gases like hydrogen sulphide and
sulphur dioxide which in turn cause putrid and musty smell in water.
(ii) Oils. Water insoluble oils and soluble oils, i.e., cutting and
degreasing oils cause enormous oil pollution in water.
(iii) Aldehydes. Acetaldehyde, benzaldehyde, furfural, formaldehyde
and vanillin etc., cause odour in water, inhibit algal growth and are toxic to
fish and other aquatic animals.
(iv) Aromatic hydrocarbons and polychlorinated biphenyls
(PCBs). PCBs are used in dielectrics, lubricants and plasticizers. In
animals, PCBs affect the central nervous system and respiratory tract.
(v) Phenolic compounds. They enter into the water system through
trade wastes and produce bitter taste in water.
Effects of Organic Pollutants.
• Organic compounds in water undergo degradation and putrefaction by
bacterial activity. They consume dissolved oxygen which is an essential
requirement for aquatic plants and animal's life in water.
• -Organic matter coming from domestic and agricultural lands contains
nutrients which nourishes algal growth. There occurs a loss of all DO
content resulting in dead pool of water.
• These pollutants present an anaesthetic scene and disturb recreational
uses of water.
3. SEWAGE
Sewage is a cloudy dilute aqueous solution containing mineral and
organic matter. About 75% of water pollution is caused by sewage, domestic
wastes, food processing plants, garden wastes and sewage sludge from cess
pools etc. Sewage contains decomposable organic matter and exert an oxygen
demand on the receiving waters. Sewage treatment deposits sludge at the
bottom while liquid waste consists of ions Ca2+, Mg2+, Na+, K+, Cl-, N02,
a
SO~-, PO~-, HCO etc.
Effects of Sewage.
• Sewage containing oxidizable and fermentable matter causes
depletion of dissolved oxygen in the receiving water bodies affecting
the aquatic flora severely.
• Oxygen deficiency also leads to the production of objectionable odours

in water. Due to the evolution of putrefied gases, the solid wastes are
buoyed up by these gases resulting in offensive foul matters floating
on water surface.
• Presence of solid matter floating in suspension, colloidal and
pseudo-colloidal dispersion in sewage creates serious water problems.
• Suspended matter present in sewage has a tendency to blanket the
stream thereby interfering with the spawning of fish and reduction of
aquatic biota.
• Sewage from different cities in India has a high value of BOD because
water used for treating flushing system is less than that used in
developed countries. In Madras sewer blockage and overflowing is
common which intensely pollute the aquatic environment. Another
problem is the high grit content in water caused by using sand for
scouring pans which create hindrances at inlets of treatment plants.
• Sewage poses major threat to water courses. Today developed countries
are fighting against thermal and chemical pollutants, while Indians
have to combat with chemicals and pathogens with their limiteq,
resources.
4. SEDIMENTS
The natural process of soil erosion gives rise to sediments in water.
Sediments include soil, sand and mineral particles washed into aquatic
environment by flood waters. In addition, large deposits of sewage sludge,
pulverised coal ash and various industrial solids are disposed off into waters.
Suspended solid loadings reaching natural waters are about 700 times as
large as solid loading from sewage discharge. Soil erosion gets enhanced 10
times as a result of agricultural development and about 100 times due to
construction activities. Bottom sediments are subjected to anaerobic
conditions and have the ability to exchange cation with surrounding aquatic
medium. Sediments are important repositories for trace metals, e.g., Cr, Cu,
Co, Mn, Ni etc.
Detrimental Effects of Sediments.
Sediments destroy aquatic organisms. Bottom sediments decrease
fish population by blanketing fish nests, spawn and food supplies. Suspension
may cause thickening of fish gills which may lead to asphyxiation of the fish.
Toxic metals like Hg, Cd and Pb present in sediments attack sulphur
bonds in enzymes causing immobilizing effect and transportation through cell
membrane.
Reduces light penetration in water. Sediments reduce direct
penetration of sunlight which lowers photosynthesis in aquatic plants.
Sediments result in less food availability and plant biomass.
Water gets clouded. Sediments increase the cost of water treatment
used for culinary purposes. Sediments passing through power plant turbines
make serious abrasion and wear. Due to turbid water, the hunting ability of
fish gets curtailed. The amount of suspended solids, which are greater from
surface run off than that of sewage discharge deteriorate the water quality
broadly.
Water bodies get easily flooded. Sediments make the rivers,
streams, channles and reservoirs to overflow. They also change the flow rates
and depths of water systems as well as destroy the life of reservoirs. However,
expensive treatments are needed to counteract these effects.
5. SYNTHETIC DETERGENTS
Detergents include ingredients like surfactants, builders and additives
such as anticorrosive sodium silicate, stabilizers and soil suspending
carboxymethyl cellulose etc. The surfactant is a surface active agent which
dissolves partly in water and partly in organic solvent because of its dual
hydrocarbon and polar character. Alkyl benzene sulphonates (ABS) are
considered as surfactants. They decrease the surface tension of water so that
they penetrate the surface and interstices of the object being cleaned. Oils and
greases also absorb the hydrophobic end of surfactants.
The builder is usually a sodium phosphate (polyphosphate) of the type
Na5P301O or Na4P207 acting as a sequestering agent. The builders form
chelated complexes with calcium and magnesium ions and react with water
forming alkaline solutions. Both the surfactants and builders of detergents
create serious pollution problems in water. Additives may consist of
enzymes, perfumes and bleaching agents. The active components of
detergents are double headed amphipathic molecules usually consisting of a
bulky water insoluble hydrocarbon chain to which is attached a highly polar
group. The hydrolytic enzymes used in detergents act by solubilizing the
biological stains by degrading the large molecules into smaller water soluble
compounds which do not adhere to fabric. Structure of alkyl benzene
sulphonate is as follows :
CHa CHa CHa CHa
I I
16
I
CH a-C-CH2 -C-CH2 -C-CH 2 -C-H
HI HI H
1#
I
SOaNa
Alkyl benzene sulphonate (ABS) showed remarkable resistance to
biodegradation (the so called hard detergents) and has been subsequently
replaced by a new surfactant called linear alkyl sulphonate (LAS) which is of
comparable cost and cleaning potential but is rapidly biodegradable. At
present, one of the most pressing problems is not the effects of surfactant
itself but the release of polyphosphate builders into natural waters.
These detergent builders contribute significantly to the phosphate
present in sewage effiuents. The main builder Na5P301O undergoes fast
biodegradation and hydrolyse to orthophosphate.
P30~O + 2H20 ~ 2HPO~- + H 2P04
These hydrolysed products do not pose any serious threat to aquatic

organisms. But the phosphates released into streams act as plant nutrient,
thus supporting eutrophic conditions. In the US, a legislation restricting the
phosphate levels in detergents was proposed and became a law in some
states. Nitrilotriacetate (NTA) was considered as a replacement but it proved
to be hazardous to human health. At present there is no acceptable substitute
available for polyphosphate builders in detergents. The best alternative is to
minimise the use of phosphates in detergents.
Currently, celluzyme obtained from Hermicola insolents, is added in
detergents. The high percentage of sodium tripolyphosphate (STPP) in
detergents may be partly replaced by enzymes. Lipases can remove lipids,
amylases remove starch, proteases degrade proteins etc. These enzymes can
be produced by microbes. Alkaline proteases, obtained from Bacillus licheni
formis remove stains of blood and protein materials. The process, however,
causes protein to precipitate on the fabric.
Effects of Synthetic Detergents.
• Detergent enzymes are potential allergens and can cause serious
complications if inhaled or when they penetrate the body through
wounds or cuts, in people allergic to them. Although enzyme
detergents were introduced in India in 1970, they had to be
withdrawn for reasons of toxicity.
• Complex formation between NTA (nitrilotriacetate) and Hg or Cd
increases the possibilities of transmission across the placental barrier
into a foetus, thereby increasing the likelihood of birth defects.
• NTA is degraded in waste treatment systems, but under anaerobic
conditions it exists in some septic tanks. These NTA may persist and
get back to a water-well system.
• Phosphate, the major ingredient of most detergents, favour the
luxuriant growth of algae which forms algal blooms. This extensive
growth of algae consumes most of the available oxygen from water.
This depletion in 02 level becomes detrimental to growth of aquatic
organisms which produce a foul smell upon decay. Such decomposing
waters are known to produce toxins as strychnine which kills animals.
• Detergents increase the concentration of soluble salts, in addition to
the toxicity caused by the surface active materials.
• The surface active ingredients which are not easily degradable
produce nuisance at sewage works by creating foam and froth. It has
been reported that about 50% of these substances occur in the final
effluents.
• The increased use of syndets, which replaced surface active agents
like soap are able to produce foams even in very low concentrations,
so aeration is not possible. As a result, the rate of re-aeration of a
river water as well as the efficiency of sewage purification is reduced.
• Laboratory experiments have proved that water plants are adversely
affected by syndets. They also produce foul tastes in water bodies.
• The aquatic flora and fauna are destroyed due to the toxicity of these
syndets.
• Although detergents are not highly toxic to fishes, they do cause

damage to gills and remove protective mucus from gills, skin and
intestine.
• Alkyl benzene sulphonates kill several species of mayfly numphs
when they are exposed to 15 ppm for 10 days.
e Aquatic invertebrates are also severely affected by the detergents.
Gray fish are reduced in numbers by 15 days exposure to 10 ppm of
toxic effluents.
6. OXYGEN DEMANDING WASTES

Dissolved oxygen (DO) is essential for sustaining plant and animal life
in any aquatic system. The optimum DO in natural water is 4 to 6 ppm.
Decrease in DO level is an index of pollution due to organic matter such as
sewage, industrial effluents, wastes from food processing plants, paper mills
and tanneries, run off from agricultural lands etc. All these materials undergo
degradation by bacterial activity in presence of DO. The net result being the
deoxygenation process and quick depletion of DO. DO is actually affected by
reaeration, photosynthesis, respiration and oxidation of wastes.
The primary cause of deoxygenation of aquatic system is the presence of
organic substances called oxygen demanding wastes. When these substances
enter a water-way, DO is consumed in their breakdown by micro-organisms,
so the organic substances can be said to exert a demand on the availability of
DO. The more the oxygen is required for the breakdown of the substance, the
greater will be the deoxygenation of waterway. Pollution results when the
oxygen demand exceeds the available oxygen. The organic wastes, in addition
to depleting DO levels produce annoying odours affecting taste and colour. DO
in the long run ends up in a dead pool of water.
Oxygen Sag Curve. The discharge of wastes into water causes
depletion of DO level as the wastes are oxidised by the bacteria. Opposing this
drop in DO is reaeration which replaces O2 through the surface, at a rate
which is proportional to the depletion of O2 below the saturation value. The
simultaneous action of deoxygenation and reaeration produces a typical
pattern in the DO concentration of the
aquatic system. This pattern is known as Point of waste discharge
the DO sag and a typical curve is shown in
Fig. 1. The sag curve initially drops as the
wastes deplete the O 2 faster than it can be DO
replaced. At the point where the dissolved
oxygen is minimum, the rate of reaeration
becomes equal to the rate of deoxygenation.
Beyond this point, the rate of reaeration tc
exceeds the rate of deoxygenation and the Distance downstream
DO level begins to increase and eventually Fig. 1. Oxygen sag curve.
returns to normal.
Biological Oxygen Demand (BOD). The degree of microbially
mediated 02 consumption in water is known as BOD. It is a measure of
oxygen utilised by micro-organisms during the oxidation of organic materials

during 5 day period. The demand for oxygen is directly proportional to the
amount of organic waste which has to be broken down. Hence BOD is a
direct measure of O 2 requirements and indirect measure of
biodegradable organic matter. If a given amount of organic matter is
introduced into a water sample and its decomposition is measured, then the
rate of oxidation of organic matter (rate of decline of BOD) can be
approximated as a first-order reaction whose kinetics may be expressed as,
dL =-k L
dt 1
where L = BOD remaining in time t-the concentration of the remammg

oxidisable material (mg/L), kI = reaction rate constant or deoxygenation
constant, day-I. However, the assumption of first-order reaction for the BOD
oxidation is an over simplification since the model does not include bacterial
cell concentration as a parameter and does not correspond to microbial growth
relationships. Drinking water usually has a BOD of less than 1 mgIL and
water is considered fairly pure with a BOD of 3 mglL. But when BOD value
reaches 5 mgIL, the water is of doubtful purity.
Table 2. Range of BOD levels of some industrial effluents.

Source BOD range (mgIL) Source BOD range (mgIL)
Dairy wastes 400-2000 Food processing wastes 500-4000
Wool scouring 500-10,000 Chrome tanning 500-5000
Paper pulping 1500-25,000 Pharmaceuticals 400-10,000
In the first stage of BOD, carbonaceous matter is degraded by bacterial

oxidation. But when nitrogenous material is also present, nitrifying bacteria
exert an additional oxygen demand. During carbonaceous stage, ammonia is
produced by the breakdown of organic nitrogen materials, but this stage is so
slow that nitrifiers do not predominate until nearly the end of carbonaceous
stage. Ammonium ion is then oxidised to nitrite and then to nitrate by
Nitrosomonas and Nitrobacter micro-organisms respectively.
2NH! + 302 ~ 2N02" + 2H20 + 4H+
2N02" + O2 ~ 2NO a
The oxidation process speeds up towards the end of the first stage and
slows down again as ammonia is oxidised. Ammonia exerts a very high oxygen
demand requiring over 4·5 times its own weight of oxygen for complete
oxidation. Thus, if nitrification is allowed to occur in the receiving stream, a
further decrease in the oxygen resource will be observed.
Example. Calculate the BOD of a water sample which contains one gm of
urea for every 100 litres of water. The reaction between urea and oxygen is
NH2 CONH2 +402 ~ CO2 +2N03+2W+H2 0
Solution. From the balanced equation it is clear that 4 moles of 02 are

required to react with one mole of urea. One mole of 02 weighs 32·0 g. and
one mole of urea weighs 60 g. We have
4(32·0 g. 02) = 1 (60·0 g. urea)
or 4 x 32 g. = 128 g. 02 = 60·0 g. urea
To react with 1·0 g. urea, we need
1·0 g. urea x 128·0 g. 02 3
60 g. urea
= 2.13 g. 02 = 2·13 x 10 mg 02
2·13 x 103 mg 02 .
Thus BOD = 100litres = 21·3 mg Oz/htre
7. DISEASE-CAUSING AGENTS
Water has been a potential carrier of toxic inorganic and organic
materials, non-biodegradable matters and pathogenic microbes which can
endanger health and life. The potable water contaminated with municipal
sewage and industrial wastes is the root cause of dangerous diseases in living
beings.
Effects of Pathogens.
• Parasites are considerably harmful for man. Egg of nematodes, hook
worms and tape worms occur mostly in crude sewage. When such
sewages are discharged into water bodies without treatment,
contamination of water occurs with resultant danger to man and
aquatic life.
• The enteric diseases are transmitted mainly by drinking
contaminated water or swallowing food. The pathogens most
frequently transmitted through water cause infections of intestinal
tract like typhoid, paratyphoid, amoebic dysentry, cholera, polio and
infectious hepatitis.
• The disease causing organisms present in the faeces of infected people
get ultimately mixed with the water supply spreading chronic
diseases.
• Intestinal helminthes, i.e., Ascaris lumbricoides and
Trichuristrichiura are also water borne. Entamoeba histolytica is the
casual agent which causes internal amoebiasis and several
extra-intestinal diseases.
• The guinea worm, responsible for dracontiasis, is transmitted through
open village wells and ponds infested with the copepod intermediate
host.
• Several human diseases whose epidemics recurrently detrimate
human population, get contaminated by water.
Infections Transmitted from Man to Man.
• Typhoid fever, bacillary dysentery, cholera, poliomyelitis and hepatitis
occur from contaminated water and food through inhalation.
• Staphylococcal and streptococcal infections occur from water, air, food

or contact and inhalation.
• Caxsackie and Echovirus diseases occur from water through
ingestion.
Table 3. Some water-borne diseases.

Pathogen Diseases Pathogen Diseases
Salmonella typhosa Typhoid fever Vibrio cholerae Cholera
S. Typhimurium Enteric fever S. choleraesuis Salmonella septicemias
S. Schottmulleri Gastroenteri tis Shigella dysenteriae Bacterial dysentery
Schistosomiasis Snail fever Mosquitoes Urban yellow fever
Anopheles mosquito Malarial fever Hook-worm Skin diseases
Index Organisms of Water Contamination.

The identification of pathogens in water requires many sophisticated
techniques. Sometimes heavy chlorination is needed to destroy the eggs and
cysts of bacteria, viruses and other parasites. The standard method involves
determination of the most probable number (MPN) of coliform organisms in
the water sample. Following parameters indicate the level of pollution.
(i) Coliform Group. The most prevalent in the coliform group are
strains of Escherichia coli and Enterobacter aero genes which occur in millions
in polluted water. These are aerobes, facultative anaerobes and non spore
forming rods that produce acidity and gases from lactose fermentation at
40°C within two days.
(ii) Bacteria. Bacteria occurring in human faeces include
Streptococcus faecalis, Clostridium mainly C. perfringes and Lactobacillus
bifidus. Their presence in considerable number suggests water pollution of
few hours or days.
8. RADIOACTIVE POLLUTANTS
Radioactivity is a physical type of environmental pollution caused by the
increase in natural back ground radiation emerging from the activities of
man.
Sources of Radioactive Pollutants in Water.
• Use of radioactive materials in nuclear weapons.
• From nuclear power plants and nuclear reactors.
• Mining and processing ores to produce radio-isotopes.
• Radioactive fall out from nuclear bombs.
• Use of radio-isotopes in medicine, industry, agriculture and research
operations.
• Uranium ore which contains 2 to 5 pounds of U 30 S per tonne.
Large quantities of uranium tailing's are produced during extraction
which find their way into water. Once these radionuclides find access into the
aquatic environment, they entirely disrupt the metabolic pathways.
Radium is the most significant waste product and is considered to be a

hazard in drinking water. Certain marine organisms have the capacity for
accumulating radionuclides from water. This biomagnification may cause
objectionable radioactivity in living organisms. Phytoplankton and fish '-
may concentrate metal radionuclides by factors of 102 to 105• Water
supplies must not contain more than 3 picocuries per litre of Ra-226, nor
more than 10 picocuries per litre of Sr-90.
Effects of Radioactive Pollutants in Water.
Living organisms are considered as the prey for radioactive
contaminants in water. As compared to organic poisons, injurious effects of
radionuclides are exceedingly high.
• Radioactive contaminants deposit on surface and ground water. This
water consumed by plants during photosynthesis acts as a medium for
radioactivity in them.
• In living organisms, radiation produces a whole host of extremely
hazardous species like H+, H 2 , H 20-, H 20+, e-, e+, H0 2 , H 30- and
H 20 2 etc., causing severe effects.
• The radioactive materials in water react with proteins of aquatic
invertebrates and appear to deactivate enzymes by breaking
S-H-S hydrogen bonds. With enzyme inhibition, cell growth may
continue, but cell division may be stopped.
• In aquatic animals, radiation damage makes cell membranes
permeable so that abnormal interchange of materials through an
imperfect cell membrane can result in temporary or permanent injury
in them.
• Ionizing radiations in water mainly result in cellular damage. When
a water molecule in the cell is irradiated, an electron is knocked out
of orbit. This ejected electron can then attach to a normal water
molecule making it unstable. It splits into H+, OH- and free radicals
H· and OH·. These free radicals react with various molecules in the
cell which can no longer function normally and ultimately die.
• Radionuclides occurring in water bodies from leaching of minerals or
reactors include Ra-226, Sr-90, K-40, Ba-140, Kr-85, Co-60, Mn-54,
Pu-239, 1-131, Cs-137, U-236, Zr-95, Y-91, Nb-95 and Ru-103. Some of
these fission products are insoluble in water and settle out with
particulate materials and concentrate in silts and sludges. The soluble
isotopes remain in solution and enter into biological cycle causing
chronic health effects.
• Reports indicate that Zn-65 accumulates in oysters at high level,
Fe-55 concentrates in fishes, while Sr-90 accumulates in certain
animals and affects their metabolic activities seriously.
• Traces of radioactive materials present in water cause cancers,
leukemia, eye cataract, DNA breakage and carcinoma in man.
• Drinking water containing Rn-222, Ra-226 and Th-232 could
accumulate dangerously in man causing somatic and genetic
disorders.
9. PLANT NUTRIENTS
Plant nutrients constitute an important limiting factor for plant growth.
Nitrogen and phosphorus are the main nutrient species which enter in to
fresh and marine systems changing oligotrophic water to intensely
productive eutrophic conditions. According to Wetzel each phosphorus
molecule promotes the incorporation of 7 molecules of nitrogen and 40
molecules of carbon in aquatic algae. These nutrients ultimately tend to
accumulate in ground water.
Why do small addition of Nand P promotes algal growth?
Continued addition of phosphorus into water may lead to nitrogen depletion
in the photic zone. Under these conditions, blue green algae fIxes sufficient
nitrogen to main eutrophic conditions. Phosphates and essential inorganic
nutrients, i.e., iron and manganese form highly insoluble compounds and are
effectively lost to the lake sediments. Carbonates and associated cations lead
to artifIcial oligotrophy. Many pathogens begin to grow on products in water
bodies under anaerobic conditions and spread fatal water borne diseases.
EUTROPHICATION
Eutrophication is a natural process, derived from the Greek word
eutrophos meaning well nourished or enriched. This enrichment leads to
other slow processes referred to as natural aging of lakes. C.H. Weber
described eutrophication as nutrient rich conditions used to determine the
flora of German peat bogs as eutrophe, mesotrophe and oligotrophe. It is a
phenomenon through which a nutrient rich bog in a shallow depression
changes to leached bog deficient in nutrients. Eutrophication escalates rapidly,
however when normally high amounts of nutrients from fertilizers, domestic
and industrial wastes, urban drainage, detergents, animal wastes and
sediments enter water streams. Eutrophication was fIrst studied in lake
located in north western Ontario.
Lake Erie in USA. Lake Erie in USA is an excellent example of
eutrophication due to human's activities. Nearly 80 tonnes of phosphates were
added in this lake daily in 1965. Each 400 g of P0 4 encourages 350 tonnes of
algal slime. Due to extensive algal growth, the lake appeared as big mounds
producing unpleasant odour, clogging pipes and interfering with fIshing and
navigation.
Types of Eutrophication.
(i) Natural Eutrophication. The process of lake aging characterised
by nutrient enrichment is called natural eutrophication. During this process
oligotrophic lake is converted into an eutrophic lake. It permits the production
of phytoplankton, algal blooms and aquatic vegetation including water
hyacinth, aquatic weeds, water fern and water lettuce which in turn provide
ample food for herbivorous zooplankton and fIsh.
(ii) Cultural Eutrophication. This process is generally speeded up by
human activities, which are responsible for the addition of 80% nitrogen and
75% phosphorus to lakes and streams. Lake Mendota and Lake Washington
have undergone rapid eutrophication due to man's activities. In India,
recreational value of Kashmir lakes is reduced while Nainital lake is
undergoing a rapid eutrophication as a result of sewage, domestic waste and
detergent addition.
Effects of Eutrophication.
Eutrophication causes several physical, chemical and biological changes
which considerably deteriorate the water quality.
• During eutrophication, algal bloom release toxic chemicals which kill
fish, birds and other aquatic animals causing the water to sink.
• Decomposition of algal bloom leads to oxygen depletion in water. Thus
with a high CO2 level and poor oxygen supply, aquatic organisms
begin to die and the clean water turns into a stinking drain.
• When 02 level falls to zero (anaerobic zone), some bacteria drive
oxygen through reduction of nitrates. On complete exhaustion of
nitrate, oxygen as a last resort be obtained by reduction of sulphate
yielding hydrogen sulphide causing foul smell and putrefied taste of
water.
• Many pathogenic microbes, viruses, protozoa and bacteria etc. grow
on sewage products under anaerobic conditions. It results into spread
of fatal water-borne diseases such as polio, dysentery, diarrhoea,
typhoid and viral hepatitis.
• Algae and diatoms attain high degree of dominance due to over
fertilization. Algae and rooted weeds interfere with the hydroelectric
power, clog the filters, retard the water flow and affect water quality
and water works.
• Macrophytes, particularly Hydrilla, Potamogeton, Ceratophyllum and
Myriophyllum assume high population densities making near shore
and shallow regions unsuited for any purpose.
• During eutrophication midge Chironomous plumosus and tubificid
worms develop extremely high populations creating anaesthetic and
economic problems in water.
" Phytoplankton communities are most sensitive to eutrophication.
Investigations on lake Wisconsin showed that their population in
eutrophic lake is smaller as compared to oligotrophic water.
• The lake undergoing eutrophication may become oxygen deficient
destroying fish habitats leading to the elimination of several desirable
aquatic species in water.
• Prolonged eutrophic conditions lead to dystrophic state. The lake
receiving huge amounts of organic matter from alloethonous source
are called dystrophic. These lakes contain bog flora and high amounts
of humic acid while planktonic productivity is very low.
• In India, Dal, Nagin, Loktak lake and Hussain sagar are seriously
chocked by aquatic weeds affecting fisheries production, utility for
aquatic flora and aesthetic value.
Control of Eutrophication.
• The waste water must be treated before its discharge into water
streams.
• Recycling of nutrients can be checked through harvest.
• Eutrophication can be minimized by removing nitrogen and
phosphorus at the source, division of nutrient rich waters from the
receiving bodies and dilution of these elements. In lake Tahoe in
California and lake Washington in Seattle, total removal of sewage
effluents produced marked reversal of eutrophication.
• Algal blooms should be removed upon their death and decomposition.
• Algal food web should be disrupted to stimulate bacterial
multiplication.
• Algal growth can be controlled by limiting the dissolved nutrients.
The most suitable, feasible and effective method involves the use of
chemicals to precipitate additional phosphorus. Such precipitants
include alum, lime, iron and sodium aluminate.
• Physico-chemical methods can be adopted to remove dissolved
nutrients. For example, phosphorus can be removed by precipitation
and nitrogen by nitrification or denitrification, electrodialysis, reverse
osmosis and ion exchange methods.
10. THERMAL POLLUTANTS IN WATER
Thermal pollution involves warming up of an aquatic ecosystem to the
point where desirable organisms are adversely affected. Coal fired power
plants, electric power plants, chemical industries as well as nuclear energy
plants discharge their heated effluents into nearby lakes or rivers. A coal fired
power plant at 40% efficiency generates 16·7 joules of waste heat for every
41·8 joules of fuel burnt. All these processes increase the temperature of water
by lOoC to 15°C. A single 100 MW power plant may use one half million
gallons of cooling water per minute. The heated waters have reduced amount
of dissolved oxygen content which results into killing of marine life.
Effects of Thermal Pollution on Aquatic Ecosystem.
• Reduction in dissolved oxygen. DO content is decreased in the
warm water. Normal, biological reduction of DO level of the
atmospherically unreplenished lower layer of water may give rise to
anaerobic conditions leading to fish mortality.
• Direct fish mortality. There appears to be particular temperature
ranges that are tolerated by fish and other related species. For
example, lethal temperature for trout is 77°F, for yellow perch 88°F
and for carp it is 85°F. Thus thermal death of fish may occur due to
the action of heat on nervous system, inactivation of enzymes and
coagulation of cell protoplasm.
• Interference with reproduction. The increased temperature
triggers deposition of eggs by female. Other activities like nest
building, spawning, hatching and migration etc. get disturbed by
rising temperature.
• Longevity. High temperature increases activities in aquatic

animals, which exhaust the organisms and shortens life. Generally
the speed of a chemical change is doubled for every lOoC rise in
temperature. Daphie lives for 40 days at BOC while 29 days at 21°C.
• Increased vulnerability to disease. Some bacteria such as,
chondroccus grow rapidly with rising water temperatures. It is
believed to be responsible for the massive kill of blue black salmon on
the Columbia river in 1946.
• Invading destructive organisms. Sometimes hot water permits
the invasion of highly destructive organisms. A best example is the
invasion of Shipworms into New Gersey's Oyster Creek.
• Destruction of aquatic animals. Power plants require enormous
amount of stream water for cooling purposes, even 500 million gallons
per day. So a large number of fish, plankton and insect larvae may be
sucked into the condenser along with the cooling water and destroyed
by thermal shock, water velocity and pressure.
• Changes in algae population. Blue green algae and diatoms have
different tolerance ranges for water temperature. Enriched nutrients
and increased water temperature promote blue-green algal blooms.
• Disruption of food chain. Heated water effluents disturb aquatic
food chain.
11. BIOLOGICAL POLLUTANTS IN WATER

Biological pollutants may be conveniently classified into Primary
pollutants and Corollary pollutants.
(i) Primary pollutants. These comprise biota that are added to water
directly due to man's activities, e.g., pathogenic bacteria or viruses. These
pollutants exist for a short time in water.
(ii) Corollary pollutants. They are not added directly to water bodies
by man, but are attributable to human endeavour e.g., algae. These pollutants
are the indigenous living materials that interfere with the beneficial uses of
water.
Addition of domestic sewage, fertilizers, pesticides and industrial
effluents etc., encourage the growth of bacteria, viruses, algae, fungi, harmful
parasites and micro-organisms, which adversely affect human health.
Effects of Primary Pollutants.
(i) Bacteria. Pathogenic bacteria are the chronic disease carriers
which pose a serious threat to human health. Several diseases are
transmitted to man by these pathogens through contaminated water supplies.
The common and severe diseases are dysentery, typhoid fever, cholera,
gastroenteritis, jaundice, leptospirosis, brucellosis and tularemia, which are
transmitted in water, polluted by animal discharges. The dangerous
pathogenic disease spreading bacteria are-Salmonella typhosa, Leptospira,
Vibrio cholera, Mycobacterium, Pasteurella and Tuberculosis etc.
(ii) Viruses. The common viruses present in polluted waters and
sewage are Adenoviruses, Enteroviruses, Polioviruses, Coxsackie viruses and
infectious Hepatitis viruses. Most dreadful viruses disease is poliomyelitis in

which body parts get paralysed due to the destruction of certain nerve cells
controlling the muscles.
(iii) Parasites. Deformities of body parts and mal-functioning of
organs like liver, kidney and intestine take place by harmful parasites.
Ascaris, round worms and helminthic parasites enter and multiply in man's
body by drinking such polluted water.
Effects of Corollary Pollutants.
• Green algae produces a nasturtium like smell at high concentrations.
Several algal species impart colour and turbidity to water.
• Algae are considered to be indirectly responsible for gastroenteritis.
Accumulation of dead planktons in sand filters offers a substrata for
the growth of Pseudomonas which causes gastroenteric troubles.
• Algae are deadly poisonous which attack epidermis and central
nervous system.
• Algae poison is considered to be one of the most virulent poison which
produces cirrhosis of the liver. It also reduces the resistant power
against diseases in man.
• Algae filaments are capable of clogging filters. They also interfere
with the water treatment processes like sand infiltration and
disinfection.
• Algae reduce the useful capacity of reservoirs by concentrating at
certain depths in water.
• They can deplete 02 or supersaturate water with oxygen causing
imbalance in oxygen content. They cause heavy fish mortality through
poisoning.
• Dead algae form a mat on the surface of water and act as oxygen
barrier.
• Excessive algal growth destroys the recreational and aesthetic value
of water bodies. They concentrate in huge mass on the shore. So if
they are not removed, they decompose and cause unpleasant septic
odour in water systems.
12. PESTICIDE POLLUTANTS

Pesticides are the organic chemicals used to control unwanted and
dangerous species of plants and animals. These are the economic poisons
employed to regulate the impact of various pests upon our life and economy.
Arsenic sulphide, lead arsenate, Paris green (copper aceto arsenite) were used
to control insects. At present more than 10,000 different pesticides are widely
used. The need of increased food production as a result of population
explosion led to manipulation of land resources causing a stress in the natural
environment. The production and protection technologies are, however, so
interwoven and interdependent that it is impossible to visualize a shoot up in
crop production individually. There was 108% increase in the yield of IR-8 and
194% in TN-1 varieties, when grown under plant protection umbrella. The
annual world production of pesticides grew from 6000 million pounds (1954)
to over 24000 million pounds (1975). Of these 6000 million pounds were
organochlorine insecticides which persist in the environment.
Classification of Pesticides.
Herbicides are used to kill weeds. Fungicides are toxic to fungi.
Insecticides are meant to kill insects. Rodenticides are used against rodents
(cats and mice). Nematicides inhibit nematodes.
Molluscicides are used to kill molluscs (snails and slugs).
Piscicides are used to control undesirable fish species.
Synthetic pesticides are more effective that penetrate into plant
tissues. Chlorophenoxy acid compounds, i.e., 2, 4-D, 2, 4, 5-T and MCPA are
known as hormone weed killer. 2,4, 5-T and picloram were used as defoliants
by US forces in Vietnam in 1968-69. Since weeds are not pests like insects,
fungi or bacteria so a broader term biocide can be used to include herbicides.
Structural Classes of Insecticides. Insecticides are :
(i) Chlorinated hydrocarbons
(ii) Organophosphates
(iii) Carbamates
(iv) Chlorophenoxyacids.
Among them chlorinated hydrocarbons and organophosphates serve
mainly as insecticides. Structure and permissible limits of pesticides are
illustrated in Table 4.
Table 4. Structure and uses of pesticides.

Trade Pennissi-
Formula Uses ble limit
name
I. Chlorinated hydrocarbon
Aldrin Cl Soil insecticide for 0·003 Ilg/L
Cl control of nuts,
beetles and cotton
pests. Use is now
Cl banned in USA.
Cl
Cl
Cl
Dieldrin
o
Cl
Cl
Chlordane Effective against O·Olllg/L
Cl Cl
termites. Potential
Cl:¢CrCl carcinogen. Use
Cl2 I banned in USA in
1975.
Cl.
Cl
Contd . ...
Trade Permissi-
Formula Uses
name hIe limit
Lindane Control of cotton 0·01 ~g/L
CI
insects and rice stem
Cl*Cl H
H
H
H
borer.
CI H Cl
CI
Hexa chlorocyclohexane
DDT H Broad 0·001 ~g/L

spectrum-cotton
soya- bean and
Cl-@-f-@-Cl
peanut pests,
CCla mosquito control.
1, 1, I-Trichloro-2, 2-bis Persistent in the
(p-chlorophenyl)-ethane environment.
Accumulates in food
chain, use banned
in USA.
Toxaphene
c£(CH'
CIs
Insect control on
crops and livestock;
widely used in USA,
5~/L
(CH a)2 ban has been

proposed for
carcinogenic
properties.
Heptachlor CI Pest control in soil; 0·001 ~g/L
Cl:¢Q
I I Cl2
use suspended due
to potential
carcinogenicity.
CI
CI CI
Endrin Effective against 0·004 ~g/L

CI
black current mud
o ~Cl
CH Cl I 2 2
mite, also used as
Zoocide. Precautions
CI to be taken to avoid
skin contact during
CI application.
Metho- Popular DDT 0·03 ~g/L
xychlor CCla .
substitute,
reasonably
CHao-@-t-@-OCHa
biodegradable,
H low-toxicity to
mammals.
Contd. ...
Trade Permissi-
Formula Uses
name hIe limit
II. Organophosphates
Malathion Control some O·ll!g/L
S 0 pests of fruits and
II II vegetable-little
(CHaOh-P-S-CH-C-OC2H5
hazard to
I ~
CH 2-C-OC2H5
mammals.
0, o-Dimethyl-s-(l, 2-dicarboethoxy ethyl)

phosphorodithioate
Parathion Larvicide for O·OOll!g/L

S
02 N -@-
0 0 - PII (OC 2H5)2
mosquito control,
also
spectrum
broad
0, o-Diethyl-o-p-nitrophenyl phosphorothionate
insecticide for fruit
and vegetable
pests.
Methyl Control of plant O·OOll!g/L
S
parathion
02 N -@-
0 0 - PII (OCHah
pests; ranks second
in
consumption
pesticide
in
USA.
III. Carbamates
Carbaryl Used on 0·005l!g/L
(Sevin) O-C-NH-CHa
crops-cotton,
oo~
00
forage, fruits and
vegetables;
and
lawn
garden
I-Naphthyl-N-methyl carbamate
insecticide; low
toxicity to
mammals.
Baygon Control of flies, O·OOll!g/L
<Q{-II
0
o O-C-NH-CHa
mosquitoes,
and cockroaches.
ants
O-CH(CHah
2-Isopropyl-N-methyl carbamate
Iv. Chlorophenoxy acids
2,4-D Herbicide-control 100llg/L
CI
of broad leave
weeds, aquatic,
Cl-@-O-CH,-COOH vegetation;
military defoliant
2, 4-Dichlorophenoxy acetic acid (May contain
highly toxic TCDD
as impurity)
Contd. ...
Trade Permissi-
Formula Uses
name hIe limit
2,4,5-T Weed control, 100llg/L
Cl
military defoliant.
Cl-!fi)-o-CH 2-COOH
CI
Sources of Pesticidal Pollutants in Water.

(i) Rain water. Pesticides contaminate in vapour phase in air, get
adsorbed onto the dust particles which ultimately reach soil or water along
with the rain water. Weibel et al., recorded 187 mglL of total DDT of which
18 mglL was DDE in rain water of Ohio, USA. Residues of BHC, DDE and
dieldrin are detected in rain water.
(ii) Spray drift. Water contaminates heavily (40%) with pesticides
during aerial spray. Wind also carry the drift to considerable distances.
(iii) Run off from agricultural fields. Caro and Taylor have reported
that 0·07% of dieldrin applied to the soil persists in run off water. DDT in
water was 70 mgIL to 440 mgIL in mud.
(iv) Industrial effluents. A huge amount of pesticides are escaped by
pesticide producing industries.
(v) Domestic sewage. DDT level in Yamuna water has risen from 0.25
ppb to 0·558 ppb after the Najafgarh drains sewage into this river at
Wazirabad in Delhi.
Other sources include suspended particulates, accidental spillage,
evaporation from soils and plants, forest cover, residues in horticultural
products.
Pesticide Residues in Water.
There is a widespread pesticide pollution of fresh water bodies. Recently
USA, Canada, Europe and CIS have set up a National Pesticide's Water
Monitoring Network with 265 sampling sites.
Permissible limits (f.lg/L) of insecticides in public water supplies are :
DDT (42), Aldrin (17), Dieldrin (17), Chlordane (3), Lindane (56),
Heptachlor (18) Methoxychlor (35), Toxaphene (5), Organophosphates (100),
Carbamates (100). DDT residues in water account for the largest followed by
lindane, dieldrin, endrln and heptachlor etc.
Persistent Pesticides.
Pesticides are found widespread in various segments of environment.
Their impact on the environment is based on (i) tendency to vapourise
(ii) tendency to dissolve in water (iii) resistance to various degradation
processes.
Pesticides in the soil act as reservoirs from where they find their way in
water bodies. In India, about 97420 tonnes of pesticides are used for
agricultural developments every year. Out of 20,000 tonnes of DDT used,
about 25% get entered to oceans. From 1 ppt (10-6 ppm) of DDT in sea water
there is more than one million biomagnification in birds of prey (10 ppm). A
man stands at the top of every food chain. The average level of DDT is
estimated at 5 to 10 ppm in human tissues. DDT and its metabolites are not
readily degraded in the environment. The stability of organochlorines, coupled
with their high solubility in fatty tissues, causes them to concentrate in the
food chain.
DDT-The Most Widespread Molecule.
DDT was first synthesized by Othmar Zeidler of Germany in 1874 and
rediscovered by Swiss entomologist Paul Mueller in 1939 who won Noble
Prize for uncovering its powerful insecticidal properties. In World War II,
Allies used it as delousing agent to replace pyrethrum. It showed spectacular
success in controlling malaria, yellow fever and typhus. Due to continuous
use, 12 insect species developed immunity in 1948 which rose to 165 by 1967
and the number is still increasing rapidly. DDT is partially soluble in water
and carried by air and rain. Disquietingly large residues of DDT are
appearing every where.
Bioaccumulation of Pesticides.
Pesticides present in water get concentrated in tissues of plants and
animals. Aquatic animals like protozoa-Blepherisma concentrate DDT by
about 3000 folds in 12 hours. The fish Gambusia affinis accumulates DDT
to 25000 folds in a few hours.
Factors. Bioaccumulation of pesticides depends on :
• Lipid and water partition coefficient.
• Nature of aquatic organisms.
• Environmental conditions, i.e., temperature, pH, salinity.
• Concentration and duration of exposure of pesticide.
Organochlorines accumulate to a greater extent than organophosphorus
. and· carbamates. They are hydrophobic and concentrate 1000 times over the
levels in water.
Measurement of Bio-accumulation. The extent of bio-accumulation
can be measured by (i) Model ecosystem (direct method). (ii) By determining
octanol per water partition coefficient of the pesticide (indirect method).
Accumulation of DDT in Food Chain.
DDT from air and water accumulates in animal tissues, soil and humus
in concentrations thousands of times higher than it is found in water through
food chain. DDT is a persistent chemical. Once introduced into the
environment, it keeps circulating for many years.
The cycle of food chain consists of plankton in sea water which contains
0·04 ppm of DDT. The clams that consume plankton concentrate it ten times,
i.e., they contain 0·4 ppm DDT. Fish feeding on clams contain 2·4 ppm DDT
while its level goes upto 76 ppm in birds which eat fishes. In man, DDT
content rose to 15 ppm which feed on fishes in India, 2·2 ppm in England and
11 ppm in United States (Table 5).
Table 5. Pesticide contamination in food-samples.

% Sample % Sample
Insecticide Insecticide
with with
Source detected and Source detected and
insecticide insecticide
range (ppm.) range (ppm.)
residues residues
Milk 80 DDT 0·25 to 0·50 Fruit 60 DDT 0·1
Dieldrin 0·01
Eggs 60 DDT 0·025-1·0 Meat 20 DDT 0·2
Lindane 0·0-0·4 Dieldrin 0·01
Vegetables 70 DDT 0·2 Cereals 38 DDT 0·25-1·0
Lindane 2·0 Lindane 0·0-0·4
Heptachlor 2·0 Endrin 1·0
Chlordane 2·0 BHC 0·0 to 0·1
Endrin 2·0
The main worry about DDT contamination in man is that since DDT
stores in the fatty tissues and there is always a risk of its being massively
released into body fluids when the fat cells are metabolised during starvation.
It is thought to dissolve in the fatty membrane surrounding nerve fibres and
interfere with ion transport in and out of the nerve membrane. DDT affects
in a remarkable fashion. If there are 10 ppm DDT in plant tissues, it would
rise to 100 ppm in cattle and 1000 ppm in man. Oysters living in sea water
with 0·001 ppm of DDT show highest DDT level upto 700 ppm in their bodies.
Biological Magnification or Biological Amplification.
Aquatic plants and animals can accumulate certain pesticides in their
body tissues in greater concentration than can in water. For instance, DDT
not only circulates in its original form in aquatic organisms but its
concentration continuously increases in successive trophic levels in a food
chain. Woodwell et al., reported that DDT level in estuary water is only
0·00005 ppm but the food chain consisting of plankton (0·04 ppm), minnow
(0·94 ppm), heron, (3·5 ppm), fish (5 ppm) increased the amount of DDT in
fish eating birds (mergansers and geills) to 75·5 ppm. Organochlorine
insecticides have the greatest magnification because they have a high affinity
for lipids and are quite persistent ecopoisons. The degree of magnification
of insecticides is usually proportional to their persistence and inversely
related to their solubility in water.
Biodegradation of Pesticides.
The biodegradation of pesticides varies depending on the chemical
nature of the pesticide itself. In soil a pesticide may be transported into
various sectors of the environment by different physical processes. It may be :
(i) absorbed by soil, (ii) leached by rain water,
(iii) picked up by plants and animals (iv) carried away by wind.
The processes that actually play important roles in reducing total
amount of pesticide residues are those mediated by micro-organisms,
actinomycetes, fungi, bacteria, plants, animals and sunlight.
Biodegradation of pesticides is extremely essential to maintain the

environmental quality. It can be achieved by the following methods :
• Pesticides can be metabolized and degraded by the living organisms.
They can be detoxified and removed from the cycle.
• By transferring the aquatic animals containing higher concentration
of pesticide to a clear water where they may unload the pesticidal
residues. For example, fresh water snail can lost 75% DDT within 24
hours in clean water.
• Both DDT and BHC can also be degraded by the process of
dehydrochlorination.
H
Cl-@+@-cn CCl3
DDT DDE
CI CI
Cl@CI
CI Cl
CI
--
-Hel
CIO
CI
Cl
Cl
BHC Para chlorocyclohexane

(peeR)
• Microbial degradation of pesticides can be achieved by chemical

methods viz., ester and amide hydrolysis, ether cleavage, oxidation,
dehalogenation and epoxide formation. For example, aldrin can be
oxidized to dieldrin by epoxide formation.
Cl Cl
Cl
O~Cl
~Cl
Micro.organisms
Cl
Cl Cl
Aldrin Dieldrin
Generally oxidation may occur by introduction of -:-OH group into

aromatic ring while reduction of pesticide occurs by converting
-N02 group into -NH2 group.
• Biodegradation of pesticides is strongly affected by soil temperature,
pH, moisture and texture etc. Enzyme system existing in soil also play
a significant role in their degradation.
However, biodegradation of pesticides does not always result in harmless
products. Degraded intermediates may be more toxic to aquatic life than are
the original pesticides. For example, biodegraded product DDD is more toxic
than DDT.
Mode of Poisoning of Pesticides. Pesticides are classified into four
categories depending upon their mode of poisoning action.
(i) Physical poisons. Pesticides penetrate percutaneously through the
epidermis of the skin and produces lethal effects.
(ii) Nerve poisons. DDT, MIC, malathion, parathion, diazinon etc.,

initiate extreme nervous excitations and cause release of excessive
neuro-active substances, disrupting nerve activity.
(iii) Protoplasmic poisons. Endrin, ziram, lead arsenate etc., cause
precipitation of protein in the body causing liver damage and death.
(iv) Respiratory poisons. Pesticides like HCN, CH3 Br, ethylene
dichloride inactivate the respiratory enzymes like oxidases, peroxidases and
reductases causing suffocation and blockage of respiratory tract.
How Pesticides Endanger Our Life.
Pesticides acutely affect man, animal, plants, soil as well as the aquatic
biota. Toxicity towards the aquatic flora and fauna is an important criterion
for a pesticide to be considered as a hazardous pollutant. Pesticides affect in
a variety of ways as follows :
Effects on Man.
e Higher concentration of DDT in body parts and blood of human beings
causes anxiety, tension, cancer, mutations, stress reactions, congenital
and impotency.
• Central nervous system is the target of DDT poisoning in man which
ultimately leads to death. It dissolves in lipids and accumulates in the
body fats (3 to 90 ppm).
• The most threatening fact is DDT concentrates and accumulates in
the food chain. It is continuously recycled in living organisms. The
level of DDT estimated in human tissues lies in range of 5 to 10 ppm.
According to an estimation DDT concentration in man's body fat
varies from 3·3 g/m 3 in UK to 25 g/m 3 in India.
• Low levels of DDT cause cancer, high blood pressure and cirrhosis of
the liver. DDT also interferes with signal transmission at the synoptic
gap.
• Insecticides like BHC, aldrin, dieldrin, chlordane and endosulfan are
extremely toxic to living beings. These are considered to affect the
vital organs, heart, brain, kidneys and liver producing chronic
disturbances.
• Pesticide 2, 4, 5-T consists of highly toxic component called dioxin or
TCDD which IS teratogen and liable to cause congenital
malformations.
• Organophosphates like ethion, fenthion, dimethoate, trithion,
monocrotophos, diazinon, dursban, phosdrin, thionazin and
metasystox inhibit the production of cholinesterase at the junction
between adjoining nerve cells with the result that cholinesterase
breaks down acetylcholine secreted by nerve cell axons. Excessive
accumulation of acetylcholine interferes with the nerve impulse
transmission.
• The organo phosphorus insecticides such as parathion, malathion and
TEPP may be absorbed by the lungs, eye membrane and skin to toxic
amounts. This excessive absorption leads to larger accumulation of
acetylcholine in the body disturbing the normal functioning of blood.
• Chronic accumulation of pesticides plays a major role in liver and

kidney malfunctioning, secretion of excess of amino acids in human
blood and urine, blood abnormalities as well as electroencephalogram
deformations of brain tissues.
• Today children born have to start their life with a body burden of
pesticides which increases with age. Pesticides are also reported to
cause chronic impotency in man.
• The 2, 4, 5-trichloro phenoxy acetic acid is teratogenic and foetocidal
in lower mammals.
• On Dec. 3, 1984 Bhopal industrial disaster was the worst ever
pesticidal MIC accident in history, taking an unprecedented and still
unaccounted death toll leaving no fewer than 50,000 quickly dwarfed
by the tidal wave of man's suffering that spread across the central city
like the poison cloud. This carbaryl (carbamate pesticide) leakage
caused an increased risk of sleeping, digestive problems, vision
problems and sterility.
Effects on Animals.
• Insecticides such as aldrin, dieldrin, chlordane, endosulphan,
heptachlor and gammexane are reported to affect the wild life by
changing their metabolic activities and body chemistry.
• Animals which have become weak and moribund as a result of
exposure to pesticides may easily be destroyed by predators.
• Chlorophenoxy acid compounds such as 2, 4-D affect embryos while
triazones may cause mutagenic effects in animals.
• Organophosphate pesticides cause extreme muscular weakness,
tremors and dizziness in poisoned animals.
Effects on Birds.
• Pesticides such as parathion, malathion, aldrin, dieldrin, heptachlor
and several other organochlorines are reported to affect severely the
metabolism in birds. Many species of hunting birds, particularly those
having high levels of DDT are threatened with extinction.
• DDT has been implicated as the main villain and the cause of thin
and fragile egg shells because of inhibition of enzymes and
interference with the hormones that control calcium metabolism.
• Pesticide pollution reduces egg laying in birds and fewer youngs are
hatched.
Effects on Aquatic Biota.
• Aquatic animals are extra ordinarily sensitive even to low
concentrations of DDT. Even one part of DDT per trillion seems to
cause death in brine shrimps.
• Acute danger arises from DDT which results in changing the
behaviour and metabolic activities in sea animals.
• It is reported that marine invertebrates can concentrate pesticides by
a concentration factor upto 70,000 or more.
• Experiments have shown that DDT causes depression of
photosynthesis in planktons. Its very low concentration as 1 ppb. has
immobilizing effects on Simocephalus species.
• Endrin is extremely toxic to fishes like blue gills and rainbow trout
with LC 50 values being 0·006 and 0·007 ppm respectively, while DDT
is much less toxic to these fishes with LC 50 values being 0·016 to
0·018 ppm. respectively.
• Even the raptorial hawks and falcons are severely affected by DDT
which causes breeding failure and death in them.
• Recent studies have proved that extremely low quantities of pesticides
which enter the aquatic environment can affect productivity of
organisms, kill eggs and larvae of clams and oysters, influence the
behavior of fishes such as schooling and feeding and deteriorate water
quality.
• Pesticides induce changes in blood chemistry and enzymatic functions
of marine invertebrates. They reduce their backbonic collagen content
and indirectly interfere with food chain.
• Since slight concentrations of organochlorine pesticides affect
reproduction in fishes, there is every possibility that these pesticidal
pollutants may adversely affect local fishery.
• Varieties of crustacea that make up the most valuable marine harvest
are badly affected by pesticides. The oysters might be susceptible
because of their tendency to concentrate and store trace chemicals
from the surrounding environment.
• Pesticides-induced mortality pattern of marine molluscs, crustaceans
and teleosts are also measurably related to various physico-chemical
environmental parameters like concentration of pesticide and
duration of its exposure.
• PCBs are reported to be concentrated in the food chain of marine
ecosystems.
• Pesticidal pollutants ranked second to all other industrial wastes.
Effects on Grains.
• Many vegetables, fruits, rice, cereals and grains such as wheat, maize,
grams and barley have been found to be contaminated with significant
amounts of DDT and BHC. The level of DDT residue vary from 1·6 to
17·4 ppm in wheat, 0·8 to 16·4 ppm in rice, 3 to 17 ppm in pulses, 3
to 19 ppm in ground nuts, upto 5 ppm in vegetables and 68·5 ppm in
potatoes.
Effects of Pesticides on Soil.
Pesticides not only pose potential hazards to man, animal, livestock, wild
life and fish but they seriously interfere with the desired use of soil and water
as follows:
• The most dangerous characteristic of pesticides, particularly
organochlorines, is their long persistence in the soil which causes
numerous adverse effects on grain quality.
• Even the accepted dosages of pesticides create deleterious effects in
the long run in the soiL
• Pesticide retained in the soil also concentrate in some crops,
vegetables, cereals and fruits which taint them to such an extent that
they are not usable.
• American Scientists observed that raddish and carrots grown on a

loamy soil treated with aldrin at one lb/acre contained 0·03 and 0·05
ppm of it respectively.
• Retention of pesticides in bottom soil of ponds renders them
unsuitable for fish culture.
• Soil contaminated by pesticides also causes pollution of water sources,
water supply and ground or surface water by rain.
Pesticides Contaminate Air Too.
• Pesticidal introduction into air may occur through spraying
operations, volatilizations, evaporation of chemicals from soil, plants
or burning of plant products.
• In a nutshell, if we continue to rely upon broad spectrum of pesticides,
the recovery of natural forms of control will become almost impossible.
CENTRAL FACILITY FOR SAFETY EVALUATION OF PESTICIDES

