20 78 37

110ml

100ml

68%

95%
99.7%
-3s -2s -1s x +2s +2 +3s
(mean)

Jagat Bahadur KC Basanta Kumar Rai

BASIC FOOD ANALYSIS HANDBOOK

JAGAT BAHADUR KC
Professor of Food Technology
Tribhuvan University

BASANTA KUMAR RAI

Lecturer (Food Technology)
Tribhuvan University

FIRST EDITION-2007
Publisher: Mrs. Maya K.C.
Anamnagar, Kathmandu – 32, Nepal

First Edition: August 2007

Number of copies: 500
Copyright: Reserved by the authors
Price: NRS 250/-

Printed at: Prompt Printers Pvt Ltd.

Anamnagar, Kathmandu – 32, Nepal
Phone No.: 01-4250655
ISBN: 978-99949-2-796-7
PREFACE

This manual is a compilation of routine analyses most often carried out in food
analysis. During the preparation of this manual, several books and manuals have been
consulted and the methods simplified (where needed) for better comprehension. A
basic concept of statistical method and its use through computer have been also been
explained. Although simplicity and clarity have been emphasized throughout the
book, many of the practicals presented here are not in ‘do-it-yourself’ form and so
will require instructor’s guidance. There are profuse illustrations, examples and cross-
references. The preparations of reagents have been presented as a footnote (rather
than in the Appendix) so that one does not need to move back and forth. Appendices
and indices are other very useful features of this book. A unique feature of this book
(which other books generally lack) is the inclusion of backgrounder in statistical
analysis of the data. We are indebted to several authors, whose published materials
we took the liberty to use. As a token of thanks, we have appended their work in the
bibliography.

The manual is basically meant for beginners, who are doing their first semester in
food analysis but the students of food chemistry, water microbiology, and other
biological sciences may also find it equally useful.

Suggestions and criticisms for the improvement of this book will be thankfully
received.

Jagat Bahadur K.C.

Basanta Kumar Rai
CONTENTS

PREFACE -------------------------------------------------------------------------------------- iii

CONTENTS ------------------------------------------------------------------------------------ v
CHAPTER I: STANDARD LABORATORY PRACTICE ------------------------------ 1
1.1. Introduction ----------------------------------------------------------------------------- 1
1.2. Some Do’s and Don’ts ---------------------------------------------------------------- 1
1.3. Use of operation manuals ------------------------------------------------------------- 1
1.4. Writing laboratory report ------------------------------------------------------------- 1
CHAPTER II: BACKGROUNDER --------------------------------------------------------- 4
2.1. The need for food analysis ----------------------------------------------------------- 4
2.3. Sampling plan -------------------------------------------------------------------------- 5
2.4. Sampling of food for analysis and some statistical treatments ------------------ 6
2.5. Preparation of sample ----------------------------------------------------------------- 8
2.6. Accuracy, specificity, precision and related terms ------------------------------- 11
2.7. Statistical treatment of data---------------------------------------------------------- 12
2.7.1. Degrees of freedom ------------------------------------------------------------- 12
2.7.2. Variance -------------------------------------------------------------------------- 12
2.7.3. Standard deviation -------------------------------------------------------------- 12
2.7.4. Standard error -------------------------------------------------------------------- 13
2.7.5. Utility of standard error -------------------------------------------------------- 13
2.7.6. Statistical hypothesis------------------------------------------------------------ 14
2.7.7. Level of significance ------------------------------------------------------------ 15
2.7.8. One-tailed and two-tailed tests ------------------------------------------------ 16
2.7.9. Confidence limit ----------------------------------------------------------------- 18
CHAPTER III: STANDARDIZATION --------------------------------------------------- 30
3.1. Calibration of measuring devices (pipettes, burettes, and vol. flasks) --------- 30
3.2. Preparation of standard solutions --------------------------------------------------- 33
CHAPTER IV: PROXIMATE ANALYSIS ----------------------------------------------- 38
4.1. Determination of crude fat by solvent extraction --------------------------------- 39
4.2. Determination of crude fiber in food sample ------------------------------------- 43
4.3. Determination of protein ------------------------------------------------------------- 48
4.3.1. Protein determination by Kjeldahl nitrogen method ----------------------- 48
4.3.2. Macro-Kjeldahl method -------------------------------------------------------- 53
4.3.3. Determination of protein in milk by formol titration ----------------------- 54
vi
4.3.4. Determination of protein by biuret assay ------------------------------------ 56
4.4. Moisture in foods --------------------------------------------------------------------- 59
4.4.1. Moisture content by IR-moisture meter -------------------------------------- 63
4.4.2. Moisture content by hot-air oven method ------------------------------------ 64
4.4.3. Moisture content by hot plate method ---------------------------------------- 65
4.4.4. Moisture content by solvent distillation method ---------------------------- 66
4.5. Determination of ash in food samples --------------------------------------------- 67
4.5.1. Determination of total ash in solid food sample by dry ashing ----------- 69
CHAPTER V: SOME ULTIMATE ANALYSES ---------------------------------------- 71
5.1. Determination of acid-insoluble ash ----------------------------------------------- 71
5.2. Determination of calcium content by volumetric method ----------------------- 72
5.4. Potassium by flame photometry (emission) --------------------------------------- 74
5.5. Determination of L-ascorbic acid (vitamin C) ------------------------------------ 76
5.5.1. The 2,6-dichlorophenol indophenol titration method ---------------------- 76
5.5.2. The 2,4-dinitro phenylhydrazine method ------------------------------------ 79
5.6. Determination of reducing sugars -------------------------------------------------- 82
5.6.1. Nelson somogyi method -------------------------------------------------------- 83
5.6.2. Dinitrosalicylic acid (DNS) method ------------------------------------------ 86
5.6.3. Lane and Eynon method-------------------------------------------------------- 87
5.7. Determination of starch by hydrolysis --------------------------------------------- 90
5.7.1. Lane and Eynon method of starch determination --------------------------- 90
5.8. Determination of acidity and pH of food ------------------------------------------ 91
5.8.1. Determination of acidity in (i) tomato, (ii) milk ---------------------------- 93
5.8.2. Determination of pH of tomato and milk ------------------------------------ 94
CHAPTER VI: NATURAL PIGMENTS AND RELATED COMPOUNDS -------- 96
6.1. Determination of chlorophyll ------------------------------------------------------- 96
6.2. Determination of carotene by solvent partition method ------------------------- 98
6.3. Tannins ------------------------------------------------------------------------------- 100
6.3.1. Volumetric determination of tannins --------------------------------------- 100
6.3.2. Colorimetric determination tannins ----------------------------------------- 102
CHAPTER VII: FOOD ADDITIVES ---------------------------------------------------- 103
7.1. Artificial colorants by thin layer chromatography ----------------------------- 103
7.2. Determination of sulfur dioxide -------------------------------------------------- 106
7.2.1. Determination of SO2 in foods ---------------------------------------------- 107
7.2.2. Determination of benzoic acid/sodium benzoate in food ---------------- 110
CHAPTER VIII: GENERAL TEST OF ALCOHOLIC BEVERAGES ------------- 113
8.1. Ethanol content by specific gravity method ------------------------------------- 113
8.2. Determination of methanol by colorimetric method --------------------------- 115
8.3. Ethanol content by oxidation method -------------------------------------------- 117
CHAPTER IX: ANALYSIS OF FATS AND OILS ------------------------------------ 121
9.1. Determination of free fatty acids (FFA) and acid value ----------------------- 122
9.2. Determination of saponification value of fat/oil -------------------------------- 124

vi
 FOOD ANALYSIS

9.3. Determination of iodine value of fat/oil by wij's method --------------------- 126

9.4. Determination of peroxide value ------------------------------------------------- 131
9.5. Melting point of fat by open-tube capillary method --------------------------- 132
9.6. Tests for the adulteration of fats and oils ---------------------------------------- 134
9.6.1. Reichert-Meissl, Polenske, and Kirschner value -------------------------- 135
9.6.2. Baudouin test ------------------------------------------------------------------ 139
9.6.3. Hexabromide test -------------------------------------------------------------- 139
9.5.4. Presence of animal fat by microscopic examination --------------------- 140
9.6.5. Test for presence of argemone oil------------------------------------------- 141
9.6.6. Kries test for rancidity in fats/oils------------------------------------------- 142
CHAPTER X: ANALYSIS OF WATER ------------------------------------------------ 143
10.1. Total dissolved solids ------------------------------------------------------------- 143
10.2. Total hardness in water ----------------------------------------------------------- 144
10.3. Calcium in water ------------------------------------------------------------------ 145
10.4. Magnesium in water -------------------------------------------------------------- 146
10.5. Alkalinity in water ---------------------------------------------------------------- 147
10.6. Chlorides in water ----------------------------------------------------------------- 148
10.7. Iron by1,10-phenonthroline method -------------------------------------------- 149
10.8. Microbiological analysis of water----------------------------------------------- 150
10.8.1. The standard plate count of water sample -------------------------------- 151
10.8.2. The presumptive coliform test by membrane filtration ----------------- 153
CHAPTER XI: ANALYSIS OF COMMON SALT------------------------------------ 155
11.1. Salt content by potasssium dichromate method ------------------------------- 155
11.2. Iodine content in common salt -------------------------------------------------- 156
11.3. Test of magnesium and calcium ------------------------------------------------- 158
BIBLIOGRAPHY --------------------------------------------------------------------------- 161
APPENDICES ------------------------------------------------------------------------------- 162
INDEX ---------------------------------------------------------------------------------------- 174

vii
CHAPTER I: STANDARD LABORATORY PRACTICE

1.1. INTRODUCTION

Standard laboratory practice is essential for safety, convenience and successful

completion of relevant works. Take your time to make the operations most
comfortable, for example, put aside the jumble of unwanted glasswares from the
table, wipe the table dry, etc. Work out the sequence of activities carefully for each
work so that you can minimize the unnecessary lag in your work. This will help you
finish the work in time, without making a mess!