Recently Industrial Toxicology Research Centre (lTRC), Lucknow
has established a Central Facility for Safety Evaluation of Pesticides with the
following objectives.
• 7b generate toxicological data on different pesticides synthesized,
manufactured or formulated by national laboratories and private
sector industries within the country.
• 7b conduct carcinogenic, teratogenic and multigeneration studies on
selected strains of experimental animals, in vivo tests using bacterial
mutants, human cell lines and lymphocytes to map mutagenic and
carcinogenic effects and interference with immune mechanism.
• To study cytogenic effects of pesticides using bone marrow cells of
intact animals.
• To develop new techniques for monitoring and predicting harmful
effects of pesticides on target and non-target organisms and their
diffusion in the environment.
• To provide expertise for diagnosing toxic hazard's symptoms and
developing methods of their prevention and control.
Methods to Minimize Pesticidal Pollution.
Fortunately, it is possible to treat persistent pesticides which
contaminate in water bodies by the following techniques.
• Less persistent and harmless pesticides can be used instead of highly
persistent ones.
• Proper utilization of persistent pesticides would minimize drifts etc.
• Pest control may be achieved biologically by the release of sterile males
and entocones.
• Chemical methods like coagulation, volatilization, irradiation,
sedimentation and filtration can be adopted which can remove upto
90% DDT.
• Industrial wastes and effluents containing pesticides can be treated
before they are released into water systems.
• Neem based insecticide holds promise as an ecofriendly substitute.
FUTURE STRATEGY FOR PESTICIDE USE

Pesticides hit the aquatic ecosystem and terrestrial organisms ranging
from acute toxicity to invisible chronic effects. So the question is how to best
regulate the pesticidal use?
• Broad spectrum chlorinated pesticides (DDT etc.) must not be used.
• Repeated application of insecticides should be avoided.
• Farmers, field workers and public must be educated regarding the use
of pesticides.
• A combined biological and chemical approach should be developed to
control pests.
• Monitoring and research should be encouraged to solve the pesticide
problems.
• Pesticides should be applied only in recommended dosages to crops
when pests population has exceeded the economic threshold limit.
• Cattle, cows and other grazing animals should not be allowed to graze
on the treated crops.
• The product should be harvested after the waiting period is over.
• Direct mixing of insecticidal dusts with food grains is to be avoided.
• Consumers should be advised to wash thoroughly with water and peel
all the vegetables and fruits before use. This can remove a substantial
proportion of pesticidal residues.
• Provisions should be made for the quantitative estimations of the
pesticide residues in various food stuffs. So it is recommended that all
the analytical laboratories should be equipped with the latest
sophisticated instruments for determining the micro quantlties of
pesticides.
One should strictly follow these suggestions so that the benefits of
pesticides are not denied on account of their negligent use.
13. GASEOUS POLLUTANTS

Gaseous pollutants include NOx , S02, CO, CO2, ozone, ammonia, free
chlorine, hydrogen sulphide and phosphine. Nitrogenous organic matters and
sewage disposed in water increase the content of ammonia to dangerous
levels. Sewage consisting of organic sulphur compounds are decomposed by
protolytic bacteria to H 2S in absence of oxygen. Hydrogen sulphide so formed
gets oxidized to sulphuric acid which corrodes water pipes. Even in very low
concentrations it causes odour nuisance as it has obnoxious and penetrating
smell. Its 0·5 to 1·0 ppm. concentration in water is lethal to fish. H 2S inhibits
oxygen utilization like cyanides. It behaves as a local irritant and acts as a
respiratory depressant causing bronchial irritation, oedema of lungs and
conjunctivitis in man. Ammonia seems to be an internal poison to fish which
enters in their gills during respiration. Inhalation of considerable amounts of
ammonia causes a spasm of glotis which may lead to death.
14. FARM WASTES

Increasing population of cows, cattles, pigs and poultry farms have
resulted in considerable water pollution. Cow's dung is washed out which
deposits on soil as wet slurry. This slurry may seep into drains and streams
polluting them. It has been reported that a cow can produce as much organic
matter as 20 persons and a pig as much as three people.
When feed lot runoff occurs, it poses severe pollution in receiving water
causing massive fish kill. Raw sewage has a BOD of 200 ppm, COD of 400
ppm and nitrogen 40 ppm, while feed lot runoff have 1000 ppm BOD, 8000
ppm COD and 700 ppm nitrogen. So treatment methods of municipal sewage
can not be applied to farm animal wastes.
15. FERTILIZERS
Modem agricultural techniques have introduced NPK fertilizers into
water systems. The excess of fertilizers not consumed by crops are washed
away from land by rain water and pollute water bodies. These fertilizers are
generally retained in the soil by crops but due to negligence in their
application, some nitrates are liable to be washed out during rainy season. If
phosphate and nitrate concentration exceeds one part and thirty parts per
hundred million part of water respectively, it can cause eutrophication and
the whole stretch of water may become chocked. The less DO content may
result in the death of aquatic biota.
16. AUTO-EXHAUST AS WATER POLLUTANT

Environmental Protection Agency (EPA) has shown that auto-exhausts
spread on road and befoul water. These pollutants include asbestos from
brake lining, rubber from tyres, lead from gasoline etc. Rain stream carry
these pollutants into water ways and deteriorate water quality.
o
7
ANALYSIS OF WATER POLLUTANTS
OBJECTIVES OF WATER ANALYSIS

The control of water pollution necessarily requires quantitative
measurements of water pollution. So different types of examinations (such
as physical, chemical, biological and microbiological) of water are
necessary due to following reasons:
1. To assess water quality.
2. To provide pure water to public for drinking, domestic and industrial
purposes.
3. To choose the most effective treatment method for water purification.
4. To determine the efficiency towards natural purification when sewage
and industrial wastes are discharged into water bodies.
5. To trace the origin and extent of pollution and to suggest a possible
remedy.
6. To check efficiency, uniformity and consistency of treatment processes.
7. To analyse whether infection by microbial organisms has occurred.
8. To find out particular micro-organism and to suggest preventive
measures and effective disinfection procedures.
9. Sewage and waste waters are analysed to determine the influence of
them on receiving waters and to protect them from contamination.
ANALYSIS OF WATI:R POLLUTANTS
The analysis of water is extremely important as it contains a large
number of impurities which are necessary to be checked before the water is
used for any specific purpose.
Water analysis is usually expressed in milligrams per litre or in
parts per million (ppm), where
One part of hardness
1 ppm: -
106 parts of water
1 ppm: 1 J.lg g-l and 1 ppb: 1ng g-l, 10 ppb: 1 ng
Parts per million (ppm) means the number of parts of substance per
million parts of water.
CHEMICAL AND PHYSICAL EXAMINATION OF WATER

For chemical and physical analysis of water, sufficient water samples
(2-litres) are collected under different conditions. Sampling involves :
Sample Container. Generally glass container or stoppered
Winchester Quartz Bottle of 2·5 litres capacity is used. When composite
(148)
ANALYSIS OF WATER POLLUTANTS 149
samples are to be collected, it is advisable to use wide mouthed bottles of

capacity 200-300 mL for subsamples.
Samplers. Point samplers, depth-integrating sampler and
displacement samplers (for the determination of dissolved oxygen) are
employed to meet the specific requirements.
Grab sample and composite sample. Liquid samples may be
instantaneous, spot, snap or grab samples and continuous or composite
samples.
Taking the Sample.
When the liquid is homogeneous, it is better to take the grab sample
in a sample bottle. In case of a heterogeneous liquid like sewage, a
composite sample is taken.
Sampling potable waters. Sample of potable waters should be taken
from a tap directly connected to the main line. If water from a well is to be
collected, then the well has to be pumped for a long time so that the sample
represents the ground water that feeds the well.
Sampling industrial effluents and waste waters. The sampling
and analysis of industrial effluents require great care. It is therefore,
advisable to study during sample collection, the character of effluents, the
waste load, major sources of wastes within a plant, recovery of useful
materials and the effect of discharged wastes on the receiving body of water.
Industrial effluents are subjected to rapid changes due to breakdowns, spills,
floor washings and numerous other causes. So suitable sampling points, the
frequency and type of sample to be collected should be selected first.
Generally, sampling points are located where flow conditions encourage a
homogeneous mixture.
Sampling sewage. Sampling sewage involves the collectio.n of grab
samples in a bottle of capacity 100 mL and combining them in a single
container after 24 hour period. ,
Sampling near treatment plant. It required sampling ofthe raw and
treated water. For water supply of constant composition samples drawn at
8-hours intervals are collected.
Time interval between collection and analysis. Generally, the
shorter the time between collection and analysis, the more accurate will be
the result. Time interval depends upon the nature of the sample conditions of
storage and the constituent to be analysed. Determinations like pH, dissolved
gases such as oxygen, carbon dioxide and hydrogen sulphide can be made
immediately while other constituents may be fixed and analysed in the
laboratory.
Preservation Methods.
Preservation is essential to protect samples from changes in
composition, deterioration with ageing, retarding biological action, reduction
of volatility of constituents and for accurate results. Water samples should be
preserved in polythene or glass bottles (Table 1).
Table 1. Preservation methods of water sample.

Parameter Sample size, mL Preservation method
pH or DO 100 Measure within 0-4 hours
COD 500 Add H 2 S04 to pH2, refrigerate
Ammonia 500 Add 0·8 mL conc. H 2 S0,JL
Nitrate + nitrite 500 Add 40 mg HgC121L, refrigerate
or phosphate
Phenol 500 Acidify with H aP04 to pH4 and add 1 g
CuS04 . 5H2OIL
Metals 500 Add 5 mL conc. HNOalL
E.coli, Total 100 Sterilise glass bottles in autoclave at 121°C at
bacteria, 15 Iblinch2 pressure for 15 minutes, collect the
Actinomycetis sample in sterilised bottle and refrigerate.
Preconcentration Methods.
1. Carbon Adsorption Method. A large volume of collected water, say
1000 gallons, is allowed to pass over activated charcoal. The organic matter
adsorbed on activated carbon is extracted from the dried charcoal with CHC13
followed by alcohol. The solvents are evaporated and the weights of the
extracts are expressed as Ilg/L of carbon-chloroform extract (CCE) and
carbon-alcohol extract (CAE). Organic pollutants, such as phenols and oils,
which impart tastes and odour to water are usually present in CCE.
Compounds such as phenols, fulvic acid, pesticides, polycyclic aromatic
hydrocarbons, carboxylic acids, sulphonic acids etc., can be characterised from
these extracts by making use of a number of separation techniques. The carbon
adsorption method, though, widely been used due to its speed, efficiency and
simplicity, but it has a number of disadvantages. For example:
• There is a possibility of a chemical reaction on the surface of activated
charcoal.
• During evaporation of solvents, some organic material may also
evaporate.
2. Freeze Concentration Method. In this process, the water sample
is allowed to freeze, as a result of which pure crystals of ice are formed and
water soluble impurities are left behind in the liquid phase of much reduced
volume. The process of freezing, also minimizes the loss of volatile
constituents in the sample.
3. Solvent Extraction Method. The liquid extraction is a technique
in which a substance dissolved in one phase (usually an aqueous phase) is
more or less completely transferred to the second phase, essentially
immiscible with the first, i.e., an organic liquid such as benzene, ether,
chloroform etc. Thus in solvent extraction, organi~ matter soluble in water is
separated by using a water insoluble organic solvent. Parts per billion
(llgiL) amounts of various metals such as Co (II), Fe (III), Pb (II), Zn (II),
Ni (II) etc., in saline water can be determined by solvent extraction method
in which metal complexes of these metals with ammonium pyrrolidine
dithiocarbamate are first extracted with methyl isobutyl ketone solvent and
then the resulting organic extract is analyzed by feeding into an atomic

absorption spectrophotometer.
4. Ion Exchange Method. Ion exchange chromatography has
extensively been used for the concentration and separation of metal ions from
natural and waste waters. The ions are first concentrated on a s'ritable ion
exchange column and then selectively eluted. Many inorganic ligands, such as
HF, HBr, HCI, HI, HNO s , H 2S04 , Na2COS and H SP04 etc., have been used
as eluting agents for the separation of metal ions.
PARAMETERS FOR WATER ANALYSIS

COLOUR.
Pure water is colourless. The presence of fine colloids, industrial
effluents, metals, tannis, peat, humus, algae, weeds, phytoplanktons, protozoa
and suspended matter impart colour to the natural water. True colour can be
estimated visually or photoelectrically.
1. Platinum Cobalt Method.
Preparation of Stock Solution. Dissolve 1 g of CaCl2 . 6H20 and
1·245 g of ~PtCI6 in minimum quantity of water. Add 100 mL conc. HCI and
dilute to 1 L with distilled water. The resulting stock solution has colour value
of 500 units.
Colour Standards. Prepare standards by diluting 0·5, 1·0, 1·5, 2·0, 2·5,
3·0, ... ,7·0 mL of the above solution with distilled water to 50 mL in standard
Nessler tubes. The solutions have colour value of 5, 10, 15,20,25,30, ... , 70
respectively. Indicate the colour value on each tube.
Method.
• Centrifuge the water sample to remove suspended matter.
• Fill 50 mL Nessler tube with clear water sample to a height equal to
that of standards.
• Compare the colour of sample and standards by looking vertically
downward through the tubes upon a white or mirrored surface placed
at such an angle that light is reflected up. Read the colour value.
• If the sample shows a colour more than 70 units, it should be diluted
with distilled water and the colour estimated is multiplied with
dilution factor
Limitation. Platinum cobalt method is valid only for potable water and
not for industrial efiluents. It is always necessary to specify pH when colour
is measured.
2. Luminescence and Colorimetric Method.
Colour is identified in terms of the dominant wavelength and degree of
brightness by luminance. Tristimulus colorimeter with three filters is also
used to measure transmittance of sample of water.
3. Spectrophotometric Method.
It is the most reliable method, wherein the absorbance of water is
measured between 400 and 700 nm.
TURBIDITY
Turbidity in water is due to suspended matter like silt, clay, organic or
inorganic matter, planktons and other-micro-organisms. It is an expression of
optical property (Tyndall effect) of water which causes light to be
scattered and absorbed rather than transmitted. Turbidity of suspension
may vary due to different optical properties, refractive indices and particle
size of matter.
MEASUREMENT
1.·By Jackson Candle Turbidimeter.
Although standard method but Jackson model permits measurement of
turbidity from 25 to 1000 JU. Lower turbidity values (100 NTU) can be
measured by Nephelometer.
2. By Nephelometer.
Standard Turbid Suspension. Dissolve 1 g of hydrazine sulphate in
100 mL of distilled water. Also dissolve 10 g of hexamethylene tetramine in
100 mL of water. Mix 5 mL of each solution in 100 mL volumetric flask and
dilute it with distilled water to the mark. This suspension gives a turbidity of
400 NTU.
Method .
• Set the nephelometer at 100 using 40 NTU (10 mL ofthe above stock
solution in 100 mL water) standard suspension. Every percent of the
scale will be equal to 0·4 NTU.
• Shake the sample thoroughly. Take the sample in nephelometer tube
and read the value on the scale. If the sample has turbidity more than
40 NTU, dilute it, so that turbidity can be read on the same scale.
Calculation.
Turbidity (NTU) = Nephelometer reading x 0·4 x Dilution factor
CONDUCTIVITY
Conductivity of water varies directly with the temperature and is
proportional to its dissolved mineral matter content. Specific conductance
can be measured by conductivity meter using dip-type cell. The instrument
and cell are calibrated with 0·005 M KCI solution (conductivity = 654
Il mho cm- l ).
1 A
Specific conductance, K=-'-
R I
where R is the observed resistance of a column of electrolyte, 1 cm long and
cross-section area A cm2 .
Electrical Conductivity (EC).
EC is a measure of water's capacity to convey electric current. EC is
directly proportional to area (a) and inversely proportional to length (I).
EC oc all or EC = K all
where K is proportionality constant called specific conductance.
Measurement of Electrical Conductivity.

The instrument consists of an AC salt bridge or electrical resistance
bridge and conductivity cell having electrodes coated with platinum black.
Solubridge (calibrated assembly) gives conductivity of the solution in milli
mhos/cm or deci Siemenlm at 25°C.
Principle.
A simple Wheatstone bridge circuit is used to measure EC by null
method. The bridge consists of known and fixed resistances rl' r2' variable
standard resistance r 4 and unknown r3' The variable resistance r 4 is adjusted
until a minimum or zero current flows through the AC galvanometer. At
equilibrium,
rl r3 rl
- = - or r3=-xr4
r2 r4 r2
Since conductivity is reciprocal of resistivity, it is measured with the
help of r3'
Reagents.
Standard KCI Solution (0·01 M). Dissolve 0·7456 g of dry KCI in
double distilled water and make up the volume to one litre. It gives EC of
1·413 deci Siemen/m at 25°C.
Method.
• Take the water sample in a beaker.
• Warm up the instrument and calibrate the meter with 0·01 M KCl.
• Adjust cell constant and temperature of the conductivity IJleter.
• Rinse the conductivity cell with distilled water and then with the
sample.
• Connect the conductivity cell to meter and dip in the sample.
• Adjust the current by rotating the dial so that maximum sensitivity
is obtained.
• Read the conductivity value in dSlm. Direct reading may be obtained
by digital meters.
Result. Observed values of EC are multiplied by the cell constant and
a temperature factor to express results at 25°C.
TOTAL SOLIDS
The term solid refers to the matter that remains as residue upon
evaporation. Total solids include both dissolved solids and suspended solids.
Potable waters contain mineral matters in dissolved conditions whereas
industrial eftluents and sewage contain huge amount of undissolved matter.
Suspended Solids.
500 mL of a sample is taken exactly in a volumetric flask and allowed
to filter through a dried and weighed Gooch crucible containing an asbestos
mat. The suspended solids retained in the crucible are washed with distilled
water to remove chloride. The crucible is finally dried, cooled in a desiccator
and weighed. The increase in the weight of the crucible is equivalent to the
suspended impurities present. The total solid contents of 500 mL sample can
also be calculated by evaporating it to dryness on a steam bath and drying at
about 100-HODC in an oven for about one hour. From this, substract the
dissolved solids to get the quantity of suspended solids.
Dissolved Solids.
Filter 500 mL sample in a Gooch crucible to free it from suspended
matter and evaporate to about 50 mL. It should be noted that any deposit on
the walls of the beaker due to evaporation of water should not touch the flame
of the burner. The 50 mL liquid is carefully transferred to a weighed platinum
dish with the help of a policeman and wash with distilled water. Evaporate
the solution to dryness on steam bath and dry the dish in an oven at about
lOO-HODC for about an hour. Cool it in a desiccator and weigh.
6
Weight of solids x ~go = ppm. dissolved solids.
Results. Disposal of industrial effluents and sewage contribute
suspended solids to the water bodies. The lSI has specified a maximum limit
of 30 mg/L for suspended solids discharged into river. Solid determination is
particularly useful in the analysis of sewage and other waste waters.
ACIDITY
Acidity is a measure of the effects of combination of compounds and
conditions in water. It is the power of water to neutralise hydroxyl ions and
is expressed in terms of calcium carbonate. Water attain acidity from
industrial effluents, acid mine drainage, pickling liquors and from humic acid.
Measurement of Acidity by Titration Method.
Principle.
Acidity of water can be determined by titration with sodium hydroxide
solution. The amount of sodium hydroxide required for the sample (pH below
4·5) to reach pH 4·5 (methyl orange end point) is a measure of mineral acidity
while the amount of sodium hydroxide to reach pH 8·3 (phenolphthalein end
point) is a measure of total acidity. Samples containing acidic wastes (pH
below 4·5) correspond to both mineral and CO2 acidity.
Procedure.
Mineral Acidity.
Take 50 mL or suitable dechlorinated aliquot of the sample in a 250 mL
conical flask. Add 2 drops of methyl orange indicator and titrate with 0·02 N
NaOH solution till faint orange colour.
Total Acidity at Boiling Temperature.
To 50 mL of the sample, add 5 drops of phenolphthalein indicator. Heat
to boil for 2 minutes. Titrate with 0·02 N NaOH solution to pink colour.
Calculation.
A .dit C CO IL =mL titrant (NaOH) x 1 x 1000
CI Y as a 3' mg mL sample taken for titration
Result. Methyl orange acidity value shows mineral acidity. In absence

of mineral acidity, total acidity is only the CO2 acidity of the sample.
Total Acidity.
Total acidity expressed as CaCOg, can also be determined in the
following manner. Take 100 mL of a sample in a tall cylinder to decrease the
surface of the sample and minimize loss of dissolved carbonic acid during
titration. Now add few drops of phenolphthalein indicator and titrate the
solution very rapidly against 0-02 N NaOH with constant stirring until a faint
pink colour is obtained.
Calculation.
mL alkali titration x normality x 0-05 x y, 1

0.0
~06samp1e = ppm
Free mineral acids. Take 100 mL of the sample in a conical flask and
add few drops of aqueous methyl orange indicator to it. Now titrate the
solution against 0-02 N NaOH till an orange yellow colour is obtained at the
end point. The calculation is exactly the same as for total acidity.
ALKALINITY
Alkalinity of water is due to the presence of carbonate, bicarbonate and
hydroxide ions.
Determination of Alkalinity by TItrimetric Method.
Principle. Alkalinity is determined by titration with 0-02 N H 2S04
using methyl orange and phenolphthalein as indicators.
Reactions.
2CaCOg + H 2S04 ~ CaS04 + Ca(HCOg)2
Ca(HCOg)2 + H 2S04 ~ CaS04 + 2C02i + 2H20
Ca(OH)2 + H 2S04 ~ CaS04 + 2H20
Reagents. :
(i) Sulphuric acid 0-02 N. Dilute 20 mL of IN-H2S04 to 1000 mL with
distilled water.
(ii) Sodium carbonate solution. Dissolve 13-25 g Na2COg in distilled
water to 250 mL.
(iii) Phenolphthalein indicator solution. Dissolve 500 mg
phenolphthalein in 50 mL alcohol and 50 mL distilled water. Add 0-02 N
NaOH solution till light pink colour appears.
Procedure. Take 50 mL of the sample in a 250 mL conical flask. Add
2 drops of phenolphthalein indicator. Titrate the pink colour with
0-02 N H 2S04 till it becomes colourless. If the sample contains waste waters,
then remove the suspended matter by filtration or centrifugation and then
determine alkalinity_
Calculations. Phenolphthalein alkalinity (as CaCOg) mg/L
mL 0-02 N H 2S04 for phenolphthalein end point x 1 x 1000
=
mL sample taken for titration
Total alkalinity (as CaC03 ) mglL

mL 0·02 N H 2S04 for total alkalinity end point x 1 x 1000
=
mL sample taken for titration
Result. Alkalinity measurements are used as the means of
evaluating the buffering capacity of waste waters and sludge. It is also
significant in determining the suitability of a water for irrigation, in the
treatments of natural and waste waters and to calculate the Langelier
Saturation Index. Alkalinity provides an idea of the nature of salts present
in water. If it is equal to hardness, calcium and magnesium salts are only
present in water. If alkalinity is less than hardness, sulphates of calcium and
magnesium must be there. Greater alkalinity shows the presence of alkali
salts of sodium and potassium in addition to those of calcium and magnesium.
Alkalinity can also be predicted in the following manner. Take
100 mL of the filtered sample in a conical flask and add few drops of alcoholic
phenolphthalein indicator to it. Now titrate the solution with 0·02N HCI until
colourless. If the sample is colourless after the phenolphthalein indicator is
added, add few drops of aqueous methyl orange indicator till an orange pink
colour is obtained at the end point. The nature of alkalinity is then predicted
from the titration as follows :
• If the titration to the phenolphthalein end point is zero, the alkalinity
may be regarded as due to bicarbonate alone.
• When there is no further titration to the methyl orange end point after
the phenolphthalein end point, the alkalinity is solely due to the
hydroxide.
• When the phenolphthalein end point titration is half the total titration,
only carbonate alkalinity is expected to be present.
• When the phenolphthalein end point titration is greater than half the
total titration, the alkalinity is due to both carbona~ and hydroxide.
• When the phenolphthalein end point titration is less than half the total
titration, the alkalinity is due to carbonate and bicarbonate.
Those waters that have been softened with orthophosphate do not obey
the above rules. All kinds of alkalinity are expressed in terms of CaC03 .
HARDNESS
Hardness indicates water quality, mainly in terms of Ca2+ and M~+
expressed as CaCOa.
Temporary hardness is due to the presence of bicarbonates of Ca2+
and M~+. It may be removed by boiling the water.
Permanent hardness is due to the presence of sulphates and chlorides
ofCa2+ and M~+.
Complexometric Titration.
Principle. During titration with EDTA (Na2H2Y)' Ca2+ first reacts to
form stable Cay2- followed by M~+ to give Mgy2- complex (indicator!
wine-red) releasing free indicator (blue). The colour changes from wine-red to
blue at the end point.
Reactions.
Ca2+ + H2y2- ~ Cay2- + 2H+
M~+ + H2y2- ~ Mgy2- + 2H+
MgD-(red) + H 2y2- ~ Mgy2- + HD-(blue) + H+
Procedure for Ca2+ and Mg2+ Hardness.
Take 20 to 50 mL sample of hard water in a conical flask. In presence
of organic matter in waste waters, add 2 to 4 drops of 30% H 20 2.
• Adjust the pH to 8, boil for 15 minutes and cool.
• Add 5 mL buffer of pH 10 (142 mL conc. NH3 + 17·5 g NH4CI diluted
to 250 mL with deionised water) and warm the solution.
• Add two drops of 1·4% Eriochrome black T in triethanolamine.
• Titrate with 0·01 M EDTA solution till the colour changes from,
wine-red to blue.
Calculations.
1 mL 0·01 M EDTA == 1·0 mg CaC03
H d (fL) Vol. of EDTA used x 0·01 M x 1000
ar ness mg = mL of Sample taken
This gives total Ca2+ and M~+.
Procedure for Ca2+ Hardness.
• To a 100 mL sample add 20% KOH solution to bring the pH to 12 and
precipitate M~+ as Mg(OH)2'
• Add 5 to 10 drops of calcon carboxylic acid indicator (0·4% in
methanol).
• Titrate with 0·05 M EDTA under magnetic stirring till the colour
changes from wine-red to pale blue.
Alternatively, add drops of mureoxide indicator (0·1 g stirred with 2·5
mL deionised water and filtered). Titrate with 0·05 M EDTA solution till the
colour changes from orange to violet.
Calculation. 1 mL of 0·01 M EDTA == 0·4008 mg Ca.
CHLORIDE
Chloride in drinking water is harmless if present below 250 ppm but its
higher content harms metallic pipes and crops.
Mohr's Method.
Chloride is determined by titration with AgN0 3 solution using ~Cr04
as an indicator. The end point is indicated by the appearance of reddish tinge.
The method is valid for 0·15 to 10 mg cr and electrical conductivity of water
is greater than 1 dS/m at 25°C.
Procedure.
• Take 100 mL sample in 250 mL conical flask. Adjust pH from 7 to 10
with H 2S04 or NaOH (0·5 g Na2B407 will keep the pH at 9).
• Add 1 mL of 5% ~Cr04 indicator with stirring.
• Titrate with 0·0282 N AgN03 solution (282 mL of 0·1 N AgN0 3

diluted to 1 litre) to a reddish tinge.
Cl- + Ag+ ~ AgCI; 2Ag+ + CrO~- ~ Ag2Cr04
Calculations. 1 mL of 0·0282 N AgN0 3 =1 mg Cl-
_ Normality of AgN0 3 X Vol. of AgN0 3 x Eq. wt. of cr
or Cl (mWL)-------------~--------~----------
- mL of Sample taken
Potentiometric titration is performed for smaller quantities of
chloride with AgN0 3 using glass and Ag-AgCl electrode system.
SULPHATE
Sulphate usually occurs in natural waters. Mine drainage wastes also
contain high content of sulphate by virtue of pyrite oxidation. The presence
of Na2S04 and MgS04 in drinking water beyond the prescribed limits may
cause cathartic action.
Gravimetric Method.
In gravimetric procedure sulphate is precipitated as BaS04 in acidic
(HCI) medium using BaCl2 solution. The precipitate of BaS04 is digested,
filtered and washed with hot water to remove Cl- ions, ignited and weighed.
Volumetric Method.
100 mL of the sample water is taken in a conical flask and add 10 mL
of benzidine hydrochloride solution (solution of benzidine in dilute HCl
containing 4 g. of the diamine base per litre of the solution). The precipitate
of benzidine sulphate is filtered and washed free of acid with minimum
amount of distilled water. The precipitate and filter paper are taken in a
conical flask and 50 mL of distilled water are added to it. Now few drops of
phenolphthalein are also added.The conical flask is well shaken to dissolve
the precipitate and the solution so obtained is titrated against N17 NaOH
until pink colour is obtained at the end point.
Calculation.
Sulphate (as N~S04) ppm = No. ofmL ofNI7 NaOH x 100.
Na2S04 ppm can be converted into CaC03 ppm by multiplying with a
factor of 0·705.
FLUORIDE
Fluoride occurs in all natural water supplies and in chemical wastes
from industries. Fluorides, if present in small concentration up to 1 ppm, are
generally considered to be beneficial in water. Excessive fluorides in drinking
water may cause mottling of teeth or dental fluorosis, which results in
discolouration of enamel, chipping of teeth in children in severe cases. Bone
fluorosis or crippling effects are observed in case the concentration of
fluorides exceeds 1·0 mg/L.
Spectrophotometric Method.
Alizarin-S Visual Method. Fluoride reacts with Zr Alizarin-S lake to
form colourless ZrF~- and the dye. The colour of the dye lake becomes
progressively weak with increase in amount of F-.
~r~o
~S03Na+
o
Zr-Alizarin red-S lake
To a 100 mL sample add 1 drop of NaAs0 2 solution (5g/L) to remove
residual CI, if any. Add 5 mL acid-zirconyl-alizarin reagent (300 mg
ZrOC1 2 ·8H20/50 mL + 70 mg alizarin red S/50 mL + 800 mL of 1·5 N
HCl-1.2 N H 2S04 made up to 1 litre). Mix thoroughly and compare the
samples and standards after 1 hour.
Spadns Method.
Spadns method is preferred to the Alizarin visual method as the latter
takes 1 hour for colour development. [Spadns is Sodium 2-(para sulphoph-
enylazo)-l, 8-dihydroxy-3, 6 naphthalene disulphonatel.
The reaction rate between F" and Zr02+ is influenced greatly by the
acidity of the reaction mixture. By increasing the proportion of acid in the
reagent, the reaction can be made practically instantaneous.
Procedure.
• Prepare a series of standard solutions ofF-, i.e., 0·5,1·0 and 2·0 mg/L.
• Add 1 drop of NaAs0 2 solution (0·5%) to remove any residual chlorine.
• Dilute sample to 50 mL. Add 5 mL of SPADNS (1·9 giL) and 5 mL of
zirconyl acid reagent (0·26 g ZrOCI2·8H20 and 700 mL of 50% cone.
HClIL).
Mix well and read absorbance at 570 nm against standard immediately.
Calculate the concentration of fluorine from a standard curve.
lon-Selective Electrode Method.
This is the most convenient method for the estimation of F-, down to
10-5 M (0·2 mg/L). It is based on potentiometric measurements with a
membrane electrode consisting of a single crystal of europium doped
lanthanum fluoride LaF3' The purpose of Eu doping is to improve electrical
conductivity. The membrane is cut as a 1-mm thick disc, a few mm in
diameter. The disc is sealed into the end of a rigid plastic tube, filled with an
equimolar solution of KCI and NaF, into which dips a AgCI electrode. A
reference electrode (saturated calomel electrode) is inserted into the test
solution along with the fluoride electrode. The potential difference is
measured.
Cell Ag/AgCI, CQO·3 M), F (0·001 M) ILaF31 test solution/reference
electrode
RT
Emeas = Const + -F log ar E = 0·058 log [F"] + constant
n SCE
Plastic beakers and flasks (polyethylene, teflon) should be used for

storing F- solution and for all measurements.
Procedure.
• Prepare a series of standards equivalent to 0·5 to 1·0 and 2·0 mg/L of
fluoride.
• Take 50 mL sample, add 50 mL buffer solutions of high-ionic strength
(pH 5·0 to 5·5) (glacial acetic acid + NaCI + cyclohexylene diamine
tetracetic acid, CDTA + NaOH-adjusted to pH 5·0 to 5·5).
• Transfer each standard and sample to a series of 150 mL beakers. Stir
test solution on a magnetic stirrer. Measure the developed potential.
SILICA
Silica is not a water pollutant but excess of silica is undesirable. It is
generally removed by the use of strongly basic anion-exchange resin in the
deionization process or by distillation. The silica content of natural water is
usually 1 to 30 mg/L. Brackish waters and brines may contain as high as 1000
mg/L of silica. The gravimetric method is useful for 20 mg/L or more of
Si0 2 and spectrophotometric method, for 0·4 to 25 mg/L of Si02.
Gravimetric Method.
Silicates and dissolved Si02 are decomposed by HCI giving silicic acids
during evaporation. Ignition completes dehydration of Si02 which is weighed
and then volatilised as SiF4 , leaving impurities as non volatile residue. The
residue is weighed and the difference gives Si02 lost on volatilisation.
Na2SiOa(Si02) + 2HCI ~ 2NaCI + H 2SiOa
H 2SiOa ~ Si02 + H 20
Si02 + 4HF + 2H2S04 ~ SiF4i + 2H20 + 2H2S04
Procedure.
1. Take a clear sample (10 mg Si02) in a 200 mL platinum dish. Add
5 mL 6N HCI and evaporate repeatedly with addition of HCI to
dryness on a water bath.
2. Bake the residue on a hot plate for half an hour.
3. Leach with 5 mL 6N HCI, warm and add 50 mL hot distilled water.
While hot, filter through an ashless filter paper. Wash the dish and
residue with hot 0·2 N HCI and then with small volume of distilled
water till the filtrate is chloride free.
4. Evaporate the filtrate and washings from the above step to dryness
in the original platinum dish and repeat steps 2 and 3 above.
5. Transfer the two filter papers and residues thus obtained to a
covered and weighed platinum crucible, dry at no°c and finally
ignite at 1200°C to constant weight.
6. Moisten the residue in the crucible with distilled water. Add 4
drops of 18 N H 2S04 and then 10 mL HF. Slowly evaporate to
dryness over a hot plate in a hood. Ignite the crucible at 1200°C to
constant weight.
ANALYSIS OF WATER POlLUTANTS 161
7. Record the weight of Si02 as the difference in these two weights

from the steps 5 and 6.
Spectrophotometric (Molybdosilicate) Method.
Principle.
Ammonium molybdate at pH 1·2 reacts with Si02 and any phosphate
present to produce heteropoly acids. Oxalic acid is added to destroy the
molybdophosphoric acid, H3PMo12040 but not the molybdosilicic acid,
H4SiMo12040' The resulting yellow molybdosilicic acid is measured at 650
nm.
.Procedure •.
1. Place 50 mL sample in a 100 mL platinum dish and 200 mg
NaHC03 and digest on a steam bath for 1 hour. Cool and add slowly
with stirring 2·5 mL H2S04,
(Note : The digestion step is necessary to convert any molybdate
unreactive silica to the reactive form). Make the volume to 50 mL and
transfer to a 100 mL separatory funnel.
2. To 50 mL treated sample, add quickly 1 mL of 6 N HCI and 2 mL
ammonium molybdate. Shake and allow to stand for 10 minutes.
3. Add 1·5 mL oxalic acid (10 gin 100 mL distilled water) and shake
vigorously. Measure the absorbance at 650 nm against a reagent
blank.
PHOSPHATE
Industriai efiluents, domestic sewage and agricultural run off are the
major contributor of phosphorus in water. Phosphates are largely used for
laundry purposes and treatment of boiler waters. Phosphorus occurs both in
inorganic and organic (85%) forms.
Principle. Orthophosphates form heteropoly acid when they react with
ammonium molybdate and potassium antimony tartrate in acid medium. The
phosphomolybdic acid reduces to molybdenum blue by ascorbic acid.
Reagents.
Standard Phosphate Solutions.
Dissolve 2·194 g of anhydrous potassium hydrogen phosphates in
deionised water and make up the vol:ume to 500 mL. Take 10 mL of this
solution and add deionised water to make 1 L of stock solution containing
1 mg PIL.
Prepare standard phosphorus solutions of various strengths from 0·0 to
1·1 mg PIL at intervals of 0·1 mg PIL by diluting the stock solution with
distilled water.
Reagent A. Take 1 g of ammonium molyb4ate and 0·02 g of potassium
antimony tartrate in 1000 mL volumetric flask. Add 16 mL of concentrated
H 2S04 slowly. Dilute with distilled water to the mark.
Reagent B. Weigh 0·88 g of ascorbic acid' and dissolve in 1 L of

reagentA.
Procedure.
Take 25 mL sample in an Erlenmeyer flask and evaporate to dryness.
Cool and dissolve the residue in 1 mL of 70% HCI04.
• Heat the flask gently so that the cQntent becomes colourless. Cool and
, : add' 10 mt.' distilled water and two drops of 1% phenolphthalein
,:', >indicator. >' "
, . ., Titrate> against l' N NaOH solution u~til pink colour appears. Make
, >up the volume to 25 niL' With distilled water.
• Transfer this solution into 50 mL volumetric flask and add 10 mL of
reagent B.
.• , Make the volume to 50 mL with water till the blue colour develops.
• Record the absorbance on spectrophotometer at 660 nm.
• Run simultaneously a distilled water blank in the same manner.
• Process the standard phosphorus solutions of various strengths in a
similar way.
• Plot a curve between absorbance and concentration of standard
p;hosphorus solution.
• Deduce the phosphorus content of the sample by comparing its
absorbance with standard curve.
Calculatioti. '
P (mgIL) = mg P in 50 mL x 1000
Volume of sample
DIFFERENT FORMS OF NITROGEN

Nitrogen occurs in water in small amo~ts and in ,bound form it is found
as NH~, NOs, N0 2 and organic nitrogen.
1. AMMONIA
Ammonia is present in surface water, ground water and domestic
sewage. In water bodies, it is produced naturally by the reduction of nitrates
under anaerobic conditions., ,Titration procedures are valid when the NH3
level exceeds 5 ppm. Distillation is necessary when the sample is coloured and
when the titrimetric method is followed.
Spectrophotometric Nessler's Method. The method is useful for
ammonia nitrogen upto 5 ppm. It is based on the reaction between NH3 and
HgIa- tetra iodo mercury (II) anion in alkaline solution
NH3 + 2HgI~- + 30Ir ~ NH2Hg2IO + 7r + 2H20
The orange-brown reaction product· tends to precipitate at higher
concentration, while at lower concentration it is measured at 420 nm
spectrophotometrically.
Procedure 1.
• To a 100 mL sample, add a little NaOH to neutralise the acid used for
storage and then add 1mL 10% ZnS04·7H20 followed by 1 mL of 10%
NaOH. Stir and filter.> Ca, Fe, Mg, 8 2- are precipitated.
• Collect the colourless middle fraction, add 1 drop of 50% EDTA

(disodium salt), mix well and add 2 mL of Nessler's reagent [70 g
KI + 160 g HgI2 + 160 g NaOH (ice-cooled) diluted to 1 litrel. Shake
well.
• Measure the resulting yellow colour at 420 nm.
Procedure 2.
• When the sample is coloured, distil a 500 mL sample with dil. NaOH
and collect the distillate in an Erlenmeyer flask containing 200 mL of
0·1 NH2S04.
• Make up the volume of distillate to 250 mL in a volumetric flask.
• Take 5-10 mL aliquot, neutralise with 0·1 N NaOH to pH 4 and add
2 mL Nessler's reagent. Proceed as above for measurement.
Procedure 3.
Distil as above and collect only 100 mL of the distillate in an Erlenmeyer
flask. Titrate with 0·02 N ~S04' using a mixed indicator (200 mg methyl red
in 100 mL 95% ethyl or isopropyl alcohol + 100 mL methylene blue in 50 mL
95% ethyl alcohol), until the indicator turns a pale lanvender. Carry a blank
through all the steps.
2. NITRATE AND NITRITE
Nitrate and nitrite are usually monitored regularly in water supplies as
they are deemed to be potentially hazardous to health if their maximum
admissible concentrations of 50 mglL and 0·1 mg/L, respectively, are exceeded.
NOs is particularly dangerous to infants less than six months old, causing
child disease methemoglobinaemia. A limit of 10 mg/L for NOs has been
imposed on drinking water.
Nitrates generally occur in traces in surface water and ground water.
Nitrites are an intermediate product, both in the oxidation of NHg to N0 2 and
in the reduction of NO g, which occurs in waste-water treatment plants,
water-distribution systems and natural waters.
Spectrophotometric Method for Total Nitrate and Nitrite.
Nitrates and nitrites are reduced to NHa by Devarda's alloy (50 Cu, 45
Al, 5 Zn) in strongly alkaline solutions, the NHa is distilled into excess
standard acid and finally estimated spectrophotometrically.
a
3NO + BAl + 50ft + 2H20 ~ BAl02+ 3NHa
• Take 500 mL sample in NHa distillation apparatus. Add 50 mL of 10%
NaOH and evaporate to about 200 mL.
, Cool the solution, add 3 g Devarda's alloy (20 mesh) and then 30 mL
of 10% NaOH and immediately connect the flask with a vertical
condenser whose outlet dips into a receiver containing 200 mL of
0·2NH2S04·
• Distil at 50 to BO°C for one hour.
• Disconnect the receiver. Make up the volume of the solution in the
receiver to 250 mL. Take 5 to 10 mL aliquot in a 50 mL volumetric
flask and neutralise to pH 4·5.
• Add 2 mL Nessler's reagent and estimate the absorbance at 424 nm

as described under ammonia.
a
• This spectrophotometric method is valid for NO concentrations
greater than 0·5 ppm.
a
Titrimetric method may be followed for NO content exceeding 5 ppm.
Here the distillate can be directly back titrated with standard alkali
(0·2 N NaOH) using methyl red as indicator.
1 mL N H 2S04 = 0·06201 g NOs
3. NITRITE
Method based on Diazotisation Reaction.
A reddish purple azo dye is produced at pH 2·0 to 2·5 by the coupling of
diazotised sulphanilamide with N-C1-naphthyl) ethylenediamine dihydro-
chloride.
• Take a 40 mL sample in a volumetric flask and adjust pH to 7·0.
• Add 2 mL of sulphanilamide solution C50 gin 500 mL of 1·2 N HCI),
shake and allow to stand for 10 minutes.
• Add 2 mL of N-Cl-naphthyI) ethylene diamine dihydrochloride (0·83 g
in 200 mL warm water, cooled, filtered and diluted to 250 mL with
glacial acetic acid), dilute to 50 mL and mix thoroughly.
• Measure the resulting purple azo dye at 543 nm within two hours,
also against standards covering the range of N02 from 1 to 25 IlglL.
4. TOTAL ORGANIC NITROGEN (TON)
Titrimetric Method.
Organic nitrogen occurs in the form of amino acids, urea and nucleic acid.
Materials.
• Micro-Kjeldahl distillation assembly.
• Digestion mixture. Dissolve 16·25 g of~S04 in 200 mL of distilled
water. Add 0·4 g of mercuric oxide and 25 mL of concentrated H 2S04
slowly. Make up the volume to 250 mL with distilled water.
• Hypo solution. Dissolve 50 g of NaOH in 200 mL distilled water
and add 10 g of sodium thiosulphate. Make up the volume to 250 mL
with distilled water.
• Mixed indicator. Mix 0·1 % methyl red solution and 0·5%
bromocresol green in 1 : 2 ratio in methanol.
Procedure.
• Take 200 mL of the sample in a dish and evaporate to dryness.
• Add 4 mL of digestion mix~ure to the residue and dissolve it in 20 mL
of distilled water.
• Heat the solution for 15 mi~utes. Cool and transfer the digest to
micro-Kjeldahl distillation assembly. Add 5 mL of hypo solution.
• Take 5 mL of 1% boric acid solution containing 2 drops of mixed
indicator in a conical flask.
• Place the flask below the condenser so that the tip of outlet of the
condenser is dropped in contents of the flask.
• Heat the Kjeldahl flask. Continue distillation for 10 minutes. Remove
the conical flask having distillate.
• Titrate the distillate against 0·01 N HCI till.the colour changes from
blue to pink.
• Run a blank using distilled water in a similar manner.
Calculation.
(8 - B) x 0·01 N x 1000 x At. wt. ofN2 (14)
TON (mg/L) = Volume of sample
where 8 = Volume of HCI used against sample.
B = Volume of HCI used against blank.
5. DISSOLVED ORGANIC NITROGEN (DON).
Take the sample and filter through millipore filter paper, Employ the
above procedure of TON. Express the result of dissolved organic nitrogen in
mg/L.
6. PARTICULATE ORGANIC NITROGEN (PON).
Determine the dissolved organic nitrogen and total organic nitrogen as
described above and calculate particulate organic nitrogen.
PON (mg/L) = TON - DON.
MEASUREMENT OF DISSOLVED OXYGEN

DO test is an important parameter in evaluating the pollution potential
of domestic and industrial waste waters.
1. Modified Winkler Method.
Principle. Kl is oxidised to 12 with the dissolved oxygen present in the
water sample after adding MnS04, NaOH and Kl. The basic manganeic oxide
formed from N aOH and MnSO 4 acts as an oxygen carrier to enable the
dissolved oxygen in the molecular form to take part in the reaction. The DO
present in the sample oxidises the Mn2+ to its higher valency which
precipitates as a brown hydrated oxide after the addition of NaOH and Kl.
On acidification, the manganese reverts back to the divalent state and an
equivalent amount of iodine is liberated from the Kl present. This liberated
iodine is titrated against standard sodium thiosulphate (hypo) solution, using
starch as indicator.
MnS04 + 2KOH ~ Mn(OH)2 + ~S04
2Mn(OH)2 + 02 ~ 2MnO(OHh
Basic manganeic oxide
MnO(OH)2 + H 2S04 ~ MnS04 + 2H20 + °
2Kl + H 2S04 + ° ~ ~S04 + H 20 + 12
12 + 2Na2S203 ~ Na2S406 + 2Nal
Interferences. Iron, nitrite and microbial mass are the chief sources of
interference in this method. The interference due to nitrite can be eliminated
by adding sodium azide.
2NaN3 + H 2S04 ~ 2HN3 + Na2S04
Sodium azide Hycirazoic acid
HN02 + HN3 ~ N 20 + N2 + H 20
Reagents Required.
• Standard N120 KtCr207 (Mol. wt. 294·21; Eq. wt. 49·035). Dissolve
2·4518 g of dry A.R. ~Cr207 powder in distilled water and make up
to 1000 mL in a measuring flask. For more accurate work, the
KaCr207 powder should be dried at 140°C for 1 hour in an electric
oven and cooled in a desiccator before weighing.
• N/20 Sodium thiosulphate solution. Dissolve 12·41 g of A.R.
sodium thiosulphate pentahydrate (Na2S203·5H20) in 1 L of distilled
water. The solution may be preserved by adding 3 drops of chloroform.
• Cone. H 2S04•
• Manganese sulphate. Dissolve 400 g of MnS04 or 480 g of
MnS04·4H20 in distilled water and make up to 1 L. This solution
should not give colour with acidified potassium iodide and starch.
• Alkaline iodide-azide reagent. Dissolve 500 g of NaOH and 150 g
of KI in distilled water. Add JO g of NaN3 in 40 mL distilled water.
Dilute to 1 L. This solution should not give colour with starch on
dilution and acidification
• Starch solution. Prepare a fresh paste of 0·5 g of starch with
distilled water and pour this into 100 ml of boiling distilled water
while stirring.
• Solid KI free from iodate, NaHC03 and cone. HCl.
Procedure.
(A) Standardization of hypo solution: Transfer 100 mL of 90iled,
cooled distilled water in a 250 mL iodine flask. Add about 2 g of KI, '2 g of
N aHC0 3 and shake until the salts are dissolved. Add 6 mL of conc. HCI
slowly while gently rotating the flask. Add 25 mL of N/20 KaCr207 solution
and mix the solutions well. Wash the inner sides of the flask with a littl~
distilled water and stopper the flask. Allow to stand for 5 minutes for the
completion of the reaction.
Cr20~- + 6r- + 14H+ ~ 2Cr3+ + 312 + 7H20
Rinse the stopper with distilled water and titrate the liberated iodine
with the hypo solution from the burette. When most of the iodine has reacted,
the solution acquires a greenish-yellow colour. At this stage, add 1 mL of
starch solution when the solution turns blue due to the formation of starch
iodine complex. Continue the titration until the greenish blue colour changes
to light green by the addition of a single drop of hypo at the end-point.
Note. If KI is not iodine free, a blank experiment has to be performed
and the corresponding correction should be made to the titre value.
(B) Titration with the water sample.· For the. 1;>0 determination, the
sample should be collected from below the surface at ,a known depth and
without aeration. Generally the sample is collected in a BOD bottle with a DO
sampler.
For labl)ratory practice, collect the water sample in a 300 m BOD bottle,
avoiding contact with air. Add with the help of a pipette 2 mL of MnS04
followed by 2 mL of the alkaline iodide-azide reagent in such a way that the
tip of the pipette should dip below the liquid surface while adding the
reagents. Stopper the bottle immediately an<~ mbnvell by inverting the bottle
3 to 4 times. Allow to stand for 2 minutes, add 1 mL of conc. H 2S04, insert
the stopper and mix well till the precipitate goes into solution. Allow to stand
for 5 minutes. Take 203 mL of the clear solution into a conical flask and
titrate against hypo solution using starch as indicator.
Note. When 2 mL of MnS04 + 2 mL of alkali iodide-azide reagent are
added into full BOD bottle, from below the liquid surface, 4 mL of the original
water sample is lost. Hence 203 mL now taken for titration will correspond
to 200 mL of the original sample.
200 x 300 =·203 L
(300-4) m
Model Calculations.
20 mL of N/20 ~Cr207 == 20 mL of N/20 hypo
Normality of hypo = 20 ;0 ;0
x x N =~
DO in 200 mL of the water sample, on treatment, liberiites iodine, which

on titration is found to be equivalent to 4·2 mL of N/20 hypo.
:. Normality of the water sample with respect to DO
1 1
=4·2 x 20 x 200
1 1
Strength == 4·2 x - x - x 8 giL == 0·0084 giL
20 200 .
= 8·4 mg/L = 8·4 ppm.
Result. Dissolved oxygen in the given water sample:;: 8·4 mg/L or
8·4 ppm ..
Alternative Method of Calculations.
For 200 mL of the original water sample used, calculate on the basis that
1 mL of 0·05 N hypo == 2 mg of DOlL
4·2 mL of 0·05 N hypo == 2 x 4·2
= 8·4 mg DOlL in water sample.
2. Polarographic Method.
02 can be reduced at various electrodes in aqueous solutions. if a small
negative voltage is applied. The magnitude of the current which -flows is
determined by the rate at which 02 can diffuse to the electrode.
3. Membrane Electrode Method.