1.2. SOME DO’S AND DON’TS

There are some Do’s and Don’ts of every laboratory. Here are some for a food
analysis laboratory:

 Wear a lab coat

 Don’t eat or drink in laboratory
 Don’t wear lipsticks during pipetting
 Read the labels on the reagents carefully
 Turn off the burners when you have finished work
 Exercise extreme care when handling corrosive chemicals
 Use precaution when weighing corrosive chemicals, e.g., silver nitrate,
iodine, NaOH
 Always ask your instructor or teacher whenever you are in doubt

1.3. USE OF OPERATION MANUALS

Every instrument and apparatus has an operation manual. The working principle may
be the same but the way in which the instrument is handled differs from manufacturer
to manufacturer. Read the operating instructions carefully before you use a new
instrument. Always place the manual back in place.

1.4. WRITING LABORATORY REPORT

Use a standard format for writing laboratory report. All lab reports typically have, in
sequence:
 STANDARD LABORATORY PRACTICE

1. Practical Number
2. Name of the practical
3. Background
4. Objectives
5. Principle
6. Materials required
7. Procedure
8. Observations
9. Results and discussion

Use standard papers meant for laboratory. Write your roll number and the dates of
performance & submission in the top right corner of the first page. Leave a page or two
for maintaining index. Use the over-leaves for observations, calculations, drawing, etc.

Be very serious in writing results and discussion. Discussion in particular needs a lot
of reading but has the advantage of enabling you to critically appraise your own work
and findings. Logical discussion is the only means that enables you to reach a valid
conclusion. Fig. I-1 in the following page suggests you a sample of the Practical
Record.

2
 Performed:…..
Practical No. 1
Submitted:….
DETERMINATION OF CRUDE FIBER
Roll No.:….
BACKGROUND

OBJECTIVES

DRAWINGS
PRINCIPLE
OBSERVATIONS

CALCULATIONS
REQUIREMENTS

Fig. I-1: Sample of practical record book

FOOD ANALYSIS

3
CHAPTER II: BACKGROUNDER

2.1. THE NEED FOR FOOD ANALYSIS

Food items need to be analyzed for a host of reasons, the more important of which are:

Academic

 Analysis to find out the profile of different constituents in a new food

 Measure activity of biologically active compounds, e.g., enzymes in milk

Quality control

 Process control: for example, analysis of citric acid yield, vinegar yield, etc.,
during fermentation; adequacy of pasteurization of milk; adequacy of
appertization of canned foods
 Test of raw materials for compliance with specification, e.g., α-acid content
in hops, diastatic activity of malt, free fatty acids in fats/oils
 For judging consistency of product quality, e.g., color, alcohol and bitterness
in beer; salt content in biscuits; fat content in ice cream; melting point of
hydrogenated fats/oils; TSS of condensed milk, etc.
 For compliance with standard set by regulatory bodies (National or
International)
 Presence of adulterants, toxicants, toxins, etc.
 Microbiological safety of foods

Research work

 Shelf life assessment of items such as fats/oils, noodles, etc

 Assessment of change in nutritional value, functional properties, etc., of food
items upon processing, storage, etc.
 Assessment of loss of biologically active compounds, viz., vitamins upon
processing, storage, etc.
 Establish correlation between different test parameters and test methods
 Trace the origin of food items, e.g., synthetic and fermented vinegar, the
source oil used for making margarine, etc
 FOOD ANALYSIS

Thus, the nature, type, and number of analysis of food items are extremely diverse.
Even then, attempts are made to analyze food items for some mean value so that the
latter can be used to describe certain property and status of the food in question.

2.2. FOOD ANALYSIS IN INDUSTRIAL QUALITY CONTROL LABORATORY

In industrial quality control lab, food items are analyzed at various stages and for
various reasons, the main of which are:

 Process control (rapid in-plant test, e.g., using brix meters, pH meters, etc.)
 Buying sample (from supplier)
 Complaint sample (from consumer)
 Finished product (to insure that the food conforms to legal standards)
 Competitor’s sample

2.3. SAMPLING PLAN

Sound sampling is a prerequisite in any analysis. Because the value obtained in the
analysis is used to interpret the characteristic for the population, it is essential that the
sample be truly representative of the population. In food analysis, one has to be
familiar with the terminology “sampling plan”. Sampling plan is composed of 3
components, viz., sampling, sample preparation, and analysis.

Sampling contributes to the largest relative error. An ideal sample should be identical
in all of its intrinsic properties with the bulk of the material from which it is taken.
The representative samples can be obtained only from truly homogenous materials.
Since this condition is seldom encountered in natural food items, composite sample is
used instead.

During sampling, individual variability of samples with respect to given

physicochemical property must be taken into account. This is true for all plant and
animal products. For example, we can find different vitamin C contents in different
portions within a single tomato! This calls for taking adequate samples for
compensating the variability. The amount of sample to be taken can be estimated
statistically when the extent of variability is available. Where the extent of variability
is unknown, it is advisable to select at least ten times the amount to be taken as a
sample for analysis.

Generally, the errors in sampling are the following:

 Failure to select the individuals composing the sample at random

 Changes in the composition of the product during sampling, such as loss or
absorption of moisture, mechanical injury, etc.
 Difficulties in obtaining a uniform sample

5
 BACKGROUNDER

2.4. SAMPLING OF FOOD FOR ANALYSIS AND SOME STATISTICAL

TREATMENTS

2.4.1. POPULATION AND SAMPLE

In statistics, population is the aggregate of objects, animate or inanimate, under study

in any statistical investigation. A finite subset of population selected from it with the
objective of investigating its properties is called a sample and the number of units
drawn is known as sample size.

Sampling is a process or method of drawing a representative group of individuals or

cases from a particular population. Sampling and statistical inference are used in
circumstances in which it is impractical to obtain information from every member of
the population, as in biological or chemical analysis, industrial quality control, or
social surveys. Sampling enables us to draw conclusions about the characteristics of
the population after studying only those objects or items that are included in the
sample.

2.4.2. TYPES OF SAMPLING

The choice of the appropriate sampling depends on the objective of the sampling and
the type of population to be sampled.

The objective of sampling can be development of a national food composition

database, determination of aflatoxin levels in a load of grain, determination of
pesticide levels in a food product, quality control in industry, comparison of
competitors’ product, nutrition surveys, sampling of samples before importing items,
etc. The definition of the objective helps to determine the most appropriate sampling
strategy. If the objective is to develop a national food composition database, then two
major questions need to be answered. What foods should be selected for analysis?
What nutrient(s) or component(s) should be measured? Food analysis projects can
estimate levels of a single component (for example, selenium, β-carotene or total fat)
in foods consumed by a population of individuals, or they may focus on a single food
(for example, beef, milk or carrot) and its major components.

The population can consist of heterogeneous or homogeneous items, which can

further be animate or inanimate.

The sampling techniques can be broadly classified as:

1. Purposive or subjective or judgment sampling

2. Probability sampling
3. Mixed sampling

6
 FOOD ANALYSIS

Purposive sampling

It entails deliberate (intentional) selection of samples so that certain character of the

item can be determined, e.g., for the evidence of aflatoxin formation in grains.

Probability sampling

It provides a scientific technique of drawing samples from the population. In this

method, each unit has equal chance of being chosen as a sample.

Mixed sampling

In this method, samples are drawn partly according to probability laws (equal chance)
and partly according to some fixed sampling rule (no use of chance). There are
several subtypes of mixed sampling although not all of them are used in food
analysis. Indeed, many of them are applied in survey-type works. Some of the
examples found in mixed sampling are:

 Simple Random Sampling

 Stratified Random Sampling
 Systematic Sampling
 Multistage Sampling
 Quasi Random Sampling
 Area Sampling
 Simple Cluster Sampling
 Multistage Cluster Sampling
 Quota Sampling

The basic sampling design is simple random sampling, based on probability theory. In
this form of random sampling, every element of the population being sampled has an
equal probability of being selected. Stratified sampling is a more refined form of the
random sampling. In it, the population is divided into classes and simple random
samples are drawn from each class., Cluster sampling is done in surveys. In it, the
unit of the sample is a group, such as a household. Systematic sampling refers to
samples taken by any system other than random choice, such as every tenth name on
a list.

Unlike other items, foods and food products are variable in composition. Plant foods
are more variable than flesh foods. Processing methods cause additional changes in
composition. In addition to differences in composition between individual fruits and
vegetables of the same variety and maturity, there is a difference in composition
between various parts of the same fruit or vegetable. Some constituents vary more
than others. In sampling of food and food products, sufficient material must be taken
to compensate for variability and requirement of certain analytical techniques. The
amount of material needed can be estimated statistically when the extent of variability

7
 BACKGROUNDER

of the individuals of samples are available. When repeated chemical analyses are
done, it is advisable to make a preliminary determination of the variability of the
sample.

Acceptance sampling

This type of sampling is widely used in the statistical quality control of the incoming
raw materials and the final products. The quality control may be related to both
microbiological and physicochemical aspects of the food.

In microbiological testing of food, the sampling scheme most commonly used is that
of sampling for attributes. The simplest of the sampling for attributes is the two-class
attributes plan. In this method, the results are assigned to two classes, viz., (i) acceptable
and (ii) defective, depending on the test result. A sample is called defective if it is
shown to contain more defects than a specified level. In the case where presence or
absence is tested, the detection of the defect alone is sufficient to make the whole lot
of item defective or unacceptable.

A two-class sampling scheme can be defined by three numbers, n, m, and c (the

notations may differ from book to book):

n: the number of sample units to be tested

m: the count above which the sample is regarded defective
c: maximum allowable number of sample units which may exceed m
before the lot is rejected

For example, in a microbiological analysis, if n = 5, c = 0 cfu/g, and m = 103 cfu/g, it

means that the number of samples to be inspected is 5 and the food will be rejected if
the number of microorganisms in it is above 103 cfu/g. Since c = 0, not a single
sample should cross the specified microbial load.

2.5. PREPARATION OF SAMPLE

Fresh fruits and vegetables: Remove the adhering soil or sand by washing or wiping
the surfaces with a damp cloth. Vitamin C, sugars, acidity, TSS, etc., must be
determined on the fresh item. Protein and lipid content are determined by first drying
the sample to less than 6% moisture content. The drying can be carried out in a
vacuum oven at 60°C. For the proximate analysis, drying to a very low level of
moisture content at 100°C has often been used.

Canned fruits and vegetables

When analyses are to be made on composite sample, mix and comminute the entire
contents. When the analyses are to be made on solid and liquid portions separately,
drain the contents on a sieve.

8
 FOOD ANALYSIS

Dried fruits

Pass the sample through a food chopper three times, and thoroughly mix after each
grinding. If needed, grind initially with a coarse cutting blade, and do final grinding
with a nut butter blade.

Pureed products

Shake thoroughly pureed products such as tomato puree, ketchup, fruit pulps and
strained fruits and vegetables before sampling.

Fruit juice beverages

Render fruit juice beverages containing insoluble matter thoroughly uniform by

blending in a high speed blender.

Powdery or granular materials

Sample the material by the technique of quartering.

It is worth remembering that the samples to be collected and prepared for

microbiological analysis need special care. There is a limitation of time period for the
sampling and storage of the sample. Special packaging materials are needed for
collecting the samples. Everything thing used during the sampling process should be
sterile. The preparation (such as homogenization of the sample) and subsequent
analysis must be done aseptically.

Preparation of sample for microbiological analysis

Solid food: solid food is generally mixed with a sterile diluent in a mechanical
blender to obtain a homogeneous suspension. To 25g of food in a sterile blender jar,
225g of sterile diluent is added, and when mixed, a 1:10 dilution of the food and
associated organisms is obtained. This 1:10 dilution is also referred to as a 1/10 or 10-
1
dilution. In routine works, the dilution needed is beyond 1/10. The preparation of
additional dilution is given in the Fig. II-1.

The diluent used is usually 1% peptone water. Sterilized distilled water is sometimes
used. The plating is carried out of dilution such that the number of colonies developed
is between 30 and 300.

Samples for any analyses should be large enough for all intended determinations but
not too large to bring about waste. A general rule of thumb is:

o Homogenous samples: 250g (or 250ml)

o Spices: 100g

9
 BACKGROUNDER

o Fruits and vegetable: 1000g

The size of the sample as well as the method of drawing sample is also dependent on
the nature, type and purpose of analysis, as also the type of the product. An example
of sampling for the analysis of aflatoxin in cereals (such as such as maize) is
described in the following paragraph.

Food
50g

Blend 2 min
10-1
Low speed
10ml
450ml diluent
10ml 10ml 10ml 10ml
90ml 90ml 90ml 90ml 90ml
Dilution 10-2 10-3 10-4 10-5 10-6

1ml 1ml 1ml 1ml 1ml 1ml

Fig. II-1: Sample preparation for microbiological analysis

For rapid screening of maize for the presence of aflatoxin, a test called BGYF (Bright
Yellow Green Fluorescence) is used and the method requires at least 4kg sample.
Sampling maize for quantitative analysis is more involved. First of all, 22kg of
sample is taken. A subsample of 2kg is drawn and aflatoxin analyzed in duplicate.
The decision to accept, resample or reject is based on the protocol shown in Fig. II-2.

22kg 2kg 1A 1A + 1B < 32 ppb, accept

SAMPLE 1 SUBSAMPLE 1B 2 > 32 ppb < 150 ppb
Run Sample 2
> 150 ppb, reject

22kg 2kg 2A 1A + 1B + 2A + 2B < 44 ppb, accept

SAMPLE 2 SUBSAMPLE 2B 4 > 44 ppb < 76 ppb
Run Sample 3
> 76 ppb, reject

22kg 2kg 3A 1A + 1B + 2A + 2B + 3A + 3B < 50 ppb, accept

SAMPLE 3 SUBSAMPLE 3B 6 > 50 ppb, reject

Fig. II-2: Sampling and analysis of aflatoxin in maize

10
 FOOD ANALYSIS

2.6. ACCURACY, SPECIFICITY, PRECISION AND RELATED TERMS

2.6.1 PRECISION

Precision in analysis includes two terms, viz., (i) repeatability, and (ii) reproducibility.

Repeatability

It is the measure of how well an analyst in a given laboratory can check himself using
the same analytical method to analyze the same test sample at the same time.

Reproducibility

It is a measure of how well an analyst in one laboratory can check the results of
another laboratory using the same analytical method to analyze the same test sample
at the same or different time.

2.6.2 ACCURACY

It is defined as the closeness of agreement between measured value and the accepted
“true”, or reference value. Accuracy is indicative of the bias of the measurement
process. The term accuracy is often confused with precision, and it is used by some
authors and organizations in the sense of a combination of bias and precision.

Methods may be precise without being accurate or accurate without being precise.
The term “accuracy” is used in the sense of “bias”. It is also important when using the
term to indicate a difference, to be sure that the subject of bias is indicated – whether
it be a single value, a mean, or a long-term averaged (accepted true value).

Accuracy of a given analytical method is often very difficult to establish. It is very

tedious, especially for naturally occurring foods. In determination of accuracy of a
method, we are basically interested in establishing the deviation of an analytical method
from an ideal one. The deviation may be due to an inaccuracy inherent in the procedure;
the effect of substances other than the analyzed one in the food sample; etc.

2.6.3 SPECIFICITY

This is the ability of a method to respond exclusively to the target analyte and not to
any degradant, impurity or other component of the matrix.

Very few methods are absolutely specific, so the term “selectivity” is often used for
this property. This parameter shows that the method can be used to quantitate the
analyte without interference.

11
 BACKGROUNDER

2.7. STATISTICAL TREATMENT OF DATA

The larger the number of samples analyzed the more accurate the result will be. In
general, for a typical analysis for food composition, three or more replicates have to
be done. Presentation of raw data or a simple arithmetic mean of the value does not
lead to any meaningful conclusion about the characteristic (e.g., protein content,
acidity, fat content, vitamin C, etc.) of the population. Hence, some statistical
treatment has to be done to the sample data before we can say something about the
population with confidence.

Some of the terms involved in statistical treatment of data are described in the
following paragraphs:

2.7.1. DEGREES OF FREEDOM

Degrees of freedom are the number of values in a set of data which are free to vary.
For example, if there are 3 values A, B, and C having a mean of 9, (A+B+C)  3 = 9.
In this case, if one of the three values is fixed, the same mean of 9 can be achieved by
assigning an unlimited number of variations to the remaining two values. For
example, if the value of A is 12, the values of B and C can be freely varied so that the
sum of the two is equal to 15 (that is, B + C = 15). It can be 12, 3; 10, 5; 7, 5; etc.
This implies that for a data of 3 values, there are 2 degrees of freedom. Generalizing,
data with n number of values will have n-1 degrees of freedom.

2.7.2. VARIANCE

It is the arithmetical mean of the squares of the deviation of the values from the mean
of the data. It is given by:

2
 (x  x )
Variance, s 2 
n 1

Where, x = value, x = arithmetic mean of the values, ( x  x )2 = square of deviation, n

= number of values, and n-1 = degrees of freedom.

To avoid errors due to biased estimate (n), n-1 degrees of freedom are used for small
sample number (<30) instead of n.

2.7.3. STANDARD DEVIATION

In calculating the variance we get the average of squared deviations. However, a more
reliable index of variability would be the original units (that is, without squaring). It
can be acquired by taking the square root of variance, to give what is called as
standard deviation. Thus, standard deviation (s) is given by:

12
 FOOD ANALYSIS

2
 (x  x )
2
s s 
n 1

Standard deviation shows the degree of spread of a normal curve of distribution (Fig.
II-3). The flattened the curve, more variability in data, and more the standard
deviation. Standard deviation indicates that nearly 68% (or two-thirds) of the values
will fluctuate 1s about the mean. 95% of values will fluctuate 2s about the mean and
99.7% of values will fluctuate 3s about the mean. See Fig. II-3.

68%
s = standard deviation

95%
99.7%
-3s -2s -1s x +3s +3s +3s
(mean)

Fig. II-3: Normal curve showing variation of standard deviation about the mean

2.7.4. STANDARD ERROR

Let us consider that samples of size n are for k number of times taken from an infinite
population. For each of the k possible samples, we can calculate a statistic (say,
mean). Let the means be x1 , x2 , x3 , x4 , x5 , x6 ,.......xk . The set of the values of
x constitutes what is called sampling distribution of the statistic x (that is, mean).
The standard deviation of x1 , x2 , x3 ....xk is now known as standard error of the
mean x , and is given by:

S .E .( x )  s 2 / n  s / n

Where, s = standard deviation and n = number means taken.

Standard errors can be calculated for different statistics, such as standard deviation,
coefficient of variation, difference of two means, sample proportion, etc. The
calculation, however, is very complex.

2.7.5. UTILITY OF STANDARD ERROR

The concept of standard error is extremely useful in the testing of statistical

hypothesis. The difference between the statistic, say t, and its true value μ (the
expected value, given by E(t)) is compared with standard error of the statistic t as
follows:

13
 BACKGROUNDER

t  E (t )
Z ~N (0,1)
S .E.(t )

Where Z is a test statistic (called standardized normal variate corresponding to the

test statistic t). The notations ~ N (0,1) implies that Z is asymptotically normally
distributed with mean = 0 and standard deviation = 1. This relation is true only when
the number of samples is large (usually  30).

Let us now use the S.E.(t) for comparing it with t  E(t) and try to conclude whether t
is significantly different from E(t) at 5% level of significance.

From the normal probability table, the absolute value of Z should be more than 1.96
in order for t to be significantly different from E(t) at 5% level. Mathematically,

Z> 1.96 implies t  E(t)> 1.96×S.E.(t)

If the above condition is true, we reject the null hypothesis (that assumes no difference,
see later) and accept the alternative hypothesis (that assumes difference between t and
E(t), also see later). However, if we find that t  E(t)< 1.96×S.E.(t), we will accept
the null hypothesis and say that the difference was just fluctuation of sampling.

We will now become familiar with the standard error of the means ( x1 and x2 ) from
two independent lots of observations in which the sample size (n) is sufficiently large
(greater than or equal to 30). Because the derivation is complicated the final
expression is directly shown below:

s12 s2 2
SE ( x1  x2 )  
n1 n2

2.7.6. STATISTICAL HYPOTHESIS

A statistical hypothesis is a definite assumption made about the population parameter,

which we want to test on the basis of the evidence from a random sample. This is an
essential step in any statistical inference. In statistics, two types of hypotheses are
used, viz.: (i) Null hypothesis, and (ii) Alternative hypothesis.