(Mackerettl Oxygen Cell for the Determination of DO).
The polarographic method is not desirable for DO analysis in domestic!
industrial waste waters as the Hg electrode gets poisoned by impurities in the
test solutions. This problem is solved by using the membrane electrode
method. Two metal electrodes, one of Ag and the other of Pb are immersed in
a saturated KHCOs solution separated from the test solution by a
polyethylene membrane. Thus, a galvanic cell can be plugged to a pH meter
to give a direct reading of DO in mg L-1 (the scale of 0 to 14 pH becomes 0
to 14 mg L-100). The current is measured for sample, for a standard (sample
after air saturation) and for a blank (sample after treatment with a little
N 82S03 to expel 02).
O2 + 2H20 + 4e- ----7 40H- (Ag electrode)
Pb + 40W ----7 Pb02 + 2H20 + 4e-
or 2Pb + 40W ----7 2Pb(OH)2 + 4e- (Pb electrode)
MEASUREMENT OF CHEMICAL OXYGEN DEMAND

Principle. The chemical oxygen demand (COD) is a measure of the
oxygen equivalent of that portion of organic matter in a sample that is
susceptible to oxidation by a strong chemical oxidant. This is an important
and quickly measured parameter for stream and industrial waste water
studies and control of waste treatment plants.
Most types of organic matter are completely oxidized by a boiling
mixture of chromic and sulphuric acid to produce CO2 and H20. A measured
quantity of the sample is refluxed with a known amount of ~Cr207 and
H 2S04,
3{C~0} + 16W + 2Cr2~- ----7 4Cr3+ + 3C02 + llH20.
The excess of dichromate remaining unreacted is titrated with ferrous
ammonium sulphate solution. The ~Cr207 consumed is proportio:p.al to the
amount of oxidizable organic matter measured as oxygen equivalent.
Reagents Required.
1. Standard 0·25 N ~Cr207 solution. Dissolve 12·259 g of pure and
dry AR ~Cr207 in distilled water and dilute to 1 litre. Add about 120
mg of sulphamic acid to eliminate any interference because of nitrites
that may be present in the waste water sample upto 6 mg/L.
2. Sulphuric acid-silver sulphate reagent. Add 5·5 g of Ag2S04 to 1
kg of conc. H2S04 and keep over night for dissolution.
3. Standard 0·1 N Fe(NH~iSO~2·6~0 solution. Dissolve 39 g of the
pure salt in distilled water. Add 20 mL of conc. ~S04 and dilute to
1 litre.
4. Ferroin indicator. Dissolve 1·485 g of 1, 10-phenanthroline
monohydrate with 0·695 g of pure FeS04·7H20 in water and dilute
to 100 mL.
5. HgS04 AR grade.
ANALYSIS OF WATER POLLUTANTS 1lS9
Procedure. Place 0·4 g of HgS04 in a reflux flask and add 20 mL of

the sample or an aliquot of the sample diluted to 20 mL with distilled water.
Mix well and add 10 mL of 0·25 N ~Cr207 solution. Drop some pumice
stones and slowly add 30 mL of H 2S04-AgzS04 reagent while continuously
swirling the flask. If the colour changes to green add more ~Cr207 and
H2S04-Ag2S04 reagent or alternatively discard the solution and take fresh.
sample with lesser aliquot. Mix the contents of the flask thoroughly. Connect
the flask to the c{)ndenser and slowly heat. Reflux for at least 2 hours. Cool
and wash down the condenser with distilled water such that the washings fall
into the flask. Dilute to about 150 mL, cool and titrate the unreacted
~Cr207 with N/10 ferrous ammonium sulphate solution using ferroin as
indicator. The colour change at the end point is from blue green to wine red.
Perform a blank experiment with distilled water instead of the water
sample.
Calculations. The COD of the sample is calculated as follows :
(V1 - V2) N x 800
COD in mg/L =---==----"'----
x
where Vl = Volume of ferrous ammonium sulphate run down in the blank
experiment.
V2 =Volume of ferrous ammonium sulphate run down in the test
experiment.
N =Normality of ferrous ammonium sulphate solution and
x =Volume of test sample taken.
Note that: '
1. Straight-chain aliphatic compounds, aromatic hydrocarbons and
pyridine are not oxidised to any appreciable extent, although this
method gives more nearly complete oxidation than the permanganate
method. The straight chain compounds are oxidised more effectively
when Ag2S04 is added as catalyst. However, silver sulphate reacts
with chloride, bromide and iodide to produce precipitates that are
oxidized only partially by this procedure. The oxidation and other
difficulties caused by the presence of chloride may be overcome by
using a complexing technique for the elimination of chloride. This is
accomplished by adding HgS0 4 to the samples before refluxing. This
ties up the chloride ions as soluble mercuric chloride complex and
greatly reduces its ability to react further.
2. The method can be used to determine COD values of 50 mgIL or more
with the concentrated dichromate. With the dilute dichromate, value
below 10 mg/L are less accurate but indicate the order of magnitude.
MEASUREMENT OF BIOCHEMICAL OXYGEN DEMAND

Biochemical oxygen demand (BOD) represents the quantity of oxygen
required by bacteria and other micro-organisms during the biochemical
degradation and transformation of organic matter present in waste water
under aerobic conditions.
170 ANALYTICAL CHEMiStRY
Principle. The dissolved oxygen content of the sample is determined

before and after five days incubation at 20°C. The BOD is calculated on the
basis of the amount of oxygen depleted.
Reagents.
(i) Calcium chloride solution. Dissolve 27·5 g of anhydrous CaCl2 or
equivalent amount of the hydrated salt in distilled water and dilute to 1 L.
(ii) Magnesium sulphate solution. Dissolve 27·5 g ofMgS04·7H20 in
distilled water and dilute to 1 L.
(iii) Ferric chloride solution. Dissolve 0·25 g FeCIg·6H20 in distilled
water and dilute to 1 L.
(iv) Phosphate buffer solution. Dissolve 8·5 g of KH 2P04, 21·75 g of
~HP04' 33·4 g of Na2HP04·7H20 and 1·7 g of NH 4CI in 500 mL distilled
water and make upto 1 L. The pH of this solution should be 7·2. Keep this
solution in refrigerator to prevent any biological growth.
(v) MgS04 solution, alkali-iodide-azide reagent, H 2S04 etc. are prepared
as in DO determination.
Preparation of Dilution Water.
Redistil water in presence of a little alkaline permanganate. Store this
double distilled water in a BOD incubator at 20°C. Aerate the required
quantity of this distilled water at 20°C with clean compressed air. Add 1 mL
each of CaCI2, MgS04, FeCIg and phosphate buffer solutions per litre to the
above aerated distilled water and mix thoroughly. This standard dilution
water should be prepared just before use.
The addition of small measured volume of water containing a good
bacterial population to the dilution water is called seeding. Seeding is not
required for sewage and sewage eftluents because they contain the bacterial
flora. Seeding is necessary for industrial eftluents which generally do not
contain any bacteria. The seeding material that is generally used is fresh and
settled raw sewage or fresh and settled final eftluent of an aerobic biological
process. The seed should be kept for 1 to 2 days at 20°C before use. The seed
concentration recommended is 1 to 2 mL per litre of dilution water.
Pre-treatment Methods.
1. If the pH of the sample is not in the range of 6·5 to 8·5, add the
necessary quantity of IN· H 2S04 or IN·NaOH to bring the pH in the
said range. Determine the amount of H 2S04 or NaOH so added on
a separate aliquot of the sample.
2. If the sample contains any chlorine, destroy the chlorine by adding
a known excess of freshly prepared 0·025 N sodium sulphite solution.
Determine the amount of the Na2S0g consumed by the iodometric
method on a separate aliquot of the sample.
3. Samples must be thoroughly shaken just before dilutions are made.
4. Samples stored in refrigerator should be allowed to reach room
temperature before making the dilutions.
5. If the BOD of a polluted river water containing algae is to be
determined, the algae must be separated by centrifugation and the
centrifugate should be used for the BOD test.
6. Some waters, e.g., those containing algae may be supersaturated

with dissolve~ oxygen. In such cases, the excess oxygen above the
saturation level should be removed by partly filling the sample in a
bottle and agitating or aerating at 20°C.
Procedure.
It is preferable to make a series of dilutions for a sample such that at least
three of the dilutions should deplete 20% to 90% of the initial dissolved oxygen
(DO). The COD value or permanganate value may be treated as guideline for
the purpose of dilution. The following guidelines are advantageous:
Vol. of sample per
% of the sample in
S.No. Sample litre of the dilution
the dilution mixture
mixture, mL.
1. Raw sewage 5 to 0·5 50 to 5
2. River waters 100 (i.e., no dilution) 1000
3. Polluted river waters 50 to 10 500 to 100
4. Industrial eftluents 20 to 0·05. 200 to 0·5
The following dilution technique may be used :

1. Transfer carefully the standard dilution water (or the seeded dilution
water in case of trade wastes) into alL graduate glass cyclinder until it is
half full without any air entrainment. Now add the appropriate quantity of
the well mixed sample into the glass cylinder without producing air bubbles.
Make up the volume to the mark using the dilution water. Mix well with a
glass rod' without air entrainment. Siphon the mixture into two BOD bottles
of 300 mL capacity. Fill the two BOD bottles carefully without allowing any
air bubble and seal the bottles.
Prepare the other dilutions of the sample by adopting the above
procedure. Also, siphon out the standard dilution water (or the seeded dilution
water) into two BOD bottles and water seal them after filling them
completely.
2. Utilize one set of the entire series of dilutions prepared above for the
immediate determination of dissolved oxygen (DO). Keep the other set in a
BOD incubator maintained at 20°C for 5 days. After 5 days determine the DO
content of all the incubated samples.
Calculations. Calculate the BOD values u~ing the following relation.
(DOo - D05 - B) x 100
BOD mg/L (5 days at 20°C) = %o 0 fth e sampIe used
where DOo =Initial DO content in mgIL
D05 =DO content after incubation for 5 days.
B =Blank correction determined by the difference between the DO
contents of the blank on the initial day and that after 5 days
incubation.
Precautions.
• The temperature of 20°C should be constantly maintained during the
5 days incubation period. Even an increase or decrease of
temperature by 1°C may increase or decrease the BOD value by

about 4·7%.
• The BOD test should be performed as soon as possible after
collecting the sample.
TOTAL ORGANIC CARBON (TOC)

Total organic carbon can be determined by TOC analyser. The oxidising
agent is ~S208 and the oxidation is promoted by the free radical HO·
generated from ultraviolet light.
~S208
Organics + HO· ) CO2, + H 20
TOC analyser employing ultraviolet irradiation is shown in Fig. 1. To the
sample, ~S208 and H 3 P04 are added. H 3P04 is sparged with air or N2 to
a
expel CO2 formed from HCO and CO~- in solution. After sparging, the
sample is pumped to a chamber containing a ultraviolet lamp emitting
radiation at 184·9 nm. The HO· formed by ultraviolet irradiation is the active
species which brings about the rapid oxidation of dissolved organic
compounds. At the end of oxidation, CO2 is sparged from the system and
measured with a gas chromatographic detector or by adsorption in ultrapure
water followed by conductivity measurement.
Septum for injecting
-
sample and reagents
_~=======::::::::;::===~~::::::::::;~ Integrating CO2
CO detector
2
Sample
Oxidiser chamber
t
Gas for sparging
unoxidised sample Gas for sparging
~===~ -::::::::()()::=:Jo.-- oxidised sample
Fig. 1. TOe analyser employing ultraviolet-promoted sample oxidation.
PESTICIDE ANALYSIS
ANALYTICAL METHODS.
Methods employed widely for the analysis of pesticides, their residues
and degradation products include :
1. Chromatographic methods are TLC, GLC and HPLC. Organo-
chlorines and carbamates are best analysed by TLC. Organophosphates are
accurately detected by GLC using flame ionisation detector.
2. Polarographic methods are used to determine pesticides containing
oxidisable or reducible groups.
3. Spectroscopic methods. JR, UV, NMR and GC-MS help to identify
metabolites and degradation products of pesticides.
[Refer to Unit 6 for pesticide pollutants, their classification and effects].
ANALYSIS OF ORGANOCHLORINE PESTICIDES BY TLC.

Separation. Walker and Beroza separated 62 pesticides in 19 solvent
systems by thin layer chromatography. They employed silica gel G layer for
chromatographing DDT, BHC, aldrins, dieldrins, endrin, isodrin, chlordane
and pentachlorophenol.
• Petroleum ether was used to separate DDT and four isomers of BHC.
• Cyclohexane was used to separate aldrin from its stereoisomer
isodrin and dieldrin from its stereoisomer endrin.
Detection. Aldrin and its isomers were detected spraying with 0·01 N
KMn04 solution. DDT and BHC were made visible by spraying with
monoethanolamine and heating to 100°C for 20 minutes. This was followed
by a spray of 0·1 N AgN0 3-HN03 (10 : 1). The sprayed plates were exposed
to UV lamp for one minute. Violet to green spots were observed.
R f values of organochlorine pesticides when the chromatoplates
are sprayed with 0·5% solution of p-dimethylamino hydrochloride prepared in
sodium ethoxide are :
Pesticide DDT BHC Aldrin Dieldrin Methoxychlor
Rfvalue 0·59 0·39 0·78 0·18 0·12
ANALYSIS OF ORGANOPHOSPHORUS PESTICIDES BY TLC.

Organophosphates can be chromatographed on silica gel G using hexane
: acetone (4 : 1) as the solvent system. The chromatoplates are sprayed with
0·5% solution of PdCl2 in HCI giving yellow spots.
Pesticide Parathion Malathion Chlorthion Diazinon Metasystox
Rfvalue 0·66 0·52 0·44 0·78 0·62
ANALYSIS OF ORGANOCHLORINE PESTICIDES BY GAS

CHROMATOGRAPHY.
The pesticidal sample is extracted repeatedly with 15% ethyl ether in
hexane. The combined ether eKtracts are evaporated on a steam bath to 2 mL
15 10 5 o
Fig. 2. Gas chromatogram of organochlorine pesticides. Column packing 5% av.
Glass column 180 cm x 4 mm i.d. Solid support Gas-chrom Q (100/128 mesh).
and diluted with hexane to 5 mL. An aliquot of this extract is injected into
the gas chromatographic column (180°C) with a micro syringe using Ar/CH4
as the carrier gas at 70 mL I minute. The pesticides are vapourised. They
move through the column at different rates and detected by electron capture
detector (Fig. 2).
Organophosphorus pesticides can be analysed by GC using
dichloromethane as the extractant and flame ionisation detector.
Phenoxy herbicides (2,4-D and 2, 4, 5-T) can be analysed by
derivatisation GC using electron capture detector. The herbicides are
extracted with ether and after hydrolytic precipitation, methyl ester is
analysed by gas chromatography.
ANALYSIS OF INSECTICIDES BY HPLC.
Dolphin and coworkers used automated HPLC pesticide analyser for the
analysis of chlorinated insecticides (Fig. 3) and fat in milk by injecting raw
milk onto a short silica precolumn where the fat was retained and the
pesticides eluted with hexane. The less polar fat eluting pesticides such as
pp' DDT, pp' DDE and (X-BHC were not separated from each other by the
precolumn and stored at the top of the column. Pesticides such as I3-BHC,
'Y-BHC and dieldrin which eluted latter were resolved from each other and
passed directly into an electron capture detector by means of a computer
controlled pnematically operated surtching valve.
Fig. 3. Separation of Insecticides by HPLC.

Column packing. L1chrosorb S.I. 60 (5 J.lm); Column dimensions 250 x 3 mm I.d.
Eluent. dry hexane; Flow rate. 4 cm 3 min-1 ; Column temperature 300 K; Detection
UV at (a) 205 nm and (b) 254 nm. (Insecticides : 1. Hexachlorobenzene,
2. Decachlorobiphenyl, 3. Aldrin, 4. Heptachlor, 5. pp'-DDE, 6. op'-DDE,
7. op'-DDT, 8. pp'-DDT, 9. Biphenyl, 10. pP'-DDD).
Organophosphorus pesticides such as phosphamidon, mono-and
dicrotophos can be detected by HPLC. The pesticide abate has been separated
and analysed on 500 x 3 mm i.d. column of permaphase ETH coupled both to
a UV detector and auto analyser sensitive to cholinesterase inhibiting
compounds. In case of carbamates, the 2-isomer of carbaryl can be analysed
by the hydrolysis of the carbamates to I-and 2-naphthol and then analysing

the naphthols in presence of unhydrolysed carbamate on a corasil II column
with hexane/chloroform (80 : 20) eluent.
In fluorigenic labelling technique, carbamates were hydrolysed in
base to their corresponding amines which on reaction with dansyl chloride
give derivatives. The latter were monitored by HPLC using fluorescence
detector, Chromatograms were obtained on corasil I and Zipax/BOP columns.
ANALYSIS OF FUNGICIDES BY HPLC.
Kirkland analysed benomyl in cucumber extract (Fig. 4) using:
Cucumber control Fortified cucumber

CD
II)
§
c.
~
BenomyVMBC
I
c
0.1 cm 3 Benomyl/MBC
injected (0.05 ppm)
0.1 cm 3
injected
\
o 10 20 30 o 10 20 30
Time (min) Time (min)
Fig. 4. HPLC analysis of benomyl in cucumber extracts.
Column packing of Zipax SCX pellicular strong cation exchanger.
Column dimension. 1 m x 2·1 mm i.d.
Eluent. 0·025 N tetramethyl ammonium nitrate/0·025 N HN03 .
Pressure. 300 psi. Flow rate 0·5 cm3 min-I, column temperature 60°C.
UV detection is carried out at 254 nm.
ANALYSIS OF HERBICIDES BY HPLC.
Herbicides such as 2,4-dichlorophenoxy acetic acid (2,4-D), 2,4-dichloro-
phenoxy butyric acid (2,4-DB), 2,4,5-trichlorophenoxy acetic acid (2,4,5-T),
2-(2, 4, 5-trichlorophenoxy propionic acid) (silvex) and their corresponding
methyl esters are separated by using gradient elution system of mobile phase.
The eluate is monitored by a UV detector at 280 nm.
Experimental Technique.
One litre of sample is quantitatively transferred to a 2 L separatory
funnel and acidified to pH 2 with H 2S04 , Water sample is then partitioned
with 60 niL of acetone by shaking for 2 minutes. Water layer is collected in
the original water sample container and the organic phase is transferred in a
500 mL Kudema Danish flask.
The extraction is repeated twice with 50 mL each of dichloromethane
and hexane. Solvent extract volume is reduced to 0·5 mL under low pressure.
Few microlitres of this concentrate is injected by stop flow injector to HPLC
for the analysis of samples. Same procedure is adopted for the analysis of
methyl esters of 2,4-D, 2,4-DB, 2,4,5-T and silvex.
The linearity of the UV detector is determined at 280 nm. The plots of
peak height versus concentration for the chlorophenoxy acids and their esters
are found to be linear at low microgram levels (0·5 to 1·0 ~g/L) and from them
the concentration of unknown herbicide calculated.
Lowrence detected linuron herbicide at 200 ppb level by using
(i) HPLC with UV detection at 254 nm.
(ii) GLC with electrical conductivity detector and
(iii) GLC with electron capture detector.
ANALYSIS OF PESTICIDES BY POLAROGRAPHY.

Organochlorine pesticides such as aldrin, endrin and BHC can be
analysed on DME in LiCI in isopropanol. The supporting electrolytes are
0·1 M HNOa, acetate buffer, NHa-NH4 CI buffer and o· 1M NaOH. The half
wave potential (E 1J2 ) in these electrolytes varies from -0·32 V to 0·72 V. In
case of parathion, the E1I2 is -0·26 V, -0·62 V and +0·74 V in 0·1 M HNOa,
acetate buffer and NHa-NH4CI buffer respectively.
ANALYSIS OF PESTICIDES BY SPECTROMETRIC METHODS

In spectrometric methods, the standardised adsorbents are generally
required for clean up before analysis. IR and UV methods have been used for
the analysis of various isomers of BHC. NMR helps to identify metabolites
and degradation products of pesticides. Carbamates, chlorinated insecticides
and their metabolites can be analysed by mass spectrometry. PCBs, lindane
and 2, 4, 7, 8-T have successfully been analysed by GC-MS technique.
WATER POLLUTION LAWS
In India, specific laws have been passed by both Central and State
Governments to control water pollution. All these laws are based on
standards, a set of parameters used to define water quality.
1. Central Enactments. Acts passed by Government of India are:
(i) North India Canal and Drainage Act, 1873.
(ii) Indian Fisheries Act, 1897.
(iii) Damodar Valley Corporation Regulation Act, 1948.
(iv) The River Boards Act, 1956.
(v) The Water (Prevention and Control of Pollution) Act, 1974
(Amended in 1988).
(vi) The Water (Prevention and Control of Pollution) Cess Act, 1977
(Amended in 1991).
(vii) The Environment (Protection) Act, 1986.
(viii) The Merchant Shipping Act, 1987.
2. State Enactments. Acts passed by State Governments are :
(i) The Orissa River Pollution Prevention Act, 1953.
(ii) The Maharashtra Prevention of Water Pollution Act, 1969.
The Water (Prevention and Control of Pollution) Act, 1974
(Amended in 1988).
• The Act defined terms like pollution, sewage effluent, trade effluent,
stream and boards. The Act also assigns the functions to be carried
out by the Central and State Boards.
• The Water Boards have power to obtain information, to take samples
of effluents from any industry and to make survey of any area and
gauge and keep record of the volume and other characteristics of any
stream or well.
• A person empowered by the Board has the right to enter, inspect and
examine any plant, record register, document or any other material
object, or for conducting a search of any place where he has reason to
believe that no offence of water pollution is comm~tted.
• The Board has wide powers to prohibit the use of any stream or well
for discharging any pollutant in it. The Board has powers to
restructure the outlets for dumping pollutants.
• The Act prohibits disposal of any poisonous, noxious or polluting
matter to the flow of water in a stream. However, dumping of any
material into a stream for the purpose of reclamation of land is not
considered an offence. The Act provides for severe and deterrent
punishments for violation of the Act which includes fine and
imprisonment.
The Water (Prevention and Control of Pollution) Cess Act, 1977
(Amended in 1991).
• The Act empowers the Central Water Board to collect cess on water
consumed by persons carrying on certain scheduled industries and by
local Authorities responsible for supplying water.
• The cess and the consent fees form the major sources of revenue to
run the Central and State Water Boards.
• The Act has been amended in 1991 with a view to augment the
resources of the Boards by removing the lacunae in the Act and to
provide rebate to the industries for complying with the consumption
and effluent quality standard.
Technical Difficulties in Controlling Water Pollution.
There are, however, several enforcement problems. Although the Water
Cess Act was passed to meet the expenses of the Central and State Boards yet
the Water Board has no power to take direct action against the erring party.
The court procedures are time consuming and delays often prevent quick and
preventive action thereby defeating the sole purpose of the Act. Because of the
problems inherent in the implementation of the Act, amendments were
proposed for strengthening the working of the State Boards. Inspite of the
legislative measures, the pollution of our water ways continues unabated. This
is due to the lack of civic sense among people and due to the lack of necessary
infra structure for enforcing implementation of the laws efficiently.
WATER QUALITY STANDARDS

The standards are based on the water quality in the streams and specify
its suitability for different purposes such as drinking, irrigation, industry,
public health and environmental safety. Indian Standard Institution (lSI)
has the principal role in specifying the norms for various effluents so that the
ambient water quality standards are maintained. Types of standards are :
1. Drinking water standards. 2. Stream standards.
3. Irrigation standards. 4. Bacteriological standards.
5. Effluent standards.
1. Drinking Water Standards. National and international authorities
have laid down various standards for domestic use of drinking water. The
main agencies include lSI, Indian Council of Medical Research (ICMR),
Ministry of Works and Housing (MWH), World Health Organisation (WHO),
United States Public Health Standards (USPHS) etc. Parameters for water
quality characterisation and standards are illustrated in Table 2.
Table 2. Drinking water standards (Domestic water supplies).

Parameter USPHS lSI Parameter USPHS lSI
Colour Colourless - pH 7·0-8·5 6·0-9·0
Odour Odourless
Taste / Tasteless
Specific conductance 300 mmho - Turbidity 25JTU 25 JTU
em-I
Suspended solids 5·0 - Total dissolved solids 500 -

Chloride 250 600 Sulphate 250 1000
Cyanide 0·05 0·01 Nitrate + nitrite <10 -
Fluoride 1·5 3·0 Phosphate 0·1 -
Sulphide 0.1 mgL- 1 - Ammonia 0·5 -
(ppb)
Boron 1·0 - Calcium 100 -
Magnesium 30 - Arsenic 0·05 0·2
Barium 1·0 - Cadmium 0·01 -
Chromium VI 0·05 0·05 Copper. 1·0 -
Iron <0·3 - Lead <0·05 0·01
Manganese 0·05 - Mercury 0·001 -
Selenium 0·01 - Silver 0·05 -
Zinc 5·5 - COD 4·0 -
Carbon CHCl3 extract 0·15 - Phenols 0·001 0·005
Pesticides (total) 0·005 - PAH 0·2 ppb -
Contd. ...
Parameter USPHS lSI Parameter USPHS lSI

Surfactants 200 - Dissolved oxygen 4·0-6·0 -
ppm
Gross beta 1000 pc I L - Radium-226 3 pc/L <5000
radioactivity
Strontium 90 10 pc I L <5000 Coliform cellsll00 mL 100 -
Total bacteria Ix 106 - Detergents 0·1 -
count/100 mL
Note. All the units, except otherwise mentioned, are in ppm or mg/L.
2. Stream Standards. The permissible limits of drinking water
parameters are 3 to 5 times higher for surface waters. The water quality
criteria are classified into different categories by epeB and SpeB depending
on the use of water for irrigation, drinking, industry, power generation and
recreation etc.
Table 3. Classification of water bodies by CPCB.

Class Best use of water
A Drinking water source without conventional treatment but after
disinfection.
B Outdoor bathing.
C Drinking water source with conventional treatment followed by disinfection.
D Propagation of wild life, fisheries, irrigation.
I Sea water including estuaries and coastal waters.
II Shell fishing, contact sport.
III Commercial fishing, non-contact recreation.
IV Industrial cooling, navigation and controlled waste disposal.
There should be no visible discharge of domestic and industrial wastes
into class A waters. In case of class Band e, the discharges shall be regulated
so as to ensure the maintenance of the stream standards (Table 4).
Surface water should be free from
(i) materials which impart colour, taste, turbidity (e.g., oils, grease,
phenols) and
(ii) toxic metals, radionuclides, debris and scum etc.
3. Irrigation Standards. The major parameters of concern are :
(i) Salinity. Salinity or total dissolved solids is the most important
parameter for irrigation water since it controls the availability of water to
plants through osmotic pressure regulating mechanisms.
(ii) Water infiltration rate. High sodium and low calcium content of
water reduces the water infiltration rate so that sufficient water can not
reach the crop roots.
Table 4. Water quality criteria for fresh water

classification by cpce.
Class Water quality criteria
A DO (6 mg/L), BOD (2 mglL), MPN of coliforms per 100 mL (maximum 50),

pH (6·5-8·5)
B DO (5 mglL), BOD (3 mglL), MPN of coliforms per 100 mL (maximum 500),

pH (6·5-8·5)
C DO (4 mg/L), BOD (3 mglL), MPN of coliforms per 100 mL (maximum 5000),

pH (6·0-9·0)
D DO (4 mglL), pH (6·5-8·5), Free NHa as N (1·2 mglL)
E pH (6·0-8·5), conductivity (2,250 mS/em), sodium absorption ratio (26), boron

(2 mg/L)
(iii) Specific ion-toxicity. The presence of toxic metals such as Cu,

Cd, As, Pb, Hg, Mn, Ni etc. in water cause crop damage and reduce
agricultural yield.
The tolerance limits for trace metals (in ppm) in irrigation water are :
Al As Be B Cr Co Cu Pb Li Mn Ni Zn V
1·0 1·0 0·5 0·75 5·0 0·2 0·2 5·0 5·0 2·0 0·5 5·0 10·0
(iv) Sodium Concentration. Sodium concentration in water can be

denoted by
• Percent sodium = 2 ....2 Tff + X 100

Ca + + MI;; + +.n. + N a
• Sodium absorption ratio (SAR) = --J 2+Na+ g2+

Ca +M 12
• Residual sodimp. carbonate =(CO~- + HCOa)- (Ca2+ + Mg2+)
The values of individual constituents are taken in meq.lL.
Suitability of water for irrigation with different values of sodium
absorption ratio (SAR) is illustrated below :
• SAR, 0-10. Suitable for all types of soils and crops.
• SAR, 10-18. Suitable for coarse textured or organic soil with good
permeability. Unsuitable for fine soil.
• SAR, 18-26. Harmful for all types of soils, requires good drainage,
high leaching and gypsum additlon.
• SAR, >26. Unsuitable for irrigation purposes.
(v) Miscellaneous constituents. Excess of nitrate ions in water
causes eutrophication which is injurious for the plant growth. Suitability of
water with different constituents is shown below.
Class TDS SO~- cr B Na EC Suitability

of water ppm ppm ppm ppm % mS/cm for irrigation
I 0-700 0-192 0-142 0-0·5 0-60 0-750 Good for
irrigation
II 700-2000 192-480 142-355 0·5-2·0 60-75 750-2250 Sui table for
moderate
leaching
Class III water is totally unfit for irrigation.

4. Bacteriological Standards. These standards are set by WHO and
Ministry of Works and Housing.
(i) E. coliform bacteria count in any sample of 100 mL water should
be zero.
(ii) Total bacterial count not more than 10/100 mL should be present in
any sample.
5. Effluent Standards. Effluent standards are related to the quality
of waste waters originated from industry, community and agriculture.
Table 5. General standards for discharge of effluents set by

cpce in 1995.
Standards
Parameter Inland Marine
Public Land for
surface coastal
sewers irrigation
water area
pH 5·5 to 9·0 5·5 to 9·0 5·5 to 9·0 5·5 to 9·0
Suspended solids 100 600 200 100
Dissolved solids 2100 2100 2100 -
BOD (5 days at 20°C) 30 350 100 100
Oil and grease 10 20 10 20
Total residual chlorine 1·0 - - 1·0
Sulphate 1000 1000 1000 -
Total N 100 - - 100
NHa-N 50 50 - 50
Chloride 1000 1000 600 -
Fluoride 2·0 15 - 15
Percent Na - 60 60 -
Phenolic compounds 1·0 5·0 - 5·0
Alpha emitters IlC/mL 10-7 10 ·'1 10-8 10-7
10~ 10~ 7 10-6
Beta emitters IlC/mL 10
Note. Maximum limits of parameters are expressed in ppm or mglL.
o
8
HEAVY METAL POLLUTION
METAL TOXICITY.
Out of 112 elements discovered so far, over 65 are metals. Almost all
metals are toxic at high concentrations and some are extremely lethal even
at low levels. Toxic metals are added in aquatic system from industrial
effluents, domestic sewage discharges, agricultural run off, street dust and
fossil fuel burning. Traces of heavy metals such as Hg, Cd, Pb, As, Co, Cr, Mn,
Fe and Se have been identified as cumulative poisons causing deleterious
effects in living organisms.
The toxicity due to metals is mainly determined by
• Its solubility, stability, biological reactivity, physical form at the site
of action and the presence or absence of a homeostatic mechanism.
• The delivery of the metal to the cell to attain a critical concentration
at the active site.
• The cellular biochemical defence mechanisms.
The actual toxic action of each metal is different but most of them bind
to the metabolically active groups such as carboxyl, phosphoryl, amino, imino,
phenolic, sulphydryl or imadazole group.
Factors Responsible for Heavy Metal Toxicity.
• Solubility, stability and reactivity of heavy metal or its complexes.
• Oxidation state and electrochemical character of the metal.
• The rate of transport of heavy metal complex in blood, its distribution
and retention in the body tissues.
• The ability of the heavy metal to chelate with various ligands in the
body tissues and reactivity of the chelate thus formed.
• The efficiency of the enzymatic and homeostatic mechanism which
controls the absorption, distribution, retention and excretion of the
heavy metal ions or complexes.
Reasons for the Adverse Effects of Metal Toxicity in Biological
Systems.
• Interaction of the metal with protein leading to denaturation.
• Interaction with DNA causing mutation.
• Effect on cell membranes and regulatory enzymes.
The adverse effects in mammals may manifest in the following
disorders.
• Pathological changes.
• Retardation of growth.
• Symptoms of chronic disease.
(182)
HEAVY METAL POLLUTION 183
• Morbidity.
• Decrease in longevity.
• Detrimental changes in the reproductive cycle causing mortality in
the offspring.
PUBLIC HEALTH SIGNIFICANCE OF HEAVY METALS

CADMIUM.
Occurrence. Cadmium, a toxic heavy metal, occurs in nature in
association with zinc minerals (1 : 200 in ZnS). The maximum permissible
concentration of Cd in water is 5 ng g-l as per the WHO guidelines. Normal
Cd levels are less than 1% ~g in blood. Tolerable daily intake of Cd is 57 to
72 ~g per day. Normal Cd content in rice is about 29 ppb.
Industrial Uses and Pollution Sources. Cd is used in the
manufacture of alloys, paint-pigments, plastics, Ni-Cd batteries and in
electroplating. Cadmium stearates are used as stabilizers in the production of
PVC plastics. CdO is used in glass manufacture, in ceramic glazes and
ceramic alloys. Cadmium selenide is used in photoelectric cells, rectifiers and
in phosphors.
Over one million kg of Cd are released into the air every year from
smelters and factories processing Cd which accounts for 45% of total Cd
pollution. About 52% of Cd pollution occurs from the incineration or disposal
of Cd bearing products like automobile tyres, motor oils, coal, plastics etc.
Toxic Effects of Cadmium on Man.
• The outbreak of Cd-poisoning occurred in Toyama city of Japan in
the form of itai-itai or ouch-ouch disease. The bones of these victims
became fragile, brittle and shattered accompanied by shrieks of
tormenting pain.
• Cadmium acts as a potent inhibitor of several enzymes like adenosine
triphosphate, amylase, peptidase, carbonic anhydrase, glutamic
oxaloacetic transminase, alcohol dehydrogenase, cholinesterase,
tryptophan oxygenase, leucine amino peptidase and aldolase etc.
• Cadmium has a strong affinity for sulphur containing ligands such as
SCH3 and SH in methionine and cysteine amino acids. It has also got
affinity for other ligands like hydroxyl, carboxyl, phosphatyl and
cysteinyl side chains of proteins, purines and porphyrin. It can disrupt
path ways of oxidative phosphorylation.
• Cadmium causes strong inhibitory effect on metalloenzymes such as
alkaline phosphatase, carboxy peptidase, thermolysin (Zn-containing
enzymes) and ceruplasmin (Cu-containing enzyme). The specificity of
the enzyme is altered when Zn 2+ or Cu2+ ions are replaced by Cd2+.
• Cadmium toxicity causes anaemia, hypertension, adrenal dysfunction,
bone marrow disorders, cancer, damage to kidney, lungs and liver.
• Cadmium is highly toxic because of the absence of homeostatic control
for Cd in human body. It is bound by body proteins, metallothionein,
present in kidneys and gradually accumulates with age causing
metabolic disorders.
• Cadmium exposure in man results in aminoaciduria (urinary

excretion of amino acids), glucosuria (excretion of blood sugar in the
urine), hypercalciuria (urinary excretion of excessive Ca), osteoporosis
(decalcification of the skeleton), proteinuria (urinary excretion of
proteins).
CHROMIUM
Occurrence. Chromium is distributed in the earth crust at about 200
ppm levels and in sea water at 3 ppb. In nature, Cr occurs as chromium iron
ore (FeO . Cr203)' The Cr concentration in man's blood is found to be 0·5 to
5 JlgL-1, in urine 5 to 10 JlgL-1 and in hair over 500 ng g-l.
Uses and Pollution Sources. Chromium is an ingredient of stainless
steel. Cr203 is used in chrome plating, copper stripping, photography and as
a corrosion inhibitor. Chromium sulphate is used as a mordant in textile
manufacture, in leather tanning, paints, varnishes, inks and glazes for
porcelain. Wastes from industrial units contain soluble chromate salts which
is a source of water pollution.
Health Effects of Chromium.
• Chromium in its trivalent form is essential to man since it plays a
vital role in insulin metabolism as the glucose tolerance factor.
Supplementation of chromium helps to improve glucose tolerance in
diabetics, older people and malnourished. Most mammalian species
tolerate about 100 times their normal body burden of Cr(lll) without
experiencing any adverse effect.
• Chromium deficiency is characterised by impaired growth and by
disturbances in glucose, lipid and protein metabolism.
• Orally administered Cr(IlI) is poorly absorbed (1% only) than Cr(VI).
Absorbed anionic Cr(VI) readily passes through the membrane of red
blood cells and gets bound to globin fraction of haemoglobin. Cationic
Cr(IlI) is unable to pass through the membrane. It combines with
~-globulin fraction of the plasma protein and transported to the
tissues bound to siderophilin.
• The chromium entering the tissues is distributed among the
subcellular fractions and is mobilized from the body. stores to glucose
administration.
Toxic Effects of Cr(lll). Chronic exposure to chromate dust has been
correlated with increased incidence of lung cancer. Oral administration of
excessive levels (50 ppm) has been associated with liver, kidney damage and
depression.
Toxic Effects of Cr(VI).
• Cr (VI) is 100 times more toxic than Cr(III). Exposure to Cr(VI) causes
allergic skin irritations, dermatitis, conjuctiva, gastro intestinal ulcers
and irritation to mucous membranes.
• Cr(VI), a teratogen, is also mutagenic and carcinogenic inducing
bronchogenic cancer.
• Higher levels of Cr(VI) causes chrome holes that is, penetrating ulcers
occur around finger nails, joints, eyelids and on forearms. Lesions on
the nasal mucosa may lead to perforation of the septum. Alkali
biochromates can be absorbed through skin lesions to cause renal
damage.
COPPER
Occurrence. Copper, an essential chalcophile, is ubiquitous in earth
crust as sulphide deposits along with Pb, Cd and Zn. Soluble copper levels in
contaminated water ranges from 0·5 to 2 j.1gL-l .
Uses and Pollution Sources. Copper is also used in the manufacture
of alloys, paints, ceramics and pesticides. Contamination of air with copper
occurs near industrial smelters, fertilizer industry, iron and steel industry.
Water pollution by copper results from the discharge of mine tailings, flyash,
disposal of municipal and industrial wastes.
Health Effects of Copper.
• Copper is necessary for the normal biological activities of amine
oxidase and tyrosinase enzymes. The former enzyme is involved in the
formation of elastin and collagen. Elastin is the major protein
constituent of the walls of large blood vessels while collagen is the"
proteinaceous component of tendons and bones.
• The Cu-containing enzyme, tyrosinase, is required for the catalytic
conversion of tyrosine to melanin, the pigment located beneath the
skin protecting it from radiation injuries. Tissues lacking tyrosinase
cannot produce melanin and thus would extremely be sensitive to
sunlight and probably be prone to early death.
• Defects in bone formation, pigmentation, reproduction, myelination of
the spinal cord, cardiac function, connective tissue formation, defects
in growth and hematopoiesis were found to be the manifestations of
copper deficiency.
• Cytochrome oxidase, ascorbic acid oxidase, laccase, tyrosinase,
urinase, amine oxidase, aminolevulinic acid dehydrase, dopamine
hydroxylase are all copper enzymes.
A daily dietary intake of 2 to 3 mg of Cu is recommended for human
adults. Copper absorption and retention depends on factors such as the
chemical form in which the metal is ingested, dietary levels of other
minerals and the acidity of the intestinal contents in the absorptive area.
Toxic Effects of Copper.
• Inhalation of air borne copper causes irritation of the respiratory tract
and metal fume fever.
• Workers involved in the spraying of vineyards with Bordeaux mixture
(a fungicidal preparation containing about 2% of copper sulphate)
develop a respiratory disorder known as vineyard sprayer's lung
disease. This condition is characterised by the development of
interstitial pulmonary lesions and nodular fibro-hyaline scars
containing deposits of copper. During the course of this disease, lung

cancer may develop.
• The ingestion of 15-75 mg of copper causes gastro-intestinal
disturbances. The intake oflarge quantities of copper salts may result
in hemolysis, hepatotoxic and nephrotoxic effects. Generally, copper
toxicity could be aggravated by low dietary zinc or molybdenum.
• Copper deficiency results in Menke's disease whereas excessive
accumulation of Cu causes Wilson's disease, thalassemia
(Mediterranian anaemia), hemachromatosis, cirrhosis, atrophy of
liver, tuberculosis and carcinoma.
• Copper content more than 470 mg in man causes coma, uremia,
hypertension, sporadic fever and damage to brain tissues.
LEAD
Occurrence. Lead occurs in nature chiefly in lead minerals galena,
anglesite and cerussite. The major source of air-borne lead is the combustion
of leaded petrol/gasoline. Lead is added in the form of tetra alkyl lead
together with scavengers, 1, 2-dichloro (or dibromo) ethane. Busy streets
contain 1000 to 4000 mg kg- 1 of Pb.
Industrial Uses and Pollution Sources.
Lead is used in the manufacture of pipes, pottery, alloys, paints,
pigments, varnishes and pesticides etc. Main sources of Pb pollution are
mining, smelting of Pb ores, emission from automobile exhausts, effluents
from storage battery industries etc.
Toxic Effects of Lead.
• Lead intake by a city dweller is from food (200 Ilg per day), air and
water (10 Ilg per day each).
• Lead interferes with heme synthesis which leads to hematological
damage. Lead is implicated in the metabolism of delta-amino levulinic
acid (ALA) to porphobilinogen (PBG) and in the formation of heme
from iron and protoporphyrin (PP). Both of these steps are mediated
by free sulphydryl dependent enzymes, viz. ALA-dehydrase (ALA-D)
and heme synthetase.
An important phase of heme synthesis is the' conversion of ALA-D to
PBG.
Porphobilinogen
Lead inhibits ALA-D enzyme (I) so that it cannot form PBG(II). The
overall effect is the disruption of the synthesis of haemoglobin and other
respiratory pigments. such as cytochromes, which require heme. Finally, Pb
does not permit utilisation of 02 and glucose for life sustaining energy
production.
• Lead can even disturb the structure-function relationships of DNA
molecules. It can combine with specific biochemical residues, like
sulphydryl, carboxyl and imidazol groups.
• Lead inhibits acetylcholinesterase, ATP, cytochrome oxidase, carbonic
anhydrase and alkaline phosphate etc.
• Elevated lead levels « 0·8 ppm) in the blood cause anaemia, facial
pallor, gradual paralysis of muscles, atrophy, insomnia, tremor,
dizziness, arthralgia, arteriosclerosis, cardiorenal manifestation.
• Chronic lead poisoning results in vertigo, dyspepsia, reticulocystosis,
porphyrinuria, sterility, anorexia, vomiting and brain damage.
• Due to chemical analogy of Pb2+ with Ca2+, bones act as
repositories for Pb. This Pb may be remobilised along with phosphates
from the bones which exert toxic effects to soft tissues.
Toxic Effects of Tetraethyl Lead (TEL).
• TEL is an inert compound and does not exert its toxic effects as such.
But its toxic metabolite R3Pb+, biotransformed by oxidases enzyme
system in the liver, is 100 times more toxic than inorganic lead.
• Brain is the target organ in TEL poisoning. The organo lead
compounds, being lipophilic, are selectively localised in the nervous
tissues resulting in CNS toxicity.
• Prolonged TEL exposure is associated with stippled RBCs and WBCs,
lead line in gums, hallucination and encephalopathy.
Lead poisoning can be treated bl giving CaNa2 EDTA intravenously,
when lead is chelated (Pb2+ displaces Ca + from the chelate) and excreted via
urine. But a person suffering from renal disorder should not be treated with
this chelating agent. In such cases d-penicillamine may be used orally.
Sodium citrate or British antilewisite (BAL) i.e., 2, 3-dimercaptopropanol can-
also be prescribed.
ZINC
Occurrence. Zinc is an essential metal occurring as zinc blende (ZnS),
zincite (ZnO), willemite (Zn2Si04) and smithsonite (ZnC03 ). It occurs as
0·004% in earth crust and 20 ppb in oceans. Zinc content in milk lies between
3 to 5 IlgmL-1, in wheat germ and bran from 40 to 120 ppm. Oysters, the
richest source of zinc contain 1000 ppm of zinc. Human body contains 300 mg
zinc distributed in muscles (65%), bones (20%), plasma (6%), RBCs (2·8%) and
liver (53%).
Uses and Pollution Sources. Zinc is used in dry batteries, pigments,
protective coatings on iron, printing processes and in construction materials.
Smelting of ores and agricultural use of pesticides like zineb and ziram are
the pollution sources of zinc. Zinc salts being astringent and antiseptic have
limited use in medicine.
Health Effects of Zinc.