A null hypothesis is denoted by H0 and is defined as a hypothesis which is tested for

possible rejection under the assumption that it is true. Such a hypothesis is usually a
hypothesis of no difference. For example, if we want to test if a particular drug is
effective, we shall take neutral attitude and set up the hypothesis that it is not
effective. For testing if out of two foodstuffs, one is better than the other, we shall set
up the hypothesis that there is no significant difference between them. In other words,
the difference is just due to fluctuation of sampling.

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An alternative hypothesis is any hypothesis which is complementary to null

hypothesis. It is usually denoted by H1. For example, if a null hypothesis, H0, assumes
that the two means, namely, μ and μo are equal (that is, μ = μo), the alternative
hypothesis can assume any of the followings:

μ  μo (that is, μ < μo or μ >μo or μ = μ1)

If the evidence supports the null hypothesis (or has no sufficient evidence against it)
we accept the null hypothesis. Otherwise we reject it, which means we automatically
accept the alternative hypothesis. Acceptance of a hypothesis is very difficult using
the statistical procedures by themselves. However, for most purposes, “not rejecting a
hypothesis” is a workable substitute for “accepting a hypothesis.”

When a hypothesis explicitly defines one specific value of parameter involved, it is

referred to as a simple hypothesis. In a testing of H0: μ = μ0 against H1: μ = μ1, both
the null and alternative hypotheses are simple hypotheses. A result of rejecting the
null hypothesis would be in favor of the alternative hypothesis and would lead to
increased belief in the alternative hypothesis.

If the alternative hypothesis had been H1: μ < μ1, it would not be simple hypothesis (it
would be a composite hypothesis). In this case the test would only be an attempt to
answer the question: is the observed value of μ significantly smaller than μ0? Such
tests can be called significance tests.

It is thus clear that we do not accept or reject hypothesis by merely looking at the raw
data or some arithmetic means. Instead, we process the data rather rigorously so that
we can have confidence in what we state. Nevertheless, there is always the chance of
making error. The two main types of errors encountered in statistical hypothesis
testing are:

(i) Type I error (reject H0 when it is true), and

(ii) Type II error (accept H0 when it is false).

In industrial quality control terminology, Type I error is rejecting a good lot and Type
II error is accepting a bad lot. The size of Type I error is denoted by α (see later)
while that of Type II error is denoted by β.

2.7.7. LEVEL OF SIGNIFICANCE

The maximum size of Type I error, which we are prepared to risk is known as level of
significance. It is usually denoted by α. In other words, the level of significance is the
weight of evidence, in terms of probability, for rejecting the null hypothesis (when in
fact it is true) in favor of the alternative hypothesis. The commonly used levels of
significance in practice are 5% (0.05) and 1% (0.01). If we adopt a 5% level of
significance, it implies that in 5 samples out of 100 we are likely to reject a correct
H0. In other words, we are 95% confident that our decision to reject H0 is correct. The

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level of significance is always fixed in advance, before collecting the sample

information.

Significance is important in accepting the validity of the conclusions derived from a

data. For example, if we wish to compare chlorophyll contents in control and after
treatment, the problem arises in deciding whether the difference between the two is
real or a chance encounter. Significance test assesses the probability that the apparent
effect could have arisen by chance. The lower this probability, the more likely is the
conclusion that the effect was real.

A significance test can be called a one-tailed test if the rejection region is at only one
extreme of the range of test statistic values; if the rejection region at both extremes,
the test can be called two-tailed.

2.7.8. ONE-TAILED AND TWO-TAILED TESTS

In any test, the critical region (significant region) is represented by a portion of the
area under the probability curve of the sampling distribution of the test statistic.

A test of any statistical hypothesis where the alternative hypothesis is one-tailed

(right-tailed or left-tailed) is called a one-tailed test. For example, a test for testing the
mean of a population H0: μ = μ0 against the alternative hypothesis H1: μ < μ 0 (left-
tailed) or H1: μ > μ 0 (right-tailed), is a single-tailed test. In the right-tailed test, the
critical region lies entirely in the right tail of the sampling distribution of the mean, x
while for the left-tailed test the critical region is entirely in left tail of the distribution
of x (see Fig. II-4 and Fig. II-5)

A test of statistical hypothesis where the alternative hypothesis is two-tailed such as:
H0: μ = μ0 against the alternative hypothesis H1: μ  μ 0 (i.e., H1: μ < μ 0 or H1: μ > μ 0)
is known as two-tailed test and in such a case the critical region is given by the
portion of the area lying in both the tails of the probability curve of the test statistic
(see Fig. II-4).

In a particular problem, whether one-tailed or two-tailed test is to be applied depends

entirely on the nature of the alternative hypothesis. If the alternative hypothesis is
two-tailed we apply two-tailed test and if alternative hypothesis is one-tailed, we
apply one-tailed test.

For example, suppose that there are two population brands of paneer, one
manufactured by standard process (with mean shelf-life of μ1) and the other
manufactured by some new technique (with mean shelf-life μ2). If we want to test if
the paneers differ significantly, then our null hypothesis is H0: μ1 = μ2 and an
alternative hypothesis will be H1: μ1  μ2, thus giving us a two-tailed test. However, if
we want to test if the paneer produced by the new process have longer shelf-life than
the one produced by standard process then we have H0: μ1 = μ2 and H1: μ1 < μ 2 thus
giving us a left-tailed test. Similarly, if the alternative hypothesis is H1: μ1 >μ 2, we

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 FOOD ANALYSIS

use a right-tailed test. Thus, the decision about applying a two-tail test or a single-tail
(right or left) test will depend on the problem under study.

Critical values or significant values of one-tailed and two tailed tests

The value of test statistic which separates the critical (or rejection) region and the
acceptance region is called the critical value or significant value. It depends on:

1. The level of significance used, and

2. The alternative hypothesis (whether it is one-tailed or two-tailed)

Level of significance ''

lower critical upper critical

value value

rejection rejection
region (/2) region (/2)

-Z Z=0 +Z

Fig. II-4: Two-tailed test

Level of significance ''

rejection
region ()

Z=0 +Z

Fig. II-5: Right-tailed test

Level of significance ''

rejection
region ()

-Z Z=0

Fig. II-6: Left-tailed test

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In the case of single-tail alternative, the critical value Zα is determined so that the total
area to the right of it (for right-tailed test) is α and for left-tailed test he total area to
the left of - Zα is α (see Fig. II-5 and Fig. II-6).

Table 1: Critical values (Zα) of Z

Level of significance (α)

Critical values Zα 1% 5% 10%
Two-tailed Zα = 2.58 Zα = 1.96 Zα = 1.645
Right-tailed Zα = 2.33 Zα = 1.645 Zα = 1.28
Left-tailed Zα =  2.33 Zα =  1.645 Zα =  1.28

In the case of two-tailed test, Zα is the value so that the total area of the critical region
on both tails is α. This implies that the area of each tail is α/2 (see Fig. II-4). Thus the
significant value of Z for a single-tailed test (left or right) at level of significance ‘α’
is the same as the critical value of Z for a two-tailed test at level of significance ‘2α’.
We give above the critical values of Z at commonly used levels of significance for
both two-tailed and single-tailed tests. These values can be obtained using normal
probability table.

2.7.9. CONFIDENCE LIMIT

It is possible that the mean obtained for a sample may not truly represent the actual
mean of the population. To express the uncertainty, confidence limits are assigned to
the observed mean, ( x ). Most often, to get satisfactory results, the value of
confidence limit is chosen as 95%. It means that the observed mean will enclose the
true mean with the frequency of this confidence limit. Confidence limits are obtained
by the following formula:

95% confidence limits  x  (test statistic ( )  standard error)

 x  (test statistic(0.05)  standard error)

If the test statistic is t (that is, when dealing with small sample size), the significant
value (also called critical value) of t can be obtained from the t distribution table by
entering appropriate degree of freedom for a probability of 0.05 (5% significance
level). When dealing with means of large sample sizes, the Z-statistic is calculated
and compared with corresponding area under normal probability curve for 5% level
of significance. The areas under the normal curve for different values of Z are
available in tabulated form.

Example 1:
Ten replicates of water samples were taken and tested for total hardness (Table 2).
Find out variance, standard deviation, standard error, and confidence limits to see the
variability in data and reliability of means.

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 FOOD ANALYSIS

Table 2: Data for example 1

Sample replicates Total hardness (mg/liter)

1 73
2 75
3 68
4 71
5 80
6 77
7 74
8 78
9 69
10 72

We see from above that n = 10, degree of freedom, d.f. = n – 1 = 9 and x  73.7

2
 (x  x )
Using calculator, variance, s 2  =15.12
n 1

Standard deviation s = s 2  15.12  3.88

s 3.88
Standard error of mean, SE ( m)   = 1.23
n 10

The value of t from the table for 9 d.f. = 2.26

95% confidence limit = x  (t  SE ( m))  73.7  2.26  1.23  73.7  2.78

The total hardness (mg/liter) in water is therefore 73.7 ± 2.78

The value of s (3.88) indicates that about 69% of the replicate has the value of total
hardness between 69.82 and 77.58mg/liter. The value of SE (m)  1.23 conveys that, if
instead of 1 mean, several means would have been taken, then about two-thirds of
them would fluctuate by ± 1.23. The 95% confidence limits shows that there are 95%
chances that the true mean of the total hardness of the source water will be
somewhere between 73.7 ± 2.78.

Testing of equality or difference between two means

In several situations, we might want to compare two means for significant difference.
The situations we come across often in food analysis are:

1. Comparison of means from two independent lots

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2. Comparison of sample mean with a given specified mean

3. Paired comparison of means

A brief description of the above situations and the test applied will be given shortly
but for now, a brief mention of the concept of large sample and small sample needs to
be made first. As such, the greater the number of samples tested, the more reliable
and true will be the mean. In statistics, a sample size of 30 or more is considered as
large sample. Sample sizes less than 30 are called small sample. There are two
separate calculations for comparing the means in large and small samples.

Large sample (n  30)

For comparing the two means ( x1 and x 2 ) of two large samples from independent lots,
a test statistic called Z score is used. It is a standardized normal variate given by:

x1  x2
Z
s12 s2 2

n1 n2

Where, s1, s2 are standard deviations of sample means, and n1, n2 sample sizes of two
independent lots.

This particular test is also called Z test for two means (unequal variance). For a two-
tailed test, if the Z score is less than 1.96 (the critical Z value at Zα = Z(0.05)), the null
hypothesis that there is no difference in the sample means at 5% level of significance
is accepted. On the other hand, if the Z score is greater than 1.96, the null hypothesis
is rejected.

Consequently, the 95% confidence limit for the statistic is:

s12 s2 2
( x1 - x2 )  Z ( ) 
n1 n2

s12 s2 2
 ( x1 - x2 )  Z (0.05) 
n1 n2

s12 s2 2
 ( x1 - x2 )  Z (0.05) 
n1 n2

Example 2:

Below are given two sets of data for moisture content of paneer from two industries.
Test the hypothesis of equality of means using α = 0.05.
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 FOOD ANALYSIS

Moisture content of paneer samples from industry A:

56, 47, 70, 52, 54, 48, 55, 52, 52, 54, 44, 58, 44, 62, 54, 71, 57, 55, 56, 55, 44, 51, 53,
56, 52, 54, 54, 68, 58, 52.

Moisture content of paneer samples from industry B

50, 46, 54, 46, 45, 45, 45, 51, 47, 54, 51, 55, 40, 52, 56, 50, 54, 46, 46, 52, 42, 47, 52,
55, 46, 51, 49, 68, 56, 54.

We will use Excel add-ins for testing the hypothesis. Assume that you are running an
Excel program. First of all calculate the variances of the sample means through: fx →
VAR→ OK→ select the single range data in the Excel file (for industry A)→ OK.

Calculate the variance of the variance for industry B in a similar manner.

Next, click tools → data analysis → z-test: Two samples for means → OK → fill up
the data required. Don’t forget to set ‘alpha’ = 0.05. Also, don’t forget to input 0
(zero) for hypothesized difference of mean (because we want to show no difference in
means). You don’t have to worry about the rest.

Below is given the actual table (Table 3) generated using Excel add-in (Analysis
toolpak) for the above data. The shaded portions have been added here only for the
purpose of explaining the details.

Table 3: Calculation in Excel add in for example 2

Variable 1 Variable 2
Mean 54.6 50.16666667
Known Variance 43.63 29.73
Observations 30 30
Hypothesized Mean Difference 0
z 2.83505411
P(Z<=z) one-tail 0.002290959
z Critical one-tail 1.644853476
P(Z<=z) two-tail 0.004581919
z Critical two-tail 1.959962787

In Table 3, Variable 1 refers to industry A (you can edit the name if you like, as
shown below) and Variable 2 refers to industry B. The ‘z’ in the shaded portion refers
to calculated value of Z score. ‘z critical two-tail’ in the shaded portion refers to
critical value of z at 5% level of significance. It is clear that the calculated value of Z
(2.85) is greater than the critical Z (~1.96), the hypothesis of no difference is rejected
at 5% level of significance.

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Small sample (n 30)

If n is small, the sampling distribution of the test statistic Z will not be normal and in
that case we can’t use the above significant values, which will be obtained by normal
probability curves. In this case (when the n is small), we use the significant values
based on the exact sampling distribution of Z, which turns out to be t, F, or 2
distribution. These significant values are found tabulated for different values of n.

The test statistic for comparing means from such two independent lots (small sample)
is given by:

x1  x2
t
1 1
sp 
n1 n2

( n1  1) s12  ( n1  2) s2 2
Where, sp = pooled standard deviation, which is equal to
n1  n2  2

It must be remembered that we use here n1  n2  2 degrees of freedom when entering

the value in the t distribution table.

As in the case of large sample, the calculated t is compared with the critical t value
obtained from the table at appropriate degree of freedom and specified probability
level. If the calculated t is smaller than the critical t, we accept the null hypothesis
that there is no difference in the two means at that particular level of significance.
Otherwise, the alternative hypothesis has to be accepted.

The 95% confidence limit of the statistic is given by:

1 1
( x1 - x2 )  t( )  s p 
n1 n2
1 1
 ( x1 - x2 )  t(0.05)  s p 
n1 n2

Thus we see that Student’s t-test is used for exact sampling distribution, in which the
number of samples is small. The statistical testing of equality or difference between
two means is one of the most important uses of t test. It can also be used to test the
mean against a specified value. The difference can be calculated at various levels of
significance. In practice a 5% level of significance is normally used. This level of
significance implies that the statement made about the difference have 95%
confidence. In a statistical term, if the two values are different at 5% level of

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 FOOD ANALYSIS

significance, the difference is too large to be ignored. On the other hand, if the
difference is not significant at the stated level of significance, the difference we see in
the data is merely due to chance and can be ignored. In other words, we can accept
with 95% confidence that there is no difference between the samples means.

We will deal with some of the aspects of this test, with the help of examples in the
paragraphs to follow but some additional uses of t mentioned in the beginning but not
discussed until now will be briefly outlined now.

Comparison of sample mean, x ; (small sample) with a specified mean, 

The test statistic t is given by:

x 
t , where the notations carry usual meaning
s/ n

Comparison of two sample means (small sample) in pair

This is also called paired t test. In this test, different pairs of measurements are
obtained, one measurement in each pair from population 1, the other from population
2. It is clear that n1 = n2. We use n-1 degree of freedom. The test statistic is given by:

d
t
sd n

Where, d = difference between the pair x1, x2

sd = standard deviation of difference, d

Example3:

The net weights of products from 2 different filling lines are given in Table 4. Find
out the whether there is any significant difference at 5% level of significance between
the two lines.

The above problem is a testing of equality of the means of two independent lots,
where the number of samples in each lot is less than 30 (small sample). We now set
up a hypothesis (the null hypothesis) that there is no significant difference in the
weights from the two lines at 5% level of significance against an alternative
hypothesis that there is difference.

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Table 4: Data for example 3

Sample Weight from Filling line 1 (gram) Weight from Filling line 2 (gram)
1 15.0 16.2
2 15.2 15.8
3 15.8 16.1
4 16.1 15.9
5 15.6 15.7
6 14.9 15.4
7 15.4 15.7
8 14.8 15.5
9 15.5 16.2
10 15.7 15.3

It is clear that the sample size is less than 30. Assuming that the variances of the
means from the two lots are identical, the formula used for calculating the value of t
in the above case will be:

x1  x2
t , where x1 , x2 are means of two independent lots; n1, n2 are
1 1
sp 
n1 n2
number of samples; and sp is the pooled standard deviation. The degree of freedom to
be used = (n1 + n2 - 2) = 10 +10 - 2 =18.

Although we can compute t – value manually, we can also use computer’s inbuilt
programs for faster result. Using analysis toolpak that comes as an add-in Excel
(Excel file→tools→data analysis→t-test, as shown in Fig. II-7) we have the result
shown below. Table 5 is an unedited form (the one you will obtain if you run the
program).

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 FOOD ANALYSIS

Fig. II-7: Using Excel Program for t-test

The hypothesis that there is no significant difference in the means (shaded row) has
been set up for the present case. We see that the calculated value of t for two-tailed
test is -2.26 at 5% level of significance. The tabulated critical value for the same
should be 2.1 to be significantly different. Ignoring the negative sign in -2.26, we see
that 2.1 (tabulated) < 2.26 (calculated). Hence we conclude that the difference is
significant at 5% level. In other words, we reject the hypothesis that there is no
difference in the means.

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Table 5: t-test for example 3 (from Excel add-in)

t-Test: Two-Sample Assuming Equal Variances

Variable 1 Variable 2
Mean 15.4 15.78
Variance 0.177777778 0.104
Observations 10 10
Pooled Variance 0.140888889
Hypothesized Mean Difference 0
df 18

t Stat -2.263759192
P(T<=t) one-tail 0.018090112
t Critical one-tail 1.734063062
P(T<=t) two-tail 0.036180224
t Critical two-tail 2.100923666

When we want to compare the mean against a hypothesized or a given mean we use
one-tailed test to find whether the observed mean is significantly less or not. We do
the same for finding out whether the sample mean is significantly greater than the
hypothesized mean. If we are interested only in the degree, regardless of less than or
more than, we use two-tailed test. An example of one-tailed test is given below in
Example 4. For further clarification, we can use following example: Suppose the
regulatory standard states that the total ash content of instant noodles should not be
greater than 4%. In a series of tests, the mean of the total ash content in the sample
was found to be 4.2%. In this case, we need to use one-tailed test because if the
sample mean is different by having less than 4% total ash content it is not a problem.
The sample mean should not differ from the standard by having total ash content
more than 4% only.

We will use one more example for the test of significance of difference between two
means. In the present example, we will test the sample mean against a specified mean.

Example 4:

The drained weight of the canned fruit from a lot is given in Table 6. Find out
statistically whether the average drained weight differs significantly from the
specified limit of 55%.

Table 6: Testing of sample mean against a specified mean

Sample 1 2 3 4 5 6 7 8 9 10 11 12 13
Drained weight 54.5 53.2 54.1 55.2 55 56.2 54.5 53.8 55.5 55.6 55 53 54.8

We first set up the hypothesis (null hypothesis) that there is no difference between the
mean and the specified mean at 5% level of significance. The alternative hypothesis

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 FOOD ANALYSIS

would be that the sample mean is less than the specified mean so that the null
hypothesis is rejected on the ground that the samples are underweight.

This example has to test the calculated mean against a specified mean. It may be
noted, the product will be considered not meeting the specification only if the
observed mean is significantly lower than the specified mean. This means that one-
tailed test must be used. The value of t can be obtained using following equation:

x 
t , where x = sample mean, μ = specified mean, and s / n = standard error
s/ n
of mean (discussed elsewhere).

Putting the respective pre-calculated values in the above equation,

54.646  55
t = -0.544.
2.345 / 13

From the standard table, the value of t for 12 degree of freedom at 5% level (for one-
tailed test) is 1.78. The calculated value of the mean, irrespective of the negative sign,
is less than the tabulated value. The sample mean is therefore not significantly
different from the specified value at 5% level of significance. In other words, the
sample is well within the specification.