• Zinc is present in the body in metalloenzymes such as carbonic
anhydrase, alkaline phosphates and dehydrogenase or as co-factors in
other enzymes (e.g., arginase and histamine diaminase), taking part
in the synthesis of DNA, proteins and insulin.
• Several zinc enzymes act as catalysts for RNA and DNA metabolism
that is, for thymidine kinase and polymerase.
• About 80 enzymes including aldolase, alcohol dehydrogenase, carboxy
peptidase, lactic acid dehydrogenase are dependent on zinc.
• Zinc is essential for the normal functioning of cell including protein
synthesis, carbohydrate metabolism, cell division and growth. It is
also required for the synthesis of tryptophan which is a precursor to
auxin biosynthesis.
• Zinc has a wound healing capacity. Zinc is present in plasma, bones,
hair, nails, blood and also in eyes, liver, kidney, muscle, pancreas and
heart etc.
• Recommended daily dietary allowance of zinc is 15 mg for adults.
• Zinc deficiency syndrome manifests itselfby retardation of growth,
anorexia, lesions of skin and appendages, impaired development and
function"of reproductive organs.
Toxic Effects of Zinc.
• Toxicity of zinc may be due to other associated metals like Cd, Pb, Sb
and As.
• Inhalation of Zn dust and ZnO fumes causes metal fume fever,
depression, cough, .vomiting, headache and salivation.
• Gastrointestinal symptoms appear within 12 hours following
ingestion of 220 to 440 mg of ZnSO 4'
• Ingestion of ZnCl2 results in acute renal failure. But the emetic action
of zinc may be a protective mechanism.
• Electrolytic imbalance, dehydration, lethargy, dizziness, pneumonitis
and uncoordination may occur due to excessive zinc.
MANGANESE
Occurrence. Manganese is an essential element but it is toxic at
higher concentrations. Pyrolusite, braunite, magnatite, tephroite are the
minerals of Mn. Human beings get their daily quota of Mn from vegetables,
fruits, nuts and germinal portions of grains. Mn is found in sea water at one
ppb level.
Uses and Pollution Sources. Manganese is used in dry battery cells,
electrical coils, ceramics, alloys and in glass and steel manufacturing
industries.
Health Effects of Mn.
• Mn at low concentrations (2-5 to 5·0 mg) is essential for cell
development.
• It acts as a cofactor in a number of enzymatic reactions, particularly
those involved in phosphorylation, cholesterol and fatty acid
synthesis.
Toxic Effects of Mn.

• Higher levels (100 ppm) of Mn accumulate in liver, kidney and bones.
Manganese pneumonia is caused by excessive inhalation of Mn.
• Permanganate is most toxic among different Mn forms. Mn2+ is three
g
times more toxic than Mn +.
• Chronic exposure to Mn may cause manganese psychosis, an
irreversible brain disease similar to Parkinson's disease. It is
characterised by uncontrollable laughter, euphoria, sexual excitement,
blindness, impulsiveness followed by impotence and speech disability.
MERCURY
Occurrence. Mercury, also named as quick silver, is extracted from
cinnabar (HgS). Its natural abundance in soil is 0·1 ppm. Fossil fuels, coal and
lignite contain 100 ppb of Hg. The ambient air concentration of mercury
prevailing in some industrial areas is found to be 0·7Ilglmg. The USEPA
limits of Hg in drinking water is 2 IlgL-1.
Natural addition of Hg to the oceans is about 5000 tonnes per annum
and a further 5000 tonnes is added via human activities. Sewage effluents
may contain up to 10 times the level of Hg in natural water (0·001 ppm).
Uses and Pollution Sources. The largest consumer of Hg is
chloralkali industry which manufactures Cl2 and NaOH by using Hg
electrodes. Production of electrical apparatus (bulbs, switches, batteries) and
the use of Hg in agricultural industry (e.g., fungicides for seed dressings)
constitute the second and third consumer of Hg. Pharmaceuticals, ointments,
dental amalgams and finger print powders also disperse Hg in air.
Once mercury is absorbed on sediments of water bodies, it is slowly
released into the water and constitutes a reservoir which is likely to cause
chronic pollution long after the original source of Hg is removed.
Toxic Effects of Hg.
Minamata Incident. The extreme toxicity of Hg was appreciated in
1953 when 52 persons living around Minamata Bay in Japan died of a
Minamata disease. The victims had eaten shell fish contaminated with
methyl mercury (27-102 ppm) containing effluent from a nearby vinyl
chloride plastic, Shin Nihon Chis so Hiryo company. Several persons became
crippled and showed genetic and neurological disorders. The causative agent,
organomercurials was identified by Prof. Takeuchi.
Biological Methylation of Hg in Food Chain.
The Minamata chemical company discharged Hg into Bay but the fish
were found to contain CHgHg+. Actually Hg is readily scavenged and
converted to CHgHg+ by anaerobic methane synthesizing bacteria
(clostridium). This conversion is facilitated by Co(lll) containing vitamin B12
coenzyme. A CHg-group bonded to Co(ll!) on the enzyme is transferred
enzymatically by methyl cobalamin to Hg2+, yielding CHgHg+ or (CHg)2Hg.
CH g
" I~ Methyl cobalamin
/Co" + Hg2+ ATP ~
I
An acidic medium promotes the conversion of (CH3)2Hg to CH3Hg+

which is soluble in water. It is methyl mercury which enters the food chain
through plankton and is concentrated by fish by a factor of 104 as it passes
through the food chain.
Toxicity of Different Chemical Species of Mercury.
1. Hg. The vapours of elemental Hg are highly toxic when inhaled. Hg,
being lipid soluble, can be absorbed through the intact skin. It attacks mainly
liver, kidneys and can combine with carboxyl, sulphydryl, phosphoryl, amide
and amino groups.
2. Hg2+. Mercuric ions are toxic but can not easily transported across
biological membranes. Hg2+ acts as potent enzyme inhibitors, protein
precipitants and corrosive agents.
Hg2+ ions can attach to sulphydryl groups present in haemoglobin and
serum albumin. It can replace H atom from the sulphydryl group to form
mercaptides of the type X-Hg-SR and Hg(SR)2 where X is an
electronegative radical and R is a protein.
• Higher levels of Hg2+ ions cause headache, diarrhoea, hemolysis,
tremors and abdominal pain.
• Mercuric chloride is corrosive and when ingested precipitates proteins
of the mucous membrane causing ashen appearance of the mouth,
pharynx and gastric mucosa.
3. Hd+. Mercurous ions form insoluble Hg2C12. Since a fairly high
concentration of CI- is present in stomach so H~+ is not so toxic.
4. RHg+. Methyl mercury is the most toxic species. It is soluble in fat,
lipid and brain tissues. The covalent Hg-C bond is not easily disrupted and
the alkyl mercury is retained in cells for prolonged time.
• The most dangerous aspect is the ability of RHg+ to move through the
placental barrier and enter foetal tissues.
• Attachment of Hg to cell membranes inhibit active transport of sugars
across the membranes and allow the passage of K to the membrane.
In case of brain cells, this will result in energy deficiency and
disorders in the transmission of nerve impulses. It also causes
irreversible damage to the central nervous system including cerebral
palsy, mental retardation and convulsions.
• Methyl mercury poisoning at 0·5 ppm level leads to segregation of
chromosomes, insomnia and retardation in cell growth.
5. R2Hg. Diorganomercurials have low toxicity but can be converted to
RHg+ in acidic medium.
Treatment of Mercury Poisoning.
• Sodium formaldehyde sulphoxide provides excellent local antidote.
• Using metal chelating agents such as N-acetyl-D, L-penicillamine etc.
• Intramuscular injection of dimercaprol should be given to chelate the
mercury and to accelerate its excretion.
ARSENIC
Occurrence. Arsenic occurs in earth crust (2 ppm), sea water (5 ppb),
soils (1 to 40 ppm), in human body tissues (18 mg) and blood (25 mg).
Uses and Pollution Sources. Arsenic oxide, known as white arsenic
is formed as a by-product in smelting of Pb, Cu, Ag ores. As 20 3 is used in
insecticides, as a preservatives and mordant in textile industry. Paris green
(copper acetoarsenite) is used as a feed additive. Arsenites are used as weed
killers. Elemental arsenic is employed in the manufacture of infrared
transmittance glass, semiconductors and oil cloth.
Toxic Effects of Arsenic.
Arsenic is a general protoplasmic poison and it affects all the systems in
man. The order of toxicity of arsenic compound is : Arsines [As(II!)] > arsenite
[As(III)] > arsenate [As(V)] > arsenic organic acids fAs(v)].
1. Complexation of As with coenzymes. As(III) exerts its toxic
action by attacking 8H group of an enzyme thereby inhibiting enzyme action.
/8H 0"" /8'-.....

Enzyme, + /As-O ~ Enzyme"" /As-O+20Ir
'8H 0 8
The enzymes which generate cellular energy in the citric acid cycle are
adversely affected. The inhibitory action is based on inactivation of pyruvate
dehydrogenase by complexation with As(Il1) whereby generation of ATP is
prevented.
H8-CH2 S-CH2
/
0- I /1
"I
-
-O-As + CH2 O=As CH2
""-0- I
Arsenite HS-CH S-CH
1 1
(CH2)4 (CH2)4
1 I
C=O C=O
I 1
Protein Protein
Dihydrolipoic acid protein Inactivated protein complex with As(III)
By virtue of its chemical similarity to P, As interferes with some bio-

chemical processes involving P. This is observed in the biochemical generation
of the key energy yielding substance ATP (Adenosine triphosphate). An
important step in ATP generation is the enzymatic synthesis of
1, 3-diphosphoglycerate from glyceraldehyde 3-phosphate. Arsenite interferes
by producing 1-arseno-3-phosphoglycerate instead of 1, 3-diphosphoglycerate.
Phosphorylation is replaced by arsenolysis which consists of spontaneous
hydrolysis to 3-phosphoglycerate and arsenate.
CH20PO~
I Additional
Phosphate H-C-OH process Glyceraldehyde
I leading to
)
3-phosphate
C=O formation of ATP
I
OPO~- As033-
1, 3-Diphospho- Arsenite
glycerate
Preventing Non enzymatic

ATP (
formation spontaneous hydrolysis
1-Arseno-3
phosphoglycerate
2. Coagulation of Proteins. As(III) compounds coagulate proteins

possibly by attacking sulphur bonds maintaining the secondary and tertiary
structures of proteins. Soluble arsenicals are absorbed from mucous
membranes. Arsenic containing ointments or lipid soluble vesicants are
absorbed through skin.
• Dermatitis and conjunctivitis are frequently observed in workers
exposed to arsenic dusts. Continued inhalation of arsenic dust may
cause perforation of the nasal septum. '
• Slow arsenic poisoning causes nausea, salivation, vomiting, vascular
failure, diarrhoea, hyperkeratosis, loss of weight, skin lesions and loss
of hairs.
• Chronic ingestion of arsenic causes peripheral arteriosclerosis, known
as black foot disease.
• Arsenic may cause peripheral neuritis resulting in motor and sensory
paralysis of the nerve extremities.
• Arsenic is toxic to liver. It produces fatty infiltration and causes
central necrosis, cirrhosis, hypoxic convulsions, vertigo and coma.
• It affects bone marrow, elements of blood and can cross placental
membranes.
• The fatal cases of arsenic poisoning are mostly homicidal, a few are
suicidal and accidental.
Antidote for arsenic poisoning are chemicals having -SH groups
capable of bonding to As(II!), e.g., 2, 3-dimercaptopropanol (British
Antilewsite, BAL).
INSTRUMENTAL TECHNIQUES FOR THE ANALYSIS OF HEAVY

METALS IN WATER
General Survey of Instrumental Techniques.
Most instrumental techniques are common to water, air, soil and
biological samples. The sensitive instrumental techniques used for heavy
metal analysis along with their sensitivities are listed in Table 1.
Table 1. Some sensitive instrumental techniques.
Method Sensitivity (mole L -1)
Anodic stripping voltammetry (ASV) 10-9 _10-10
Atomic absorption spectrophotometry (AAS) 10-0 _10-7
Optical and emission spectroscopy 10 5 _ 10-0
Classical polarography 10-5 _10-0
Potentiometry with ion-selective electrodes 10--4 _10-0
Other techniques like inductively coupled plasma emission spectroscopy

(lCPAES), GC-MS, HPLC and ion chromatography are also used for heavy
metal analysis. The detection limit for some metals are given in Table 2.
Table 2. Comparative detection limits [(ppb) C(JlglL)]
for some heavy metals.
Flame Graphite Flame Graphite ICP
Metal ICPAES Metal
AAS AAS AAS AAS AES
As 100 0·06 15 Cd 1 0·008 1
Co 5 0·03 2 Cu 2 0·008 2
Cr 3 0·005 2 Fe 5 0·003 1
Mn 3 0·004 0·5 Ni 8 0·02 5
Pb 10 0·03 15 Ti 50 0·30 1
Zn 0·6 0·0007 1 Se 100 0·10 15
ANALYSIS OF HEAVY METALS USING ATOMIC ABSORPTION

SPECTROPHOTOMETRY
Principle.
Analysis of Cu, Fe, Zn, Mn metals using AAS was given by Lindsay and
Norvell. Diethylene triamine penta acetic acid (DTPA), a chelating ligand,
combines with free metal ions (Cu2+, Fe2+, Zn2+, Mn2+) in the aqueous system
to form soluble complexes. Stability constant for the simultaneous complexes
of Cu, Fe, Zn, Mn shows that DTPA is a most suitable extractant. The
concentration of these metal cations is then determined on AAS.
Apparatus.
1. Atomic absorption spectrophotometer.
2. Hollow cathode lamps of Cu, Fe, Zn, Mn.
3. Volumetric flasks and pipettes etc.
Reagents.
1. DTPA Solution (0·005 M). Take 1·967 g of DTPA in 1 L volumetric
flask and make up the volume to the mark with deionised water.
2. Stock Standard Solution. Dissolve 0·1 g of the metal foil in dilute
HCI (1 : 1) and make the volume to one litre with deionised water to obtain
100 ).lg/mL (i.e., mg/L or ppm) of solution of every metal ion. Alternatively,
dissolve separate
0·3928 g of CuS04·5H20 0·4964 g of FeS04·7H20
0·3076 g of MnS04·H20 0·4398 g ofZnS04·7H20
in 1 L of deionised water to prepare 100 ).lg / mL of stock solutions of Cu, Fe,
Mn and Zn respectively. Add 5 mL of H 2S04 to the solutions.
3. Working Standard Solutions. Transfer 0, 1, 2, 4, 6 and 8 mL of
stock solution (100).lg M2+/mL or 100 ppm of M2+, where M2+ = Cu2+, Fe2+,
Mn2+, Zn2+) to a series of 100 mL volumetric flasks. Add DTPA solution and
make up the volume to the mark. This will give standard solutions having
metal ion concentration of 0, 1, 2,4,6 and 8 ).lg/mL (or ppm).
Procedure.
1. For the determination of Cu2+, Fe2+, Zn2+ and Mn 2+ in aqueous
sample, set the atomic absorption spectrophotometer to zero using
blank solution.
2. Feed standards of the metals to be determined to AAS to standardise
the instrument to read absorbance or concentration in the sample
having the given metal within the standardised range.
3. Feed the DTPA extract and record the absorbance or concentration of
the metal.
4. Repeat the above steps for every metal.
5. In case the AAS shows a sign of over for some metal in a particular
sample indicating thereby that the sample has a concentration out of
the range for which the instrument has been standardised then make
further dilution of the sample 2-5 times. Feed the sample and record
absorbance or concentration.
Calculations.
Most of the modern AAS are calibrated to display the concentration of
a metal in ppm directly in the sample. In such cases, the concentration of a
given metal in the sample is calculated by multiplying the displayed reading
by the dilution factor, 2.
If the AAS displays the reading in absorbance, then a standard curve
has to be prepared for the known standards on a graph paper. Convert the
absorbance readings into concentration ().lg/mL) from the standard curve. The
amount of given metal is then calculated as follows :
Volumelweight of the sample taken= 10 mL
Volume ofDTPA extractant added = 20 mL
Dilution = 2 times
Absorbance shown by AAS = A
Concentration of the metal as read from the standard curve against
A = C ).lg/mL (or mg/kg = ppm).
Content of the metal cation in the sample = C x 2 mg/kg or ppm.
Precautions.
• Apparatus (glasslpolythene) to be used for analysis must be
thoroughly washed with acidified water and deionised water.
• Turbid solutions should not be used for feeding since they may block
the capillary of AAS.
ANALYSIS OF COPPER
(i) Cuprethol method. Cupric ions yield a yellow chelate when react
with cuprethol (bis-2-hydroxy ethyl) dithiocarbamates at a pH of 5 which is
maintained by HCI and CHaCOONa solution. The interference from Fe is
removed by adding pyrophosphate. The yellow colour is measured
spectrophotometrically.
(ii) Neocuproine method. Copper reacts with
~) {~
neocuproine (2, 9-dimethyl 1, 10-phenanthroline) in
weakly acidic solution (pH 3 to 8) to form a complex
which can be extracted as a yellow coloured solution
into a mixture of CHCla and CHaOH. The yellow HaC CHa
coloured solution is measured spectrophotometrically at Neocuproine
457 nm.
Procedure. To 100 mL sample in a 250 mL beaker add 1 mL conc.
H 2S04 and 5 mL conc. HNOa. Evaporate to white dense fumes of SOa. Add 5
mL conc. HNOa and heat to fumes until the solution becomes colourless. Cool
the solution, add 70-80 mL of distilled water and again boil. Cool and filter
the solution in a 100 mL ~olumetric flask and make the volume to 100 mL by
distilled water.
Transfer 50 mL of this solution into a 150 mL separatory funnel and
dilute it with 50 mL of distilled water. Add 5 mL of 1% hydroxyl amine
hydrochloride, 10 mL of 40% sodium citrate and 10 mL of neocuproine reagent
(100 mg/100 mL alcohol, CHaOH), maintaining the pH between 4 to 6. Extract
the solution with CHCla and repeat the extraction of aqueous layer with 10
mL chloroform. Transfer the CHCla extract in a volumetric flask containing
CHCla extract. Dilute the combined extract to 25 mL with methanol. Measure
the absorbance of the coloured solution at 457 nm against a reagent blank.
2. By Salicylaldoxime. Dissolve 1 g salicylaldoxime in 5 mL of alcohol
and dilute to 100 mL with water. The neutral solution OH
of the sample reacts with a drop of 10% acetic acid and r(3Y
a drop of salicylaldoxime, yielding green precipitate. ~ CH - NOH
The sensitivity is 0·0005 mg of Cu in 10 ppm -
concentration. Salicylaldoxime
3. Atomic Absorption Method. Aspirate Cu2+ solution into an
air-acetylene flame of an atomic absorption spectrophotometer and measure
the absorbance at 325 nm.
Differential Pulse Polarography (DPP) for the Determination of Copper
and Zinc in Tap Water.
Most tap water mains contain sufficient Cu and Zn for them to be
determined directly using DPP. Since the sample contains very less
interfering species, the experiment may be conducted without any sample

preparation other than the addition of a supporting electrolyte to reduce the
resistivity of the solution. The experiment may be conducted by comparing
the response of the sample against a calibration curve prepared from
standard solutions of the two metals or it may use the method of standard
additions made directly to the sample.
Typical conditions. Drop time 1 sec., starting potential + 0·1 V (Vs
SCE), scan range 1·4 V negative, scan rate 2 mVs- 1, pulse modulation
amplitude 50 m V, current range 2 !lA full scale deflection.
Method. Prepare solutions which contain both Zn2+ and Cu2+ in the
range 0·5 to 20 Ilg g-l in a supporting electrolyte of 0·01 M KCl. Other
supporting electrolyte such as an ethanoate or citrate buffer at 0·05 to 0·2 M
can be used but the material must be low in heavy metals. Place a sample of
the lowest concentration in the polarographic cell and degas for the
recommended time, usually 2 minutes. Wait for one minute so that the
solution becomes stationary and record the DPP trace. The copper peak may
well be on the side of a large peak at positive potentials but should be fairly
well resolved, whereas the zinc peak should be completely symmetrical. If the
peak heights are satisfactory, record traces for each of the other standard
solution in the same way.
Construct as calibration curve of peak height against concentration
for the two metals. (Fig. n If the peaks are too large or small, adjust the
sensitivity of the apparatus so that all the traces can be conveniently
observed. Obtain traces for the tap water sample under the same conditions
and with the addition of sufficient 0·1 M KCI solution to the sample to give
the same final concentration of supporting electrolyte as the standard
solutions. Measure the peak heights for both copper and zinc in this solution.
By comparison against the standard curve, estimate the concentration of
copper and zinc in tap water.
DPPTRACE
calibration curve Z112+
iii
c:
Cl
u;
1
O.21lA
f
..0.10 0 -0.20 -040 -0.60 -O.BO -1 00 -1.20
Potential (V vs SCE) Concentration

(a) (b)
Fig. 1. (a) Copper and zinc in tap water.

(b) Calibration graph of peak height against concentration.
Square Wave Polarography for the Determination of Copper and

Zinc in Tap Water.
Typical experimental conditions. Drop time is for single hanging
drop. Scan increment 2 m V. Square wave frequency 100 Hz. Starting potential
+ 0·1 V (Vs SCE). Scan rate 200 mV s-1. Scan range 1·4 V negative. Pulse
modulation amplitude 50 m V. Current range 2 !lA full scale deflection.
This experiment will take 7s per scan compared with 700s or nearly 12
minutes for the standard DPP scan.
ANALYSIS OF LEAD IN WATER

1. Atomic Absorption Method.
Lead can be measured by directly aspirating the sample into an air
acetylene flame and measuring the absorbance of Pb at 283·3 nm. For lower
concentration of lead follow the procedure of complex formation with
ammonium pyrrolidine dithiocarbamate, extraction into methyl isobutyl
ketone and then aspiration of the methyl isobutyl ketone complex into the
flame (air-acetylene) of an atomic absorption spectrophotometer.
2. Polarographic Method.
The half wave potential (El/2) values are - 0·37 to - 0·40 V, - 0·43 to
- 0·47 V and - 1·13 to - 1·17 volts in 0·18 M (NH4)2S04, 0·4 M NH 40H + 0·18
M (NH4)2S04 and 0·44 M NH 40H + 0·18 M (NH4hS04) + EDTA respectively.
Procedure. Take 20-50 mL sample in a flask and add 10 mL of
KMn04 solution. Add 3 mL of conc. H 2S04 and reflux the mixture for at least
3 hours. Now cool and add 40% hydroxylamine hydrochloride (NH20H·HCI)
solution in order to reduce KMn04. Filter and dilute the filtrate to about 100
mL by adding distilled water.
Now take 20 mL aliquot portion of the solution in a separatory funnel
and add 10 mL of 40% sodium citrate solution and then add NH 40H solution
to adjust the pH to 8·5. Extract it with 0·01% dithizone in CCl4 solution. Back
extract with 1% HNOg and transfer to a 25 mL volumetric flask. Add
2·0 mL of 1 MKCI solution and HNO g to adjust pH to 6. Make up the volume
to the mark. Measure lead polarographically at El/2 = - 0·4 volts.
3. Spectrophotometric Dithizone Method.
Lead ions react with dithizone in chloroform to form a complex which is
soluble in chloroform and imparts cherry red colour. The red extract IS
measured at an absorbance of 515 nm against a reagent blank.
H 5 CS-NH-N-Pb-N-NH-C6H 5
............ NH-NH-C6H 5 I tt I
Pb 2+ + 2SC - C=SS=C + 2H+
"'-N=NH-C6H 5 I I
H 5 C6 -N=N N=N-C 6H 5
4. Differential Pulse Stripping Method.
Reagents. For the analysis of lead in tap water, prepare standard
0·01 M lead solution by dissolving 1·65 g lead nitrate in redistilled water in
500 mL flask.
• Prepare a supporting electrolyte solution (0·02 M KN03) by

dissolving 10 g Aristar or similar pure material in 500 mL of
redistilled water.
• Confirm that this solution does not contain lead by carrying out the
preconcentration and stripping procedure (given below) using 10 mL
of the solution and 10 mL of redistilled water. If a significant lead
peak is obtained, the solution must be discarded.
Procedure.
Place 10 mL of the tap water and 10 mL of supporting electrolyte in the
electrolysis vessel. Mount the vessel on a magnetic stirrer. Insert an SCE, a
platinum plate auxiliary electrode, a nitrogen circulating tube and the
capillary of the mercury electrode. Operate the dispensing mechanism to form
a mercury drop at the end of the capillary. Pass oxygen free N2 through the
solution for 5 minutes and adjust the nitrogen to pass over the surface of the
liquid. Make the connections to the polarographic analyser. Adjust the applied
voltage to - 0·8 V, i.e., a value well in excess of the deposition potential oflead
ions. Set the stirrer in motion.
After 15s simultaneously switch on the electrolysis current and start a
stop clock. Allow electrolysing for 5 min. Turn off the stirrer but leave the
electrolysis potential applied to the cell. After 30s allow the liquid to become
quiescent. Replace the electrolysis current by the pulsed stripping potential
and set the chart recorder in motion. When the lead peak at 0·5V has been
passed, turn off the stripping current and the recorder. A suitable scan rate
for stripping is 5 m V s-1. Clean the electrolysis cell and change it with 10 mL
of supporting electrolyte, 10 mL of redistilled water and 1 mL of the standard
lead solution.
Set up a new mercury drop, pass N2 for 5 minutes. Record a new
stripping voltammogram by repeating the above procedure. Repeat with three
further solutions containing 2, 3 and 4 mL of the standard lead solution.
Calculation.
Measure the peak heights of the five voltammograms and deduce the
lead content of the tap water. In some commercially available equipment, the
concentration calculation can be performed automatically using built-in
nomograms.
o
9
SOIL ANALYSIS
INTRODUCTION
Soil is a dynamic natural body developed as a result of pedogenic
processes during weathering of rocks. The soil is in fact the very heart of the
life layer called biosphere. It consists of mineral and organic constituents,
exhibits definite physical, chemical and biological properties, has variable
depth over the surface of earth and provides a suitable medium for plant
growth. Soil mainly consists of 50% pore space (air and water) and 50% solid
phase. The solid phase is broadly composed of 45% mineral matter and 5%
organic constituents. The liquid phase of the soil (40 to 50%) is an aqueous
solution of salts. The soil acts as a reservoir for supplying water to plants.
The gaseous phase of soil consists of same nitrogen and oxygen contents as
of air but carbon dioxide concentration is much higher (0·1%). All these phases
of the soil system have a definite role to play. The solid phase provides
mechanical support and nutrients to the plants. The liquid phase supply
dissolved nutrients to plant roots. The gaseous phase satisfies the aeration
needs of plants. These three phases share the soil's responsibility to sustain
the plant growth.
COMPONENTS OF SOIL
1. Inorganic Components (Mineral matter) of the Soil (45%).
The soil is essentially a silicate mineral. The composition of common
elements in the soil is ; ° 46·6%, Si 27·7%, Fe 5%, Al 8·1%, Ca 3·6%, Na
2·8%, K 2·6%, Mg 2%. Finely divided quartz, Si02, commonly occurs in soil.
Among the silicates, orthoclase KAlSigOS, albite NaAISigOS and epidote
4CaO·3(AlFe)20g. 6Si02·H20 are common components of soil minerals. In
some soils, iron oxides FeO(OH), magnetite Feg04, Mn20g, TiO and CaCO g
are relatively abundant. The clay minerals in soil are secondary minerals,
essentially hydrated aluminium and iron silicates, which serve to bind cations
such as Na+, K+, Ca2+, Mg2+ and NHt. These elements are not leached by
water and are available as plant nutrients. Rocks which form the earth crust
(Petrology i.e., science of rocks) are made up of minerals.
2. Organic Components of the Soil (5%).
The organic matter content of soil varies from 3·5% by weight in a top
soil. In addition to the partly decayed plant and animal residues, soil organic
matter contains microbially synthesized compounds. Organic matter
functions as a granulator mineral particles. It is responsible for the loose,
easily managed conditions of productive soils.
(199)
Importance of organic matter in soil. , ,.,; ,

• The organic matter provides food for micro-organisms.
• It takes part in chemical reactions such as ion-exchange, governs
physical properties of soil and increases water holding capacity of soil.
• It is the major source of nutrients (N, P, K) and energy for microbes.
• It improves aeration, contributes to the weathering of mineral matter
and reduces soil erosion.
Major classes of organic compounds in soil.
(i) Humus (Heart and soul of soil). Humus is formed by microbial
degradation of plants and animals remain. Humic materials constitute the
most important classes of complexing agents. They show high-uptake of heavy
polyvalent cations. The process of humus formation is called humification.
Humic substances have an elementary composition: C 45 to 55%, 0 30
to 45%, H 3 to 6%, N 1 to 5%, S 1%. Humin, humic acid and fulvic acid
comprise a wide range of compounds. In general, humic substances are high
molecular weight polyelectrolytic macro molecules. They consist of a carbon
skeleton with a high degree of aromatic character and a large percentage of
functional groups containing oxygen. They also contain carbohydrate fraction
and a protein like materials. These fractions can be easily hydrolysed from
the aromatic nucleus.
Some typical decomposition products of humic acid are catechol,
syringaldehyde and 3, 5-dihydroxybenzoic acid. Humic substances are usually
classified on the basis of solubility. If a lunatic substance is extracted with a
strong base and the resulting solution acidified, the products are :
(a) non-extractable plant residue called humin
(b) a material which precipitates from the acidified extract, known as
humic acid and
(c) a soluble material called fulvic acid.
The insoluble humin and humic acid affect the water quality through
exchanges of cations, organic materials with water and can accumulate
enormous quantities of metals. On the basis of stages of decomposition,
humus formed from litter may be classified as
• Raw humus. It consists of partially decomposed organic matter.
• Mor. The layer of humus is not mixed with mineral soil.
• Mull. A completely decomposed humus is intimately mixed with
mineral soil.
The dark brown humus is very beneficial for the soil and exhibits
following properties.
• Humus constitutes the most abundant organic matter. It improves
physical properties of the soil, increasing its water holding capacity
and the rate of circulation of air and water through it.
• Humus is a reservoir of fixed N and constitutes the much needed food
for soil microbes.
• It reduces pore size and makes the clumps of soil.
• Growth promoting humic acid is present in the humus.
• It makes aeration better, enhances percolation and easy penetration of
roots.
SOIL ANALYSIS 201
(ii) Saccharides in organic matter. Saccharides include cellulose,

starches, gums, hemicellulose and sugar etc. Sugars are water soluble
compounds and constitute available source of C, N and energy for the
micro-organisms. When the nutrient or oxygen supplies are restricted,
different oxidised compounds (such as CO2, H 20, HCOOH, CH3COOH,
hydroxy acids and alcohols) are formed by starch. Soil micro-organisms
through ammonification convert amino acids, amides etc., into ammonia.
Nitrification (conversion of NH3 to N02 and then to N03) and
denitrification (N03 to N2) is also accomplished in organic matter of soil.
The micro-organisms break. up cellulose into cellubiose and glucose.
The latter gets converted into organic acids and then to CO2 and H 20. The
freshly synthesized hemicelluloses also form a part of humus. Saccharides
are significant as they constitute the major food source for micro-organisms
and help in stabilising soil aggregates.
(iii) Fats, resins and waxes in organic matter are lipid extractable.
Fats degrade by the enzyme lipase into glycerol and fatty acids and then to
CO2 and water. Resins, waxes and fats are phyto-toxic and adversely affect
soil properties by repelling water.
(iv) Nitrogen containing organics in soil include nitrogen bound to
humus, amino acids and amino sugars. These compounds provide N for soil
fertility.
(v) Phosphorus compounds in organic matter occur as phosphate
esters, phospho-lipids and are sources of plant phosphate.
(vi) Some organic compounds contribute to the weathering of mineral
matter. Calcium oxalate, produced as a soil fungi metabolite, dissolves
minerals accelerating weather process and involving complexation of Fe and
AI in minerals.
M(OH)3(s) + 2CaC20 4 (s) + 3H+ ~ M(C204)~(aq) + 2Ca2+(aq) + 3H20
Where M =AI or Fe.
Some soil fungi yield citric acid and other chelating organic acids which
react with silicate minerals and liberate K+ and other nutrient metal ions
held by these minerals.
(vii) The biologically active components of organic matter consist of
polysaccharides, nucleotides, amino sugars, organic Sand P compounds.
3. Soil Water (25%).
Soil water does not only act as solvent and transporting agent but also
maintains soil texture, arrangement and compactness of soil particles. It
makes the soil a perfect habitat for plants and animals. It is the major
constituent of plant protoplasm. Soil water is essential for photosynthesis and
conversion of starches to sugars. It is a main regulator of physical, chemical
and biological activities in the soil.
4. Soil Air (25%).
Soil air occupies the pore space between soil particles which is not water
filled. The exchange of CO2 and 02 between the soil pore space and the air is
called soil aeration. The crumb and intercrumb pores are involved in soil
aeration. A loam soil with humus containing 34% of air and 66% of water is
considered best for majority of crops. Most of the gaseous interchange in soil
occurs by diffusion.
MICRO AND MACRO PLANT NUTRIENTS

Micronutrients.
The essential micronutrients for plants are B, Na, Cu, Fe, Mn, Zn, V,
Mo. These elements are required at trace levels and, if present at higher
amounts exert a toxic effect. Most of these elements act as components of
essential enzymes. Elements such as Mn, Fe, Zn, V and CI are likely to take
part in photosynthesis. Several other elements like Cr, Ni, Fe, Mn have been
found to stimulate plant growth and regarded as potential micronutrients.
Out of 35 elements, 16 elements promote metabolic activities in plants.
Soil is a reservoir of primary nutrients (N, P, K), secondary nutrients
(Ca, Mg, S) and micronutrients (Fe, Mn, Zn, Cu, Mo etc).
Functions of the Micronutrients.
(i) Micronutrients help in the synthesis of chlorophyll and vitamin A.
(ii) They act as catalysts in oxidation reduction reactions within the
plants.
(iii) They help in protein synthesis in chloroplast.
(iv) Micronutrients enhance symbiotic N-fixation and maintain K and
Ca ratio of the plants.
(v) These are required for proper utilisation of N, P and K from the soil.
Deficiency of Micronutrients.
(i) Growth of plant is hampered.
(ii) Rate of photosynthesis get reduced thereby decreasing crop yield.
(iii) It may cause shedding of flowers, improper utilisation, poor seed
setting and attack of diseases.
Macronutrients.
The essential macronutrients are C, H, 0, N, P, S, Ca, Mg, K. The
atmosphere and water are the sources of C, H, O. Nitrogen may be obtained
by some plants, directly from the atmosphere, through N-fixing bacteria. N,
P, K are commonly added to soil as fertilizers. Mg is made available to plants
through ion-exchange of organic matter or clays which hold magnesium
strongly.
Nutrient Functions.
Nitrogen. The nitrogen content in soil is mostly of organic nature
(90%) resulting from the decay of dead plants (biomass) and animals, plant
residues and excreta of animals etc. Nitrogen fixing bacteria, rhizobium
convert atmospheric N to organic form that can be used by host plant.
When nitrogen is applied to soils as NH! (fertilizer), nitrifying bacteria
convert it in to NOg for use by plants. Certain leguminous plants, e.g., alfalfa,
clover, soyabeans, ground nut, pulses and guar etc., exhibit the unique ability
to fix atmospheric N through nitrogen fixing bacteria on their nodules.
Legumes can add considerable quantities of N to soil.
Functions of Nitrogen.
(i) Nitrogen is not only an essential constituent of protein and
chlorophyll but it is also involved in photosynthesis, respiration and
protein synthesis.
SOIL ANALYSIS 203
(ii) Nitrogen bound to humus is responsible for maintaining soil

fertility.
(iii) N increases the tillering of cereal crops and plumpness of grains in
cereals
(iv) N promotes vegetative growth and improves the quality of food
crops.
Excessive supply of nitrogen may cause several adverse effects.
(i) It delays ripening by encouraging vegetative growth.
(ii) Excess N deteriorates the quality of some crops such as barley,
sugarcane, potato.
(iii) The leaves acquire dark green colour, become thick, leathery and
wrinkled.
(iv) Straw and grain ratio is increased and the straw become weak.
Deficiency of Nitrogen.
CD Plants become yellowish and leaves drop prematurely.
(ii) Fruits ripe earlier and crop gives poor yield. The seeds and kernals
of cereals become shrivelled and contain less protein.
(iii) Root growth is severely affected.
Phosphorus. Phosphorus is an essential constituent of several
enzymes, structural component of membrane system of cells, the chloroplast
and mitochondria, nucleic acids, phytins and phospholipids. Phosphorus
stimulates root and plant growth and increases maturity. At the soil pH,
H 2P04 and HPO~- are the phosphate species utilised by plants. In acidic
soils, the orthophosphate ions are either precipitated or sorbed by Al3+, Fe3+
etc. In alkaline soils, the following reaction occurs, with CaC03 whereby
hydroxypatite is precipitated.
6HPO~- + lOCaC03 + 4H20 ~ CalQ(P04>S<0H)2J.. + lOHCO + 2011 a
Thus, very little phosphorus, added as fertiliser, leaches from the soil.
This is in favour of the use of phosphorus fertilisers.
Functions of Phosphorus. It increases the number of tiller in cereal
crops and ratio of grain to straw. It influences cell division and stimulates
flowering, fruit setting, seed formation in crops. It induces nodule formation
in leguminous plants. Phosphorus develops resistance to certain diseases in
plants.
Excessive phosphorus, however, has some harmful effects such as
profuse root growth in case of lateral and fibrous rootlets. It may cause iron
and zinc deficiency in some cases.
Deficiency of Phosphorus.
(i) Root and shoot growth is restricted and plants become spindly.
(ii) Shedding of leaves, delaying in flowering and fruiting.
(iii) Stunted growth even under abundant supply of Nand K
(iv) Potato tubers show rusty brown lesions.
Potassium. Potassium is required at high levels by growing plants. It
activates some enzyme and plays a key role in the water balance in plants
and for some carbohydrate transformation. Though potassium is abundant in
the earth crust, most of it is not available to plants. Only clay minerals in soil
containing excessive potassium make it available to plants. Crop fertilisers

contain 6% N, 12% P (as P 20 5) and 8% K (as ~O).
Functions of Potassium.
(i) It is an essential constituent of chlorophyll and photosynthesis.
(ii) It is necessary for tuber development.
(iii) It strengthens the cereal plants and reduces lodging in them.
(iv) Potassium exerts a balancing effect on both Nand P.
(v) It increases formation of carbohydrates and promotes metabolism in
plants.
Deficiency of Potassium.
(i) Deficiency of potassium may cause chlorosis and decreases the rate
of photosynthesis.
(ii) Its deficiency is responsible for drying back tips of shoots and
reduced growth of shoots.
(iii) Potato plants show abnormal dark green or brown colour of foliage.
Calcium. Calcium acts as a plant nutrient and as a soil amendment to
correct soil acidity. It occurs as calcium pectate in the cell walls of leaves and
in the ionic form in cell sap. Application of calcium to the soils in the form of
CaCOa corrects the soil acidity. It is a structural component of chromosomes.
Functions of Calcium.
(i) Calcium is involved in root elongation and cell division.
(ii) It is an essential cofactor of various enzymes including lipase and
amylase.
(iii) It acts as·a
\
buffer in plant systems and ameliorates the toxic effects
of other nutrients.
(iv) It enhances nodule formation in leguminous plants.
(v) It is helpful in translocation of sugars in plants.
(vi) It induces stiffness of straw and prevents undesirable lodging of
plants.
(vii) It improves intake of plant nutrients such as Mn, Cu, Zn, Fe and B
by correcting the pH of the soil.
Excessive amounts of calcium, however, decreases the availability of
many micronutrients.
Deficiency of calcium. Calcium deficiency in soil is due to calcium
uptake by plants, leaching by H2 COa in acid soils and competitions with high
levels of Na, K, Mg in alkaline soils. Its deficiency causes several adverse
effects such as :
(i) Normal growth of plant is arrested.
(ii) Roots become short, stubby and brown.
(iii) Acidity of cell saps increases abnormally and it hampers the
physiological functions of the plants.
(iv) Leaves become wrinkled and buds die.
Magnesium. Magnesium acts as a carrier of phosphate and plays a
significant role in the formation of phospholipids and nucleoproteins. It is a
mineral constituent of chlorophyll (2·7%). Several photosynthetic enzymes
present in chlorophyll require Mg as an activator. Magnesium is required by
plants in small quantities. Mg deficiency in soil is caused by high levels of Ca,
SOIL ANALYSIS 205
K, Na. Dolomitic limestone (MgCOg) is used to supplement the deficiency of

magnesium.
Functions of magnesium.
(i) Mg is required for the formation of chlorophyll which is
indispensable for the photosynthesis in plants.
(ii) It plays a key role in the synthesis of carbohydrate, proteins, fats
and oils.
(iii) Its deficiency may cause premature defoliation.
Boron. Boron is required by plants in trace amounts. Its function is
obscure. However, it is required in protein synthesis.
Main functions of boron include : Boron plays a key role in the
development and differentiation of tissues, carbohydrate metabolism and
translocation of sugar in plants. It makes up the calcium deficiency to certain
extent.
Deficiency of boron causes several adverse effects.
(i) Plant growth is retarded and the leaves turn yellow.
(ii) Its deficiency is generally associated with sterility and
malformation of reproductive organs.
(iii) Tendency of water absorption and translocation of sugar in plants
decreases.
Iron. Iron is needed for energy transfer, plant enzyme functions and
photosynthesis. It is used by plants in some of its respiratory enzyme
systems, especially catalyse cytochrome and peroxidase. Iron deficiency
retards the growth of plants, causes chlorosis, leaves become papery and turn
brown and necrotic.
Functions of Iron.
(i) Iron is essential for the synthesis of proteins in chloroplast.
(ii) It acts as a catalyst in the activities of several enzymes.
(iii) It regulates photosynthesis and respiration and reduction of nitrates
and sulphates.
Zinc. Zinc is involved in the enzyme systems, particularly carbonic
anhydrase and carboxylase. It is associated with Mn and Fe for the synthesis
of chlorophyll.
Functions of Zinc. Zinc is needed for RNA synthesis, nitrogen
metabolism and formation of seed. It is involved in the biosynthesis of plant
growth hormone.
Deficiency of Zinc.
(i) Zinc deficiency causes intervenial chlorosis of foliage with size
reduction in young leaves.
(ii) New leaves of maize plant emerge as white bud.
(iii) Shortening of internodes, premature falling of leaves occur.
Manganese. Mn is an essential factor in photosynthesis, nitrogen
metabolism and respiration. It influences the uptake and utilisation of other
nutrients in the plants. Deficiency of Mn occurs in alkaline, highly acidic and
organic soil. Mn deficiency leads to chlorosis and results in grey speck of oats.
Functions of Mn. It helps in the protein synthesis in chloroplast and
supports movement of iron in plants. It acts as a catalyst in the
oxidation-reduction reactions within the plant tissues.
Copper. Copper is associated with plant enzyme systems such as

polyphenol oxidase and ascorbic acid oxidase. It acts as electron carrier in
enzyme systems.
Copper is essential for the synthesis of vitamin A and chlorophyll. It acts
as a catalyst in respiration.
Copper deficiency is evident as chlorosis, weathering and distortion of
leaves. Food synthesis is hampered. Gum pockets under the bark and dead
patches of shoots in citrus are some symptoms of copper deficiency. Deficiency
is induced by heavy liming and excessive applications of nitrogen and
phosphates.
Sulphur. Sulphur exists in methionine (21·5%) and cystine (26·5%)
which are the components of proteins. It is an essential nutrient for cereals,
sugar and pulses. Sulphur promotes nodule formation, root growth and seed
formation. Pungent odour of onions and garlic is due to sulphur. It may be
called as master nutrient for oil seed formation.
Deficiency of Sulphur. The stem and leaf petioles become brittle and
may collapse. Plants produce less protein and oil.
ANALYSIS OF SOIL
Soil analysis is essential for getting the maximum yield as it provides
the knowledge of soil components, nutrients and their deficiency in a
particular part of soil.
SOIL MOISTURE MEASUREMENT.
Soil moisture can be measured in a variety of ways.
1. Gravimetric Method. In this method soil is taken in a container,
weighed in the moist condition, oven dried and weighed again after cooling.
The drying in the oven is carried out at 110°C to constant weight. Drying
takes about 2 hours for small samples, but as much as 24 hours for bulky
clayey soil samples.
The mass water percentage is calculated on the basis of dry soil weight.
Weight of moist soil = W 1 g.
Weight of oven dried soil =W 2 g.
Weight or mass of moisture =(Wl - W2 ) g.
(Wl - W2 ) x 100
Percent moisture = W
2
2. Electrical Conductivity Method. This method is based on the
change in electrical conductivity with the variation in soil moisture. In this
method, a gypsum block, which is provided with two electrodes at a definite
distance, is used. The gypsum block requires calibration for uniformity before
use. The blocks are buried in the soil at desired depth and the conductivity is
measured with modified wheatstone bridge.
The electrical conductivity between two electrodes set at a fixed distance
apart inside a small block is an indirect measure of soil moisture from the
field capacity to the wilting point. The blocks may be made of nylon, fibre
SOIL ANALYSIS 207
glass or porous material. It can be buried in the soil at any desired depth with
wires from each block extending to the surface of the soil for easy reading of
conductivity with the modified Bouyoucos wheatstone bridge. However,
electrical conductivity method is unsuitable for soils containing high salt
contents.
3. Tensitometric Method. Tensitometer can effectively measure the
moisture content of sandy soils because of their high matric potentials. In
tensitometer, a porous cup is attached to a glass tube which is connected to a
mercury manometer. The tube and cup are filled with water and cup is inserted
in the soil. As the soil dries, water moves out through the porous cup, creating
a suction or vacuum on the water column. Thus water flows through the porous
cup into the soil until equilibrium is maintained. These tension readings
(suction or vacuum readings) in the manometer, expressed in terms of cm or
atmosphere, measure the tension or suction of the soil. If the soil is drier, water
moves through the porous cup, setting up a negative tension (Fig. 1).
Stopper
r----,.- Water
- ---- - -+-Unbalanced
- column
Water
Mercury
Soil surface
..._-_-_-_-_-_- Porous cup
Fig. 1. Tensltometer for determining soil moisture tension.

However, tensitometer can not measure soil matric potential value as
low as the usual wilting values.
4. Neutron Scattering Method. Neutron probes look like flash light
cylinders attached with long cords. The probe is provided with radioactive
substance such as radium or beryllium that emit rapidly moving neutrons. As
the neutrons emitted from the probe collide with hydrogen ions (moisture is
a good source of these ions), they are slowed down and deflected. Some of the
slowed down deflected neutrons are directed back to the probe, where a
counter measures them. Only slowed neutrons are counted. The more the
slowed neutrons that return, the greater is the moisture content of the soil.
DETERMINATION OF SOIL pH
The pH of soil suspension can be determined by electric pH meter and
colorimetric methods.
1. Electric pH Meter Method.