Testing equality between more than two means

When several similar items are analyzed for a particular characteristic (protein
content, acidity, etc.), we might want further want to see whether the mean values of
the characteristic analyzed for different samples are different or not. Looking merely
at the mean value will not provide much information about the difference. Hence a
statistical must be used for this. The two widely used tests are the t-test and the F-test.

F-test is used for testing difference in more than two sample means. This test is also
called Analysis Of Variance (ANOVA). Generally, the observations from repetitive
analysis or experiments, products made using different raw materials or machines, use
of number of machines of the same type, operators producing the product, product
produced on different days, etc., cause variation. Using additive property of the
variance between different observations, the observed variance can be broken down
into components, attributable to different criteria which are of importance. Using
ANOVA of these components, it is possible to estimate how much of the total
variation is due to one or more assignable causes of variation; the remainder which
cannot be attributed to any assignable causes of variation is classed as due to chance
causes which produce the residual or error variation.

Both t-test and F-test can be calculated manually as well as using computer software.
Manual calculation is very tedious and requires use of several tabulated values.
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Microsoft Excel also contains inbuilt system for both t-test and F-test (and many other
tests).

We will deal with an example for carrying out the ANOVA and try to draw inference
about the sample. The calculation can be done manually but it is too tedious. The
calculation presented in this example has been done using the Excel add-in analysis
toolpak (Excel file → tools → data analysis → two-way ANOVA without replication
→ ..).

Example5:

The vitamin C contents of 4 varieties of tomatoes were analyzed by 3 methods and

data given in Table 7 were obtained. Find out whether there are significant
differences with respect to vitamin C content between the varieties and the methods
employed.

Table 7: Data for example 5

Vitamin C content of tomatoes, mg/100g

Test method
Variety 1 Variety 2 Variety 3 Variety 4
Method 1 23.4 24.2 25.1 23.0
Method 2 24.1 25.0 26 23.9
Method 3 22.8 23.9 24.0 22
Mean value 23.43 24.37 25.03 22.97

If you input the above data in Excel’s data analysis for ANOVA (2-factor without
replication) you will get following output at 5% level of significance. The row refers
to methods and the column refers to variety.

The main things you will really need to know are the values of F and Fcrit (the shaded
portion in the above output table). You will notice that Fcrit refers to the critical F-
value. An F value greater than Fcrit implies that the observed means are significantly
different at the given level of significance (usually 5%). You will see that the
calculated F values for both the rows and columns are greater than the corresponding
Fcrit values. This implies that the vitamin C contents of tomatoes are significantly
different at 5% level of significance (and this much amount of difference cannot be
due to chance!).You will also notice that there is significant difference between the
methods determining vitamin C employed in the present example.

In the following table (Table 8), you need very little to worry about values like P-
value, MS, df, etc. The column heading source of variation refers to variations that
can be ascribed to, for example the variation in the variety of tomato, the testing
method itself, etc. A concise interpretation of the above table is given in Table 8.

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 FOOD ANALYSIS

Table 8: ANOVA for example 4 (from Excel add-in)

SUMMARY Count Sum Average Variance

Row 1 4 95.7 23.925 0.8625
Row 2 4 99 24.75 0.923333
Row 3 4 92.7 23.175 0.909167

Column 1 3 70.3 23.433333 0.423333

Column 2 3 73.1 24.366667 0.323333
Column 3 3 75.1 25.033333 1.003333
Column 4 3 68.9 22.966667 0.903333

ANOVA
Source of Variation SS df MS F P-value F crit
Rows 4.965 2 2.4825 43.59512 0.000267 5.143249
Columns 7.7433333 3 2.5811111 45.32683 0.000162 4.757055
Error 0.3416667 6 0.0569444

Total 13.05 11

ANOVA
Source of Variation SS df MS F P-value F crit
Method 4.965 2 2.4825 43.59512 0.000267 5.143249
Variety 7.7433333 3 2.5811111 45.32683 0.000162 4.757055
Error 0.3416667 6 0.0569444

Total 13.05 11

29
CHAPTER III: STANDARDIZATION

3.1. CALIBRATION OF MEASURING DEVICES (PIPETTES AND BURETTES)

BACKGROUND

Every measuring device has some errors (manufacturing or inherent). In some

devices, the errors remain constant, for example accuracy of a pipette. In other
devices, the accuracy changes constantly because of wear and tear, such as in a
mechanical balance, an electronic balance, etc. These devices warrant periodic
restandardization. Similarly, no two similar devices have the same accuracy. For
example, two 10-ml pipettes can never deliver exactly the same amount. This is also
true of volumetric flasks, measuring cylinders, etc. When using for the first time,
these apparatus need to be calibrated.

An electronic balance can be calibrated using standard weights, which in turn are
calibrated against yet another standard weight. The volumetric capacity of pipettes
can be readily calculated by weighing the amount of water delivered at 4°C (at this
temperature, the density of water is 1000kg/m3 and the volume equals the weight). A
routine electronic balance can easily measure up to 0.1mg but it is difficult to imagine
any routine pipette that will measure the equivalent 0.0001ml (= 0.1L)! The
accuracy of different volumetric flasks and burettes can similarly be carried out.

The accuracy of volume measuring devices is generally of the following order:

 Bulb pipette > graduated pipette > small burette > large burette
 Volumetric flask >> measuring cylinder

Therefore, during the preparation of standard solutions, measuring cylinders should

never be used. The accuracy of pipettes and volumetric flasks is due to the small
neck. In them, even a small fluctuation of volume will significantly change the level
of the solution. On the other hand, measuring cylinders have large diameters and even
a significant change in volume is hardly visible.

If the volumetric measurement and weight have been carried out at temperatures other
than 4°C, the weight must be divided by density of water at that particular
temperature to obtain the true volume. For example, the densities of water at 0, 4, 10,
 FOOD ANALYSIS

20, 40, 60, 80 and 100°C are 999.86, 1000.00, 999.72, 998.23, 992.00, 983.00, 972.00
and 958.00kg/m3 respectively. The volume change due to expansion of the glass
apparatus can be considered negligible.

PRINCIPLE

The accuracy of static volume-measuring devices can be readily tested by comparing

the stated volume (at the stated temperature) with the actual volume. The actual
volume can be obtained by dividing the weight of equal volume of water with density
of water at that particular temperature.

REQUIREMENTS

 Electrical or electronic balance, ± 0.0001g  Distilled water: 1000ml

 Graduated pipette: 10ml, 5 pcs  Clean beaker: 50ml, 1 pc
 Bulb pipette: 10ml, 5 pcs  Density chart
 Mono pan balance: 1kg capacity, ± 0.1g  Volumetric flasks: 100ml and
250ml, 5 pcs each

PROCEDURE

1. Categorize pipettes, burettes and volumetric flasks according to their stated

capacity
2. Note down the details about volume and temperature inscribed on the
apparatus
3. Note down the temperature of distilled water
4. Place a clean dry 50ml beaker on an electronic balance and re-zero the weight
5. Pipette out 10ml distilled water with a 10ml bulb pipette into the beaker and
note down the weight of water
6. Empty the beaker, dry it with a piece of muslin cloth or blotting paper and re-
zero the weight
7. Pipette out 10ml distilled water with the next 10ml bulb pipette into the
beaker and note down the weight of water
8. Repeat steps 5 and 6 for all the 10ml bulb pipettes
9. Repeat steps 5 and 6 for all the 10ml graduated pipettes
10. Take a dry 100ml volumetric flask and tare it in a mono pan balance
11. Fill the flask with distilled water up to the mark and weigh again
12. Calculated the weight of water by difference and note it down
13. Repeat steps 9 and 10 for all the 100ml volumetric flasks
14. Repeat steps 9 and 10 for all the 250ml flasks
15. Enter the observation in a tabular form

A sample of how reports need to be prepared is as follows:

31
 STANDARDIZATION

OBSERVATIONS (example)

Temperature of distilled water, t°C

Density of water at that particular temperature, ρt g/cm3

Measuring device Device code Stated volume, Weight of water, Calculated volume,
ml g ml
10-ml bulb pipette 1 10ml a1 a1 ρt
2 10ml a2 a2 ρt
3 10ml a3 a3 ρt
4 10ml a4 a4 ρt
5 10ml a5 a5 ρt
10-ml graduated 1 10ml b1 b1 ρt
pipette 2 10ml b2 b2 ρt
3 10ml b3 b3 ρt
4 10ml b4 b4 ρt
5 10ml b5 b5 ρt
100-ml volumetric 1 100ml c1 c1 ρt
flask 2 100ml c2 c2 ρt
3 100ml c3 c3 ρt
4 100ml c4 c4 ρt
5 100ml c5 c5 ρt
250-ml volumetric 1 250ml d1 d1 ρt
flask 2 250ml d2 d2 ρt
3 250ml d3 d3 ρt
4 250ml d4 d4 ρt
5 250ml d5 d5 ρt

CALCULATION (example)

Measuring Device Stated Calculated % error Standard deviation

device code volume, ml volume, ml of errors
10-ml bulb 1 10ml + or 
x  x 
2
pipette 2 10ml 
3 10ml n 1
4 10ml
5 10ml
10-ml 1 10ml
graduated 2 10ml
pipette 3 10ml
4 10ml
5 10ml

32
 FOOD ANALYSIS

Measuring Device Stated Calculated % error Standard deviation

device code volume, ml volume, ml of errors
100-ml 1 100ml
volumetric 2 100ml
flask 3 100ml
4 100ml
5 100ml
250-ml 1 250ml
volumetric 2 250ml
flask 3 250ml
4 250ml
5 250ml

n = no of observation;  = standard deviation; x = % error; x = arithmetic mean

RESULTS AND DISCUSSION (example)

1. For each category of volume measuring device, express the range of errors as
% error ± 3×standard deviation
2. Arrange the categories of measuring devices in the decreasing order of
accuracy
3. Mention the most likely sources of errors in the collection of data

3.2. PREPARATION OF STANDARD SOLUTIONS

BACKGROUND

When the concentration of a solution is known it becomes a standard solution. The

concentration (also called strength) can be expressed in various units, e.g.,
percentage, normality, and parts per million (ppm). The operational definitions of the
above-mentioned units are:

Percentage: Gram of pure substance per 100ml of final solution.