The glass electrode with calomel reference electrode gives accurate pH
of soil. This instrument being a potentiometer requires to be calibrated with
buffer solutions of known pH value.
Principle.
The glass electrode in contact with hydrogen ions of the soil suspension
acquires an electrical potential (emf) and gives H+ ion concentration or pH of
the sample.
Preparation of Standard Buffer Solutions.
Buffer solutions may be of pH 4·0, 7·0 or 9·2 in pure water. A buffer
tablet is dissolved in 100 mL of distilled water to obtain buffer solution.
Saturated solution of potassium hydrogen tartrate may be used which gives
a pH of 3·6 at 25°C.
(i) pH in Saturated Soil Paste.
• Prepare a soil paste in distilled water.
• On saturation, the soil paste glistens and flows slightly when the
container is tilted.
• Allow it to stand for about four hours.
• There should be no free water on the soil surface and also paste
should not stiffen.
• The glistening soil paste is ready to determine pH.
(ii) pH in 1 : 2 soil water suspension. Weigh 40 g of soil in 250 mL
flask and dissolve in 80 mL of distilled water. Shake the mixture
on the reciprocating shaker for one hour.
(iii) pH in 1 : 2 Soil and CaCl2 Solution Suspension.
• Weigh 10 g of air dry soil in 100 mL beaker.
• Add 20 mL of 0·01 M CaCl2 solution. The pH should be between 5·0
to 6·5. Adjust pH with Ca(OH)2 or HC!.
• Allow the soil to absorb CaCl2 solution without stirring. Then
thoroughly stir for 30 minutes and allow to settle.
Method.
• Take either saturated soil paste or 1 : 2 soil water/soil CaCl2
suspension to determine the pH.
• On the pH meter, set the temperature compensating knob and confirm
that the electrode is completely filled with the saturated KCI solution.
Allow the pH meter to warm up for 15 minutes to eliminate
asymmetric potential of the instrument.
• Place known standard buffer solution in a beaker of pH 7. Immerse
glass and calomel electrode or single (combined) electrode into the
buffer solution.
• Adjust the instrument reading at the known pH (7) of the buffer.
• The buffer is removed and the electrodes are flushed with distilled
water.
• Take another buffer solution of known pH say 9·2. Note the reading
after immersing the electrodes in it. The pH meter must read 9·2.
SOIL ANALYSIS 209
• The second buffer is removed and the electrodes are again flushed
with water.
• The electrodes are then immersed in a beaker containing soil paste or
soil suspension. Read the pH and record it.
2. BDH universal indicator is used to obtain approximate pH. For
this, take some soil and a little BaS04 in a flat dish and knead the mixture
with a few drops of the indicator. Tilt the dish and note the colour of the
supernatant liquid. It will show the approximate pH of the soil.
3. Lovibond comparator instrument is also used for pH measurement.
It has an opal glass screen and two glass tubes, one containing the sample
and the other containing the sample as well as the indicator. A circular disc,
having a set of nine standard coloured glasses, fits into the front portion and
can be easily rotated in order to bring each coloured glass in front. The disc
is rotated so that the colour matches with the standard from where the pH is
noted.
DETERMINATION OF TOTAL NITROGEN

Nitrogen occurs in soil samples in bound forms as NHg, N02, NOg and
organic nitrogen (viz., amino acids, urea etc). The nitrogen content in soil is
mostly of organic nature (90%). It is hydrolysed to NH! ion which can be
oxidised to NOg by natural process in soil. Total nitrogen in soil varies from
0·01 % to 0·03%. In plants, it ranges from 0·2 to 0·4% depending on the species
and the plant part. Nitrogen bound to soil humus maintains soil fertility. Total
nitrogen can be determined by following methods.
1. Modified Kjeldahl Method.
Principle. The soil sample is digested with sulphuric salicylic acid.
The organic Nand NOg-N is converted to ammonium sulphate and the
ammonia gas is distilled into boric acid. It is then titrated with a standard
solution of H 2 S04.
Reagents.
(i) Sulphuric salicylic acid is prepared by dissolving 1 g of salicylic
acid in 50 mL of conc. H 2S04.
(ii) Sulphate mixture is prepared by mixing 10 parts of ~S04' 1 part
of FeS04 and 0·5 part of CUS0 4. The mixture is grinded to pass
through a 40 mesh sieve.
(iii) 45% Aqueous solution of NaOH.
(iv) 2% Aqueous solution of HgBOg.
(v) Aqueous solution of 0·01 N H 2S04 solution.
(vi) Bromocresol green-methyl red indicator.
(vii) Dried powdered sodium thiosulphate.
Prepare 0·1% solution of bromocresol green by adding 2 mL of 0·01 N
NaOH per 0·8 g of indicator. Prepare 0·1% methyl red in 95% ethanol and mix
bromocresol green indicator with 25 mL of methyl red and dilute to 200 mL
with ethanol.
Procedure.
Take 10 g of soil which should pass through a 20 mesh sieve. Add 50 mL
of sulphuric-salicylic acid and shake. Add 5 g of sodium thiosulphate and heat
the contents gently for 5 minutes. Cool and then add 10 g of sulphate mixture
and digest in Kjeldahl flask at full heat. Cool and then add 300 mL of distilled
water and fit in the distillation apparatus. Add 100 mL of 45% NaOH and a
large piece of zinc. Connect to distillation head, and distil off 150 mL into 50
mL of 2% aqueous boric acid solution. Add few drops of the bromocresol
green-methyl red indicator. Titrate to the faint pink colour with standard
solution of 0·1 N H 2S04 . Repeat the titration with the blank.
Calculations.
Weight of soil sample = 10 g.
Volume of 0·1 N H 2S04 used in titration = V mL
Weight of nitrogen in 10 g soil = V x 0·1 x 0·014 = 0·0014 V
. 0·0014 x V x 100
Percent mtrogen = 10 = 0·014 x V percent
Milliequivalent of nitrogen in 10 g soil = V x 0·1 = 0·1 V
Milliequivalent percent of nitrogen in soil= O·lV x 10 0 = V
10
"t " "I Percent nitrogen x 106
ppm 0 f m rogen m sOl = 100
= 0·014 V x 104 = 140 V.

2. Kjeltec Auto 1030 Analyser Method.
Principle. NH 4-N, liberated by distillation of the digest with strong
alkali, is absorbed in H 3 B03. Ammonium borate so formed is titrated back to
H 3B03 by titration against standard HCl.
Reagents.
(i) 40% NaOH solution.
(ii) Receiving solution. Dissolve 100 g H 3B03 in 10 L water. Add 10 mL
Bromocresol solution (100 g in 100 mL methanol). Add 70 mL
methyl red indicator (l00 mg in 100 mL methanol) and 5 mL of
40% NaOH.
(iii) Standard 0·01 M HC!.
Method.
Bring the digest to 100 mL. Follow instructions for the operation of
Kjeltec Auto 1030 analyser.
• Set the alkali pump to deliver 30 mL of 40% NaOH.
• Titrate with 0·01 M HCl.
Calculations.
fJ7 N· S ·1- (A-B)xO·01MHClx1·401
70 In 01 - ur . h tof oven dyrSOl
vveIg
· l samp1e (g)
where, A = Volume of standard HCl (mL) for titration of the sample.
B = Standard HCl for titration in blank (mL).
SOIL ANALYSIS 211
DETERMINATION OF NITRATE NITROGEN OF SOIL

Nitrate nitrogen of soil ranges from less than 1·0 ppm to several hundred
ppm.
Principle.
The soil extract is clarified by using copper sulphate, calcium hydroxide
and magnesium carbonate. Nitrate is determined by adding
phenoldisulphonic acid with which a yellow coloured compound is formed
with nitrate.
Reagents.
(i) 1N copper sulphate solution.
(ii) Dry calcium hydroxide and dry magnesium carbonate.
(iii) NH40H (1 part) + H20 (2 parts).
(iv) Phenoldisulphonic acid can be prepared by first dissolving 25 g
pure phenol in 150 mL of conc. H2S04 . Then 75 mL of fuming
sulphuric acid containing about 15% S03 is added. The contents are
heated for two hours and stored in brown coloured bottle.
(v) Dissolve 0·7216 g of pure KN0 3 in 1 litre of water to prepare
standard nitrate solution. This solution contains 100 ppm
nitrogen as nitrate.
(vi) Dissolve 3·12 g Ag2S04 in one litre water to prepare 0·02 N
Ag2S04 solution.
Procedure. I
Place the sample of the soil in an oven at 70°C for 4 hours. Grind and
pass it through a 2 mm sieve. Take 50g of soil in a 500 mL flask and add 250
mL of distilled water containing 5 mL of 1 N copper sulphate solution and
shake for 10 minutes. If the soil is not very acidic and does not give a coloured
extract, add 0·4 g of Ca(OH)2 and 1·0 g of MgC03 to the soil suspension and
shake for 5 minutes to precipitate copper. Filter on a dry filter paper and
discard the first 20 mL of filtrate.
If the soil gives a coloured extract, allow it to settle. Decant 125 mL
of the supernatant liquid into a flask. Add 0·2 g of Ca(OH)2 and 0·5 g of
MgC03. Shake the contents well and filter. Pipette out 10 mL of the filtrate
in dish and evaporate to dryness. Cool and rapidly add 2 mL of
phenoldisulphonic acid directly to the centre of the evaporating dish. Rotate
the dish in such a way that the reagent comes in contact with all the residue.
Allow it to stand for 15 minutes and then add about 15 mL of cold water and
stir with a glass rod until the residue dissolves to form a solution. Add
NH40H to make the solution slightly alkaline. Transfer this solution to a
Nessler tube, dilute and compare with standard solution containing 1 ppm
nitrogen as nitrate using a colorimeter or Nessler tube.
Note. (i) If the soil contains more than 15 ppm of chloride, the latter is
precipitated by adding 10 mL AgN03 solution in the 250 mL of test solution.
This will remove 80 ppm of chloride calculated on the basis of dry soil. If the
soil contains high concentration of chloride, add solid Ag2S04 to the soil
suspension before shaking.
lit
(ii) In the case of highly coloured soil extract, add 1 g of chloride free
carbon black to 100 mL of the supernatant liquid and shake for 15 minutes
before adding Ca(OH)2 and MgC0 3 to the solution. If the soil is calcareous,
add 5 mL of IN copper sulphate also to the soil extract with the carbon black
in order to ensure complete removal of colloidal carbon with copper hydroxide.
Note that copper sulphate is used for the clarification and
decolourisation of the soil extract. MgC0 3 is used to remove the excess of
Ca(OH)2'
DETERMINATION OF TOTAL PHOSPHORUS
Phosphorus in soil ranges from 0·01 to 0·3% and occurs in the form of
apatite and hydroxy phosphates of Fe, AI, Mn, Ca and Hg.
Olsen Method.
Principle. Phosphorus in the form of phosphate is extracted from the
soil by NaHC03 (pH 8·5). PO~- ion reacts with ammonium molybdate in acidic
medium to form molybdophosphoric acid which is reduced to blue coloured
complex by ascorbic acid.
H 3P04 + 12H2Mo04 ~ H3P(M03010)4 + 12H20
Molybdophosphoric acid
Absorbance readings are taken on spectrophotometer at 730 nm. A
standard curve between absorbance and concentrations of standard
phosphorus solutions helps to deduce the total phosphorus content of the
sample.
Reagents.
1. NaHCOa (0·5 M) solution. Dissolve 42 g NaHC03 in 1 L distilled
water. Adjust the pH to 8·5 with 1 M NaOH solution.
2. Ammonium molybdate solution. Dissolve 40 g ammonium
molybdate in 1 L of distilled water.
3. Darco G-60 or phosphorus free charcoal. 80 g Charcoal or Darco
G-60 is slurried in distilled water. Keep it over night with 1 L of 6 M HCl in
60 mm diameter column (250 mL capacity). Add deionised water till the
leachate is chloride free. Dry at 110°C.
4. Ascorbic acid solution. Dissolve 26·4 g of L-ascorbic acid in 500 mL
distilled water.
5. Antimony potassium tartrate solution. 1.454 g of antimony
potassium tartrate is dissolved in 500 mL distilled water.
6. H 2 S04 (2·5 M). Add 140 mL concentrated H 2S04 in 1000 mL distilled
water.
7. Murphy-Riley developing solution. It consists of 250 mL of
2·5 M H 2 S04, 75 mL ammonium molybdate solution, 50 mL ascorbic acid, 25
mL antimony potassium tartrate solution and 100 mL distilled water.
s. Standard stock phosphorus solution. Dissolve 0·439 g KH 2P04
in 500 mL distilled water. Add 25 mL of 7 N H 2S04 and make the volume to
one litre. It forms 100 ppm stock solution of P. From this take 5 mL solution
in a 100 mL flask to obtain 5 ppm of P solution .
•
SOIL ANALYSIS 213
Method.
• Take 2·5 g air dried soil sample in a 125 mL flask. Add a little
phosphorus free Darco G-60.
• Add 50 mL NaHC03 solution at 25°C and shake for 30 minutes.
• Similarly run a blank without soil.
• Filter the extract using Whatman No. 40 filter paper.
• Pipette 10 mL aliquot in 50 mL volumetric flask. Add 10 mL distilled
water and one drop of p-nitrophenol indicator. Acidify the content to
pH 5·0 by adding 2·5 M H 2S04 till the colour disappears.
• Add 8 mL of Murphy-Riley solution and make the volume to 50 mL
with water.
• Wait for 10 minutes and record the absorbance on spectrophotometer
or colorimeter at 730 nm.
• Process the standard phosphorus solution of different concentration in
a similar manner.
• Plot a standard curve between absorbance and concentration of
standard phosphorus solutions.
• Deduce the total phosphorus content of the soil sample by
comparing its absorbance with standard curve.
Calculations.
Weight of soil taken = 2·5 g
Volume of NaHC03 extractant added = 50 mL
Volume of extract taken for colour development= 10 mL
Reading of spectrophotometer/colorimeter = x
Concentration of P in 50 mL extract = ClIO Ilg mL-1.
Concentration of P in 1 g of soil
C 50 -1
= 10 x 2.5 Ilg mL
:. Available P in kg ha- 1 = ~ x ;.~ x 2·24

= C x 4·48 (ppm x 2·24 = kg ha- 1)
or Aval·1 a ble P -_ C x volume of"tITextractant!
• ht f ·1
aliquot x 2· 24
vveIg 0 SOl
where C = Ilg P in the aliquot obtained from standard curve.
DETERMINATION OF PO~- ION
Total phosphorus (85%) is usually present in organic form. Inorganic
phosphorus as orthophosphate plays a dynamic role in aquatic ecosystem.
Phosphorus, the most important micronutrient, is utilized by plants in the
form of H 2P04and HPO~- species. In acidic soils, the orthophosphate ions are
either precipitated or sorbed by cations, viz. AI 3+, Fe 3+ etc. In alkaline soil,
reaction occurs with CaC03 and hydroxypatite CalO(P04)6(OH)2 is
precipitated out.
Reagents.
(i) 70% HCI04 and IN NaOH solution.
(ii) Ammonium molybdate and SnCl2 solution.
(iii) Phenolphthalein indicator.

(iv) Standard phosphate solution. Dissolve 4·388 g of dried
potassium hydrogen phosphate in distilled water to make the volume
1 litre. Take 10 mL of this solution and add distilled water to make
1 litre of stock solution containing 1 mg PII... Prepare standard
phosphorus solution of various strengths (0·0 to 1·0 mgIL of P) by
pipetting out 1·0 mL to 10·0 mL of stock solution and diluting to one
litre.
Procedure.
Preparation of soil extract.
• Take 1 g of air dried soil in a 500 mL flask.
• Add 200 mL of 0·002 N sulphuric acid to it and shake for about half
an hour.
• Filter the suspension through a filter paper (Whatman No. 50).
1. Inorganic Phosphate Determination.
• Take 25 mL of the soil extract in a flask and add to it 1 mL of
ammonium molybdate solution and 3 drops of stannous chloride
solution. A blue colour will appear. Wait for 10 minutes and record
absorbance (A) on spectrophotometer or colorimeter at 690 nm
(filter no. 67).
• Run a distilled water blank in a similar way.
• Process standard phosphorus solutions of different strengths in
similar manner and plot a standard curve between absorbance and
concentrations. Deduce the content of PO~- in soil extract solution
by comparing its absorbance with standard curve and express the
result in mg phosphate-phosphorus per litre.
2. Organic Phosphate Determination.
• Organic phosphate content of soil extract can be determined using
following relation :
• Organic phosphate (mg/L)
=Total phosphate (mg/L) - Inorganic phosphate (mg/L)
Formula used.
P043- - P (mgll't) PS x V
1 re = 1000 x W
where, PS =PO~- -P estimated in suspension (mgIL).

V = Total volume of suspension (mL).
W = Mass of air dried soil taken (g).
DETERMINATION OF SILICA
Silicon, the most abundant element, is present in nearly all the
minerals. Most of the minerals in igneous rocks occur as silicates, hence soil
is largely composed of silicates. Si is required for the growth of rice, barly and
cucumber etc.
SOIL ANALYSIS 215
Total Silicon.
• Soil is digested with perchloric-nitric acid and heated for 15 minutes.
• Filter the silica from the diluted digest through Whatman No. 41
filter paper. Wash the residue with warm dilute HCI (1: 20) and then
• Evaporate the combined filtrate and washings to dryness. Heat the
residue at 110°C for an hour to recover traces of silicic acid from the
solution.
• Extract the residue with dilute HCI, filter and wash as before.
• Ignite the combined residue and filter paper to a constant weight in
a platinum crucible using a muffle furnace and record the result as
crude silica.
• Crude Si02 can be converted to Si by the factor 0·4675.
Water Soluble Silicon.
Molybdosilicate Method.
Principle. Ammonium molybdate at pH 1·2 reacts with silicon and any
phosphate present to produce heteropoly acids. Oxalic acid is added to destroy
molybdophosphoric acid, HsPMo12040. The remaining molybdosilicic acid,
H4SiMo12040 is measured at 650 nm.
Reagents.
1. Standard silicon solution.
2. Ammonium molybdate solution (10% w/v).
3. Sodium sulphite (17 gL-1) solution.
Procedure.
• Extract the soil with water and filter.
• Pipette 25 mL of the clear filtrate into a 150 mL conical flask and add
15 mL dilute HCI.
• Add 15 mL ammonium molybdate solution. After 5 minutes, add 2 mL
oxalic acid and 25 mL sodium sulphite solution.
• Read the optical density at 650 nm exactly one minute after adding
the reductant.
• The standard curve should cover the range 0·1 to 5·0 Ilg per mL Si.
DETERMINATION OF LIME
Lime is mainly used for the correction of soil acidity, to increase the soil
fertility and to supply nutritional calcium to crops. Agricultural liming
includes CaO, Ca(OH)2 and CaCOs . These compounds are generally derived
from chalk or limestone e.g., calcite CaCOs and dolomite [CaMg(COs)21. Lime
reactions in soil depend upon the nature and the fineness of the liming
material. Lime or lime stone should not contain silica. Burnt lime or
quicklime is commercially available with a known content of CaO.
Principle.
The lime sample for agricultural U3e generally contains CaO, CaSiOs,
MgCOs , Fe20s and Al20 S' This sample is finely ground, suspended in water
and treated with a solution of cane sugar. As a result, soluble calcium sucrate,
C12H220U·CaO is obtained, whereas CaC03 and CaSi03 remain insoluble.
The solution containing calcium sucrate is titrated as CaO against a standard
solution of HCl using phenolphthalein as an indicator.
Reagents. (i) 0·05 N HCl (ii) 10% cane sugar solution.
Procedure.
Accurately weigh 5·0 g of the powdered sample and transfer it to a 500
mL flask. In order to prevent the possibility of caking, moisten it with 10 mL
of alcohol. Add 10 mL of 10% solution of cane sugar and make up the solution
to the mark with water. Immediately stopper the flask and shake the flask
atleast for 4 hours. Filter the solution through a filter paper into a dry beaker.
Titrate 50 mL of the filtrate with 0·05 N HCI using phenolphthalein as an
indicator.
1 mL of 0·05 N HCI= 0·0014 g. CaO.
Calculations.
. X x 0·0014 x 500 x 100
Percent of CaO In the sample = 50 x 5
where X is the volume of 0·05 N HCI used to titrate 50 mL of calcium sucrate.
DETERMINATION OF MAGNESIUM
In soils, magnesium occurs in the clay minerals, micas, vermiculites and
chlorites. Mg, a macronutrient, is essential for the plant growth. It affects
translocation of phosphorus and helps to increase sugar, starch, vitamin and
inulin in root crops.
Mg can be determined by titrimetric method, atomic absorption
spectrophotometric method and gravimetric method after removing calcium
salts.
Titrimetric Method.
Principle. Mg in solution can be titrated with 0·01 N EDTA using
Eriochrome black T dye as indicator at pH 10 in presence of
NH4 CI-NH40H buffer. At the end poipt, colour changes from wine red to
blue.
Reagents.
1. 0·01 N EDTA.
2. NH4 CI-NH40H buffer. Dissolve 67·5 g NH4CI in 570 mL NH3
solution and make it to one litre.
3. Eriochrome black T indicator.
Method.
• Pipette out 25 mL aliquot containing not more than 0·1
milliequivalent (me) of Ca and Mg.
• Add 2 to 5 crystals of carbamate and 5 mL NH4CI-NH40H buffer.
Add 3 drops of Eriochrome black T indicator.
• Titrate this solution with 0·01 N EDTA solution till colour changes
from wine red to blue or green.
SOIL ANALYSIS 217
Observations. If N 1V 1 are normality and volume of aliquot

(Ca2+ + Mg2+) and N2V2 are the normality and volume of EDTA used, then
N1V1 =N2V 2
N = N 2V 2 = Volume of EDTA x Normality of EDTA
or
1 V1 mL of aliquot taken
Here Nl = Normality = equivalents ofCa2+

+ Mg2+ present in 1 L of aliquot,
Ca2+ + Mg2+ Normality of EDTA x Vol. of EDTA x 1000
H ence me 0 f = .
, L mL of ahquot taken
Milliequivalent of Mg2+ = me (Ca2+ + Mg2~ - me of Ca2+ + Mg2+ giL
Since me x eq. wt. _ K
L 1000-L
_ Normality of EDTA x Vol. of EDTA x Eg. wt. ofCa2+ + Mg2+
- mL of aliquot taken
Mg2+ (giL) = [Ca2+ + Mg2+ (giL)] - [Ca2+(g/L)]
DETERMINATION OF TOTAL MANGANESE
Principle. Mn in the soil is oxidised with 0·1 N phosphoric acid and
0·3 N nitric acid to give permanganate and the optical density of the latter is
measured. Interference from chlorides can be avoided by adding mercuric
sulphate.
Reagents. 1. Conc. HF, conc. H 2S04 and 85% phosphoric acid.
2. Ammonium persulphate salt.
3. Preparation of standard manganese solution. Prepare 100
ppm solution from manganese by dissolving 0·100 g in dilute nitric
acid. Boil, cool and dilute to one litre.
4. Mercuric sulphate solution. Dissolve 75 g of mercuric sulphate
in 400 mL of conc. HN03 and 200 mL distilled water. Add 200 mL
of phosphoric acid and 0·035 g AgN0 3 . Dilute to one litre.
Procedure.
Take 0·5 g of 100 mesh soil in a platinum. dish and add 4·0 mL HF and
2·0 mL conc. H 2S04 , Heat gently on a low flame and then increase the
temperature till fumes of sulphuric acid are given off. Cool the contents and
add 3 mL conc. H 2S04 and again heat till the fumes of acid are given off.
Cool and dilute the contents by adding 10 mL of distilled water.
Take a small known portion of the solution and add 1·0 mL of conc.
H 3P04 . Boil the mixture to a suitable small volume and cool. Add 1·0 g
ammonium persulphate and boil for 2 to 3 minutes. Cool the solution and
dilute to 100 mL and find its optical density at 525 nm in a
spectrophotometer. Compare its optical density with a standard curve and
calculate the results.
Determination of Manganese Using DTPA.

Principle.
Diethylene triamine penta-acetic acid (DTPA), a chelating ligand,
combines with free Mn2+ ions to form soluble complex.
Reagents.
1. Stock standard solution. Dissolve 0·1 g Mn foil in dilute HCI
(1 : 1) and make the volume to one litre.
2. Extracting solutions. 0·005 M DTPA, 0·01 M CaCI2·2H2 0 and
0·1 M TEA (triethanol amine) at pH 7·3.
3. Working standard solution. Transfer 0, 1, 2, 3, 4, 6 and 8 mL of
the stock solution to a series of 100 mL flasks. Dilute each to the
mark with DTPA extracting solution. Standard solutions so prepared
have Mn concentration of 0, 1, 2, 3, 4, 6 and 8 Ilg/mL (ppm).
Extraction of Soil Samples.
• Take 10 g air dried soil sample in a 100 mL conical flask.
• Add 20 mL DTPA extracting solution. Stopper the flask and shake for
2 hours at 25°C.
• Filter the content through Whatman No. 42 filter paper.
• Analyse Mn with an atomic absorption spectrophotometer.
• Also keep a blank with each set following all the steps except the soil.
Analysis of Extracts.
• Mn in soil extracts, obtained by the above procedure can be
determined by AAS.
• Set zero of the AAS instrument with blank.
• Feed standards into the AAS to standardise the instrument and read
absorbance.
• Feed the DTPA extract and record the absorbance or concentration of
Mn.
• Draw a standard curve between readings of absorbance and
concentration of Mn in ppm.
Calculations.
Weight of soil taken = 10 g
Volume of DTPA extractant added = 20 mL
Dilution = 2 times
Absorbance shown by AAS = A
Concentration of the micronutrients from the standard curve against
A=C
Mn in soil sample =C x 2 mg/kg or llg/mL or ppm.
DETERMINATION OF SULPHUR
Sulphur occurs in soil in both organic and inorganic forms but only a
fraction of it is available for crop growth. Direct uptake of sulphur by plants
occurs largely as inorganic sulphate. Sulphate may be present in soil solution.
adsorbed on soil surface or as insoluble compounds such as gypsum or
SOIL ANALYSIS 219
associated with CaC03 . Sulphur in soil can be determined by heat treatment

method or colorimetric method.
1. Heat Treatment Method.
Principle. Inorganic sulphate and a portion of organically held S-S04
are quantified during gentle hydrolysis as a result of, heat treatment and
extraction with 1% NaCl. BaCl2 crystals along with stabilising reagent are
added to convert sulphur-sulphate into BaS04 suspension which causes
turbidity. This turbidity is measured by spectrophotometer.
Reagents.
(i) NaCI solution, 1%
(ii) BaCI2·2H20 solution, 20 to 30 mesh crystals.
(iii) Stabilising solution 75 g NaCI, 30 mL conc. HCI, 100 mL absolute
alcohol, 50 mL glycerol and 250 mL distilled water.
(iv) Standard sulphate solution. Dissolve 1·080 g ~S04 in 100 mL
distilled water.
Method.
• Weigh 5·0 g soil into a silica basin and add 20 mL distilled water.
• Evaporate the content to dryness on a water bath. Then heat in a hot
air oven at 102°C for one hour.
• After cooling, transfer the soil to a 50 mL centrifuge tube and extract
it with 33 mL of 1% NaCl.
• Pipette 25 mL of the aliquot into a silica basin and evaporate to
dryness with 2 mL of 3% H 20 2. Heat it in a hot air oven at 102°C for
one hour.
• Cool it and dissolve the residue in 25 mL water. Centrifuge the
content to settle suspended matter.
• Take 10 mL aliquot in a 250 mL flask and add 20 mL water.
• Add 2·5 mL stabilising solution, 0·2 g BaCl2 crystals to the standard
and extract sulphur.
• Shake the flask for 5 minutes and measure the turbidity by
colorimeter using a blue filter or by spectrophotometer at 340 nm.
• Prepare standard curve between sulphur concentration in standard
solutions and colorimeter readings.
Calculations.
SO --S ( gIkg) =S (mg/L) in the aliquot x Vol. of extract
4 m Vol. of aliquot x Oven-dry soil
where S (mgIL) is obtained by standard curve.
2. Colorimetric Palaskar Method.
Principle. Sulphur from soil is extracted in 0·15% CaCI2'~0 solution
(1; 5 soil to solution ratio), filtered and estimated.
Reagents.
(i) Barium chromate solution 0·1 N in 1·5 N HCl.
(ii) Standard sulphate solution (100 ppm. S). Dissolve 0·5435 g dry
~S04 is 1 L distilled water to obtain 100 fJ.g SlmL.
Method.
• Pipette out 5 mL BaCr04 solution into 100 mL volumetric flask. Add
1·2 mL of 5 N NH 40H in it to reduce free acidity to about 0·05 N.
• Measured aliquot of soil extract (5 to 10 mL) is poured into the flask
maintaining sulphate-sulphur content below 2000 Ilg. Shake and
allow to stand for half an hour.
• Add 1 mL NH 40H to precipitate unreacted BaCr04 and make up the
volume to 100 mL with distilled water.
• The flask is stoppered and inverted twice for thorough mixing. Filter
through Whatman No. 42 filter paper.
• The intensity of the yellow coloured filtrate is measured III
photoelectric colorimeter using blue (420 nm) filter.
• Concentration of S is calculated from the standard curve.
Note. The test solution should have a pH around 2 or 3.
Preparation of Standard Curve.
Take 0, 4, 8, 16 and 20 mL of working solution of sulphate-sulphur (of
100 ppm) in 100 mL volumetric flask. Make up the volume to 100 mL with
the distilled water. This gives standard solution having 0, 4, 8, 16 and 20
ppm S concentration. Prepare a blank sample without soil. Measure colour
intensity by photoelectric colorimeter using blue filter.
DETERMINATION OF SALTS IN SOILS

All soils contain varying amounts of salts in the soluble form as CO§-,
HCOs , SO~-, Cl- and NOs in association with Na+, K+, Ca2+ and Mg2+. The
soil may be saline or alkaline depending on the nature and quantity of salt
present. Water soluble salts in soil can be determined using soil extracts.
1. Preparation of Saturation Extract.
• Weigh 200 g of soil in a container and record the total weight of the
container and the soil sample.
• Add sufficient distilled water while mixing to saturate the soil sample.
• Allow the sample to stand for 4 hours. If free water has been
accumulated on the surface, add a weighed amount of soil and remix.
If the soil has stiffness and does not glisten, add water and mix well.
• Weigh the container with contents. Record the increase in weight,
which corresponds to the amount of water added.
• Calculate saturation percentage (SP) gravimetrically,
SP = Water content of saturated soil paste 100
Oven dried weight of soil x
• Again allow the paste to stand for 4 hours. Transfer it to Buchner
funnel with highly retentive filter paper. Apply vacuum and collect the
extract until air passes through the filter.
• Add 1 drop of 0·1% (NaP03)6 solution per 25 mL of extract to prevent
precipitation of CaC03 .
• Store the soil extract at 4°C until analysed.
SOIL ANALYSIS 221
2. Preparation of Soil Water (1 : 2) Extract.

• Weigh 25 g of soil in a 100 mL beaker. Add 50 mL distilled water with
stirring. Filter the suspension through Whatman No.1 filter paper
and collect the clear filtrate.
• The saturation extract or soil water (1 : 2) extract can be used for the
determination of soluble cations and the anions in soil as per the
requirement.
Conductivity Method.
Principle. For determining soluble salts in soils, the soil is preferably
kept at the field conditions and its conductivity is measured. The conductivity
or the concentration of soluble salts in 1 : 2 soil water suspension multiplied
by 2 gives the values for the original soil sample.
Method.
• Rinse the conductivity cell first with distilled water and then with soil
water suspension. Place the cell in the solution in such a manner that
electrodes are well immersed.
• Balance the galvanometer or the magic eye of the conductivity meter
and read directly the specific conductivity of the soil solution.
Observations.
• If conductivity lies between 0·0 to 0·8 m. mhos, the approximate salt
concentration is below 0·05%. If the conductivity iw between
0·8 to 1·6 m. mhos, the concentration of salts would be between 0·05
and 0·15%, and if the conductivity lies between 1·6 and 3·2 m. mhos,
the concentration of salts would be between 0·15 and 0·25%.
• For saline alkali soil, the conductivity of saturation extract is
greater than 4 m mhos/cm at 25°C, while for non-saline alkali soil,
conductivity is less than 4 m mhos/cm at 25°C. Exchangeable sodium
percentage is more than 15 in both these soils.
• The conductivity can also be determined by making use of Wheatstone
bridge operated by alternating current employing a cathode ray tube
as the null indicator.
Quantitative Methods.
The cations viz., Ca2+, M~+, Na+, K+ present in aqueous extracts of soil
are quantitatively determined by complexometric titration, atomic absorption
(or emission) spectrometric methods. The anions viz., cr, NOs, CO§-, HCOs ,
NOs in soil extracts can be determined by titrimetric or atomic emission
spectrometric methods.
DETERMINATION OF SODIUM IN SOIL BY FLAME PHOTOMETRY
Principle.
The emission of sodium is based on the fact that electrons are excited
from the ground state to the higher energy state which then return back to the
original state with the emission of light. Trace amounts of sodium can be
determined by flame photometry at a wavelength of 589 nm. The sample is
sprayed into gas flame and excitation is carried out under controlled
conditions. The desired spectral line is isolated by the use of interference

filters or prism or grating. Intensity of light is measured by a phototube
potentiometer. The intensity of light at 589 nm is nearly proportional to the
concentration of the element. After calibration of photometer with solution of
known composition, it is possible to correlate the intensity of a given spectral
line of unknown solution with the amount of an element present that emits
the particular radiation.
Reagents.
1. Sodium stock solution. Dissolve 2·54 g dry NaCI in 1000 mL
distilled water at 140°C. (1 mL = 1 mg Na).
2. Working sodium solution. Dilute 10 mL of stock solution to 1
litre. (1 mL = 0·10 mg Na).
Procedure.
Preparation of soil extract. Take 50 g of air dry soil in a flask and
add 100 mL of 40% ethanol. Shake well and wait for 10 minutes. Filter the
suspension through Whatman filter paper no. 50. Wash the soil residue with
40% ethanol and finally with absolute alcohol. Transfer the residue to a
beaker. Add 100 mL of ammonium acetate solution, stir and allow to stand
over night. Filter and note the total volume of the soil extract.
Determination of Sodium.
• Start the electrical supply and switch on the air supply. Stabilise the
air and the needle should be steady at the mark.
• Switch on the gas and maintain the gas fuel mixture so that the blue
flame is seen through the viewing window. Set the filter (No. 60) for
reading at 589 nm.
Aspirate distilled water and adjust the galvanometer reading to zero.
Calibrate the instrument by using standard and adjusting the galvanometric
reading. Plot a standard curve between concentration and emission of
standard sodium solution. Use distilled water to bring the reading to zero
mark. Note galvanometric reading of the sam~le solution.
Put off the fuel supply first followed by air and then main switch.
AxV
F ormuIa us. eel Sodium (mg/g) = W x 10000
where A = Sodium content of soil extract (mg/litre).

V = Total volume of soil extract (mL).
W = Mass of dry soil (10 g) taken for extraction (g).
Precautions.
• Operate in the lowest practical sodium range.
• Add radiation buffers to suppress ionization and anion interference
like CI-, SO~-, HCO s.
• Introduce identical amount of the same interfering substances present
in the sample into the calibration standard.
SOIL ANALYSIS 223
DETERMINATION OF POTASSIUM IN SOIL BY

FLAME PHOTOMETRY
Principle. Trace amount of potassium can be determined in either a
direct reading or internal standard type of flame photometer at a wavelength
of 766·5 nm.
Reagents.
• Standard potassium solution. Dissolve 1·91 g of potassium chloride
in distilled water to make the volume 1 litre. This stock solution
contains 1 g/litre. Prepare various standard potassium solutions of
different strength by diluting original stock solution. (1 mL = 1 mg
ofK).
• Deionized water.
Procedure.
• Prepare the soil extract as described in the determination of sodium.
• Set the filter of flame photometer for reading at 766·5 nm and proceed
for determination of potassium in soil extract following the method
described for the determination of sodium.
• Use standard potassium solution for preparation of standard curve.
Formula used. Potassium (mg/g) = W ~ ~o~oo
where A = Potassium content of soil extract (mg/litre).
W =Mass of soil used for extraction (g).
o
10
FUEL ANALYSIS
FUELS
Fuels may be defined as naturally occurring or manufactured
combustible organic substances which on burning supply heat energy for
practical applications without the formation of objectionable byproducts.
According to the modern concept, a fuel is any fissionable material or
reactant which produces energy in a form that can be used for producing
power. Fuels contain carbon as the main constituent associated with small
amounts of N, S, H, 0, moisture and mineral matter. Coal, wood, petrol,
diesel, kerosene, oil gas etc. are some common fuels.
Characteristics of a Good Fuel.
• A good fuel should have a high calorific value.
• It should have a low non-combustible matter content.
• Its ignition temperature should be low so that it can burn smoothly.
• Moisture content should be low because it reduces the combustion
process of a fuel.
• It should be safe, cheap, readily available, easy to handle and
transport.
• The products of.combustion should be non-polluting.
• Solid fuels should have low ash and uniform size to achieve regular
combustion.
• Liquid fuels should have correct surface tension, boiling range,
volatility and free from objectionable odour and acid components.
• A good fuel for internal combustion engines should have high octane
number, no ash and sulphur and high antiknock characteristics.
CLASSIFICATION OF FUELS
Fuels may be classified into two categories :
1. Primary or Natural Fuels. These fuels occur freely on earth's crust
and may be solid (wood, coal, peat, dung), liquid (crude oil, petroleum) or
gaseous (natural gas) fuels.
2. Secondary or Derived Fuels. The fuels are manufactured from
primary fuels and may also be solid (coke, charcoal), liquid (kerosene, petrol,
gasoline, LPG) and gaseous (oil gas, water gas, bio gas) fuels.
SOLID FUELS
Solid fuels consist of combustible organic matter and incombustible
matter. Combustible matter contains C, H, S whereas incombustible matter
contains moisture and minerals such as carbonates, phosphates, silicates,
(224)
FUEL ANALYSIS 225
sulphides of Ca, Fe, Mg, AI, K, Na etc. After burning, solid fuels form ash.
Solid fuels are classified into natural, artificial and industrial solid fuels.
1. Natural Solid Fuels.
(a) Wood. Wood derived from plants contains lignocellulose which
consists of three primary precursors viz.
(i) Cellulose, a polysaccharide, (C6HlQ05)n,
(ii) Hemicellulose and
(iii) Polyphenolic lignin (C19HlS05)x
Wood also contains resins and proteins. Ultimate analysis of dry wood
shows C = 49·65%, H = 6·25%, N = 0·92% and 0=43·20%. Calorific values
varies from 4000 BTU to 6480 BTU. On destructive distillation, wood yields
charcoal (23%), wood tar (5·3%), pyroligneous acid (44·3%) and gases (26·9%).
Wood is a convenient fuel because of its low ignition temperature and low ash
(0·3%) content.
(b) Bagasse. It is crushed sugarcane residue.
(c) Coal. Coal is a primary fuel formed from fossil remains of
terrestrial plants by the combined action of high temperature and pressure.
It consists of cellulose, lignin and small amounts of resins, fat and water. The
progressive transition from .
Vegetation ~ Peat ~ Lignite ~ Bituminous ~ Anthracite ~ Graphite
is accompanied by increase in aromatisation, calorific value, hardness and
decrease in ° content, reactivity and moisture. C 13 NMR and X-ray studies
have shown coal to be composed of macromolecules (mol. wt. 100,000) which
are predominantly aromatic and some hetrocyclic rings. The ring
macromolecules are linked together by methylene bridges or longer aliphatic
chains which may contain oxygenated functional groups. Typical composition
of coal is C 100 H S5 S 2 .1 N 1.5 09.5'
Grading of Coal.
Coal can be graded on the basis of heating values, coking properties,
geological age, chemical composition and commercial applications etc.
However, the most accepted system is used by ASMT based on the ranks
which signifies the degree of coalification, maturation or metamorphism
(change in form and structure under the influence of heat, pressure and
water). Coal is graded in the following ranks.
(i) Peat (Precoal).· Peat is formed by gradual decomposition of
vegetable matter in moist places. It is the initial transformation stage during
the formation of coal. It is not an economic fuel but can be used as fertilizer.
It has low calorific value (7700 BTU per pound).
(ii) Lignite. Lignite is obtained from finely divided plant tissues. There
are two types of lignite. Brown coal lignite is unconsolidated while black
lignite is consolidated. It has high moisture content but sometimes it may
spontaneously ignite by absorbing oxygen. Its calorific value is 6000-9900
BTU. Ash content is about 4%.
,(iii) Sub-bituminous Coal or Black Lignite. It ignites very easily.
Calorific value is 7000-15000 BTU and moisture content 20%.
(iv) Bituminous Coal (C15aHu5NaOlaS2)' It is hard, dense, burns

with smoky flame. It is of two types. Low volatile coals have fuel ratio less
than 2 and high volatile coals have fuel ratio more than 2. Calorific value is
12600 BTU per pound.
(v) Super Bituminous Coal or Semi-anthracite. Bituminous coal of
light grade is known as super bituminous coal. Its calorific value lies between
12000 to 15000 BTU. It burns with smokeless flame and has the best heating
power among all coals.
(vi) Anthracite. Anthracite, obtained from fossil coal, is lustrous, low
ash, sulphurless coal. Its calorific value ranges between 14000 to 15000 BTU.
It has highest percentage (60-80%) of fixed carbon and fuel ratio is 10·2.
(vii) Pulverised Coal. It is obtained from fossil coal after crushing,
drying and grinding of coal. This coal has low ignition temperature due to the
presence of volatile matter.
Coking and Non-coking Coals. A coal is called coking coal, if it has
a tendency to soften, swell and stick together during combustion. Bituminous
coals have coking properties while lignite, anthracite are non-coking coals.
;2. Artificial Solid Fuels.
(a) Coke. Coke is prepared by destructive distillation of wood. It is
produced by a low -600°C and high 1400°C temperature carbonisation.
Carbon content is 85% and ash content 9%.
(b) Charcoal. Charcoal is formed either by burning of wood in a
limited air supply or by destructive distillation of wood. Charcoal consists of
5% tars and oils, 25% gas, 10% moisture. It is a good fuel for producer gas.
(c) Briquette. It is prepared from powdered coal, peat and lignite in
presence of some binding materials under pressure. Briquettes are used as
domestic fuels. Its calorific value is 8000 BTU per pound.
3. Industrial Solid Fuels.
Industrial fuels are fossil coals, peat, furnace slags and oil shales. Boiler
slags contain upto 40% combustible matter. Oil shales have high ash content
and 70% volatile matter.
LIQUID FUELS
Liquid fuels are more popular and economical than solid fuels. The most
important source of liquid fuel is petroleum, which is known as rock oil,
mineral oil or liquid gold. According to its composition, petroleum is
paraffin based, asphaltic based and mixed based.
Fractions Obtained by Fractional Distillation of Petroleum.
1. Petroleum Gas. The molecular composition of petroleum gas ranges
from C 1 to C4 hydrocarbons. It is used as a fuel in the form of LPG.
Polymerisation of petroleum gas yields gasoline or petrol also known as motor
spirit. Its boiling point is below 40°C.
2. Gasoline or Petrol (C 5 to C 12 hydrocarbons, b.p. 40°C to 170°C).
Gasoline or petrol is used as a fuel in vehicles.
3. Kerosene Oil. (ClO to C 12 hydrocarbons, b.p. 170°C to 250°C). A
special grade of kerosene oil is used as an aviation fuel in aeroplanes.
FUEL ANALYSIS 227
4. Diesel Oil (C 13 to C I5 hydrocarbons, b.p. 250°C to 350°C). It is used

as a fuel for heavier vehicles.
5. Fuel Oil (C 15 to C I8 hydrocarbons, b.p. 350°C to 400°C). It burns
completely without leaving any residue.
6. Lubricating Oil (C 17 to C 20 hydrocarbons, b.p. above 400°C). It is
used for lubricating machinery etc.
7. Paraffin Wax (C 20 to C30 hydrocarbons, b.p. above 400°C).
8. Asphalt (C 30 to C50 hydrocarbons). Asphalt is used for paving road
surface.
Other liquid Fuels.
(i) Benzol (ii) Power alcohol (iii) Colloidal fuel
(iv) Coal tar (v) Liquefied petroleum gas.
GASEOUS FUELS
Advantages of Gaseous Fuels.
Gaseous fuels have distinct advantages over solid and liquid fuels. For
instance,
(i) Many gaseous fuels have high calorific value.
(ii) Smoke and ash are eliminated.
(iii) Rate of combustion is high.
(iv) Temperature can be easily controlled.
(v) Much higher fuel economy because of regenerative heating.
(vi) Uniform heating is possible.
(vii) Beside industrial and domestic use, they can be used in internal
combustion engines for direct production of power.
Classification of Gaseous Fuels.
(i) Producer gas (ii) Water gas (iii) Coal gas
(iv) Coke oven gas (v) Ethylene gas (vi) Acetylene gas
(vii) Natural gas
PRODUCER GAS
Producer gas is a mixture of CO + H2 and CO2 + N 2 . Producer gas is a
cheap industrial fuel because it can be obtained from low grade coal. Bagasse,
peat, lignite, anthracite, highly volatile bituminous coals, saw dust and coke
can be used for its production.
Gas Producer for the Manufacture of Producer Gas.
Producer gas can be prepared by passing a mixture of air and steam over
red hot coal or coke in a special type of furnace, called gas producer. A
typk:al producer (Fig. 1) consists of a refractory-lined cylindrical vessel (3 m
diameter, 4 m height) made of steel. The solid fuel enters the producer at the
top, through cup and cone feeder and is burnt on an iron grate.
Producer Gas Production Reaction Zones.
1. Ash Zone.
It is the lowest zone containing a layer of ash (0·5 m thick) on the grate.
It helps to
(i) Preheat the incoming air/steam mixture, thereby improving the

thermal efficiency of the producer.
(ii) Protect the grate from intense heat of the combustion zone.
~--Coke
::-.:c-".::-r..---CUp and cone feeder
===--+-Producer gas outlet

Gas space
Reduction
Pyrolysis zone =111111~illll~~= Refractory brick lining
Combustion zone Coke at 1000°C
Air mixed with -----.=]~~:;=~::::J

a little steam Exit for ash
Fig. 1. Typical gas producer.

2. Gasification Zone.
(i) Oxidation or Combustion Zone. Here carbon burns in oxygen to
form CO2 and CO. Temperature in this zone rises to 1200°C.
C + 02 - - t CO2 + 96· 7 kcals
C + 11202 - - t CO + 29·5 kcals
N 2 of the air remains unaffected, hence producer gas consists of mainly
CO and N 2.
(H) Primary Reduction Zone. CO2 and steam, rising from the
oxidation zone, react with carbon to produce CO and H 2.
CO2 + C - - t 2CO - 38·5 kcals
C + H 20 -~ CO + H2 - 28·3 kcals
C + 2H 20 - - t CO2 + 2H2 - 18·5 kcals
The reactions being endothermic so the temperature falls to 1000°C.
(iii) Secondary Reduction Zone. CO forming reaction becomes more
prominent. In addition, water-gas shift reaction occurs to some extent.
CO + H 20 - - t CO2 + H2 + 10 kcals
The temperature falls to 800°C.
3. Pyrolysis Zone.
Hot gases rising from the reduction zone cause carbonisation of the down
coming coal adding byproducts (oils, methane, tars, phenol) to the rising
producer gas. Temperature in this zone varies from 400°C to 800°C.
FUEL ANALYSIS 229
4. Coal Preheating and Drying Zone.

This is the gas space (400°C) above the fuel bed. The fresh incoming coal
gets dried and preheated. The producer gas so obtained has the lowest
calorific value (1300 kcal/m 3) of any gaseous fuel.
Average composition of producer gas is : CO = 27%, H2 = 12%,
N2 = 55·3%, CO2 = 2·5%, CH 4 = 2·5%, C2H 4 = 0·4%, 02 = 0·3%.
Lurgi Process.
Lurgi process is used for the production of producer gas having high
calorific value (4000 k callm 3 ). Here oxygen gas (not air) and super-
saturated steam are allowed to pass through a bed of coke or coal at 30
atmospheric pressure. Producer gas so formed contains CO + H2 and little
CO2 + N2 and CH4 ·
CO + 3H 2 -----t CH 4 + H 20
CO 2 + 4H2 -----t CH 4 + 2H 20
WATER GAS (CO + H2)

Water gas is often called blue water gas because of the blue flame
when it is burnt. It is produced by passing steam over red hot bed of coke
supported on a suitable grate in a refractory lined or water cooled steel
reactor (3 m diameter, 5 m height).
C + H 20 -----t CO + H2 - 29 kcals.
The reaction is endothermic, hence the temperature falls from
1400°C to 1000°C. At this lower temperature, steam reacts with C to form
CO2 ,
C + 2H20 -----t CO2 + 2H2 - 19 kcals.
CO2 has no calorific value so it is undesirable. Actually, the formation
of water gas depends upon the decomposition of steam by carbon. It has been
observed that at 758°C, 1000°C and 1125°C, the percentage of steam
decomposition is 25·3%, 93% and 99·4% respectively. Composition of water gas
formed at 1125°C corresponds to H2 = 50·9%, CO = 48·5%, CO2 = 0·6%.
Hence the rate of decomposition of steam and the formation of water gas
are maintained by alternate run and blow periods. Thus as soon as the
temperature falls below 1000°C, the steam blast is stopped and air blast is
passed through the red hot bed of coke to raise the temperature of coke to
1400°C. When this temperature is attained, the air blast is stopped and the
steam blast is again resumed.
C + 02 -----t CO2 + 97 kcals
2C + 02 -----t 2CO + 59 kcals
Since air forms an explosive mixture with water gas, the water gas
remaining in the generator is removed by passing steam for one minute. The
steam passed for sweeping off the water gas is called purge steam. The
period (1 minute) for which air is passed is called the hot blow. When coke
is enough hot, steam is passed for 1-5 minutes. This period is known as cold
blow. In modern plants, attempts have been made to decrease the hot blow
period and to increase the run period for the generation of much water gas.
Average composition of water gas is : CO = 41%, H2 = 51%, N2 = 4%
and CO2 = 4%. Calorific value of water gas is 2800 kcallm 3 .
Carburetted Water Gas.
To enhance calorific value of blue water gas, the latter is carburetted by
adding gaseous hydrocarbons obtained by cracking petroleum oils.
Plant producing carburetted water gas consists of a generator,
carburettor and a superheater along with purifiers and scrubbers (Fig. 2).
Chimney :~-++--Cooler
Carburettor
- ------
~=:~:~~=~=~=~
Water box
Coal
Superheater
Steam ~~
Fig. 2. Manufacture of carburetted water gas.

The carburettor and superheater are tall refractory lined steel vessels
filled with checker brick work to provide intensive heat transfer surface.
Carburettor is equipped with an atomiser for spraying the oil.
During the run period (steam blow), the water gas from the generator
passes to the carburettor and the atomiser delivers spray of oil on the red hot
fire brick. The sprayed oil in to the carburettor is cracked or pyrolysed. The
products of cracking together with the water gas passing down the
carburettor enter the superheater. The carburettor water gas containing some
tar passes through the outer tube of the superheater to the tar separator.
The gases after cooling are led to the purifiers and finally to the gas
holders. Purifiers contain hydrated ferric oxide and lime impregnated on
wood shavings which remove H 2S as FeS.
In automatic reverse flow plant, the flow of gas passing through the
carburettor and superheater is reversed by elevating the carburettor and
changing the flues. This arrangement causes better cracking of the
carburettor oil as it is sprayed counter current to the hot blue gas.
FUEL ANALYSIS 231
Average composition of carburetted water gas is : 23-28% CO,

34-38% H 2, 17-21% saturated hydrocarbons, 13-16% unsaturated
hydrocarbons, 0·2-2·2% CO2, 2-5% N 2. Its calorific value is 4500 kcal/m 3 .
NATURAL GAS
Natural gas is a mixture oflower alkanes (C l to C5 ), CO2 and N2 etc. It
accumulates in under ground reservoirs either with or without petroleum oil.
Natural gas obtained from oil wells is of two types.
1. Dry variety. When there is no oil but only natural gas exist in a
petroleum well, it is said to be dry. A typical dry gas contains 83·5% CH4 ,
12·5% C2H 6 , 0·2% CO2 and 3·8% N2. Calorific value is 1000 BTU/cu. ft.
2. Wet variety. When natural gas occurs along with petroleum in oil
wells, it is called wet gas. It consists of a mixture of methane along with
n-propane, n-butane, isobutane and isopentane etc. Wet gas is suitably
treated to remove propane, propene, butane, butene and is used as LPG. It
gives out low boiling gasoline known as casing head gasoline.
Helium is also found in natural gas. The calorific value of natural gas
lies between 950 and 1100 BTU/cu. ft. The gas obtained by fermentation of
organic matter in sewage (or cowdung) is also called natural gas. It contains
70% CH4 , 30% CO2 and has calorific value 62·5 BTU/cu. ft.
Properties and uses. Natural gas can be liquefied by pressure and
cooling to -121°C. It is used
(i) as a clean domestic and industrial fuel
(ii) in the manufacture of carbon black.
CALORIFIC VALUE OF FUEL

Calorific value of a fuel is defined as the amount of heat liberated by
the complete combustion of a combustible material with oxygen and the
condensation of the products to the desired temperature.
Units of calorific value. Calorific value of a solid or liquid fuel is
usually expressed in calorieslg or kcal/kg or British Thermal Unit per pound.
In case of gaseous fuels, the calorific value is expressed as kcal per cubic
metre or BTU per cubic foot.
1 kcal / kg = 1·8 BTUllb
1 kcal / m 3 = 0·1077 BTU/ft3
1 BTU / ft 3 = 9·3 kcalslm 3
1. High or Gross Calorific Value.
It is defined as the total amount of heat liberated when one unit of
a fuel is burnt completely and the products of combustion have been cooled
at room temperature (i.e., 15°C or 60°F).
In such a case, water vapour produced by the combustion of hydrogen
and evaporation of moisture will get cohdensed and the heat evolved is also
taken into consideration. However, the heat evolved due to the formation of
H 2S04 and HN03 (from S and N present in fuel) during combustion are
subtracted from the heat evolved.
2. Low or Net Calorific Value.

It is defined as the amount of heat liberated when one unit of fuel is
burnt completely and the combustion products are allowed to escape. In
actual practice, water vapours are not condensed easily but escape along with
hot combustion gases. Thus a less amount of heat is available. This is known
as net or lower calorific value.
Net calorific value (CV) = Gross CV - Latent heat of water vapour formed
One part by weight of H2 produces 9 parts by weight of water.
Net CV = Gross CV - Mass of H2 present in fuel x 9 x Latent heat of steam
Mean calorific value of any fuel containing carbon and hydrogen can
be calculated by
,." t I l _ (%C x 8137) + (%H x 34500)
~o a ca s. - 100
Determination of Calorific Value by Bomb Calorimeter.

The oxygen bomb calorimeter consists of following parts.
1. Combustion Bomb. It is made up of chromium-nickel-molybdenum
steel which is resistant to acids and corrosion. The capacity of the bomb is
250-300 mL and it weighs to 3 kg. The bomb is provided with an air tight
screw cap to which a couple of steel electrodes and a release valve are fitted.
2. Copper Calorimeter Vessel. Calorimeter is polished from outside.
It contains 2L of water when the bomb is placed inside. A space should be left
between water and the cover.
Beckman
Oxygen
valve
. _ - - - - - Electrically
operated stirrer
Ebonite cover
Electrodes to which --I~II~t5
a ring is attached j+---r.=+--Copper calorimeter
Mg fuse wire -+=i---M~=t>t-+-.
Weighed pallet -F--=1~-§~*--I~

of given fuel ~~~?i??~~~~-f=:t-Stainless steel bomb
sample
1:E?,;~~~~tt~~~~-~}-Stainless steel crucible
=::=::=::-=::=::=::=::=::=::=::=E=::=::=::=::=::=====::=::=::===::=::=::=::=
. Water jacket
Fig. 3. Bomb calorimeter.

FUEL ANALYSIS 233
3. Water Jacket. The jacket is in the form of a wide annular vessel

around the calorimeter vessel.
4. A mechanical stirring device and Beckman thermometer.
5. Crucibles should be preferably made of fused silica, Ni or steel.
6. Firing wire should be of platinum or nickel-chromium.
Procedure.
A weighed quantity of the fuel is placed in the crucible and supported
over the ring. A fine platinum wire is tightly stretched across the pole pieces
of the bomb and one end of the thread is tied around the wire. The crucible
is adjusted and the loose end of cotton is so arranged that it remains in
contact with the coal. Place 10 mL distilled water in the bomb with the help
of a pipette to absorb vapours of H 2S04 and HN0 3 during combustion.
Connect the bomb to an oxygen cylinder and raise the pressure to 25
atmospheres. The bomb is then placed inside the calorimeter which contains
a known mass of water in which the bomb is submerged but the terminals
remain above the water level.
Make the electrical connections, place the cover in position and switch
on the stirrer. Note the initial temperature of water with the help of Beckman
thermometer. Connect the electrodes with battery and complete the circuit so
that the sample burns and heat is liberated. Uniform stirring of water is
continued and the maximum temperature attained after the firing is
recorded. Leave the bomb for one hour to ensure that all the acid mist formed
has settled down.
Dismantle the apparatus and transfer the bomb to the holder. Release
the oxygen from the bomb carefully. Open and examine the inside portion to
see whether any unburnt carbon is present. If the combustion is comple.te,
wash the contents with distilled water quantitatively into a beaker. Preserve
this solution for estimation of H 2S04 and HN03 formed during combustion in
order to determine the corresponding corrections.
Calculation of Calorific Value.
From the rise in temperature of water in the calorimeter in which the
bomb is immersed, the calorific value of fuel is calculated as follows :
(w + W) (t2 - tl + tc> - (Cs + CN + CF + Cc )
HCV=------~-=--~--=-~~~--~
m
where, HCV = High calorific value
w = Weight of water in the calorimeter
W = Water equivalent of calorimeter
t 1, t2 = Initial and final temperature of water in the calorimeter
tc = Cooling correction
m = mass of fuel burnt.
Cs , CN, CF , Cc =Corrections for H 2S04, HN03, fuse wire and cotton
thread respectively.
LCY = [HCY - (O·09H x 587)] cals/g

where LCY = Lower or net calorific value
H = % of Hydrogen present in coal
587 cals/g = latent heat of steam formed.
Corrections.
1. Corrections due to the formation of H 2S04 , (C S) and HNOa
(CN ). Owing to high pressure, temperature and excess of oxygen present in
bomb, S and N in coal get oxidised to H 2S04 and HN0 3 .
2S02 + 02 + 2H20 ~ 2H2S04 + 144 kcals
2N2 + 502 + 2H20 ~ 4HN03 + 57·16 kcals
C s Correction. For every gram equivalent of H 2S04 formed (i.e., for
every 49 g H 2S04 ), 144/4 kcals = 36 kcals of heat are liberated.
.. 1 L of 1 N H 2S04 == 49 g (1 g equivalent) of H 2S04
which evolves 36 kcals of heat.
.. 1 mL of N/I0 H 2S04 == 49 X 10-4 g H 2S0 4
which evolves 3·6 cals of heat.
Hence, it is necessary to subtract from the total heat evolved 3·6 cals for
every 1 mL of N/I0 H 2S04 formed in the bomb after the combustion
experiment.
C N Correction. For every gram equivalent of HN03 formed (i.e., for
every 63 g HN03 ), 57·16/4 = 14·3 kcals of heat are liberated.
1 L of IN HN03 == 63 g (1 g equivalent) HN03
which evolves 14·3 kcals of heat.
1 mL of N/I0 HN03 == 63 x 10-4
g HN03
which evolves 1·43 cals of heat. Hence, it is necessary to subtract from the
total heat evolved, 1·43 cals for every 1 mL ofN/10 HN03 formed in the bomb
after the combustion experiment.
2. Cooling Corre~tion etc) by Regnault and Pfaundler method.
(V' - V)
tc =n V + (t' _ t)
[n L- 1 (t) + (to +2 tn)- nt ]
=nV+kS
where S = Whole expression in the main bracket.
_ (V' - V) _ .
K - (t' _ t) - Coolmg constant
n = The integral number of minutes elapsing between to and tn (that is,
between the time of firing and first reading after the temperature begins to
fall from the maximum). V, V' = Rate of fall of temperature in °C per minute
during the initial (before firing) and final periods.
t, t' = Mean temperature during the initial and final period.
n-l
L (t) = Arithmetic sum of temperature readings during the combustion
period.
FUEL ANALYSIS 235
to = Last temperature reading in the initial period when the charge

is ignited.
tn = First temperature reading after which the rate of fall of tempe-
rature is rather uniform.
For the bomb calorimeter with a water equivalent of 2500 g, the value
of K should not exceed 0·0025, if the heat insulation is adequate. The cooling
correction is to be added to the observed rise of temperature.
3. Fuse Wire Correction. The heat evolved for every 1 g or 1" of the
fuse wire burnt in the experiment should be subtracted from the total heat
evolved.
4. Cotton Thread Correction. Contribution of heat from the burning
of cotton thread (identical to cellulose) used can be calculated on the basis
that 4140 cals of heat are evolved per gram of the cotton thread dried at
HO°C.
PROXIMATE ANALYSIS OF COAL
Proximate analysis of coal involves determination of moisture, volatile
matter, ash and fixed carbon in coal. This provides quick information
regarding classification and suitability of coal for a particular use.
1. Moisture. Take 1 g of air dried coal sample in a weighed silica
crucible and again weigh it accurately. Heat the crucible in an oven at
HO°C for 1 hour. Cool the crucible and weigh it. The loss in weight
corresponds to the moisture.
. _ Loss in weight
MOIsture % -"tIT • ht f i t k
vveig 0 coa a en
x 100
2. Volatile Matter. Take 1 g of finely powdered coal in a weighed silica
crucible and fit the lid. Introduce the crucible with the lid on, in a muftle
furnace at 925°C. After 7 minutes remove the crucible from the furnace and
keep on a cold iron plate to ensure rapid cooling, thus avoiding oxidation of
the contents. Transfer the warm crucible to a desiccator and cool to room
temperature. Weigh the crucible and contents again to calculate volatile
matter.
Loss in weight due to removal of
Percentage of volatile matter = volatile matter x 100
Weight of coal sample
3. Ash. Weigh 1 g of the powdered coal sample in a silica crucible and
heat it on a Bunsen burner. The lid is removed and content strongly heated
to burn any tarry matter. Place the crucible without lid in a muftle furnace
at 750°C and heat for one hour to complete the combustion. Cool and weigh
with the lid. The weight of the residue in the crucible corresponds to the ash
content of the coal.
_ Weight of ash left
Percentage of ash - WeIg. ht 0 fcoasamp
l l e x 100
4. Fixed Carbon. The sum total of the percentages of moisture,
volatile matter and ash subtracted from 100, gives the percentage of fixed
carbon (Table 1).
Percentage of fixed carbon = 100 - [% moisture

+ % volatile matter + % ash]
Table 1. Proximate analysis of air dried coal (percentage by weight).
Volatile Fixed
Coal Moisture
matter
Ash carbon
CV(BTUllb)
Peat 20 50 02 27 7700
Lignite black 16 42 12 32 10200
Bitwninous 02 35 13 49 12500
Semibitwninous 01 35 04 84 15000
Anthracite 01 08 03 88 15000
Significance of Proximate Analysis.
Each constituent determined under proximate analysis has its own
importance in the assessment of the coal quality.
1. Moisture. Moisture decreases the calorific value of coal. A
considerable amount of heat is wasted in evaporating the moisture during
combustion. However, moisture content upto 10% produces a more uniform
fuel bed and less fly ash.
2. Volatile Matter. The volatile matter percentage gives some idea
about coking property of coal and denotes the proportion of the coal which will
be converted into gas and tar products by heat. High volatile matter coals
burns with a smoky flame and have low calorific values. For the manufacture
of metallurgical coke, a coal with low volatile matter and fixed carbon is
preferred. High volatile matter content is desirable in coal gas manufacture
and in carbonisation plants.
3. Ash. Ash reduces the heating value of coal. There is a heat loss of
about 1·5% for each 1% ash present in coal. Ash consists of Si02 (50%),
Al 20 3 (30%), Fe203, CaO, Ti02, MgO etc. Clinkers (lumps of ash) cause
uneven temperature on the grates and reduce air supply.
Fused particles of ash may stick to the boiler tubes and affect heat
transfer. Ash with low melting point forms molten slag which is absorbed in
the pores of the refractory lining of boiler furnace. Difference in the
coefficients of expansion and contraction of the slag (ash) and refractory
material causes spalling of the refractory lining thereby reducing its life.
Thus coals used in boilers should have high ash fusion temperature.
4. Fixed carbon. It is the fixed carbon which burns in the solid state.
Higher percentage of fixed carbon increases the calorific value of fuel from
lignite (low ranking coals) to anthracite (high ranking coals).
ULTIMATE ANALYSIS OF COAL

Ultimate analysis includes' estimation of ash, C, H, S, N, 0. This
analysis is essential in calculating heat balances in any process in which coal
is used as a fuel.
1. Carbon and Hydrogen. 2 g of the coal sample is burnt in a
combustion apparatus to convert C and H into CO2 and H 20. These are
absorbed separately in KOH and CaC1 2 absorption tubes of known weight.
FUEL ANALYSIS 237
Percentage of C and H is calculated from the increase in weight of the

respective absorption tubes.
C + 02 ~ CO2 ; H2 + 112°2 ~ H 20
12 44 2 18
2KOH + CO 2 ~ ~C03 + H 20
CaCl2 + 7H20 ~ CaCI2 · 7H20
Increase in weight ofKOH absorption tube 12 100
P ercent age 0 f C = . x- x
Weight of the coal sample 44
Increase in wt. of CaCl2 tube 2
Percentage of H = Welg
. ht 0 fth e co al sampIe t a k en x 18 x 100
2. Nitrogen. Nitrogen can be determined by digesting 1 g of the coal
sample in Kjeldahl flask with concentrated H 2S04, ~S04 and HgS0 4
(catalyst) so that nitrogen present in the sample is converted into ammonium
sulphate. The sample solution is then made alkaline with NaOH and liberated
NH3 is distilled over into a measured amount of standard acid solution, where
it is absorbed. The unused acid is then determined by back titration with
standard NaOH solution. From the volume of the acid neutralised by liberated
NH3, the percentage of N in coal can be calculated as follows.
"t _ Volume of acid used x Normality x 1·4
P ercent age 0 f nl rogen - WeIg
" ht 0 fcoasamp
l l e t a k en
3. Sulphur. Sulphur is determined from the washings obtained from
the known mass of coal used in bomb calorimeter for the determination of
calorific value. The washings contain sulphur in the form of sulphate which
is precipitated as BaS04 using BaCI2. The precipitate is filtered, ignited and
weighed to calcuate percentage of sulphur.
Wt. of BaS04 obtained 32
Percentage of sulphur = Wt. 0 fcoa
I t a k en In
. b omb x 233 x 100
Sulphur can also be determined by heating the coal with Eschka
mixture (2 parts of MgO + 1 part of Na2C03) and estimating S produced as
BaS04·
4. Ash. Ash is determined as described under proximate analysis.
5. Oxygen. Percentage of oxygen is determined by the difference.
Percentage of oxygen = 100 - % of (C + H + N + S + ash)
Table 2. Ultimate analysis of dry peat and coal

(percentage by weight).
Coal C H N S 0 Calorific value, BTUllb
Peat 60 06 1·5 0·5 32 8000
Lignite black 74 5·4 01 0·6 19 10000
Bituminous 77 05 1·0 1·0 16 13400
Sub bituminous 92 04 1·5 0·5 02 15800
Anthracite 94 03 01 0·0 02 15600
Significance of Ultimate Analysis.

• Higher the percentage of C and H, better is the quality of coal and
higher is its calorific value.
• Hydrogen is mostly associated with the volatile matter of the coal
and thus influences the use of coal for the by-product manufacture.
• Sulphur present in coal contributes towards the heating of coal but
its combustion products (802, 803) have corrosive effects on the
equipment in presence of moisture. Further 802 and 803 act as
severe air pollutants. 8ulphur containing coal is not suitable for the
preparation of metallurgical coke, as it adversely affect the properties
of coal.
• Nitrogen present in the coal (1%) does not contribute any useful value
to coal.
• The lower the oxygen content, the more is the maturity of coal and
greater is its calorific value. As the oxygen content increases, the
capacity of the coal to hold moisture increases and the caking power
decreases.
Theoretical Determination of the Calorific value (CV) of Coal using
Proximate and Ultimate Analysis.
The calorific value is determined by burning 1 g of coal in an oxygen
bomb calorimeter and measuring the rise of temperature thus produced in
the water content of the calorimeter. Theoretically CV can be determined by
estimating the thermal efficiency of a process.
Formulae Based on Proximate Analysis.
1. Gouthal Formula.
Calorific value (BTUllb) = 147·6 C +aV
where C = % of carbon
V = % of volatile matter
a = constant depending on V
2. Nakamura Formula.
Calorific value (BTUllb) = a (V - % ~Sh ) + 140·4 C
where a depends on the percentage of volatiles and caking propensity.
Formulae based on Ultimate Analysis.
1. Dulong Formula.
Calorific value (BTUllb) = 14544 C + 62028 (H - ~) + 4050 8

where C, H, 0, 8 are their respective percentages in coal.
Gross CV = 1~0 8080 C + 34500 (H - ~) + 2240 8 kcals/kg

2. Davies Formula.
c 0-8)
Calorific value (BTUllb) = (6·543 H + 403)
(3" + H - - 8-
FUEL ANALYSIS 239
where C, H, 0, S are their respective percentages in coal.

3. Seyler's Formula.
Calorific value CBTUllb) =223·1 C + 698·6 H -7684 + 0·45 °
°
where C, H and are their percentages in coal.
Example 1. A bomb calorimeter experiment gave the following data.
Weight of coal sample taken (m) = 1·1 g. Weight of water taken (w) = 2460 g.
Water equivalent of the calorimeter (W) = 550 g
Observed rise in temperature (t 2 - t 1) = 2·51°C
Cooling correction (tc) = 0·043°C
Correction due to H 2S04 (C s) = 24·2 cals
Correction due to HNOg (CN ) = 35·8 cals
Fuse wire correction (C F ) = 8·0 cals.
Cotton thread correction (C c) = 4·0 cals
If the fuel sample contains 5% H, calculate the gross and net calorific
value of fuel.
Solution.
HCV =[[(2460 + 550) (2·51 + 0·043)] - [24·2 + 35·8 + 8 + 4]]

1·1
= (3010 x 2·553) -
72 = 6920.482 cals
1·1
LCV = HCV - (0·09 H x Latent heat of steam)
= 6920·482 - (0·09 x 5 x 587)
= 6656·332 cals/g
Example 2. A coal sample on ultimate analysis gave : 66·2% C, 4·2% H,
1·4% N 2 , 2·9% S, 6·1% 0, 9·7% moisture and 9·5 ash. Determine the quantity
of products of combustion if 1 kg of the coal is burnt with 25% of excess air.
Solution. Let the basis of calculation be 100 kg of coal which contains:
Weight Mols. MoIs.of02 Products and
Constituent
kg WtJMol. wt. required Mols.
e 66·2 5·516 5·516 e02,5·516
H 4·2 2·100 1·050 H 2O,2·100
N 1·4 0·050 - -
8 2·9 0·090 0·090 8°2 ,0.090
° 6·1 0·191 0·191 -

H 2O 9·7 0·539 - H 2O,0·539
Ash 9·5 - - -
Total 6·465 mols.
100 kg of coal requires 6·465 x 10 0 = 30·79 mols of air

21
(21 mols of 02 come from 100 mols of air)
If 25% of excess air was to be used, then air supplied would be
125
= 30·79 x 100 = 38·49 mols
.. Actual excess air used = 38·49 - 30·79 = 7·70 mols.

These 7·70 mols of excess air contain
21
7·70 x 100 = 1·617 mols of 02
N2 coming from total air= 38·49 x 1~0 =30·40 mols.

:. Products of combustion per 100 kg of coal are:
CO2 ~ 5·516 mols = 5·516 x 44 = 243·3 kg
N2 ~ (30·4 + 0·05) = 30·45 mols = 30·45 x 28 = 852·6 kg
H20 ~ (2·1 + 0·539) = 2·639 mols = 2·639 x 18 = 47 kg
802 ~ 0·09 mols = 0·09 x 64 = 5·760 kg
02 ~ 1·617 mols = 1·617 x 32 = 51·71 kg
Ash ~ = 9·5 kg
.. Products of combustion per kg of coal are :
CO2 ~ 2·433 kg, N2 ~ 8·526 kg, H 2 0 ~ 0·470 kg,
802 ~ 0·0576 kg, 02 ~ 0·5171 kg, Ash ~ 0·095 kg.
FLASH AND FIRE POINT OF LIQUID FUELS

Flash point is defined as the minimum temperature at which the
combustible liquid gives off sufficient vapours to ignite momentarily when
a flame of standard dimension is brought near the surface of liquid for a
prescribed rate in an apparatus of specified dimensions. That is, the flash
immediately disappears for want of more vapours at low temperature.
At a slightly higher temperature, the heat from the flash becomes
sufficient to evaporate more liquid and maintain combustion. This lowest
temperature (usually 5°C to 40°C higher than the flash point) at which an
oil gives off sufficient vapours which when ignited continues to burn for at
least five seconds, is called the fire point of the oil.
Liquids having flash point less than 140°F are known as flammable
liquids and those with flash point more than 140°F are called combustible
liquids.
Determination of Flash Point.
1. Cleveland's open cup apparatus is used for fuel oils having flash
point below 175°F. The oil is heated with its surface exposed to air to
determine its flash point.
FUEL ANALYSIS 241
2. By Abel's Closed Cup Apparatus.

Features of the apparatus. Thermometers
• Abel's apparatus consists of a
cylindrical brass cup surrounded
by double jacketed copper water
bath which is enclosed in a
copper casing mounted on an
iron tripod. The space between
water bath and oil cup form an ~===~~ Oil cup
air bath by means of which the
oil can be heated uniformly.
• The oil cup is provided with a ff!:_=_=_~_~_~_?_H-- Air bath
brass cover having arrangement - --------
------
- -------
for a small test flame, a - -: -:-: -: -:.-..-!:=-",,:-=J-If-- Water bath
- --------
-------
sliding shutter covering three -------
--------- Jacket
small openings in the lid and
openings for a paddle stirrer
and a thermometer immersed
in the oil (Fig. 4).
Procedure. Fill the oil cup with
the oil under test up to the point of
gauge. Replace the cover. Fix the oil cup Fig. 4. Abel's flash point apparatus.
into the apparatus and assemble the paddle stirrer and thermometer with its
bulb dipping into the oil. Fill the water bath with cold water. Close the sliding
shutter and light of the standard flame. Switch on the heating device. Adjust
the rate of heating in such a way that temperature of the oil increases at a
rate of 1°C per minute. Stir the oil continuously by turning the paddle stirrer.
Stirring should be discontinued only during the introduction of the test flame
over the oil surface.
At every degree rise of temperature, open the sliding shutter and
introduce the test flame over the oil surface through the central opening to
see whether the ascending current of oil vapour gives a flash. Record the
minimum temperature at which a distinct flash appears as the flash point
of the oil. Abel's apparatus is best suited for oils having flash point below
120°F.
3. By Pensky-Marten's Closed Cup Apparatus.
Pensky apparatus is most commonly used for the determination of flash
points of oils having flash points between 50°C to 370°C.
Features of the apparatus.
Brass cup which is supported by its flange over a heating vessel. The
cover for the cup is provided with four openings of standard dimensions,
which are meant for
(i) stirrer,
(ii) standard thermometer,
(iii) an air inlet and
(iv) a device for introducing the standard flame (Fig. 5).
I+---I'f-- Shutter control

Test flame burner
:--r::::Il~~=---+1-- Oil level pointer

Opening--r-,~~
:..VA-f---\--\,-----I+- Heating vessel
Stirrer
Fig. 5. Pensky Marten's apparatus.

The shutter provided at the top of the cup has a lever mechanism.
When the shutter is turned, openings for the test flame and air are opened
and the flame exposure device dips into the opening over the surface of the
oil. The test flame gets extinguished when it is introduced into the opening
for the test but as soon as it returns to its original position on closing the
shutter, the flame is automatically lighted again by the pilot burner.
Procedure.
The oil sample under test is poured into the oil cup up to the mark. The
cover is fixed on the top. The test flame is lighted and adjusted to the bead
size of 4 mm. The apparatus is heated so that the oil temperature increases
by 5°C per minute while the stirrer is rotated at about 60 revolutions per
minute. When the temperature rises to about 15°C of the anticipated flash
point, the test flame is dipped into the oil vapour for 2 seconds at every degree
rise of temperature. This is done by twisting the knob which lowers the test
flame and opens the shutter. The flash point is taken as the minimum
temperature at which a distinct flame is observed on introducing the test
flame into the oil cup.
Oils containing traces of volatile substances are liable to flash below the
true flash point of the oil. The minimum flash point required for insulating
oils is 145°C and that for turbine oils is 165°C.
Significance of Flash Point Determination.
Flash and fire point ensures safety against fire hazards during storage,
transport, handling and use of oil.
• Flash point of an oil is often used as a means of identification and also
for detection of solvent contamination of lubricating oils.
• Flash point provides a rough guide to the base of an oil since it is
higher for the oils of paraffin base than those of naphthenic base.
FUEL ANALYSIS 243
ANILINE POINT OF LIQUID FUELS

The tendency of a lubricant to mix with aniline is expressed in terms of
aniline point of a liquid. Aniline point (or standard aniline point) is the
lowest temperature at which the oil is completely miscible with an
equal volume of freshly distilled aniline. Alternatively, aniline point is
the minimum equilibrium solution temperature for equal volumes of aniline
and the oil.
Mixed Aniline Point.
Certain lubricants with high aromatic content remain completely
miscible with equal volume of aniline and separation into different phases
may not be observed even at solidification.
To determine such low aniline point, 1 volume of the sample is mixed
with 2 volumes of aniline and 1 volume of a diluent like n-hexane or
n-heptane. Diluent lowers the solubility of aniline with the sample and so
with the decrease in temperature, separation of phases can be easily achieved.
The equilibrium solution temperature observed under these conditions is
called mixed aniline point.
Determination of Aniline Point of an Oil.
Equal volumes of aniline and oil are heated to effect complete dissolution
and then cooled under controlled conditions. The temperature at which the
entire mixture becomes cloudy throughout is called the aniline-point of the
lubricating oil.
Procedure. The apparatus (Fig. 6) is thoroughly cleaned and dried at
110°C. About 10 mL of pure and dry aniline (dried over pure KOH pellets,
filtered and freshly distilled) are taken in a heat resistant pyrex test tube
(2·5 em, 15 em) and an equal volume of the lubricating oil is added. The tube
is fitted with a cork which also holds an electrically operated stirrer and a
thermometer in such a way that the bulb of the thermometer is about 5 mm
above the bottom of the test tube. The
tube is inserted into an outer jacket
(4 em, 17·5 em). The stirrer is started [ 3 + - - - Aniline point
thermometer
and the aniline-oil mixture is observed to
find whether the miscibility is complete
at room temperature itself. If so, the Cork
jacket holding the tube is immersed in a Test tube
non-aqueous cooling bath. Cooling is Air gap
continued slowly at a rate of 0·5 to 1°C Outer jacket
per minute, until the entire aniline-oil :~::-iN-+-- Equal volumes of
mixture suddenly becomes cloudy aniline and oil

throughout. This temperature is
recorded as the aniline-point of the Fig. 6. Aniline point apparatus.
lubricating oil.
If the aniline-lubricating oil mixture is not completely miscible in the
tube at the room temperature, then slowly heat the jacket, holding the tube
either directly or by immersing in a hot bath. Stirring is continued while
raising the temperature, until the aniline-oil mixture is completely miscible.
Then the jacket holding the tube is withdrawn from the hot bath. Now, while
the stirring is continued, the temperature is allowed to fall at a rate of 1DC

per minute. A cold bath may be used for this purpose, if necessary. The
temperature, at which the entire aniline oil mixture becomes cloudy or hazy
throughout and the outline of the thermometer bulb is obscured, is taken as
the aniline-point of the oil sample.
Precautions.
• Aniline used should be perfectly pure and dry.
• The whole apparatus should be perfectly dry, because even traces of
moisture may give errorneously high results.
• Aniline is hygroscopic and hence water should not be used even in
cold and hot baths. Non-aqueous, non-volatile and transparent liquids
should be used.
• Aniline is highly toxic and hence pipettes provided with a rubber
suction bulb or an aspirator should be used.
• Stirring should be done at such a rate that undue splashing of the
liquid or formation of air bubbles are avoided.
• If the expected aniline-point is below the dew-point ofthe atmosphere,
the space above the aniline-oil mixture in the tube should be filled
with dry N 2 .
• The true aniline-point is characterised by a turbidity which increases
sharply as the temperature is lowered.
Significance of Aniline Point Determination.
Aniline point of a lubricating oil is a measure of its aromatic contents.
Hence it gives an indication of the possible deterioration of an oil when it
comes into contact with rubber seals, used in the system to prevent
leakages. Thus, lower the aniline point of a lubricating oil, the more severe
will be its attack on the rubber sealing. Obviously, a higher aniline point is
desirable for a lubricating oil, because it means that the oil contains lower
percentage of aromatics.
CARBON RESIDUE OF LIQUID FUELS

The carbon residue of a lubricating oil is expressed in terms of
percentage of carbon that is left on evaporating a known quantity of oil
under specified test conditions in a specified apparatus.
Carbon deposition in internal combustion engines results both from
incomplete combustion of the fuel as well as carbonisation of the
lubricating oil. Excessive build-up of carbon deposits in the combustion
chamber results in decreased volume of the charge at the end of the
compression stroke giving increased compression ratio which leads to
detonation.
Determination of Carbon Residue.
1. Conradson Method.
Take two glass beads (2·5 mm) in the silica crucible and weigh. Also
weigh 2 g of the oil sample in the crucible and place it in the centre of
skidmore crucible. The skidmore crucible is put in the centre of sheet iron
FUEL ANALYSIS 245
crucible containing the levelled layer of sand (Fig. 7). It is provided with a lid
having a small opening for the escape of vapours. The whole assembly of the
crucibles is covered with the hood in Bridge
order to distribute the heat •. - - - - (Flame
uniformly. The outer iron crucible is height guide)
heated strongly till the smoke
appears above the chimney. When
the vapours cease to burn and no
further smoke is observed, the
Iron crucible
bottom of the crucible is heated to
Skidmore
redness. After 10 minutes, the crucible
burner is put off and the whole Silica
apparatus is allowed to cool until no :~f--I-- crucible
smoke is seen. The silica crucible is Asbestos
transferred to a desiccator, cooled insulation
and weighed. From the weight of )+---\-\-- Meker burner
carbon deposited in the crucible, the
percentage of carbon residue in the
oil can be calculated. Fig. 7. Conradson apparatus.
Calculations. Weight of the crucible + beads = WIg
Weight of the crucible + beads + oil = w2fJ
Weight of the crucible with carbon residue = wgg
Weight of the oil taken = (w2 - WI) g
Weight of the carbon residue obtained = (w3 - WI) g
w3- w l
Percentage of carbon residue in the oil = x 100
w2- w l
2. Ramsbottom Method.
1 to 4 g ofthe oil sample is taken in a stainless steel bulb which is placed
in a sheath consisting of an iron tube having a flat closed end. The sheath is
immersed in a bath of molten lead which is heated to 550°C for 20 minutes.
The bulb is taken out, cooled and weighed. The result is reported as 2%
carbon residue to the weight of oil taken for the experiment.
Calculations. Weight of the oil taken = WIg
Weight of the carbon residue obtained = w2fJ
Percentage of carbon residue in oil = w2 x 100

WI
Note. Expected percentage of carbon residue is 2% for the 4 g of oil
sample taken in the coking bulb.
OCTANE NUMBER OF LIQUID FUELS

Octane number is an index of knocking of a gasoline in the internal
combustion engine. It may be defined as the percentage of iso-octane present
in a mixture of n-heptane and iso-octane, that produces the same knocking
characteristics as the gasoline operated in a standard one-cylinder engine.
Iso-octane or 2, 2, 4-trimethyl pentane is adopted as a standard

antiknock fuel with octane number 100. n-Heptane has maximum
knocking property with octane number zero.
CHs CHs
I I
HsC-C- CH2-C- CHs 2, 2, 4-Trimethyl pentane or iso-octane
I I (octane no. = 100)
CHs H
HsC-CH2-CH2-CH2-CH2-CH2-CHs
n-Heptane (octane no. = 0)
In alkane series, octane number decreases as the carbon chain is
lengthened and increases with branching of chains like n-pentane (61),
2, 2-dimethyl pentane (83), 2, 3-dimethyl butane (95), 2, 2, 4-trimethyl
pentane (100). Ordinary gasoline containing large amount of straight chain
paraffins, and also small amounts of olefins, naphthens and aromatics have
octane number betwen 20 to 73. Octane number of benzene is 106 and that
of ethyl alcohol 95.
Iso-octane and other branched paraffins igniting under high
compression, have much less knocking tendency, hence the gasoline which
contains larger amount of branched paraffins has high octane value.
Isomerisation, alkylation, aromatisation, reformation increase the octane
number of fuels. The cracked gasoline containing high octane constituents
have octane number nearly 80. Alkylates formed from isobutane and gaseous
olefins have high octane number. Hence aviation gasoline with octane
number 100 is made artificially by blending straight chain run gasoline,
cracked gasoline and alkylates. Motor method, temperature method and
pressurisation methods are used to determine octane number of motor and
aviation gasolines.
Points to Note.
• Triptane (2, 2, 3-trimethyl butane) with octane number 124 is
superior than iso-octane. It is also a better antiknocking agent than
TEL.
• Ethyl fluid, a mixture of TEL (60%), ethylene dibromide (26%),
ethylene chloride (9%) and a red dye (2%), is added to improve
antiknock property of fuel petrol.
• Octane number of regular gasoline is 74 and that of premium
gasoline 81. The higher the octane number, better is the quality of
a fuel.
CETANE NUMBER
Cetane number, which expresses ignition quality of a fuel, is defined as
the percentage of cetane (cetane is n-hexadecane, C16H s4 , value = 100) in
a mixture of cetane and ex-methyl naphthalene that will have the same
ignition characteristics as the fuel under examination. The ignition quality of
hydrocarbons decreases in the order :
n-alkanes > naphthenes > alkenes > branched alkanes > aromatics.
Diesel engines require a fuel of cetane number more than 45 which can
be increased by adding ethyl nitrite and amyl nitrite etc.
o
11
CLINICAL CHEMISTRY
COMPOSITION OF BLOOD
Blood is a fluid that circulates in a closed system of blood vessels. Whole
blood can be divided into two main components as follows :
1. Plasma (55%).
Blood plasma, the non-cellular fraction, contains about 92% water and
8% solids. Solid portion consists of following constituents.
(A) Organic Substances (7% to 8%). Organic substances include
(i) Proteins (7%). Proteins are the main constituents among the
non-diffusible compounds. Proteins present in blood include
haemoglobin, albumin, globulin, fibrinogen and
prothrombin.
These plasma proteins help in regulating blood viscosity and blood
volume and can exert an osmotic pressure of 25-30 mm. Fibrinogen
helps in blood clotting.
(ii) Non-Protein Nitrogenous Substances. These are the products
of tissue activity and are transported from tissues to kidneys or
skin for excretion. Non-protein substances include urea, uric acid,
creatinine, ammonia, free amino acids, bilirubin, xanthine, choline,
thyroxin, ATP, neutral fats such as glucose, pentoses, cholesterol
and phospholipids.
(iii) Hormones. Hormones or internal secretions are secreted by
endocrine glands and act as chemical messangers.
(iv) Antibodies. Antibodies maintain blood viscosity and are
important in immunity reactions.
(v) Enzymes. Enzymes act as catalysts and include lipase, amylase
and sucrase etc.
(vi) Organic Acids. Traces of organic acids such as lactic acid, pyruvic
acid, malic acid, citric acid, acetoacetic acid, ~-hydroxybutyric acid
have also been found in blood.
(B) Inorganic Substances (0.99%). The diffusible electrolytes present
in plasma include Na+, K+, Ca2+, Mg2+, cr, SO~-, PO~- and HCOg etc.
(C) Respiratory GaSes. 02 and CO2 are also the main constituents of
plasma.
Note. Plasma is thus the liquid portion of circulating blood. The cells
are separated from the plasma by centrifuging whole blood. If blood is
allowed to clot, the fibrinogen is removed with the cells, leaving serum. The
(247)
majority of clinical analyses are performed on whole blood, plasma and

mainly on serum.
2. Cellular Elements.
Cellular elements constitute about 4.5% of the total volume of blood
containing following free blood cells or corpuscles.
(A) Erythrocytes or Red Blood Corpuscles. RBCs contain
haemoglobin and transport 02 and CO2 from one tissue to the other. These
are produced in bone marrow of flat bones, ribs, heads of femur and humerus
etc. RBCs do not have nucleus and range to 7.2 micron in diameter. Normally
50,00,000 per cubic mm erythrocytes are present in cellular fraction with life
of 120 days.
(B) Leucocytes or WBCs. A normal adult man has 5000 to 10,000 per
cm WBCs in blood. There is only one WBC for every 500 RBCs. These cells
do not contain any pigment. The main function of leucocyte is body defence
against infection by the phagocytes and production of antibodies by the
lymphocytes. Leucocytes are of two types.
(i) Granulocytes such as basophil, eosinophil and neutrophil
polymorph.
(ii) Non-granular leucocyte like monocyte and lymphocyte.
(C) Platelets or Thrombocytes <thromb = clotting, cyte = cell).
Platelets are non-nucleated fragments of cytoplasm which prevent bleeding
by coagulating the blood when injuries occur to the blood vessels. These are
produced in spleen and bone marrow and varies from 2,50,000 to 5,00,000 per
cubic mm in blood. Platelets are flat cells with 2-4 microns in diameter.
Table 1 summarizes information pertinent to blood samples for clinical
examinations.
Table 1. Normal range of concentrations of clinically important
constituents in human blood.
Constituent Sample Nonnal range
Albumin Serum 4-5 g/dL
Amino acid nitrogen Blood 4-6 mgldL
Ammonia Blood 40-125 J.1g!dL
Amylase Serum Up to 150 mgldL
Bilirubin Serum 0·4 to 1·0 mgldL
Calcium Serum 4·5-5·5 meqIL
Carbon dioxide content Serum 25-32 meqlL
Chloride, serum Serum 100-108 meqlL
Cholesterol, total Serum 140-250 mgldL ,
Cholesterol, esters Serum 50-65% of total
Creatinine Serum 07-1·7 mg.ldL
Creatinine clearance 100-180 mLlmin
Lipase Serum Up to 1·5 units
Contd . ...
CLINICAL CHEMISTRY 249
Constituent Sample Normal Range

Lipids, total Serum 350-800 mg/dL
Fatty acids Serum 200-400 mg/dL
Globulins, total Serum 2·5-3·5 g/dL
alpha-1 0·1-0·4 g/dL
alpha-2 0·3-0·7 g/dL
beta 0·4-0·9 g/dL
gamma 0·6-1·3 g/dL
Iron, serum Serum 50-180 ~g/dL
Magnesium Serum 1·5-2·5 meqlL

Nonprotein nitrogen (NPN) Blood 25-40 mg/dL
Phosphatase, acid Serum Up to 4 Gutman units
Phosphatase, alkaline Serum Up to 4 Bodansky units
Phospholipids Serum 100-250 mg/dL
Phosphorus, inorganic Serum 3-4·5 mg/dL
Potassium Serum 3·8-5·6 meqlL
Protein, total serum Serum 6·5-8 ~g/dL
Protein-bound iodine (PBI) Serum 3·5-8 ~g/dL
Sodium Serum 138-146 meqlL

Sugar, blood (glucose) Blood 65-90 mg/dL
Transaminase(S~) Serum Up to 40 units
Uric acid Serum 3-6 mg/dL
Urea nitrogen (BUN) Blood Up to 20 mg/dL
FUNCTIONS OF BLOOD
Blood transports oxygen from the lungs to the tissues and CO2 from the
tissues to the lungs. Blood is thus responsible for respiration.
• Blood performs nutritive function by transporting absorbed dietary
materials to all the body tissues.
• Blood also performs excretory function by transporting metabolic
wastes to kidneys, lungs, skin and intestine for removal.
• It provides the temperature regulating and defensive mechanism. The
ability of the body to maintain constant temperature is mainly due to
high specific heat of water.
• The efficient buffer system present in blood helps in maintaining a
constant pH in and around tissues.
• Along with the lymph and tissue fluids, blood provides the connecting
link between individual cells of organs and tissues.
COLLECTION AND PRESERVATION OF SAMPLES

Blood and urine samples are often collected after the patient has fasted
over night, particularly for glucose and cholesterol analysis. Studies indicate
that an average breakfast has no significant effect on concentration of the

blood contents of CO2 , Na+, K+, Ca2+, CI-, urea nitrogen, uric acid, creatinine,
albumin and total proteins.
Collection of Blood for Plasma or Whole Blood Analysis.
If plasma or whole blood is required for analysis, then the blood is
collected in a tube containing an anticoagulant. Heparin (sodium salt) is
frequently used. But its effect is temporary and it is expensive. Potassium
oxalate (1 mg/mL) is more widely used. Oxalate precipitates blood calcium
and the calcium is required in the clotting process.
The usual practice in preparing whole blood or plasma sample is to
add the required amount of oxalate in solution form to the collection tube and
then to dry the tube in an oven at 110°C. By this procedure, the collected
blood is not diluted. For example, 0.5 mL of a 2% potassium oxalate solution
would be taken and dried for a 10 mL blood sample.
Potassium oxalate causes RBCs to shrivel, with the result that the
intracellular water diffuses into the plasma. Hence plasma should be
separated immediately. Ammonium oxalate, salts of EDTA, trisodium citrate,
and acid-citrate dextrose (in blood banking) are also used as anticoagulants.
Collection of Blood for Serum Analysis.
When serum is required for analysis, the blood is collected in a clean and
dry tube to prevent contamination and hemolysis. Hemolysis is the reduction
of red cells with the liberation of haemoglobin and other cell constituents into
the surrounding fluid (serum or plasma). When hemolysis occurs, the serum
is red instead of its straw colour. Hence blood samples should be centrifuged
as soon as possible to separate serum or plasma from the cells.
Collection of Blood for Glucose Analysis.
Sodium fluoride is ~idely used as a preservative (along with
anticoagulant) for samples to be analysed for glucose. NaF is an enzyme
inhibitor that prevents the break down of glucose. 1 mg of NaF per mL of
blood is adequate. However, NaF should not be added to samples to be
analysed for enzymes or for urea.
Collection of Blood for CO 2 Analysis.
Mineral oil is added to the collection tube. This oil is lighter than blood
and will cover it. The sample must be kept anaerobically.
Collection of Blood of an HIV Infected Patient.
The blood from HIV or AIDS patients should be collected by wearing
gloves and masks.
Storage of Blood Samples.
Blood samples can be stored for one or two days by refrigerating them.
This slows down enzymatic and bacteriological processes but does not
eliminate them, so it is better to analyse fresh samples. Samples are always
brought to room temperature before analysing them. Whole blood should
not be frozen because the red cells will be ruptured. Plasma and serum
samples fractionate into layers of different composition when frozen and so
should be shaken gently after thawing. Samples are more stable if a
protein-free filtrate (PFF) is prepared. Glucose is stable in PFF because the
glycolytic enzymes are removed.
CLINICAL ANALYSIS
SERUM ELECTROLYTE.
Physiologically serum electrolytes (ionic solutes) include Na+, K:, Ca2+,
s.
M~+, pot and HCO All higher life forms require a subtle and complex
electrolyte balance between intracellular and extracellular millieu. The
maintenance of precise osmotic gradients of electrolytes is extremely
important to regulate blood pH, hydration of the body and for nerve and
muscle functions. Electrolyte drinks containing Na+, K+ salts and Gatorate
(sports drink) are used to replenish dehydration caused by exercise,
starvation, diarrhoea and diaphoresis etc.
. Electrolyte balance is regulated by hormones, generally by the kidneys
flushing out excretory products. In humans, hoemostatis is regulated by
antidiuretic hormone, aldosterone and parathyroid hormones. Serious
electrolyte disturbances may lead to cardiac and neurological
complications.
Clinical analysis of serum electrolytes is performed by blood
testing, estimating blood chloride, Ca2+, Na+, K+, RCO) and urine analysis.
ESTIMATION OF BLOOD CHLORIDE (SERUM ELECTROLYTE)

Chloride, the major extracellular anion of the body, plays an important
role in several physiological processes such as osmotic control, acid-base
s
balance, CI--HCO shift etc. About 33% of total chloride is present in RBCs
and 67% in the serum.
1. Schales and Schales Titration Method.
Principle. When mercuric nitrate solution is added to a chloride
solution, un-ionised mercuric chloride is formed.
Hg(N03)2 + 2Cr ~ HgCl2 + 2NO s
The first excess of mercuric ions yield blue-violet colour at the end point
with diphenyl carbazone indicator. Titration can be carried out directly with
serum but strongly coloured or jaundice specimens obscure the end point. In
such cases a Folin-Wu or tungstic acid method is used to obtain the filtrate.
Here 1 volume of serum is mixed with 7 volumes of water and 1 volume of
0·33 M H 2S04 and allowed to turn brown. Then 1 volume of 10% sodium
tungstate (Na2W04.2H20) is added. After 2 minutes, the precipitated matter
is filtered. '
Reagents.
(i) 10% Sodium tungstate.
(ii) 0·66 N H 2S04 ,
(iii) Mercuric nitrate solution. Dissolve 3g Hg(N03)2 in 500 mL of

distilled water. Add 3 mL of concentrated HN03 and make up the
volume to 1 L.
(iv) 0·1% Diphenyl carbazone solution in ethanol.
(v) Sodium chloride solution. Dissolve its 585 mg in 1 L to obtain
10 meqlL solution.
Procedure.
(a) Standardisation of mercuric nitrate solution. Take 2 mL of
standard NaCl solution in a 50 mL flask. Add 4 drops of diphenyl carbazone
indicator and titrate it with mercuric nitrate solution till the colour changes
from light yellow to purple. Note the burette reading.
(b) Titration of the Sample.
(i) To 1 mL of serum or plasma or whole blood, add 7 mL distilled
water, 1 mL of 10% sodium tungstate and 1 mL of 0·66 N H 2S04 ,
Stir the mixture for 5 minutes and then filter.
(ii) Take 2 mL of filtrate in a 50 mL conical flask and titrate it with
mercuric nitrate as described earlier. Record the burette reading.
Calculation. Let the concentration of Cl- (expressed as NaCl) in the
sample be X meqIL. Serum is diluted 10 times during protein precipitation.
~= Volume of mercuric nitrate used for sample x 10
10 Volume of mercuric nitrate used for standard
X IL = Vol. of mercuric nitrate used for sample 100
meq Vol. of mercuric nitrate used for standard x
2. Automatic Coulometric Titration.
Chloride is most widely determined by automatic coulometric titration
with electrogenerated silver ion.
Normal Values. Serum plasma = 98 - 106 meqIL
Whole blood = 77 - 86 meqlL
Clinical Interpretation. Increased plasma chloride is observed in
nephritis, hence dietary salts should be restricted. Decreased plasma chloride
may occur in gastro-intestinal disturbances associated with vomiting or
diarrhoea. Chloride content is also depleted in pneumonia and Addison's
disease.
ESTIMATION OF SODIUM AND POTASSIUM