Molarity: Number of gram-mole of pure substance per liter of final solution. It is

denoted by M.

Normality: Number of gram-equivalent of pure substance per liter of final solution.

Normality is denoted by N.

Parts per million (ppm): Number of milligram of pure substance per liter of final
solution. It is generally denoted by ppm. The concentration is also expressed as
mg/dm3, and mg/liter (mg/L).

33
 STANDARDIZATION

Sometimes, milligram percent (mg%) is also used for expressing strength of

solutions. The relation between %, mg%, and ppm is: 1% = 1000mg% = 10000 ppm.

The preparation of a standard solution can be carried out using either stock solutions
or other concentrated forms. When the latter is used, the specification, viz., percent
purity and density must be noted and included in the calculation.

A substance that is obtained in pure state and is stable indefinitely can be used for the
preparation of standard solution by direct weighing. In chemistry, such a chemical is
referred to as a primary standard. Sodium carbonate, oxalic acid, potassium
dichromate, etc., belong to this class. In the laboratory, however, there are many
reagents that tend to change during handling (e.g., due to hygroscopic nature, ability to
react with handling materials, environmental gases, etc.). Such reagents cannot be
prepared directly as standard solutions. They must be restandardized with a primary
standard after a rough strength has been arrived at. Furthermore, there are some
chemicals that do not keep well under any set of storage conditions: they gradually
deteriorate or degrade with time. Some of such solutions can be maintained by using
certain preservatives or stabilizers while others must be restandardized before each use.

PRINCIPLE

Oxalic acid, H2C2O4·2H2O (equivalent wt = 63), being a primary standard can be

prepared by directly weighing the required amount of the chemical. The amount of
100% pure oxalic acid dihydrate needed to make 1000ml of 1.0N solution is 63g.
Since the chemical is not available in 100% pure state, the purity factor must also be
considered. Therefore the true amount of H2C2O4·2H2O needed = [63 g / purity
factor] gram.

NaOH (equivalent wt = 40) is a hygroscopic chemical and is not a primary standard.

1000ml NaOH solution of ~ 1N can be prepared by quickly weighing slightly more
than 40g of the reagent and dissolving in distilled water to make 1000ml. To establish
the true strength (standardize, that is), the NaOH solution thus prepared must be
restandardized with standard acid, such as oxalic acid using phenolphthalein as the
indicator.

To adjust the strength of the standard solution to a lower strength, calculated amounts
of water and the reagent solution can be mixed. The amount of each component
needed can be calculated by the usual formula of S1V1 = S2V2, where S refers to
strength and V refers to volume. When two or more solutions of different strengths
need to be mixed to make a standard solution of intermediate strength, Pearson
Square method can be used (Fig. III-1). If S1, S2 and SR denote strength of solution 1,
strength of solution 2 and the required intermediate strength respectively, the
proportions or parts (P1 and P2) of the two solutions to be mixed are obtained as
follows:

34
 FOOD ANALYSIS

S1 P1 = SR - S2 (absolute value)

SR

S2 P2 = SR - S1 (absolute value)

Fig. III-1: Pearson square

REQUIREMENTS

 NaOH pellets or stock solution  Electronic balance

 Phenolphthalein indicator, 1% solution
 Pipette, 10ml
 Burette, 50ml
 Volumetric flask
 Oxalic acid (dihydrate), AR1 grade
 Volumetric flasks, 250ml, 4pcs
 Mono pan balance
 Conical flasks, 250ml, 2 pcs
 Reagent bottles, 250ml, 4pcs
 Beakers, 250ml, 2pcs
 Glazed paper or watch glass

PROCEDURE

1. Note the purity factor given on the label of oxalic acid

2. Weigh 15.75  purity factor (decimal)g of oxalic acid (dihydrate) in an
electronic balance. Use glazed paper or clean watch glass for weighing
3. Transfer the acid quantitatively to clean 250ml beaker
4. Add some distilled water and stir the solution with a glass rod to insure
complete dissolution of oxalic acid. Keep the rod immersed and don’t put it
aside (this may incur loss of oxalic acid)
5. Transfer the solution quantitatively to 250ml volumetric flask
6. Complete the transfer by repeatedly washing the beaker and glass rod with
distilled water and pooling the solution
7. Add more distilled water to make up the volume
8. Stopper the flask and shake well, turning upside down two to three times
9. Transfer the contents to a dry, labeled reagent bottle
10. Weigh about 12g of NaOH pellet in a mono pan balance. Use glazed paper or
watch glass for weighing. Don’t spill the chemical on the balance (corrosive!)
11. Transfer the chemical to a 250ml beaker
12. Add about 200ml water. Stir the chemical with a glass rod to insure solution
13. Cool the solution to room temperature
14. Transfer the solution to a 250ml flask
15. Add more distilled water to make up the volume
16. Stopper the flask and shake well (as above)
17. Transfer the chemical to a labeled reagent bottle

1
Analytical Reagent Grade (very high purity)

35
 STANDARDIZATION

18. Take 10ml of standard oxalic acid in a 100ml conical flask

19. Transfer some NaOH solution to a 50ml burette (avoid air gap at the tip of the
burette)
20. Titrate oxalic acid with NaOH solution using phenolphthalein indicator
21. Replicate the titration until a concordant reading is obtained
22. Calculate the strength of NaOH using the formula S1V1 = S2V2
23. If the strength of NaOH is greater than 1.0, adjust the strength to 1.0 using the
usual formula S1V1 = S2V2 again
24. Make 10-fold dilution of each solution with distilled water to give 0.1N
solution (25ml  250ml). Restandardize the new NaOH solution with 0.1N
oxalic acid using phenolphthalein indicator.
25. Store the standard chemicals in labeled reagent bottles

A sample of observations, calculations, and results and discussion is shown below:

OBSERVATIONS AND CALCULATION (example)

Purity factor of oxalic acid dihydrate = e.g., 99.5%

Weight of pure oxalic acid needed to make 250ml of 1.0N oxalic acid = 63g
Corrected weight of supplied oxalic acid needed to make 250ml of 1.0N oxalic acid =
63 / 0.995 = 63.32g

First titration data (for stock solution, oxalic acid = 1.0N)

Volume of oxalic acid, Volume of Concordant titer, Strength of NaOH

ml NaOH, ml ml stock
10 
10  e.g., 9.2ml 1.087N
10 

Adjustment of strength (only if the strength is greater than 1.0N).

The volume of 1.087N NaOH needed to make 250ml of 0.1N NaOH =

(250×1.0/ 1.087)ml  230ml.

Distilled water needed = (250-230)ml = 20ml.

Second titration data (for working solution, oxalic acid = 0.1N).

Volume of oxalic Volume of NaOH, ml Concordant Strength of NaOH

acid, ml titer, ml stock
10 
10  e.g., 9.5ml 0.105N
10 

36
 FOOD ANALYSIS

RESULTS AND DISCUSSION (example)

Using oxalic acid as the primary standard, the strength of stock NaOH was found to
be 1.087 N. By calculation, the adjustment of strength required mixing of 230ml of
NaOH stock and 20ml of water.

The strength of working solution of NaOH was found to be 0.105N. There is scope
for further adjustment of strength (for < 0.1N, there is no scope for adjustment).

The slight difference between the expected (computed) and observed values might
have resulted from inherent errors in the volume-measuring devices. It must also be
noted that NaOH pellets contain appreciable amounts of sodium carbonate, the
quantitative determination of which requires double indicator (phenolphthalein and
methyl orange) method of titration. Since such a method was not used, the result does
not represent the exact concentration of NaOH.

37
CHAPTER IV: PROXIMATE ANALYSIS

The term “proximate” is context-dependent. In food and feed analyses, this refers to
the determination of major components (moisture, minerals, carbohydrates, lipids,
crude fiber, and proteins) and hence called “proximate analysis”. The components
analyzed are called “proximate constituents”. The proximate constituents are not
limited to the components stated above. In the case of acid food, for example, the acid
content has to be considered as a proximate constituent. Similarly, alcohol in beer has
to be considered as a proximate constituent.

Proximate analysis gives inexpensive yet very useful information, particularly from
the nutritional and biochemical points of views. The result is normally expressed in
percentage, and because of the fairly general nature of test employed for the
determination, the term ‘crude’ is usually used as a modifier, for instance, crude
protein, crude fat and crude fiber. Proximate constituents therefore represent only a
category of compounds present in a biological material. Analysis of particular
element or compound, such as vitamins, reducing sugars, etc., is termed ultimate
analysis. In general, ultimate analysis is a more detailed analysis of proximate
constituents or the analysis of components found in very small amounts, e.g.,
vitamins.

Despite widespread use, there are many inaccuracies inherent in proximate analysis
which limits its usefulness. Some examples that clarify the reasons for inaccuracies are:

 Ether extract removes not only fats but also waxes and other fat soluble
materials, which may result in erroneously high estimates of fat content.
 Use of Kjeldahl analysis of food/feed-nitrogen to derive the estimated total
crude protein is based on several assumptions, for example:
o All proteins contain 16% nitrogen
o All nitrogen in a food/feed is in the form of protein
o The protein in the food/feed is totally digestible

It is worth remembering that proteins are not the sole source of nitrogen. If fact, there
are many compounds (e.g., nucleic acids, non-protein amines, etc.) which also contain
significant amounts of nitrogen. Since these are neglected in Kjeldahl method of
analysis, the results are only approximate.
 FOOD ANALYSIS

4.1. DETERMINATION OF CRUDE FAT BY SOLVENT EXTRACTION

BACKGROUND

Crude fat determination is a very important routine activity in food laboratory and
food industries. As an example to cite, fat determination is required in an ice cream
industry for formulating a given type of ice cream (low fat, high fat, etc.). Fat content
is determined in cheese industry for similar reason. In an oil mill, it may be required
to determine fat content in the press cake to get an idea about the efficiency of the
expeller. In the dairy industry, fat content is used as the pricing index. The higher the
fat content in the milk the higher the price it will fetch.