(SERUM ELECTROLYTE)
In the whole blood N a and K are present in the ionic form. About 90%
of the total blood sodium is found in plasma, whereas :I(+ is present in the
cells. Na+ and K+ ions maintain osmotic pressure and acid base balance in the
body.
Flame Spectrophotometric Method.
The sample in solution form (1 : 50) is introduced in the form of a fine
continuous spray into a non-luminous gas using either acetylene, propane or
butane gas. Light is emitted by the use of colour filter or diffraction grating.
This emitted light of a wavelength characteristic for N a + and K+ is isolated

and focussed on a photoelectric cell. The electrical response to the
photoelectric cell is measured on a meter previously calibrated to convert the
electrical impulse to ion concentration either by direct reading or by reference
to a calibration curve. Or measure at 766 nm for K+ and at 589 nm for Na+.
Concentration Range. Na+ concentration in serum ranges from 300
to 350 mg/100 mL or 130 to 154 meqlL. K+ concentration ranges from 14 to
22 mg/100 mL or 3·6 to 5·5 meq/L.
Clinical Interpretation. An increased concentration in serum
potassium causes bronchial asthma, fever and Addison disease. A decreased
concentration in serum potassium is due to severe vomiting or diarrhoea.
ESTIMATION OF SERUM CALCIUM

Calcium is present in serum as
(i) Ionised calcium,
(ii) Calcium bound to proteins and
(iii) Calcium bound to other organic substances. Most' of the
physiological functions of Ca depend on the ionised fraction but for
routine analysis, only total Ca in serum is estimated.
1. Atomic Absorption Spectrophotometric Method.
For the estimation of Ca, dilute1: 20 sample solution with 10000 ppm
Na2 EDTA and measure absorbance at 422·7 nm.
2. Clark and Collip Method.
Principle.
Serum is treated with ammonium oxalate to precipitate calcium oxalate.
The precipitate is washed with NHg to remove excess oxalate and then
treated with H 2S04 to convert calcium oxalate into oxalic acid. The oxalic acid
is titrated with standard KMn04 solution.
Reagents.
(i) Ammonium oxalate, 4%.
(ii) NHg , 2%.
(iii) H 2S04, 1N, 28 mU1000 mL H 20.
(iv) KMn04' 0·01 N. Standardised against -0·01 N sodium oxalate. 1 mL
of 0·01 N KMn04 is equivalent to 0·2 mg of Ca.
(v) Stock KMn04' 0·1 N. 23-16 g KMn04 in 100 mL water. Filter
through asbestos.
(vi) Sodium oxalate. 0·01 N (0·67 g of sodium· oxalate is dissolved in
water. 5 mL concentrated H 2S04 is added. Make the volume to
1000 mL with water). 1 mL of 0·01 N sodium oxalate = 1 mL of 0·01
N KMn04.
Procedure.
Mix thoroughly 2 mL of serum, 2 mL of water and 1 mL of 4%
ammonium oxalate and centrifuge for 15 minutes in a centrifuge tube. Pour
off the supernatant fluid and drain the tube. Add 3 mL of 2% NH g, shake,
centrifuge and drain. Add 2 mL of 1 N H 2S04 and shake. Keep the tube in
boiling water bath until the precipitate dissolves. '
Titrate the hot solution with 0·01 N KMn04 until a pink colour develops.
Let the volume of 0.01 N KMn04 solution is X mL. For blank, take 2 mL of
1 N H 2S04 and titrate it with 0·01 N KMn04' Suppose the volume of 0·01 N
KMn04 used is Y mL.
Calculations.
0·2
Serum Ca (mg/dL) = (X - y) x 2 x 100
=(X - y) x 10
Clinical Interpretation. Serum calcium ranges from 9-11 mg/dL in
healthy persons. The product of serum Ca and serum P is about 40 in adults
and 50 in children. Excess of serum calcium causes hyperparathyroidism,
multiple myeloma, sarcoidosis, hypervitaminosis, idiopathic infantile
hypercalcemia, alkali syndrome and polycythaemia. Decrease in serum
calcium results in hypoparathyroidism, osteomalacia, nephrotic syndrome,
renal failure, acute pancreatitis, starvation and rickets.
Estimation of Calcium in Urine. Dilute urine in water (1 : 10) and
determine Ca as described above. Multiply the result by 10.
ESTIMATION OF SERUM BICARBONATE

Titrimetric Method.
Principle. HCOg in blood is determined by adding excess of 0·01 M
HCI to volatilise it as CO2 , Swirl to allow CO2 to escape. Back titrate the
excess HC! with 0·01 M NaOH.
HCOg+H+ ~ H 20 + CO2 i
Excess
Reagents.
(i) 1% NaCI (saline solution) in CO2 free water.
(ii) Phenol red solution, 0·1% in 0·003 M NaOH.
(iii) Antifoam A.
(iv) Prepare and standardise 0·1 M HCI and 0·1 M NaOH.
(v) Freshly prepare 1 L of 0·01 M HCI and 0·01 M NaOH solutions by
diluting 100 mL of 0·1 M solutions to 1000 mL with saline solution.
Saline helps in volatilisation of CO 2 from the acidified solution by
decreasing its solubility.
Preparation of the Sample.
To 10 mL of the fresh blood sample, add NaF to prevent glycolysis of
glucose which can change the pH. Keep the sample tube anaerobically.
Preparation of Comparison Solution.
• Take 6 mL of 1% saline solution in a 25 mL flask and add 0·10 mL of
serum.
\
• Add 2 drops of phenol red indicator and shake the contents vigorously.
• Because of the buffering action of the blood, yellow to red colour
change occurs from pH 8·4 to 6· 7.
Procedure.
• The pooled serum or plasma sample should be prepared by touching
the end of a stirrer to Antifoam A and rotating it in the pooled sample.
This will prevent excess foaming when the sample is swirled.
• Place 0·1 mL serum or plasma in a 25 mL Erlenmeyer flask and add
1 mL of 0·01 M HCI and 4 mL of 1% saline. Swirl the flask to escape
CO2 ,
• Add 2 drops of phenol red and titrate with 0·01 M NaOH dropwise
until pink colour appears.
Calculations. Since 0·1 mL of serum was taken for analysis, it should
consume 0·26 mL of 0·01 M HCI. Hence 0·74 mL of HCI should remain
unreacted. Back titration should consume 0·7 mL of 0·01 M NaOH. Normal
s
value of HCO in blood is 26 meqlL.
ESTIMATION OF BLOOD GLUCOSE

Blood contains, in addition to glucose, many other reducing substances
like glutathione, creatinine, lactose, galactose, pentoses, amino acids and uric
acids. If these substances are not removed from the blood prior to estimation,
they will cause over-estimation of true glucose level.
Different methods remove these substances to different extent, so that
the normal value of blood glucose depends upon the method of its estimation.
1. Folin and Wu (1920) was the first most widely used method for the
reduction of alkaline cupric sulphate to cuprous oxide.
2. Astor and King Method. Blood is placed in isotonic sodium
sulphate-copper sulphate solution so that no further copper sulphate is
required. Sodium tungstat.e precipitates proteins, copper tungstate and
non-glucose reducing compounds. Glucose reduces alkaline Cu2+ ions to Cu+
state. The cuprous oxide reduces phosphomolybdic acid to molybdenum blue
which is measured photometrically.
Reagents.
(i) Isotonic sodium sulphate-copper sulphate solution. Dissolve
30 g Na2S04.10H20 and 6 g CuS04.5H20 in distilled water. Make
up the volume to one litre.
(ii) Sodium tungstate solution (10%). Dissolve 10 g
Na2W04.2H20 in 100 mL of water.
(iii) Phosphomolybdic acid reagent. Take 35 g molybdic acid, 5 g
sodium tungstate and 20 g NaOH in one litre beaker. Add 300 mL
water and boil for 30 minutes to drive off ammonia present in
molybdic acid. Cool and transfer to a 500 mL volumetric flask. Add
125 mL of 85% H 3P04 and make up the volume to 500 mL with
water.
(iv) Alkaline tartrate solution. Dissolve 25 g NaHC03, 20 g
anhydrous Na2C03 in 500 mL water in one litre beaker. Weigh 18 g
of potassium oxalate, dissolve it in warm water and add into the
carbonate mixture. Add 12 g of dissolved sodium potassium tartrate

to the carbonate solution and make the volume to one litre.
(v) Stock standard glucose solution. Dissolve 100 mg of pure
glucose in 100 mL of isotonic sodium sulphate-copper sulphate
solution.
(vi) Working standard glucose solution. Pipette accurately 2 mL of
the stock standard glucose solution in a 100 mL volumetric flask
and make up to the mark with isotonic sodium sulphate-copper
sulphate solution.
1 mL = 0·02 mg glucose
Procedure.
To 0·1 mL of blood serum or plasma, add 3·8 mL of isotonic
Na2S0rCuS04 solution and 0·1 mL of 10% sodium tungstate, mix and
centrifuge for 5 minutes. Transfer the supernatant to a clean tube and discard
the precipitate. Set three test tubes and mark them as test, standard and
blank. Add 1 mL isotonic Na2S0rCuS04 solution and 2 mL alkaline tartrate
solution to each tube. Add 1 mL supernatant to tube marked test, 1 mL
standard glucose solution to tube marked standard and 1 mL distilled water
to tube marked blank. Mix well and heat in a boiling water bath for 10
minutes. Cool and add 3 mL of phosphomolybdic acid to all the tubes. Mix
well and read the absorbance after 5 minutes against blank at 680 nm.
Calculations. mg of Glucose in testl(0·25 mL blood/plasma/serum)
= °pOptical density of test

t'lCaI d enSI'ty f st and ard x
0
0·02 (cone. of std/tube)
ODT 0·02 ODT

Blood/serum/plasma glucose (mg/dL) = ODs x 0.25 x 100 = ODs x 80
Normal Fasting Values.

Plasma or serum = 70-110 mg/dL
Vepous or capillary whole blood = 60-100 mg/dL
Renal threshold for glucose = 180 mg/dL
3. O-Toluidine method was also used for the determination of true
glucose but it is now no longer used because of its carcinogenic properties.
• Prepare protein free filtrate (PFF) with barium hydroxide and zinc
sulphate which removes most interfering nonglucose-reducing
substances.
Ba(OH)2 + ZnS04 ~ BaS04 + Zn(OH)2
Glucose is stable in a protein-free filtrate because the glycolytic
enzymes are removed.
• Oxidise sugar (glucose) with alkaline Cu (II) to form CU20.
• CU20 is determined by allowing it to reduce phosphomolybdic acid to
form molybdenum blue which is measured at 420 nm to estimate
glucose in blood or serum.
5. Enzymatic Method.
Enzymatic determination of glucose is an established method. The
enzyme glucose oxidase catalyses the aerobic oxidation of glucose to gluconic
acid and H 2 0 2 .
Glucose
C6 H 120 6 + 02 + H 20 --~
Oxidase
Actually, this enzyme shows almost complete specificity for ~-D-glucose,
a-D-glucose reacts at a rate 0·64 relative to 100 for the ~-form. In the ~-form,
all the hydrogens are axial and the hydroxyl groups are equatorial, allowing
the molecule to lie down flat on the enzyme active site and form the
enzyme-substrate complex. The a-form can not lie flat on the enzyme. Thus
the aerobic conversion of glucose (36% a, 64% ~) depends on the mutarotation
of the a-form to ~-form. Mutarotation is shifted as the ~-form is removed,
thereby analysing the glucose in blood sample.
6. Biosensor Autoanalysers to Estimate Blood Glucose.
The diabetic simply places a small drop of blood (from a sterile
lancet-prick) on a test strip. Glucose oxidase on the strip catalyses the
oxidation of glucose to produce H 2 0 2 , which is measured either
electrochemically or photometrically.
Clinical Interpretation. Blood glucose is increased in hyperthyro-
idism, hyperadrenalism, hyperpituitorism, diabetic mellitus and decreased in
Addison's disease, glycogen storage and excessive insulin secretion.
Estimation of blood sugar helps to detect cases of border line-frank diabetes
and to prescribe doses of antidiabetic drugs used to treat diabetic patient.
Blood sugar can generally be estimated as
(i) Fasting blood sugar,
(ii) Post prandial blood sugar (two hours after meal) and
(iii) Glucose tolerance test.
Here the blood samples are collected at half an hour interval for 2·5
hours after administration of 75 g of glucose orally and analysed for sugar.
Examination of urine sample is also conducted simultaneously for 2·5 hours.
Note that glucose disappears from blood on standing due to glycolysis
at the rate of 15 mg/100 mUhour at 35°C. An antiglycolytic agent such as NaF
must be present in blood samples to be used for glucose estimation.
ESTIMATION OF BLOOD UREA

Urea is the main end product of protein catabolism. In liver, amino acids
are deaminated to form urea which enters into the blood and excreted by
kidneys with the urine. Plasma contains 15 to 40 mg of urea per 100 mL.
Blood urea increases if excretion is impaired.
1. Fearon Method.
Serum urea nitrogen can be determined by direct interaction of urea
with diacetyl monoxime.
2. Crocker Method.
Principle. Urea reacts with diacetyl monoxime (DAM) and
thiosemicarbazide (TSC) in presence of Fe3+ ions to form pink complex which
is measured colorimetrically at 520 nm.
Reagents.
(i) 0·1 % Benzoic acid.
(ii) 10% H 2 S04 .
(iii) Stock diacetyl monoxime. Dissolve 6·25 g DAM in 250 mL of
distilled water.
(iv) Stock thiosemicarbazide. Dissolve 1·25 g TSC in 250 mL of
distilled water.
(v) Stock FeCla.HaP04 reagent. Dissolve 3·34 g FeCI3.6H2 0 in
65 mL H 3P04 (85%). Make up the volume to 100 mL.
(vi) Working acid FeCls • Dilute 1·3 mL stock FeCl3 to 1 L with
10% H 2S04 .
(vii) Working DAM. Mix 67 mL of stock DAM with 67 mL stock
thiosemicarbazide and dilute it to 500 mL with distilled water.
(viii) Stock urea nitrogen standard (1·0 mg/mL). Dissolve 0·2143
g pure urea in 100 mL of 0·1% benzoic acid.
(ix) Working urea nitrogen standard (1 mg/dL). Dilute 1 mL of
stock standard to 100 mL with 0·1% benzoic acid.
Procedure. Dilute 0·25 mL serum/plasma to 5 mL with 0·1% benzoic
acid. Set the experiment as illustrated below.
Reagent Test Standard Blank
Diluted serum/plasma 1mL - -
Working urea nitrogen standard - 1mL -
Working DAM-TSC reagent 2mL 2mL 2mL

0.1% Benzoic acid 1mL 1mL 2mL
Working acid FeCl3 3mL 3mL 3mL
Mix the contents and place all the tubes in boiling water bath for 10
minutes. Cool the tubes and read the absorbance of the tubes against blank
at 520 nm.
Calculations.
ODT
mg of Urea Ntrube (0·05 mL blood) = ODs x 0·01 (conc. of std.ltube)
OD = Optical density
ODT 0.01
Blood urea nitrogen (BUN) (mg/dL) = ODs x 0.05 x 100
ODT
Conc. of urea N = OD x 20
s
Clinical Interpretation. Protein contents in diet influence urea level.
It is lower in people with low protein diet. In elder persons, urea level may
be slightly higher even without significant renal dysfunction. In pregnancy,

blood urea level ranges between 15-20 mg/l00 mL of blood. Urea level also
decreases in blood in case of acute liver diseases. Blood urea level over 50
mg/l00 mL may be due to following impaired renal functions.
Pre renal causes include dehydration, diarrhoea, vomiting, diabetic
coma, intestinal obstruction, extreme hypotension etc.
Renal causes may be defective kidneys, glomerulonephritis, congenital
cystic kidneys, tubular necrosis.
Post renal causes may be obstruction in urine flow, tumour of urinary
bladder, enlarged prostate gland etc.
Since urea diffuses very readily through body fluids, so similar results
are obtained if the estimation is carried out using cerebrospinal fluid, plasma,
serum or blood.
ESTIMATION OF BLOOD UREA NITROGEN (BUN)

BUN test is a measure of the amount of nitrogen in blood that comes
from urea.
Spectrophotometric (Enzymatic) Method.
(i) Preparation of Thngstic acid Protein Free Filtrate (PFF).
1 volume of blood, serum or plasma is mixed with 7 volumes of water and 1
volume of 0·33 M H 2S04 and allowed to tum blue. Then 1 volume of 10%
sodium tungstate added. Mter 2 minutes, the precipitated protein is filtered
or centrifuged.
(ii) Incubate the above tungstic acid PFF with urease enzyme at pH 6·8
to produce NH 3 . Determine the NHs with Nessler's reagent and measure
absorbance at 480 nm.
Calculation of Blood urea nitrogen.
BUN = 60 (Mol. wt. of urea) x NPN
28 (Mol. wt. of 2N)
Non-protein nitrogen (NPN) = 2·143 x BUN
Normal values. BUN = 7 - 18 mg/dL
NPN = 20 - 40 mg/dL
Non-protein nitrogen, NPN refers to all N containing substances other
than proteins such as creatine, creatinine, peptides, uric acid and amino acids.
Clinical Interpretation. Normal human adult blood contains 7 - 25
mg of urea nitrogen per 100 mL of blood. Elevated BUN can be due to
gastrointestinal hemorrhage, dehydration or shock. Excess blood proteins are
reabsorbed by the gut thereby increasing BUN in the urea cycle. Heart attack
also raises BUN. A greatly elevated BUN (> 100 mg/dL) indicates renal
failure. A low BUN may be due to liver problems, poor nutrition, over
dehydration possibly from intravenous fluids.
Note. Estimations of BUN, NPN or serum creatinine level are
commonly used instead of blood urea for assessing kidney functions.
ESTIMATION OF URIC ACID IN SERUM

Nucleotides, resulting from degradation of nucleic acids, undergo
enzymatic hydrolysis to yield free purines and pyrimidine bases. These free
bases degrade further and the end products are excreted from the body. Uric
acid is the product of purine metabolism in humans.
Colorimetric Method.
Principle. Oxidise tungstic acid protein free filtrate with alkaline
phosphotungstic acid and measure the blue reduction product of
phosphotungstate at 680 nm.
Reagents.
(i) 10% Sodium tungstate.
(ii) 0·66 N H 2S04 , Slowly add 18·8 mL of concentrated H 2S04 to 500
mL water and dilute to 1 L.
(iii) 7% Na2C03'
(iv) Phosphotungstic acid reagent. Dissolve 40 g sodium tungstate
in 300 mL water. Add 30 mL H 3P0 4 . Reflux for 2 hours, cool and
dilute to one litre. Mix 32 g lithium sulphate in the reagent and
store in a cool place.
(v) Uric acid stock standard (1 mg/mL). Dissolve 100 mg uric acid
and 60 mg lithium carbonate in 50 mL of distilled water. Heat to
60°C, cool and dilute to 100 mL in a volumetric flask.
(vi) Uric acid working standard (0·02 mg/mL). Dilute 2·0 mL of
stock standard uric acid solution to 100 mL with distilled water.
Procedure.
To 1 mL of serum, add 8 mL of distilled water, 0·5 mL of 10% sodium
tungstate and 0·5 mL of 0·66 N H2S04 , Mix thoroughly and centrifuge. Use the
supernatant for uric acid estimation. Set the experiment as follows :
Reagent Test Standard Blank
Supernatant 2mL - -
Uric acid standard - 2mL -
Distilled water - - 2mL
Sodium carbonate 2mL 2mL 2mL
Phosphotungstic acid 3mL 3mL 3mL
Mix the contents thoroughly and allow to stand for 15 minutes. Read the
absorbance against blank at 680 nm using red filter.
Calculations.
ODT
mg of Uric acidlfube (0·2 mL blood) = ODs x 0·04 (conc. of std/tube)
ODT 0·04
Serum uric acid (mg/dL) = ODs x 0.2 x 100
ODT
=--x20
ODs
Normal value of uric acid in serum is between 2 to 7 mg/dL.
Clinical Interpretation. Elevated uric acid level (7 to 12 mg/100 mL

of serum) is found in gout. It is a disease of joints in which urates get
deposited as crystals. High level of blood uric. acid (4 to 20 mg/100 mL of
serum) is also observed in impaired renal functions, leukemia, toxemia of
pregnancy, lobar pneumonia and large abcesses accompanied by increased
excretion of uric acid in urine. The excessive break down of cells in these
conditions results in increased metabolism of nucleoproteins which are
oxidised to uric acid in urine. Decreased level of uric acid results in Fanconi
syndrome and Wilson disease.
ESTIMATION OF TOTAL SERUM PROTEIN

1. Gel Electrophoresis.
Starch gel electrophoresis can be used to separate protein fractions
(a, ~,y globulins) in blood serum. There exists two classes of blood proteins,
i.e., albumin and globulin. These are almost equal in proportion but
albumin is much smaller and slightly negatively charged, leading to its
accumulation on the electrophoretic gel.
Interpretation. A small band before albumin represents transthyretin
(pre-albumin). On the basis of band pattern, globulins are classified into
following types.
(i) The alpha band consists of aI-antitrypsin, <Xl-acid glycoprotein.
a2-haptoglobulin, a2-macroglobulin and ceruloplasmin.
(ii) Beta band is due to transferrin and LDL.
(iii) Gamma band contains immunoglobulin (lgA, IgD, IgE and .IgM)
and paraproteins.
2. Micro-Kjeldahl technique for the estimation of serum protein
(mainly N) is used when highly accurate data are required.
3. Lowry method is employed for estimating tyrosine in protein.
4. Biuret Method of Reinhold.
Biuret method is most commonly used in clinical practice and In
autoanalysers.
Principle.
Substances which contain two-CONH2 groups joined together and those
which contain two or more peptide links, give a purple colour with alkaline
copper sulphate solution. The reaction takes its name from the fact that
biuret (NH 2-CO-NH-CO-NH2) formed by heating of urea gives the
purple colour with cupric ions. One Cu-atom is complexed with 4 molecules of
biuret, involving linkage to the central N atom. Colour varies with different
proteins and carbohydrate contents in complex protein.
Reagents.
(i) Stock Biuret reagent. Dissolve 9 g sodium potassium tartrate in
500 mL of 0·2 N NaOH and add 3 g ofCuS04.5H20 stirring continuously. Add
5 g KI and make the volume to 1 L with 0·2 N NaOH.
(ii) Working Biuret reagent. Dilute 200 mL of stock reagent to 1 litre
with 0·2 N NaOH containing 5 g KI per L.
(iii) Standard protein solution. Bovine serum albumin 0·5 g/100 mL.
Procedure. Set the blank (B), test (T) and standard (S) tubes as
follows:
Reagent (mL) Blank Tl T2 SI S2 S3
Serum (1 : 10 diluted) - 1·0 1·0 - - -
Standard protein solution - - - 1·0 1·5 2·0
Distilled water 3·0 2·0 2·0 2·0 1·5 1·0
Biuret reagent working - - - - - 3·0
Mix and keep in water bath at 3TC for 10 minutes. Measure absorbance
at 550 nm using green filter.
Calculation. Plot graph between absorbance and concentration of
protein. Calculate the total protein content in serum by measuring the
absorbance of sample from the standard curve.
Normal Values. Total serum protein = 6-8 g percent
Albumin = 3·5-5·5 g percent
Globulin = 1·5-3·0 g percent
Clinical Interpretation. Total protein content may be altered by
changes in plasma volume without altering the albumin/globulin ratio. An
increase in protein concentration may be due to dehydration and a decrease
due to excess intake of water. Hyperproteinemia occurs due to excess
synthesis of globulin, chronic liver disease, acute infections like kalazar and
multiple myeloma. Hypoproteinemia is due to low protein intake,
malnutrition, starvation, diabetes and nephrotic syndrome.
ESTIMATION OF SERUM ALBUMIN AND GLOBULIN

Colorimetric Method.
Principle.
Serum albumin binds with bromocresol green at pH 4·1 to form green
coloured complex, the intensity of which is proportional to the albumin
concentration that can be measured colorimetric ally at 640 nm using a red
filter.
Reagents.
(i) Albumin reagent is prepared by dissolving 8·85 g of succinic acid,
108 mg of bromocresol green, 100 mg of sodium azide and 4.0 mL of Brij 35
in 900 mL of distilled water. The pH 4·1 is adjusted using NaOH. Make up
the volume to 1 litre.
(ii) Albumin standard. Add 4·0 g/dL of albumin in normal saline and
0·1 g/dL sodium azide.
Procedure. Perform the experiment as follows :
Reagents (mL) Test Standard Blank
Serum (1 : 10 diluted) 0·5 - -
Albumin reagent 5·0 5·0 5·0
Albumin standard (1 : 10 diluted) - 0·5 -
Distilled water - - 0·5
Mix the contents thoroughly and measure absorbance at 640 nm.

Calculations. Serum albumin (g)trube (0·05 mL serum)
ODT
= - - x 0·002 (conc. of standard/tube)
ODs
ODT 0·002
Serum albumin (gldL) = ODs x 0.05 x 100
ODT
=--x4
ODs
Normal value = 3·5 to 5·2 g/dL
Serum globulin (gldL)= Total protein - Albumin
. . . _ Serum albumin (g/dL)
Albumm-GlobulIn ratio - Serum globulin (g/dL)
Fibrinogen is present in plasma but not in serum.
Clinical Interpretation. Serum albumin is increased in dehydration
due to hemoconcentration and decreased in renal dysfunction, severe
malnutrition, liver diseases etc. A fall in albumin reduces total protein.
Serum globulin is increased in autoimmune diseases, macroglobulinemia
and multiple myeloma.
ESTIMATION OF SERUM BARBITURATES

1. Ultraviolet Method.
Barbiturates, which are drugs not normally present in the blood, are
extractable (un-ionised) from blood into methylene chloride. They can then be
back extracted into 0·45 M N aOH as the ionised form. The ionised form
absorbs in ultraviolet region, whereas unionised form does not absorb.
Plotting an absorption spectrum can qualitatively confirm the presence of
barbiturates. At pH 13 to 14, the ionised form exhibits an absorption
maximum between 252 and 255 nm with a minimum between 234 and 237
nm. At pH 9·8 to 10·5, a different ionised form exhibits a maximum at 240
nm.
2. Spectrophotometric Reading and Wallwork Method.
Principle.
Serum is shaken with CHCI3 . The barbiturates in the chloroform extract
are re-extracted into 0·0125 N NaOH. The NaOH solution is added to borate
buffer at pH 10 and barbiturates are estimated by measuring the change in
optical density caused by acidifying the solution.
Reagents.
(i) Chloroform.
(ii) Stock 1·25 N NaOH solution. Dissolve 5 gin 100 mL water.
(iii) 0·0125 N NaOH. Dilute 1 mL stock solution of NaOH in 100 mL
water.
(iv) 0·05 M borax solution is prepared by dissolving 19·07 g of sodium
tetraborate in 1 L of distilled water.
(v) Borate buffer. 50 mL of 0·05 M borax and 43 mL of 0·2 N NaOH

diluted to 200 mL with distilled water and pH adjusted to 10.
(vi) Stock phenobarbitone solution. 75 mg of the compound is dissolved
in 100 mL of alcohol.
Procedure.
(i) Test. Add 3 mL serum sample into a 50 mL tube containing 30 mL
of chloroform. Shake and allow the solution to stand for separation
of layers.
(ii) Working serum standard. It contains 2·5 mg of phenobarbitone
per 100 mL. Add 0·1 mL of stock phenobarbitone solution to 3 mL
of pooled serum and treat like test solution.
(iii) Transfer 25 mL portion of each of the extract into a fresh 50 mL
stoppered tube containing 5 mL of 0·0125 N NaOH. Mix the
contents. Pipette off aqueous layer into a centrifuge tube for
spectrophotometric estimation.
(iv) Now add 3·5 mL portion of the alkaline barbiturate extracts to
1·5 mL portion of borate buffer in a dry test tube. Record the
spectra of each alkaline extract between 200 and 300 nm against a
blank consisting of 3.5 mL of 0·0125 N NaOH and 1·5 mL of borate
buffer.
(v) After recording the test and standard spectra, add 4 drops of 6 N
H 2S0 4 to the sample and blank cuvettes. Record each spectrum on
the same sheet as the original spectrum. Measure the difference in
optical density between the alkaline and acid solutions at 239 nm.
The difference is proportional to the barbiturate concentration.
Calculation. Serum barbiturates (as Phenobarbitone) (mg/100 mL)
OD T
=--x2·5
ODs
Normal value of serum barbitone = 0·5 mg/100 mL
ESTIMATION OF SERUM ACID PHOSPHATASE

Acid phosphatase hydrolyses organic phosphomonoesters at pH 5 to 6.
Appreciable amounts of this enzyme are found in prostate and seminal fluid
and in many other tissues like red cells, spleen, liver, platelets, kidney and
bones. Average serum levels are 1·0 to 3·5 King Armstrong units per 100 mL.
Acid phosphatase is an extremely labile enzyme at alkaline pH.
Principle.
Incubate serum acid phosphatase with sodium glycerophosphate for 1
hour at pH 4·9 to liberate phosphate. Or treat serum with citrate buffer and
disodium phenyl phosphate. Measure absorbance of the reddish brown colour
at 510 nm.
Reagents.
(i) Citrate buffer (pH 4.9). Dissolve 42 g of citric acid in water and
add to 376 mL of 1 N NaOH solution. Make the volume to 1 litre
(ii) 1 M Tartrate solution. Dissolve 15 g of L( +) tartaric acid in 70

mL water and add to 18·5 mL of 10 N NaOH, adjusting the pH to
4·9. Make the volume to 1 L with distilled water.
(iii) Substrate solution (0·01 M disodium phenyl phosphate).
Dissolve its 2·18 g in one litre of distilled water. Add 4 mill of
chloroform to preserve the solution.
Procedure. Label the test tubes as test, control, standard and blank
and add reagents as follows :
Reagents (mL) Test Control Standard Blank
Citrate buffer 1 1 1·2 1·2
Substrate 1 1 - -
Serum 0·2 (Incubate for 1 h) 0·2 - -
0.5 N NaOR 1 1 1 1
Distilled water - - - 1
1% Phenol standard - - 1 -
Add 1 mL of 0.5 N Na2C03' 1 mL of 0·6% amino antipyrine solution and
1 mL of K3Fe(CN)6 solution to all tubes. Mix well after each addition.
Compare the reddish brown colours produced at 510 nm.
Calculations.
ODT-.oD C
Serum acid phosphatase (K.A. Units/100 mL) = ODs _ OD x 5
B
To obtain tartrate labile acid phosphatase, use the formula to calculate
results of two tests, one without and the other with added tartrate solution.
Difference between the results gives the phosphatase that has been
inactivated by tartrate.
ESTIMATION OF SERUM ALKALINE PHOSPHATASE

Alkaline phosphatase is an enzyme which releases phosphates from
certain monophosphoric esters and from pyrophosphates. It shows maximum
activity at pH 10. This enzyme is intracellular and only a small amount is
present in plasma. However, in some diseases which disrupt the cells, this
enzyme escapes into the plasma thereby increasing its level.
King and King Method.
Principle.
Phenyl phosphate, at pH 10, is hydrolysed to phenol when' incubated
with alkaline phosphatase. The condensed product of phenol with 4-amino
antipyrine is oxidised by alkaline potassium ferrocyanide to yield a red
coloured complex which can be measured at 520 nm.
Reagents.
(i) Disodium phenyl phosphate (0·1 N) solution. Dissolve 2·18 g
of disodium phenyl phosphate in 1 L of distilled water. Boil, cool
and preserve the solution with 1 mL of CRC1 3 at 4 ·C.
(ii) 0.1 N Carbonate buffer (pH 9·5-10·0). Dissolve 3·36 g of

NaHCOs and 6·36 g of anhydrous Na2COS in one litre of distilled
water.
(iii) 4-Amino antipyrine (0·6%). Dissolve 600 mg of 4-amino
antipyrine in 100 mL of distilled water.
(iv) Stock phenol standard (1 mg/mL). Dissolve 100 mg of pure
phenol in 100 mL of 0·1 N NaOH solution.
Procedure. Add reagents in test, control, standard and blank tubes as
follows:
Reagents (mL) Test Control Standard Blank
Disodium phenyl phosphate 1·0 1·0 - -
0.01 N carbonate buffer 1·0 1·0 1·1 1-1
Incubate for 15 minutes in water bath at 3TC
Serum 0·1 - - -
Phenol standard - - 1·0 -
Distilled water - - - 1·0
Incubate for 15 minutes in water bath at 3TC
0.5 NNaOH 0·8 0·8 0·8 0·8
Serum - 0·1 - -
0.5 N NaHCO a 1·2 1·2 1·2 1·2
4-Amino antipyrine 1·0 1·0 1·0 1·0
Potassium ferricyanide 1·0 1·0 1·0 1·0
Total volume (mL) 6·1 6·1 6·1 6·1
Measure absorbance at 520 nm against the blank.

Calculations. Serum alkaline phosphatase (KA unitsldL)
ODT-OD c
= ODs - OD x 10
B
One King Armstrong unit is the production of 1 mg of phenol liberated
by 1 dL of serum under assay conditions. (1 KA Unit = 7·1 UIL)
Normal Values. 3 to 14 KA unit or 22 - 98 UIL in normal adult. The
value is 2·5 times higher in children.
Clinical Interpretation. Serum alkaline phosphatase activity
increases in (i) bone diseases associated with osteomalacia, rickets, tumours,
(ii) Liver diseases (jaundice) and of biliary tract diseases.
Serum alkaline phosphatase activity decreases during anaemia, scurvy,
Kwashiorkor syndrome, cretinism and hypophosphatemia.
IMMUNOASSAY
Immunoassay techniques are important for the specific determin.ation of
drugs, vitamins, hormones and other compounds at nanogram and smaller
levels. These techniques involve a competitive reaction between an analyte
antigen and a specific antibody to form a complex.
PRINCIPLES OF RADIO IMMUNOASSAY

Radioimmunoassay combines the sensitivity of radiochemistry and
fluorescence (or enzymatically tag) with the specificity of immunology.
IIhmunology is the study of antigens and their reactions with antibodies that
is, an organism's defense mechanism to foreign bodies through antibodies.
Antigen. An antigen (e.g., a hormone) is a foreign substance capable of
enhancing antibody formation in the body and is able to react with that
antibody. An antigen is always a large molecule such as a protein.
Antibody. An antibody is endowed with the capacity to recognize, by
stereospecific association, a substance foreign to the organism it has invaded,
e.g., bacteria and viruses. An antibody is a high molecular weight (150000)
y-globulin protein. When the protein exhibits antibody activity, it is referred
to as an immunoglobulin (lg). There are 5 major immunoglobulins in
human blood (lgA, IgD, IgE, IgG, IgM), but IgG is most abundant. The Ig
consists of two light polypeptide chains of about 214 amino acid residues and
one heavy chain of about 430 residues. These are linked via disulphide
bridges.
When treated enzymatically with papain, three fragments of molecular
weight of about 50,000 ~ach are formed. Two fragments are identical and
retain the ability to bind antigens, hence are referred to as Fab (Fragment,
antigen binding). The third fragment does not bind antigen by itself but can
be crystallized from solution, hence it is called Fc (Fragment, crystallizable).
The Fc fragment is of fairly constant composition. The Fab fragments have
portions of variable composition and specifically bind to a given antibody. All
antibodies are similar in structure except for the variable antigen-binding
portions of the Fab fragments.
Antigen-Antibody Complex. The antibody specifically reacts with an
antigen to form an antigen-antibody complex. It is produced in the organism
only after the organism has had at least one exposure to the intruder (through
vaccination, either spontaneous or artificial). An antibody is produced for use
in immunoassay by injecting the antigen into an animal species to which it is
foreign and by recovering the serum that contains the resultant antibody
(antiserum).
The strength of the antigen-antibody complex is called the
affinity or avidity. Affinity refers to the intrinsic association constant
between an antibody and a univalent antigen, while avidity refers to the
9verall binding energy between antibodies and a multivalent antigen. Overall
binding reaction can be given as :
Ab+Ag=AbAg
_ [AbAg]
And the formation constant K- [Ab] [Ag]
The formation constants are quite large, typically 108 to 1010. The
binding forces are weak vander Waals, electrostatic and hydrophobic. The
bonds are broken by addition of salts or by increasing pH, temperature or
solvent polarity.
Radioimmunoassay Procedures.
All RIA procedures are based on the original discovery by Rosalyn
Yalow and Berson (awarded Nobel Prize in Physiology, 1977), that low
concentrations of the antigen hormone insulin could be detected
radiochemically by their ability to bind radiolabeled (1131) insulin. The
determination of unknown concentration of antigen is based on the fact that
radiolabeled antigen and unlabeled antigen (from the sample of standard)
compete physiochemically for the binding sites on the antibodies (Fig. 1).
Ab Ag*-Ab Ag*-Ab
(antibody) and Ag-Ab and Ag-Ab
bound bound
Ag* Incubation, Separation,
(radiolabeled
antigen) Ag* and Ag
(free)
Ag (antigen
in sample or
standard)
+
Ag*-Ag
(free)
Fig 1. Principles of radioimmunoassay.

The initial reaction vessel contains antibody solution (antiserum) labeled
antigen, and the serum sample that may contain unlabeled (natural) antigen
(the substance to be determined). Upon incubation, the antibody (Ab) will
form an antigen-antibody immunocomplex (Ag-Ab). In the absence of
unlabeled antigen, a certain fraction of the labeled antigen Ag* is bound (as
Ag*-Ab). But when increasing amounts of unlabeled antigen (Ag) are added,
the limited binding sites of the antibody are progressively saturated and the
antibody can bind less of the radiolabeled antigen. Following incubation, the
bound antigens are separated from the unbound (free) antigens and the
labeled portion (radioactivity, fluorescence, etc.) of either or both phases is
measured to determine the percent bound of the labeled antigen.
The antibody solution is initially diluted so that, in the absence of
unlabeled standard or unknown antigen, about 50% of the tracer dose of
Ag* is bound. When the sample is added, the diminished binding of labeled
antigen indicates the presence of unlabeled antigen.
Calibration Curve. A calibration curve is prepared using antigen
standards of known concentration by plotting either the percent bound
labeled antigen or the ratio of the percent bound to free (BIF) as a function of
the unlabeled antigen concentration. From this, the concentration of unknown
antigen can be calculated.
Specificity of Radioimmunoassay.
No antiserum used in RIA is completely specific for a particular antigen.
The specificity is influenced by
(i) heterogeneity of the antibody,
(ii) cross-section with other antigens and
(iii) possible interferences of the antigen-antibody reaction from low
molecular weight substances that may alter the environment of the
reaction.
A given antigen induces the formation of multiple antibodies. It can
combine with multiple antibodies to various degrees depending on the
respective equilibrium constants.
The problem of heterogeneity has been diminished with the
development of improved techniques for antisera purification. Also, the
synthetic production of monoclonal antibodies provides high specificity.
Non-specific factors that may modify the rate of antigen-antibody
reaction include high temperature, pH, ionic strength, composition of the
incubating medium, buffer, urea, heparin and high bilirubin concentrations.
Antigen standards and unknown should be prepared in antigen-free plasma
to swamp out differences in composition.
APPLICATIONS OF RADIO IMMUNOASSAY

• RIA has become a major tool for the estimation of hormones, proteins,
vitamins, drugs, antibiotics, nucleic acids and peptides etc. Now a
number of systems are available for automatic
radioimmunoassays.
• Substances that are routinely determined using radioiodine labeled
antigens include thyroid hormones (thyronine, thyroxine, thyroid
stimulating hormones) digestive hormones (gastrin), sex hormones
(follicle stimulating hormone, luteinizing hormone, human chorionic
gonadotropin), other hormones (insulin, adrenocorticotropic hormone,
parathormone, cortisol), vasoactive substances (renin, angiotensin,
norepinephrine) and other compounds (morphine, digoxin,
immunoglobulins, hepatitis-associated antigens).
• RIA technique using H3 or C 14 labeled antigens is used to
determine drugs (morphine, digoxin, digitoxin, LSD, marijuana,
barbiturates), hormones (serotonin, testosterone, aldosterone,
progesterone, cortisol, prostaglandins) and folate, vitamin D3 and
DNA etc.
• RIA has been used to assay anti-DNA antibodies in systemic lupus
erythematosus and in blood banking.
ELISA (enzyme linked immuno sorbent assays) has also been used in
the determination of hormones, proteins, antigens and antibodies. The
pregnancy test for the detection of human chorionic gonadotropin in urine is
based on ELISA. Non-competitive, competitive binding ELISA, sandwich
assays are performed to detect AIDS and cancer.
Indirect ELISA is mostly applied in clinical assays because a
universal antibody conjugate may be used as the secondary antibody against
all primary antibodies used from same immunoglobulin class of appropriate

species. Hence in this sensitive technique individually labeled primary
antibodies are not needed for each antigen analyte.
Drawbacks of RIA.
• RIA is expensive and hazardous in preparing and handling the
radioactive antigen.
• Both 1125 and 1131 emit gamma radiation that requires special
counting equipments.
• Body concentrates iodine atoms in the thyroid gland where they are
incorporated in thyroxine.
BLOOD GAS ANALYSIS

Introduction. Blood gas analysis, also called arterial blood gas
(ABG) analysis is a test which measures the partial pressures of 02 and
CO2 in the blood as well as 02 content, 02 saturation, HeO) content and blood
pH.
An ABG analysis evaluates how effectively the lungs are delivering 02
to the blood and how efficiently they are eliminating CO2 from it. The test
also assess the adequacy of lungs and kidneys interaction to maintain the
normal blood pH (acid-base balance). The acid-base components of the blood
provide information of kidney functions.
Blood gas studies are usually performed to assess respiratory diseases,
functioning oflungs and to manage patients receiving oxygen therapy. In case
the central venous catheter blood is low in 02' it means that lungs have not
oxygenated the' arterial blood well.
Preparation for Obtaining Arterial Blood.
Blood gas analysis is performed on blood from an artery. The patient
should breathe normally during the test. Patients have no restrictions on
drinking or eating before the test. If the patient is receiving 02' the 02
concentration must remain the same for 30 minutes before the test.
Procedure.
The blood sample is obtained by arterial puncture in the wrist, groin,
arm or fem:oral artery. The skin is properly cleaned to prepare antiseptic site
(using betadine) for puncture.
The technician then collects the blood with a small needle attached to a
disposable syringe. The pulsations of blood into the syringe confirm that
arterial blood has been obtained. The venous blood can be readily obtained,
but it usually reflects acid-base status of an extremity, not the body as a
whole. The syringe should be capped, rotated gently to mix heparin with blood
and analysed.
After blood collection. After the blood has been taken, place the
absorbent bandage over the puncture site and maintain pressure with two
fingers for 5 minutes to stop bleeding. The patient should rest quietly while
applying pressure to the punctured site.
Risks are very low when the test is done correctly. There may be
bruising or delayed bleeding from the site. Very rarely, there may be a
problem associated with circulation in the punctured area.
Blood Gas Analyser. Blood gas analysers automatically or manually
measure pH, Po and Peo of blood. The oxygen is measured with a
conventional amperometric 2 membrane oxygen electrode. The CO2 is
measured by a pH glass electrode covered with a plastic membrane that
allows diffusion of only gases. Chemical sensors have been developed for blood
gases, electrolytes and glucose.
DETERMINATION OF OXYGEN CONTENT

Oxygen in the lungs is carried to the tissues through the blood stream
but only a small amount of 02 can actually dissolve in arterial blood.
Procedure. Collect arterial or venous blood sample as described above.
Calculation.
02 Content = Haemoglobin x 1·34 x So x 0·003 x Po
2 2
where So = percent saturation of haemoglobin with 02
2
Normal Value. 02 content of arterial blood = 15-22 mL/100 mL
02 content of venous blood = 11-16 mU100 mL
Clinical Interpretation. Low value of arterial blood 02' which is
associated with high levels of arterial blood CO2 may be due to obstructive
lung disease, neuromuscular impairment and post operativ.ely respiratory
complications.
DETERMINATION OF PARTIAL PRESSURE OF OXYGEN

The partial pressure of oxygen Po is the quantity of 02 passing from the
2
pulmonary alveoli in to the blood. The Po is a measure of pressure that the
amount of O 2 dissolved in blood plasma ~xerts on the walls of the arteries.
Testing Po is actually measuring how much 02 the lungs are delivering to
the blood. 2
Procedure. Introduce a small quantity of arterial blood sample in a
blood gas analyser and measure Po by using amperometric membrane
2
oxygen electrode or polarographic clark electrode.
Normal Level.
Partial pressure of arterial blood = 80 Torr
Partial pressure of venous blood = 30 - 40 Torr
Clinical Interpretation. Increased levels of Po are associated with
2
polycythaemia, hyperventilation. Decreased levels are linked with anaemia,
cardiac decompensation, insufficient 02' hypoventilation, neuro muscular
diseases.
DETERMINATION OF TOTAL C02

In normal blood plasma, about 95% of total CO2 is contributed by
a
HCO (regulated by kidneys) and 5% by dissolved CO2 and H 2C0 3 . The test
is a measure of acidity or alkalinity of venous, arterial or capillary blood.

CO2 can be measured from dissolved CO 2, total H 2 C03 , HCOg, carbamino
CO2 ,
Procedure. Arterial or venous blood is first collected in a heparinised
syringe and then determined.
Calculation. a
Total CO2 = HCO + 0·03 x Peo .
2
HCOg in the extracellular spaces exists as CO2 , H 2 C03 and then
converted to NaHC03 by the buffers of plaf>ma and RBCs.
Normal level of total CO 2 is 24-30 meq/L.
Clinical Interpretation. Increased CO2 levels occur In severe
vomiting, aldosteronism. Decreased CO 2 content is due to diarrhoea,
starvation, renal failure and diabetic acidosis.
DETERMINATION OF PARTIAL PRESSURE OF C02 (P C0 2)

This test is the measurement of the pressure exerted by dissolved CO 2
in the blood and is proportional to Peo2 in the alveolar air. The test is
generally employed to detect respiratory abnormality, acidity or alkalinity of
the blood. It directly reflects how well the air is exchanging with blood in the
lungs.
Procedure. Introduce small quantity of arterial blood sample into a
blood gas analyser and measure CO2 tension or Peo by a pH glass electrode
covered with a plastic membrane or Ag-AgCI (Sev~ringhaus) electrode.
Normal value..Peo of arterial blood = 35-45 Torr
2
Peo of venous blood = 38-50 Torr.
2
Clinical Interpretation. A Pco of 63 torr in arterial blood increases

2
alveolar ventilation ten-fold. Reduction in Peo through its effect on plasma
2
bicarbonate concentration decreases renal HC03' reabsorption.

Failure of Peo to achieve predicted levels defines the presence of
superimposed respir~tory acidosis or alkalosis.
Increased Peo is due to emphysema, chronic bronchitis, head trauma,
lung diseases, hypov~ntilation. Decreased Peo level may be due to hypoxia,
anxiety, pulmonary emboli, pregnancy and hyp~rventilation.
DETERMINATION OF BLOOD pH
Measurement of pH of blood gives a ratio of acids to bases. The
respiratory response to changes in blood pH is instantaneous. In acidosis,
CO2 is retained and pH decreases and it stimulates ventilation. In alkalosis,
CO2 is blown off and pH rises.
Procedure.
1. Direct Method. Arterial blood sample is introduced into a blood gas
analyser and pH is measured.
2. Indirect Method. Henderson-Hassel batch equation is used to
determine pH of blood.
H 2C03", Major blood base

pH = pK + log --=---=-------
H 2C03", Major blood acid
Normal Value.
pH of arterial blood = 7·35 - 7·45
pH of venous blood = 7·32 - 7·43
Clinical Interpretation. The pH of blood decreased in acidemia which
is associated with renal failure, diabetes, ketoacidosis. The pH gets increased
hi alkalemia which is associated with acute pulmonary disease, myocardial
infection, heart failure, anxiety, neurosis and CO poisoning.
TRACE ELEMENTS IN THE BODY

A number of trace elements in the body are essential to the vital life
processes. These are involved in vitamins, hormones, RNA, activation (or
deactivation) of enzymes, skeletal and other controls. Essential trace elements
are : Ca, Mg, N a, K, Zn, Cu, Cr, Co, Fe, Se, 12 etc.
CALCIUM
Calcium Content. Calcium, the most abundant element in the body,
constitutes about 2% of the total body weight. About 90% Ca is present in
bones and teeth. Ca content in human serum, tissues and urine is 90-100
ppm, 60-90 ppm and 96-100 mg/day respectively.
Dietary sources of Ca are milk, cheese, cabbage, lentils, nuts, egg yolk
etc.
Calcium Requirement. Adult 800 mg, children 1·2 g, infants 300-500
mg/day.
Biochemical Functions.
• Activation of enzymes. Ca2+ ions are required for the direct
activation of lipase, adenosine triphosphate, ATPase and succinate
dehydrogenase enzymes.
• Release of hormones. Ca2+ ions facilitate the release of insulin,
calcitonin and parathyroid hormones.
• Calcium as intracellular messenger. There are certain hormones
which exert their action through the mediation of Ca2+ ions. Ca is
regarded as a second messenger for the hormonal action of
epinephrine in liver glucogenolysis and third messenger for
antidiuretic hormone through AMP.
• Regulates secretory processes. Ca2+ regulates microfilament and
microtubular mediated processes like endocytosis, exocytosis and cell
mortality.
• Calmodulin mediated action. Calmodulin is a calcium binding
regulatory protein. Ca-calmodulin complex activates adenylate cyclase
enzyme .
., Development of bones and teeth. Ca and P are the two
non-protein body building elements. Ca exists in bones as
CaC03.2Ca3(P04)2' Calcium along with phosphate is required for the
formation of hydroxypatite and physical strength of skeletal tissues.

Bones act as reservoirs of calcium.
• Muscle contraction. Ca interacts with troponin C to trigger muscle
contraction. Ca increases the interaction between actin and myosin
causing muscles to contract.
• Nerve transmission. Ca2+ ions are necessary for the transmission
of nerve impulse. Acetylcholine acts as a neurohuman transmitter
from nerve to muscle and Ca2+ ions help in the release of
acetylcholine.
• Blood coagulation. Most of the Ca2+ is confined to plasma and a
small amount is present in RBCs. The reactions in the cascade of
blood clotting depend on Ca2+ ions.
• Membrane integrity and permeability. Ca2 + ions decrease the
membrane permeability and this effect balances the opposite action of
N a and K capillary permeability.
• Electrical excitability of heart. Ca2+ ions act on myocardium and
prolongs systolic activity.
• Excitability of nerves. Ca is essential for the excitation of nerves.
• High contents of dietary phosphate may form insoluble oxalates and
phytates which interfere with Ca absorption.
• Rickets is a disorder of defective calcification of bones. This may be
due to dietary deficiency of Ca, P and vitamin D. The activity of
alkaline phosphate increases in rickets.
MAGNESIUM
Magnesium Content. Adults contain 20 g of Mg, 70% of which is
found in bones in combination with Ca and P. Remaining 30% is present in
soft tissues and body fluids. Mg content in human serum, tissues and urine
is 22 ppm, 300-500 ppm and 60-120 mg/day respectively. About 75% of serum
Mg is diffusible and the rest is bound to plasma protein.
Dietary Sources. Main sources of Mg are nuts, meat, cereals, fruits,
milk, cauliflower and cabbage.
Magnesium Requirement. Adult man requires 350 mg/dL and
woman 300 mg/dL of mg.
• Cofactor and activator of enzymes. M~+ ions serve as a cofator
for various enzymes requiring ATP, e.g., hexokinase, glucokinase,
phosphofructokinase. It acts as an activator for enolase, phosphory-
lase, peptidase, RNA and DNA pol:tmerase.
• Neuromuscular functions. M~+ ion is required for proper
neuromuscular functions. In the body, Mg and Ca act as antagonists
to one another. For example, the depression of central peripheral
nervous system due to hypermagnesium can be reversed by
intravenous administration of Ca.
• Mg is required for the development of bones and teeth.
• Mg is absorbed by the intestinal cells through a specific carrier
system. Its absorption decreases by the consumption oflarge amounts
of Ca2+, PO~- and alcohol.
• Mg Deficiency. Mg deficiency causes neuromuscular irritation,

convulsions and weakness. Alcoholism, malnutrition and cirrhosis of
liver may cause Mg deficiency. Low levels of Mg are observed in
abnormal pregnancy, uremia and rickets.
SODIUM
Sodium Content. About 50% of body sodium is present in bones, 40%
in extracellular fluid and 10% in soft tissues. Na content in human serum,
tissues and urine is 3200 ppm, 0·07 g/gN and 1000-5000 mg/day respectively.
Dietary Sources. Common salt (NaCl), nuts, whole grains, bread,
milk, eggs and vegetables.
Sodium Requirement. Sodium requirement for an adult is 4-5 g/day
and for a patient of hypertension about 1 g/day.
• Sodium is required to maintain the osmotic pressure inside the cell,
to prevent its collapse and also to balance the electrical charges
associated with negatively charged organic macromolecules in the
cell.
• Na+ ion produces electrical potential across cell membrane which is
essential for the smooth functioning of nerve and muscle cells.
• The movement of glucose into cells is associated with Na+ ions.
Hypernatremia is characterised by an increase in serum sodium level.
It may occur because of hyperactivity of adrenal cortex, cortisone and sex
hormones or by dehydration. In pregnancy steroid and placental hormones
cause N a and water retention in the body. Hypertension and blood volume
also increases.
Hyponatremia is due to decrease in serum sodium level by
diarrhoea, vomiting, renal diseases and adrenocortical deficiency (Addison
disease). The manifestations of hyponatremia include reduced blood pressure,
retarded growth, nausea, loss of appetite, headache and muscular cramps.
POTASSIUM
Potassium Content. Potassium content in human serum and tissues
is 120-214 ppm and 20-200 ppm (dry) respectively.
Dietary sources are grains, cereals, milk, vegetables, banana, orange,
beans, potato, coffee, fish, chicken and liver etc.
Potassium requirement for an adult man is 3 g/day.
Biochemical Functions. K and Na salts form the chief buffer system
which play vital roles in the regulation of pH of body fluids.
• Potassium is required for the transmission of nerve impulse.
Extracellular K+ ion influences cardiac muscle activity.
• Potassium is necessary for the biosynthesis of proteins and ribosomes.
• The optimal activity of enzyme pyruvate kinase of glycolysis depends
on ~ ions.
Increased level of serum potassium is observed in adrenocortical
insufficiency (Addison disease), renal failure, diabetic coma and severe
dehydration.
Symptoms of hyperkalemia (increased K level) include depression of

central nervous system, bradycardia, numbness, reduced heart sounds and
cardiac arrest.
Hypokalemia (decreased K level) is due to hyperactivity of adrenal
cortex, prolonged cortisone therapy, treatment of diabetic coma with insulin,
vomiting and diarrhoea.
COPPER
Copper Content. Copper content in human serum and tissues is 1·10
ppm and 5 to 20 ppm respectively.
Dietary sources of copper include coconut, almonds, nuts, papaya,
oranges, grapes and vegetables.
Copper requirement is 2 to 3 mg/day.
Biochemical Functions. Copper is an essential constituent of several
enzymes. Copper is important in
(i) Lysine oxidase which affects the elasticity of walls.
(ii) Dopamine hydroxylase, that affects brain function.
(iii) Tyrosinase, which affects skin pigmentation.
(iv) Ceruloplasmin, which plays an important role in iron metabolism
and conversion of Fe2+ to Fe3+. It transports transferrin to plasma.
• Copper can convert dietary iron into haemoglobin. It is a vital part of
the antioxidant enzyme super oxide dismutase.
• Copper containing proteins azurin and plastocyanin act as electron
transfer agents by means of Cu2+ICu+ couple.
• Metallothionein protein facilitates copper absorption.
• Copper Deficiency. Copper deficiency results in Menke's disease
whereas its elevated level causes Hodgkin disease.
• Wilson disease is a disorder of abnormal copper metabolism causing
its accumulation in liver, kidney and brain.
ZINC
Zinc content in human serum, tissues and urine is 1-2 ppm, 12-100
ppm and 0·3-0·6 mg/day respectively.
Dietary sources include cereals, nuts, oil seeds, grains, soyabeans,
wheat, peas, potatoes, onion, almonds etc.
Zinc requirement for an adult is 10-15 mg/day.
• Zinc is an essential constituent of various enzymes.
• Zinc is a crucial nutrient for immune and brain function, nervous
system, blood sugar and optimal health.
• Zinc guards against infection and is required for healthy skin and
hair.
• Zinc is needed to transport vitamin A to the retina and this improves
vision.
• Gusten, a zinc containing protein is important for taste sensation.
• The absorption of zinc depends on a transport protein
metallothionein. Its absorption is hampered by fibre, Ca, Cu,
phytate and phosphate in pulses. Amino acids and peptides increase

absorption of zinc.
• Deficiency. The deficiency of zinc is associated with neuro
psychiatric disorders including anorexia, nervous depression,
schizophrenia and Alzheimer disease. Deficiency may cause growth
retardation, anaemia, hair loss, vision loss. Wound healing is slowed
and protein metabolism impaired. Reduced zinc level has been found
in HIV patients.
• Hypozincemia (low Zn-level) accompanies hepatitis, cardiac
infection, oral contraception and stagnant skeletal growth. Zincurea
(elevated serum zinc) accompanies albuminuria and cirrhosis.
• Zinc toxicity includes nausea, gastric ulcer, anaemia, excessive
salivation and pancreatitis.
MANGANESE
Mn Content. Mn content in human serum, tissues and urine is 0·02
ppm, 0·2-1·7 ppm and 0·05 mg/day respectively. Total body content of Mn is
15 mg. Mn is mainly concentrated in liver, muscles, bones and kidneys.
Sources of Mn. Nuts, cereals, fruits, tea, leafy vegetables.
Mn requirement for an adult is 2-9 mg/day.
Biochemical Functions. Mn acts as a cofactor of several enzymes
such as organiase, pyruvate carboxylase, isocitrate dehydrogenase, dismutase
and peptidase.
• Mn as Mn2+ activates liver arginase, choline esterase, mitochondrial
respiratory enzymes.
• Liver arginase converts nitrogenous wastes into urea in the
ornithine-argininecitrulline cycle which is excreted in urine.
• Mn is necessary for cholesterol biosynthesis and also for the synthesis
of glycoproteins and mucopolysaccharides.
• Mn in the serum is bound to a carrier protein transmagnanin (a
p-globulin).
• Mn Deficiency. Mn deficiency causes retarded growth, impaired
haemoglobin regeneration, accumulation of fat in liver and testicular
degeneration etc.
IRON
Iron Content. Average iron content is 1·25 ppm in human serum and
0.1-0.3 mg/day in urine.
Dietary Sources. Liver, meat, fish, poultry are rich sources of heme
iron. Cereals, legumes, nuts, oil seeds, dry fruits, leafy vegetables constitute
non-heme iron.
Iron requirement for an adult man and pregnant woman are 10-14
mg/day and 40 mg/day. An adult body contains about 3 g of iron About 70%
of the total iron present in adult body occurs in erythrocytes of blood and
5% in myoglobin of muscles. Rest is stored in ferretin.
• Heme is the important constituent of several proteins and enzymes.
Haemoproteins include haemoglobin, myoglobin, cytochromes,
catalase, xanthine oxidase, tryptophan. Proteins like transferrin,
ferritin and hemosiderin contain non-heme iron.
• Haemoglobin and myoglobin are required for transport of 02 and
CO2 , Muscles store O2 in combination with myoglobin which contains
iron.
• Cytochromes and certain non-heme proteins are necessary for
electron transport chain and oxidative phosphorylation.
• Iron is associated with effective immunocompetence of the body.
• Peroxidase, the lysosomal enzyme is required for phagocytosis by
neutrophils.
• Absorption of iron is promoted by ascorbic acid, small peptides,
amino acids and decreased by tea and eggs.
• Iron, when present in excess, can actually stimulate free radical
formation.
• In hemosiderosis, excessive iron is deposited in ferritin and
haemosiderin.
• In hemochromatosis, iron is directly deposited in tissues (liver, spleen,
pancreas) causing bronze diabetes.
• Deficiency. Iron deficiency causes anaemia which may also be due
to chronic blood loss, defective absorption of iron and hook worm
infection.
IODINE
Iodine Content. Human body contains about 20 mg of 12 , About 80%
12 is stored in the form of iodothyroglobulin (a glycoprotein) in the thyroid
gland.
Sources of 12 , Richest sources are sea foods, shell fish, fish oil. Others
are fruits, vegetables, cereals, meat, milk, eggs, iodised salt.
Requirement of 12 for adults, women and children are 100-150 Ilg, 150
Ilg and 80 Ilg respectively.
Functions. 12 is required for the synthesis of thyroid hormones such as
thyroxin and tri-iodothyroxine. Thyroxin controls metabolism, utilisation of
sugars, regulates energy production and aids growth. It improves cognition
and makes skin, hair and teeth healthier.
Deficiency of 12 may cause cretinism in children. Dwarf child is
mentally retarded with enlarged thyroid gland.
In adults, thyroxine production may be hampered and cause
myxoedema. Symptoms of the disease are slower rate of metabolism, loss of
hair and enlarged thyroid glands.
o
12
DRUG ANALYSIS
INTRODUCTION
The drug, derived from drogue (dry herb), is defined as any substance
used in medicine to diagnose, cure and prevent the occurrence of diseases and
disorders and prolong the lives of patients suffering from serious or incurable
diseases. WHO defines drug as any substance or product which is used to
modifY or explore physiological system or pathological states for the benefit of
the recipient.
Pharmaceutical chemistry is the study of the chemical and physical
properties of drugs, their behaviour, preparation, composition, structure, their
influence on an organism, conditions of their storage, shelf life preservation,
identification and their therapeutic use.
SOURCES OF DRUGS
Majority of the drugs used in therapeutic action are synthetic but many
plant products also provide important therapeutic drugs.
• Plants yield morphine, atropine, quinine, reserpine, streptomycin etc.
• Synthetic drugs are aspirin, procaine, sulphonamides.
• Micro-organisms produce penicillin, bacitracin etc.
• Animals yield insulin, heparin etc.
• Genetically engineered drugs include human growth hormones,
human insulin etc.
NARCOTICS
Narcotic, that refers to opium or opioid, is a drug which produces
stupor, insensibility or sleep. It may be natural, semi-synthetic and synthetic
that behave pharmacologically. Narcotics can be administered orally,
transdermally or by intravenous injections.
Early Symptoms of Narcotics. The addict may suffer from running
nose, watery eyes, irritability, restlessness, yawning, loss of appetite, severe
sneezing, tremors, hypertension, vomiting, depression, pain in muscles and
bones etc. However, administration of a suitable narcotic can dramatically
reverse these early symptoms.
Effects of Narcotics.
Effects of narcotics depend mainly on dose, route of administration,
previous exposure to the drug and expectation of the user. Following effects
are observed : Nausea, vomiting, apathy, drowsiness, constriction of pupils,
dialation of the subcutaneous blood vessels causing flushing of the face and
(279)
neck, respiratory complications, decreased physical strength, slurred speech,

endocarditis, hepatitis, AIDS etc. Body organs like lungs, heart and brain are
adversely affected. Repeated use of narcotics decrease intensity of analgesia,
euphoria and sedation.
Uses of Narcotics.
Narcotics help in the treatment of several body diseases clinically.
• Morphine is a strong pain alleviating agent during the post operative
period.
• Apomorphine is used as an emetic and expectorant in poisoning.
• Fentanyl or synthetic heroin is used as surgical anaesthetic.
• Barbitone is a powerful hypnotic that causes natural sleep.
• Tranquilizers, neuroleptics or antipsychotics suppress mania and
psychotic activity. Tranquilizers act as anti-anxiety agents, CNS
stimulants or antidepressants.
• Rauwolfia serpentina's root contains over 40 alkaloids out of which
reserpine, serpentine and yohimbine are pharmacologically most
active.
• Reserpine is used in hypertension to lower the arterial blood
pressure.
• Haldol is employed for prolonged treatment of acute schizophrenia.
• Pipradol is used in the treatment of fatigue and depression.
• Covatin and hydroxyzine, non-hypnotic and antispasmodic drugs are
used to cure insomnia, tension and anxiety.
• Caffeine is administered as a CNS stimulant and a cardiotonic.
DANGEROUS DRUGS
Dangerous drugs exert adverse side effects on the various body organs
of the addict. These illegal drugs have the following effects :
• Psychotogenic drugs produce psychosis, depersonalisation, changes in
mood, behaviour and retarded memory.
• Hallucinogens or psychotomimetics like marijuana, peyote and LSD
cause alterations in normal thoughts, perceptions and moods.
• Overdoses of barbiturates may cause poisoning, respiratory failure
and death.
• Excess of cltffeine can result in exhaustation of nerve cells.
• Iproniazide, is a toxic drug which may produce necrosis of liver.
• Cannabis or marijuana, hashish, charas, ganja are dangerous abuse
drugs which may induce mutation, damage to chromosomes and
disrupt the growth of faetus.
• Mascaline, a psychotogenic drug, has mutagenic and teratogenic
effects.
CLASSIFICATION OF DRUGS
(A) Classification of Drugs According to their Effects.
1. Anti-infective Drugs.
Anti-infective drugs interfere selectively with the functioning of
micro-organism while leaving the host unharmed. These are of following
types:
DRUG ANALYSIS 281
(i) Antibacterial drugs. Antibiotics such as sulpha drugs, penicillins,

cephalosporins etc., either kill bacteria directly or prevent them from
multiplying so that the body immune system can destroy invading bacteria.
(ii) Antifungal drugs. Antifungal drugs can cure or may only suppress
a fungal infection.
(iii) Antiviral drugs. Antiviral drugs prevent the penetration of virus
iuto host cell or block the synthesis of new viruses. With some viruses, such
as HIV which cause AIDS, antiviral drugs can only prolong life but can not
cure the disease. Vaccines are used as antiviral drugs against poliomyelitis,
influenza and mumps etc.
2. Endocrine Drugs.
Endocrine drugs correct the hyperactivity and hypo activity of body's
natural hormones. For example,
(i) Progesterone or progestin hormone is used for the osteoporosis
and atherosclerosis.
(ii) Estrogen and progesterone are used in birth control pills.
(iii) Insulin is employed to treat diabetes.
(iv) Androgen is used to relieve from hot flashes and mood swings.
3. Central Nervous System Drugs.
CNS drugs are used to treat neurological and psychiatric problems. For
example, antiepileptic drugs reduce the hyperactivity of excited brain nerves
and eliminate seizures.
(i) Antianxiety drugs or tranquilizers treat anxiety centres of the
brain. Tranquilizers such as benzodiazepines, diazepam, chlordiazepoxide,
meprobamate are used as anxiolytics, anaesthetics and anticonvulsants.
(ii) Antimanic drugs like lithium dampens extreme mood swings in
patients. These are used to treat manic depressive (extreme excitement and
lethargy, i.e., bipolar nature) disorders.
(iii) Antidepressant drugs alleviate mental depression. These drugs
include pargyline, amitriptyline, and sertaline.
(iv) Antipsychotic drugs regulate certain brain mechanisms called
neurotransmitters which do not function properly in people with psychosis,
hallucinations or major mental disorders.
(v) Analgesic drugs. Narcotics such as codeine, morphine, heroine,
meperidine relieve pain by acting on receptors located on the nerve cells of
brain or spinal cord. Non narcotic analgesics like ibuprofen, aspirin,
acetaminophen reduce pain or fever by inhibiting the formation of nerve
impulses at the site of pain.
(vi) Sedatives are CNS depressants that are capable of reducing
nervous tension and promote relaxation without producing sleep. Simple
bromides act as good sedative with no hypnotic action.
(vii) Stimulants like caffeine, nicotine, ephedrine increase neuronal
activities, reduce fatigue and appetite.
(viii) Psychomotor stimulants such as cocaine and methamphe-
tamine stimulate sensory motor functioning and are used to treat attention
deficit hyperactivity disorder (ADHD) and narcolepsy.
(ix) Stimulatory hallucinogenics produce a mixture of psychomotor

stimulant and hallucinogenic effects.
(x) General anaesthetics depress brain activity to such an extent that
all sensitivity to pain is lost thus causing unconsciousness during surgery.
(xi) Local anaesthetics like novocaine, dicaine, benzocaine make a
particular organ insensitive. They prevent nerves from transmitting impulses
signalling pain.
4. Anticancer Drugs.
- Anticancer drugs eliminate cancer from specific tissue or organ.
Alkylating agents are cytotoxic and can alter DNA of cancer cell while vinka
alkaloid prevents cancer cell division.
5. Cardiovascular Drugs.
Cardiovascular drugs affect heart and blood vessels.
(i) Antihypertensive drugs like losacar, loram, amlopin reduce blood
pressure by dilating blood vessels and reduce the amount of blood pumped by
the heart into the vascular system.
(ii) Antiarrhythmic drugs normalise irregular heart beats and
prevent cardiac malfunction.
6. Drugs that Affect the Blood.
(i) Antianaemic drugs such as iron capsules and vitamins enhance the
formation of red blood cells.
(ii) Anticoagulants like heparin reduces blood clot formation to ensure
free blood flow in the body.
(iii) Thrombolytic drugs dissolve blood clots, which can block blood
vessels leading to heart stroke.
(B) Classification of Drugs According to Therapeutic Action.
1. Chemotherapeutic Drugs. These drugs are used to cure specific
diseases such as malaria, sphilis and tuberculosis etc. Chemotherapeutic
drugs include antibiotics, antiseptics, antineoplastics etc.
2. Pharmacodynamic drugs assist in the recovery from specific
bacteria or viral infection. These drugs include tranquilizers, anaesthetics,
antipyretic, antihistamines etc.
(C) Classification According to Chemical Structure.
Drugs can be classified according to their chemical structure and
properties, regardless of their pharmacological actions. The group of cardiac
stimulants include representatives of heterocyclic series (caffeine,
strychnine, pentetrazole), terpenes (camphor) and cardiac glycosides
(steroids).
Drawback. Substances similar in structure have absolutely different
pharmacological action.
(D) Drugs can be classified by the substance from which they are derived
(plant, animal, mineral) and by the form they are taken (capsule, syrup or
gas).
DRUG ANALYSIS 283
(E) Drugs can be classified as

(i) Barbiturates such as barbital, butabarbital, pentobarbital
(nembutal), secobarbital (seconal), methyprylon (noludar) etc.
(ii) Alkaloids like cocaine, methadone, morphine, quinine, codeine,
heroine and phenacetin etc.
(iii) Amphetamines such as benzedrine, dexosyn and dexedrine etc.
(iv) Hallucinogens like marijuana, LSD and mescaline.
(F) Miscellaneous Classifications.
Many other categories of drugs also exist such as antiallergic, antiworm
(anthelmintic), antiparkinson, diuretic, pulmonary and muscle relaxant drugs.
A drug of one category can be used to cure disease of other category. For
example, lidocaine can be used both as a local anaesthetic or as a cardiac drug.
METHODS OF SCREENING THE DRUGS

Drugs can be screened by the following five methods.
1. Physical Methods. Physical methods involve the study of physical
property of drug like determination of solubility, colour, density, melting,
freezing and boiling points etc.
2. Chemical Methods. Estimation of potency of the active principal
(functional group) of a drug by chemical methods is known as chemical assay.
For example, drugs having -OH or -NH2 group can be estimated by
acetylation or diazotisation. Penicillin can be determined directly by
iodometry.
3. Instrumental Methods. Instrumental methods like spectroscopy,
fluorimetry, photometry and chromatography are currently applied to screen
the drugs.
4. Biological Methods. These methods characterise the pharma-
ceutical effect of a drug on an organism.
5. Immunological Methods. RIA methods are used for the estimation
of certain hormones. It is based upon the fact that hormone is an antigen and
can react with its specific antibody.
SCREENING OF DRUGS BY GAS CHROMATOGRAPHY

Gas chromatography is well suited for analysing numerous drug samples
in blood, urine and other body fluids. Screening procedure involves following
steps.
Solvent Extraction prior to Drug Isolation. Liquid-liquid solvent
extraction is used to separate drugs from biological fluids and pre concentrate
them before GC measurement.
• Different classes of drugs can be extracted from an aqueous solution
at different pH into a solvent such as methylene chloride or methanol.
• Alkaloids, antihistamines, barbiturates and tranquilizers can be
extracted into a mixture of ether and acetone. Alkaloids extract at
about pH 9 and amphetamines and phenothiazines above pH 10.
• Marijuana can be extracted from dried and ground leaves with
CH2CI2·
• Lysergic acid diethylamide (LSD) can be extracted into CH2 Cl2 after
solubilizing with a carbonate buffer.
• Mascaline can be extracted with ethanol.
• Serum or urine is adjusted to pH 4 to 7·5 to extract many of the drugs.
• Extractions at pH 3 and 9 yield acidic or basic components of the
drugs.
• Barbiturates (in micro samples 0·01 to 0·05 Ilg) may be methylated
with dimethyl sulphate before extraction into hexane.
• Drugs in powder or pill form are generally dissolved in aqueous
KOH or HCI followed by extraction at appropriate pH.
• Drugs in tissues are usually protein bound. So it is necessary to
precipitate protein before extraction. Macerate (grind) the tissue and
treat it with sodium tungstate followed by hydrolysis with hot aCid. It
forms tungstic acid which is a protein precipitating agent.
Methodology.
Detector. Flame ionization detector is mostly used in drug screening.
Column. OV-17 column (phenyl methyl silicone fluid).
Other columns are :
• PPE 20 (medium polarity)
• Carbowax 20 M (high polarity).
• Glass column with direct column injection or glass injection port.
General Screening of Pills and Powder for a Mixture of Drugs.
To separate, screen and analyse the mixture of amphetamines (basic),
barbiturates (acidic) and alkaloids ( basic), three separate chromatograms are
run using different required temperatures. It is also possible to analyse a
combined extract with a single chromatogram by using temperature
programming to reduce the measurement time from 45 minutes to 15
minutes. The peaks are then identified by comparing with those of known
standards of drugs.
Screening of Methadone Drug in a Urine Sample.
The urine sample of a local methadone treated person was worked up by
the Dole technique (a combined ion-exchange-solvent extraction isolation)
and subjected to gas chromatography (Fig. 1).
Column Conditions. Glass column, medium polarity OV-17 on 80/100
mesh. High performance chromosorb W at 215°C.
Identification of Drugs.
Several drugs identifiable in the sample are methadone, cocaine,
morphine, monoacetyl morphine and quinine. The peaks are positively
identified by comparing with those of known standards of the drugs.
Head Space Technique for Drug Screening.
In head space technique, the drug sample is contained in a closed
container and the volatile constituents are allowed to equilibrate with the
atmosphere. An aliquot of the atmosphere is taken with a syringe, injected
into the gas chromatograph and peaks identified.
DRUG ANALYSIS 285
Peak no. Peak identified as

1 Methadone
2 Cacaine
3 Morphine
4 Monoacetyl morphine
5 Quinine
2
5
L -__~~-L~~~~~~~~~
12 4 6 8 10 12 14 16 18
Retention time (minutes)
Fig. 1. Gas chromatogram from a urine sample containing methadone.
SCREENING OF DRUGS BY GC-MS

Gas chromatography-mass spectrometry is a sophisticated instrumental
technique used to detect drugs in blood, urine and body fluids.
Instrumental Features.
(i) Perkin Elmer GC-MS.
(ii) Gas chromatograph features full programmable pneumatic control
with integral autos ampler.
(iii) Quadrupole mass spectrometer is ideally suited as a GC detector.
A complete scan is achieved in the duration of a GC peak, simply
by scanning a voltage.
An electron multiplier detects the separated ions at nanogram
level.
r
(iv) All the instrumental features of GC-MS are under single point of
control.
Column Characteristics.
GC column is of Elite series PE-5 of 5% phenyl methyl polysiloxane.
Column diameter = 30 m x 0·25 mm
Column flow = 1·5 mUmin.
Injection volume = 1·0 ilL
Injector temperature = 200·C.
Oven tePlperature = 75·C for 1 min, 10·C/min to 275·C.
Mass scan range = 45 to 400 Da.
Scan speed and scan mode = 0·5 s, full scan.
Ion source and transfer line temperature = 200·C.
Identification of a Drug Metabolite in a Blood Sample.
A blood plasma extract from a drug overdose victim was subjected to GC.
The gas chromatogram showed two peaks (Fig. 2a). The retention time for
peak 1 corresponds to the retention time of glutethimide. The sample was
then subjected to GC-MS analysis. The peak at mass to charge ratio of 217
also matched the ratio for the glutethimide molecular ion and was identical
with the peak 1 from a known sample of glutethimide (Fig. 2b).
The retention time for peak 2 did not match with any known drug. The
GC-MS of peak 2 however showed a molecular ion peak at rn/z ratio of 233
(Fig. 2c).
~
'iii Peak 2
c
CD
~
Iii
c
Cl
en
Time
(a)
~ ~ 233
en 'iii
c c
.Sl .Sl
£ .!:
170 200 mlz 170 200 m/z

(b) (c)
Fig. 2. (a) Gas chromatogram of a blood plasma extract from a drug overdose victim.
(b) and (c) Mass spectra due to peak 1 and peak 2.
This showed a difference of 16 mass units from the molecular ion of

glutethimide. Several other peaks in the mass spectrum from GC peak 2
differed by 16 mass units from those of glutethimide. This indicates the
incorporation of oxygen in glutethimide which corresponds to its hydroxy
metabolite.
Structure of glutethimide and its 4-hydroxy metabolite.
IDENTIFICATION OF COCAINE BY GC-MS

GC-MS provides a positive identification of cocaine in a suspected
powder sample, dissolved in methanol. Gas chromatogram of the drug sample
DRUG ANALYSIS 287
is obtained from the total ion current (TIC) monitoring mode (Fig. 3). The
peak at 11·5 minutes corresponds to the retention time expected for cocaine.
(I)
c: 6.0E6 -
0
as
"0
c: 4.0E6 -
::l
.0
« 2.0E6 -
I I I L J I I
0 4 6 8 10 12 14 16
Time (min.)
(a)
1.2E6 - 82 Scan 459 (11.541 min.)
182
1.0E6 -
(I)
0
8.0E5 -
c:
as
"0
c: 6.0E5 -
::l
.0
« 94
4.0E5 -
303
2.0E5 - 199
~ ,ll,~"
152 212
111,111.1,,, ~l. Jl, I II I III
o 100 150 200 250 300
Mass/Charge
(b)
82
1.2E6
1.0E6
~ 182
~ 8.0E5
"0
c:
::l
~ 6.0E5
94
4.0E5
303
2.0E5
o 100 150 200 250 300

Mass/Charge
(c)
Fig. 3. Confirmation of cocaine by GC-MS. (a) Total ion current gas chromatogram of
cocaine in a urine sample. (b) Mass spectrum taken from peak at 11·5 min. (c) Mass
spectrum taken from GC peak of cocaine standard at same elution time.
The middle figure is the mass spectrum corresponding to the compound at

that peak and the bottom one is the mass spectrum of a cocaine standard.
The mass spectrum of the drug sample is essentially the same as for the
cocaine standard. Furthermore, the parent ion peak is present at mJz
corresponding to M+ for cocaine (mol. wt. 303·35). There is also a small peak
at mJz 304 corresponding to MW, which is often formed in the ion chamber.
The mass spectrum of each molecule detected is stored in system's
computer and so the MS corresponding to a given GC peak can be read out.
Spectral computer searches can be made to match an unknown spectrum.
SCREENING OF DRUGS BY THIN LAYER CHROMATOGRAPHY

TLC is used to identify various unknown drugs in a drug sample.
Principle.
In TLC, certain adsorbing substances such as silica gel, alumina and
powdered cellulose are supported as thin layers on glass plate or plastic strip.
The mobile phase run up the plate by capillary action. Drug separation is
achieved by partition, adsorption, reverse phase, gel filtration or ion exchange
techniques.
Theory.
The drug sample under examination moves along the surface of the
adsorbent. The three fold competitive interaction among solute, solvent
and adsorbent establishes the relative rates at which the solvent front and
the solute ascend the layer of adsorbent on the glass plate. A more polar
solute is attracted to the adsorbent more strongly than a less polar solute.
Reference standards. Cocaine, heroin and methamphtamine.
Procedure.
Preparation of sample solution. Dissolve 125 mg of unknown
sample in 4 mL of methanol toluene (1 : 1) solution. The reference standards
have been prepared at a strength of 25 mglmL.
Application of the Sample.
Here commercially prepared fluorescent silica gel TLC plates supported
on plastic sheets (8·5 x 3·5 cm) are used. These sheets have been activated by
heating at 120°C for 30 minutes. The sample solutions of the three reference
compounds and one unknown are spotted on a single plate using fine capillary
tube. The spots should be 2·5 cm from one edge of the plate and 1 cm apart.
Use a fresh applicator for each sample.
Developing solvent.
. It consists of toluene (120 mL), acetic acid (18 mL), ether (60 mL) and
methanol (1 mL). Place 5 mL of this solvent mixture in a developing chamber.
Development of the Chromatogram.
Dip the chromatoplate with spotted end down into the developing
chamber. Since R f values are affected by the degree of saturation of the
DRUG ANALYSIS 289
atmosphere, so a paper impregnated with the solvent should be placed round

the sides of the chamber to ensure that the air in the tank is saturated with
the solvent vapour. The tank is closed firmly with the lid. When the solvent
has moved to about 10 cm above the origin, the plate is removed and the
solvent front is carefully marked.
Detection of the Spots.
The thin layer plates contain a trace of fluorescent dye, so the spots can
be detected by shining an ultraviolet
light on the plate. Exposure to iodine r ' - - ' .... Cocaine
vapour often produces a colour with

)+---+- Heroin
colourless solutes (Fig. 4). u . - - - - t - Methamphtamine
Result. Calculate Rf value of the Unknown drug
reference standards and the unknown
compound. Identify the known drug Fig. 4. Thin layer chromatograms
component from the number, position of drugs.
and appearance of spots in the reference
drug samples.
Screening of Vitamins by TLC.
Vitamin A, Da and E can be separated on silica gel using 80% cyclohexane
and 20% diethyl ether. Observe the chromatogram under UV light of 254 nm.
Vitamin A turns blue, Da gradually changes to yellow orange and vitamin E
becomes visible when the plate is heated at 373 K for 5 minutes.
Screening of Aspirin, Phenacetin and Caffeine in a Mixture by HPLC.
HPLC can be used for the determination of aspirin, phenacetin and
caffeine in common analgesic tablets using phenacetin as internal standard.
Sample Mixture. Weigh accurately 0.601 g of aspirin, 0·076 g of
phenacetin and 0·092 g of caffeine. Dissolve the mixture in 10 cm3 absolute
ethanol. Add 10 cm3 of 0·5 M ammonium formate solution and dilute to 100
cm a with de-ionised water.
Mobile Phase (Solvent). Ammonium formate (0·05 M) in 10% (v/v)
ethanol-water at pH 4·8 is used. Use a flow rate of 2 cm3 min- 1 with inlet
pressure of 117 bar (1 bar = 105 Pa).
Column. 15 cm x 4·6 mm packed with a 5 IJ-m silica SCX (strong cation
exchanger) bonded phase.
Detector. UV absorbance at 244 nm or 275 nm.
Method. Inject 1 x 10-3 cm3 of the sample solution and obtain a
chromatogram. Compounds are separated in about 3 minutes. Elution
sequence being
(i) Aspirin (ii) Phenacetin (iii) Caffeine.
Measure peak areas with an integrator. Normalise the peak area and
express each peak as a percentage of total peak area. Compare these results
with the known composition of the mixture. Determine the response factors
(r) for the detector relative to phenacetin (= 1) as internal standard by
performing three runs, using 1 x 10-3 em3 injection and obtaining the average
value of r.
Relative response factor,

r = Peak area of compound/Mass of compound
Peak area of standard/Mass of standard
Correct the peak areas initially obtained by dividing with appropriate
response factor and normalise the correct values. Compare this result with
the known composition of the mixture.
ANALYSIS OF DRUGS BY FLUORIMETRIC METHOD

Principle. The alkaloid codeine and morphine in a mixture can be
determined independently by fluorimetric method because whilst both
fluoresce strongly at the same wavelength in dil. H2S04 solution, morphine
gives a generally negligible fluorescence in dil. NaOH. The fluorescence
intensities of the two compounds are assumed to be additive.
Reagents required. Prepare standard solutions of codeine and
morphine, each of which should cover the range 5-20 mg per dm 3 :
(a) Codeine in H 2S04 (0·05 M.)
(b) Codeine in NaOH (0·1 M)
(c) Morphine in H 2S04 (0·05 M).
(d) Morphine in NaOH (0·1 M).
Prepare solutions of weighed sample (codeine morphine mixture) III
H 2S04 (0·05 M) and in NaOH (0·1 M).
Procedure.
Measure the fluorescence intensities of each of series of standard
solutions at 345 nm, with excitation at 285 nm. Construct calibration curve
for each of the four series a, b, c and d. Measure the fluorescence intensities
of the sample in NaOH solution using emission (345 nm) and excitation (285
nm) wavelengths.
Read off the codeine concentration from the calibration graph (b).
Calculate fluorescence intensity that corresponds to this concentration of
codeine in H 2S0 4 using calibration graph (a). Now measure the fluorescence
intensity of the sample in H 2S04 solution and subtract the fluorescence
intensity due to codeine. This value gives the fluorescence intensity due to
morphine in H 2S0 4 , Its concentration can be deduced from graph (c). The
calibration graph (d) may be used to correct for the small fluorescence
intensity due to morphine in NaOH. It is not negligible when morphine
concentration is high and codeine concentration is low.
ANALYSIS OF DRUGS BY UV SPECTROPHOTOMETRIC METHOD

Principle.
A mixture of drugs such as phenacetin, caffeine and aspirin are analysed
by UV method. Phenacetin exhibits UV maxima at 250 nm, caffeine at 275
nm while aspirin has maxima at 277 nm.
DRUG ANALYSIS 291
~RO-C~O
o ..........
CR g
$ NR-C~
0
'CR,
o
o
R'C"Njc~
A I /)R
N
CR 3
N
OC 2R 5 I
CR 3
Aspirin Phenacetin Caffeine
Reagents required. Methylene chloride, NaHC0 3 , 4% (w/v),
1 M H 2S04 and HCl.
Separation of Drugs by Solvent Extraction.
Powdered tablet is dissolved in CH2 CI 2 . Aspirin is separated from
phenacetin and caffeine by extracting it into aqueous NaHC03 solution. The
aqueous layer is acidified and aspirin is separated by back extraction into
methylene chloride. It is measured spectrophotometrically at 277 nm.
Phenacetin and caffeine, which are remaining in the original methylene
chloride layer are determined in the mixture.
Preparation of Standard Solutions.
Dissolve 100 mg/L aspirin, 20 mg/L phenacetin and 10 mglL caffeine in
methylene chloride. Weigh 25 mg of each drug in a flask and make up the
volume to 100 mL with methylene chloride. Since aspirin decomposes in
solution, so analysis should be performed as early as possible.
Procedure.
A tablet may contain 220 mg aspirin, 160 mg phenacetin and 30 mg
caffeine. Grind quarter (114) part of the tablet to a fine powder.
• Add 20 mL methylene chloride into the powder. with constant stirring.
Transfer this mixture to a 60 mL separatory funnel.
• Extract aspirin from methylene chloride solution with two 10 mL
portions of cold 4% NaHC0 3 containing two drops of HCl and then
with 5 mL portion of water.
• Wash the combined aqueous extracts with three 10 mL portions of
CH2CI2 . Add this wash solutions to the original methylene chloride
solution.
• Leave the aqueous extract in the separatory funnel.
• Filter methylene chloride solution into a 50 mL volumetric flask and
dilute to the mark )Vith methylene chloride.
• Again dilute 1 mL aliquot of this solution to 50 mL with methylene
chloride in a volumetric flask.
• AcidifY aqueous bicarbonate solution with 6 mL of 1 M H2S04 to pH 1
to 2 in the separatory funnel to prevent hydrolysis of aspirin.
• Extract the acidified solution with eight separate 10 mL portions of
CH2CI2 , filter into a 100 mL volumetric flask and make up the volume
to 100 mL.
• Dilute further a 5 mL portion of this solution to 25 mL with CH2 Cl 2
in a volumetric flask.
Result. Record absorbance versus wavelength curves for the standard

solutions and unknown solutions between 200-300 nm.
• Using the absorbance of the standard and the unknown aspirin
solution at 277 nm, calculate the percent aspirin in the tablet and the
milligrams of aspirin per tablet.
• Read the absorbances of phenacetin and caffeine standards and
methylene chloride extract of the sample at 250 nm and 275 nm.
U sing these absorbances, calculate the percent phenacetin and
caffeine in the tablet and the content of each per tablet.
ANALYSIS OF DRUGS BY IR SPECTROPHOTOMETRIC METHOD

Principle.
Infrared method can be used for the analysis of aspirin, pbenacetin
and caffeine (APe) in analgesic tablets. The quantitation is solely based
on the intensities of the carbonyl bands.
Materials required. APC tablets, chloroform.
Procedure.
The drug contents of an appropriate number of tablets are directly
extracted into chloroform, filtered if necessary so as to remove the insoluble
tablet components. The final concentration of chloroform solution is made in
such a way so that it should contain 90 mg/cm3 of aspirin; 64 mg/cm3 of
phenacetin and 13 mg/cm3 of caffeine. The IR spectrum is recorded in 0·1 mm
NaCI-cell between 1400-2000 cm-1.
Result.
The intensities of carbonyl bands were observed at 1764, 1511 and 1665
cm-1 for aspirin, phenacetin and caffeine respectively.
o
INDEX
Accuracy 44 Cetane number 246

Acid and alkaline phosphatases 264 Classical methods 3
Adulterants in food 85 Classification of:
Analysis of: drugs 280
fungicides 175 pesticides 135
heavy metals 193 Cleaning of glasswares 37
insecticides 91,174 Clinical chemistry 247
organophosphates 90
Clinically important constituents 248
pesticides 173
reducing sugars 79 CNS drugs 281
water pollutants 148 COD 168
Analytical balance : Collection of blood samples 249
"electrochemical quartz 36 Complexing ligands in water 97
electronic 35
Components of soil 199
single pan mechanical 33
Composition of blood 247
Analytical chemistry 1
Conductivity 152
Analytical methods :
classical 3 Contamination of food stuffs 87
instrumental 4 Coprecipitation 25
non-destructive 7 Crude fibre 76
Antibody-antigen complex 267
Aniline point of liquid fuels 243 Dangerous drugs 280
Applications of RIA 269 Determination of :
accuracy 45
Barbiturates 263 blood pH 272
calcium 81
Biuret method 73, 261
calorific value of coal 238
Blood gas analysis 270 CO 2 and O2 content 271
Blood glucose 255 partial pressure of CO 2 272
Blood urea 257 partial pressure of O2 271
total nitrogen 209
Blood urea nitrogen 259 total phosphorus 212
BOD 169 Different forms of N2 162
Bomb calorimeter 232 Differential pulse polarography 195
Buoyancy effects 37 Disposable filter funnel 29
Dissolving the sample 18
Calibration of glasswares 38 Drug analysis by :
fluorimetric method 290
Calorific value of fuel 231, 238
IR method 292
Cellular elements 248 UV method 290
(i)
(U)
Dry ashing 67 Gas chromatography for organo-

Drying methods 64 phosphates in food 92
Gaseous pollutants 146
Effects of errors 43 GC-MS of drugs 285
Electroanalytical methods 4 Gerber method for milk fat 75
Errors and evaluation 40 Grading of coal 225
Errors: Gravimetric techniques 24
absolute 44 Gross errors 43
additive 42
gross 43 Handling of reagents 11
methodic 41
operational 41 Hardness 156
personal 40 Heavy metal pollution 182
random 42 Heavy metals :
relative 44 analysis of 193
systematic 40 instrumental techniques for 193
Estimation of : public health significance of 183
blood chloride 251 HPLC 91, 174,288
serum albumin 262 HPrLC 94
serum barbiturates 263
serum calcium 253
se~ electrolytes 251 Immunoassay 266
sodium and potassium 252 Important relations 61
Eutrophication 130 Incineration 31
Industrial effiuents 113
Farm wastes 147 Instrumental methods 4, 5, 193
Fertilizers 147 Iodine 278
Fluxes 19
Food adulterants 85 Karl Fischer titration method 65
Food analysis of: KFR equivalence 65
ash 66 Kjeldahl method 71
carbohydrates 78 Kjeltec Auto analyser method 210
fat and crude fibre 74
moisture 63
phosphorus 82 Laboratory note book 15
protein 70 Laboratory operations and practices 16
starch 80 Liquid fuels :
Fuels: aniline point of 243
calorific value of 231 flash and fire point of 240
gaseous 227 carbon residue of 244
liquid 226
solids 224 Marine pollution 105
Functions of blood 249 Mean 51
(iiI)
Mean deviation 53 Producer gas 227

Measurement of : Proximate analysis of coal 235
BOD 169 Public health significance of heavy
COD 168 metals 183
DO 165
TOC 172
Radioactive pollutants 128
Median 54
Radio immunoassay 267
Micro and macro plant nutrients 202
Rejection of results 59
Microscopic examination of food 89
Reliability of results 58
Microwave decomposition 21
Reporting of analytical data 48
Minimisation of errors 46
Rules for computation 48
Moisture analysis 63
Mojonnier method 74
Safety in analytical laboratory 14
Munson and Walker method 78
Sample decomposition 19
Sample preparation 16
Narcotics 279
Neatness and cleanliness 10 Sampling statistics 17
Screening of drugs by :
GC 283
Objectives of water analysis 148
GC-MS 285
Octane number 245 HPLC 289
Organic reagents 12 TLC 288
Origin of waste water 98 Selecting an analytical method 9
Oxygen demanding wastes 125 Selecting reagents 11
Serum :
Parameters of water analysis : acid and alkaline phosphatase 264
acidity and alkalinity 154 albumin 262
chloride 157 barbiturates 263
colour 151 bicarbonate 254
fluoride 158 calcium 253
silica 160 electrolyte 251
protein 261
Pesticide analysis 90, 172 uric acid in 260
Pesticides : Significant figures 48
bioaccumulation of 139
biodegradation of 140 Soil analysis of :
chlorinated 94 magnesium and manganese 216
classification of 135 moisture 206
persistent 138 pH 207
sources of 138 salts 220
structures of 135 silica 214
sodium and potassium 221
Precision 45 sulphur 218
Principles of RIA 267 total nitrogen 209
(iv)
Sources of water pollution: Uses of statistics 60

agricultural and domestic 111 UV methods 73, 290
industrial 113
radioactive and thermal 112
Variables 62
Standard deviation 55 Volumetric glasswares :
Statistical evaluation of data 50 burettes 24
Stoichiometry 23 flasks 24
pipettes 24
Storage of blood samples 250
Structure of pesticides 135
Water gas 229
Water pollutants :
Techniques of weighing 36 disease causing agents 127
Theander Marlett method 76 inorganic 117
organic 120
TLC for pesticides in food 93, 173 radioactive 128
Total organic nitrogen 164 sediments 122
Total serum protein 261 synthetic detergents 123
thermal 132
Total solids 153
Water pollution:
Toxicity of metals 182 Laws 176
Trace elements in the body 273-278 sources of 111-116
Turbidity 152 standards 178
types of 9~110
Types of analysis 2
Washing 30
Types of water pollution 95-110
Weighing errors 36
Typical fluxes 20 Wet ashing 68
Ultimate analysis 236 X-ray methods 7

Urea nitrogen 259
Uric acid in serum 260 Zinc 187, 205, 276

Analytical Chemistry Guide for M.Sc. Students

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ANALYTICAL CHEMISTRY

For M.Sc. Students of Various Universities.

[COMPREHENSIVELY COVERING THE UGC SYLLABUS.]

2. ERRORS AND EVALUATIONS . ...................... 40-61

4. TYPES OF WATER POLLUTION .................... 95-110

5. SOURCES OF WATER POLLUTION . ................ 111-116

6. WATER POLLUTANTS AND THEIR EFFECTS . ........ 117-147

8. HEAVY METAL POLLUTION . ...................... 182-198

9. SOIL ANALYSIS ................................. 199-223

10. FUEL ANALYSIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 224-246

11. CLINICAL CHEMISTRY . . . . . . . . . . . . . . . . . . . . . . . . . .. 247-278

12. DRUG ANALYSIS . .............................. . 279-292

INDEX G" • • • • • • • • • • • • • • • • " • • • • • • • • • • • • • • • • • • • • • • • • • • • (iHiv)

• The nutritional value of food is determined by chemical analysis for

1. Macro involves the analysis of quantities of 0·1 g or more.

Table 1. Classification of instrumental methods of analysis.

2. Spectroscopic Interaction of matter with Radiant energy of a

S.No. Method Principle Property measured

3. Mass Spectroscopy Ionisation of atoms, ions and Position and intensity

• With instrumental methods it is necessary to carry out a calibration

opposed to the more commonly applied destructive methods involving

NEATNESS AND CLEANLINESS IN LABORATORY

• Solid reagents must be sufficiently dry.

ORGANIC REAGENTS USED IN INORGANIC ANALYSIS

Dithizone is specially used for the estimation of Pb which produces a

• Reagent bottles and apparatus should be placed properly after use.

• The printed data or any chart should be permanently attached within

LABORATORY OPERATIONS AND PRACTICES

SAMPLE PREPARATION FOR ANALYSIS

instruments (electrochemical sensors) and techniques (IR, GC-MS) are

are a measure of precision. For normal statistical manipulations it is often

WEIGHING THE SAMPLE

DISSOLVING THE SAMPLE

• Some inorganic substances will not dissolve in acids, so fusion with

Table 2. Typical fluxes.

on to a shaped piece of paper (Fig. Ib)

The wick of the sample paper ---}----:---

• Other non-stoichiometric methods include mass spectrometry,

precipitate during its formation. Co-precipitation may be due to isomorphous

(i) filter paper (ii) crucibles (iii) filter pulp mats

Table 3. Characteristics for Whatman series of hardened

The size of the filter paper selected for a particular operation is

Fusion samples are readily removed from these crucibles. At high

(iii) Filter Pulp.

(iv) Synthetic Filter Membrane.

hydrolysis of ferric or similar salts. Some precipitates tend to oxidise during

DRYING AND IGNITING PRECIPITATES

COOLING AND DRYING IN DESICCATOR

WEIGHING OF THE PRODUCT

TYPES OF ANALYTICAL BALANCES

Fig. 6. Schematic diagram for a typical single pan balance.

Since results are available as an electrical signal, they can be processed

TECHNIQUES OF WEIGHING USING ANALYTICAL BALANCE

2. Changes in the weight of weighing vessel.

CLEANING OF GLASS WARES

Methods for Cleaning Glass Wares.

Table 4. Volume of 1 g of water at various temperatures.

In all calibration operations the apparatus to be calibrated must be

Volumetric Flask Calibration.

TYPES AND SOURCES OF ERRORS

(vi) Ignition of precipitate at incorrect temperatures.

slow potential stabilization, undesirable electrode polarisation, side reactions