The method used for fat determination depends on the type of analyzed material and
nature of subsequent analytical problem. Crude fat from flour items can be readily
extracted quantitatively by relatively simple method (in a special tube). Fat extraction
from cocoa is also very simple. On the other hand, fat extraction from egg yolk and
dried whole egg is a bit complicated in that the sample needs to be pretreated with
HCl. Fat determination in milk is entirely different: it is based on acid digestion.

Lipids are characterized by their sparing solubility in water and their considerable
solubility in organic solvents. The successful extraction of lipids requires that bonds
between lipids and other compounds be broken so that the lipids are freed and
solubilized. Generally, such solubility is attained when polarities of the lipid and the
solvent are similar. For example, the non-polar triglycerides are dissolved in non-
polar solvents such as hexane and petroleum ether (low boiling point distillate of
petroleum). Polar compounds, such as glycolipids, and even free fatty acids, are
markedly soluble in alcohols. There is therefore no single standard solvent for lipid
extraction.

The extraction of crude fat by solvent depends on many factors. Moisture content of
the sample can be taken as one important factor. Only a part of the lipids can be
extracted with ether from moist material, as the solvent cannot penetrate the tissues
and the extractant becomes saturated with water. This problem can be avoided by
drying the sample. However, drying should not be too harsh. Drying at elevated
temperatures is undesirable because some lipids become bound to proteins and
carbohydrates and are rendered inextractable.

The particle size of the sample is also very important. In general, the finer the particle
size of the sample the more efficient will be the extraction.

In the laboratory, the most common solvents used for the extraction of lipids from
solid foods are diethyl ether and petroleum ether. Hexane, another solvent, is used on
a commercial scale, e.g., for the extraction of bran oil.

39
 PROXIMATE ANALYSIS

PRINCIPLE

In the laboratory, crude fat in solid samples is determined using soxhlet apparatus.
The apparatus extracts crude fat from the sample by recycling hot solvent, usually
petroleum ether. The apparatus consists of 3 easy-to-fit parts, namely, the extraction
tube (into which sample in a thimble is kept immersed in solvent for fat extraction),
the receiving flask (which receives through a siphon system the solvent + extracted
fat from the extraction tube and vaporizes the solvent selectively for recycling), and
the condenser (which condenses the vaporized solvent onto the sample placed in the
extraction tube). The recycling is done for a certain number of times (until the
extraction is complete) and the fat is recovered by evaporating away the solvent.

REQUIREMENTS

 Diethyl ether or petroleum ether  Sample

 Soxhlet assembly  Acetone
 Desiccator  Thimble
 Heating arrangement  Balance

PROCEDURE

1. Grind about 250 g of sample into fine particles in mortar and pestle
2. Mix well, spread on a sheet of paper and quarter it (see Fig. IV-1)
3. Mix the opposite quarters, for example 1 and 3
4. Quarter the mixture again as in steps 13
5. Prepare thimble (see Fig. IV-2) of the right size2 and tare it

Cotton plug

Quartet
1 2 Sample
Sample Thimble
3 4
Paper

Fig. IV- 1: Quartering of sample Fig. IV- 2: Thimble with sample

6. Weigh by difference 5-8 g of powdered sample obtained in steps 14

7. Stuff the thimble by lightly tapping it. Leave at least an inch of space above the
sample
8. Pack fat-free cotton wad over the space as shown in Fig. IV-2

2
The size that will easily enter the reflux

40
 FOOD ANALYSIS

9. Lightly drop the thimble (in upright position) in the reflux 3 (fat extraction
tube)
10. Fit the reflux in the receiver flask (see Fig. IV-3)
11. Pour solvent4 slowly onto the sample until the solvent starts siphoning to the
receiver. Add 50-75ml in excess
12. Place the assembly on a water bath (or temperature-controlled heating
mantle)
13. Connect the condenser on top of the reflux tube and open the tap water to run
it
14. Turn on the heat and adjust the temperature to allow light boiling of the
solvent. The solvent vaporizes, condenses to the extraction tube, and siphons
down after the volume of collected solvent reaches a critical level. During the
residence of the solvent in the reflux (extraction tube) the fat from the sample
gets slowly extracted. The extracted fat siphons down to the receiver along
with the solvent. Because of continuous of boiling, the solvent soon begins to
vaporize and the fat-free vapor begins to condense once again into the reflux
15. Carry out at least 25 cycles before terminating the extraction process. It may
take several hours. In very persistent cases, 15-16 hours may be needed
16. Add more solvent from the top of the condenser to compensate the loss of
solvent that might occur during the prolonged boiling
17. After extraction is complete, allow the last siphoning for emptying the reflux
18. Immediately turn off the heat, and if possible, remove the assembly from the
heating arrangement
19. Take out the thimble (you will need this later for crude fiber determination)
and reset the assembly (using empty reflux this time)
20. Heat gently to allow one more siphoning. This will clean the reflux
21. Continue to collect the solvent in the reflux. This time don’t allow the solvent
to siphon down. This is done to remove most of the solvent from the
extracted fat.
22. Dismantle the assembly and collect the solvent in a clean beaker (care! Don’t
tilt the reflux lest you should activate the siphon while handling)
23. Filter the extract (collected in the receiver) quantitatively5 through filter paper
into clean, tared beaker
24. Rinse the receiver with the recovered solvent repeatedly and transfer the
washings to the filter
25. Elute the residual fat in the filter paper with the recovered solvent
26. Evaporate the solvent from the extracted fat in a water bath (or temperature-
controlled heating mantle) as completely as possible. Keep the temperature of
the heater below 100°C
27. Elute the residual fat in the filter paper with the recovered solvent

3
The reflux should be thoroughly cleaned with cleaning solution (chromic acid)
4
The preferred solvent is Petroleum ether (b.pt.: 35-45°C) but hexane can also be used in times of crisis.
Anhydrous ether may also be used
5
Without loss of material

41
 PROXIMATE ANALYSIS

28. Evaporate the solvent from the extracted fat in a water bath (or temperature-
controlled heating mantle) as completely as possible. Keep the temperature of
the heater below 100°C

Water out

Condenser

Water in

Extraction tube

Overflow tube
Thimble with
sample Siphon tube

Solvent Receiver

Heat

Fig. IV- 3: Soxhlet extractor

29. Add 5ml of acetone and evaporate to completeness once again

30. Cool the beaker in desiccator
31. Wipe the external surface of the beaker with a clean muslin cloth and weigh it
to the nearest 5mg

CALCULATION

Wt. of crude fat (g)  100  100

% Crude fat (dry basis) 
Wt of sample (g)  Dry matter %

42
 FOOD ANALYSIS

4.2. DETERMINATION OF CRUDE FIBER IN FOOD SAMPLE

BACKGROUND

“Crude fiber” is the general term used to imply materials insoluble in dilute acid and
alkali under specific conditions (that simulates the human digestion process). The
residue from crude fiber determination contains about 97% cellulose and lignin. It
does not represent, however, all cellulose and lignin present initially. Typically, it
represents only about 50-80% of cellulose, 10-15% of lignin, and 20% of the
hemicellulose of the original food.

Crude fiber has been defined as the sum of all those organic components of the plant
cell membrane and supporting structures which in chemical analysis of plant
foodstuffs remain after removal of crude protein, crude fiber, and nitrogen-free
extractives.

The term crude fiber appears somewhat narrow in terms of present view of the
subject. In terms of food value, emphasis has been placed on the determination of
dietary fiber, rather than crude fiber. Dietary fiber is defined as a complex group of
plant substances that are resistant to mammalian digestive enzymes. Dietary fiber
includes following three fractions from plant foods:

1. Structural polysaccharides – associated with the plant cell wall, including

cellulose, hemicellulose, and some pectins
2. Structural non-polysaccharides – mainly lignins
3. Non-structural polysaccharides – non-starchy polysaccharides such as the
gums and mucilages

The main part of dietary fiber in foods therefore originates in the plant cell wall but is
not limited to it because dietary fiber also includes water-soluble polysaccharides
such as pectins, plant gums and mucilages. The cell wall components and dietary
fiber components are related thus:

Protein
Lipids
Inorganic constituents
Plant cell wall Lignin
Cellulose
Hemicellulose
Pectins Dietary fiber
-glucans
Gums
Mucilages
Algal polysaccharides
Modified cellulose

43
 PROXIMATE ANALYSIS

There is no fixed relationship between crude fiber and dietary fiber because plant cell
walls vary in the proportions of their basic constituents. Unfortunately, the database
on the dietary fiber content of foods is very sketchy at present. Also, methodology for
analyzing dietary fiber is still being developed, and no consensus on single method
for use with human foods has yet emerged. Therefore, it is necessary to continue to
use crude fiber values when discussing fiber in human nutrition.

It must be remembered, however, crude fiber determinations are greatly affected by

manipulations and procedures. Particle size is important: the finer the material
ground, the lower the determined crude fiber content. The method is highly empirical
and every step carried out is prone to affect the result. There are at least 100
modifications of the original crude fiber determination developed by Hennenberg,
Stohmann, and Rautenberg in 1864 in Germany.

Dietary fibers are not in the physical sense truly fibrous. In fact, a number of them are
water-soluble. Dietary fibers are present in large amounts in vegetarian diets, such as
cereal grains, pulses, beans, fruits, and vegetables. Dietary fiber contents of some
common foods are as follows:

Food item Dietary fiber, % wet basis

Polished rice 0.1
Pulses 5.0
Apple 1.0
Grapes 2.8
Peas 4.0
Potato 0.4
Green vegetables 1.0

Dietary fibers are of two main types: (a) Water-soluble, and (b) Water-insoluble.
Cereal grains and vegetables are good sources of water-soluble dietary fibers. Fruits
are rich in both water-soluble and water-insoluble fibers. Water-insoluble fibers are
represented by cellulose, most hemicellulose, and lignins. Water-soluble fibers are
represented by pectins and gums.

Both types of dietary fibers have very specific physiological roles in humans. Water-
insoluble fibers increase stool bulk and reduce transit time in the gastrointestinal tract.
Most components of dietary fiber are capable of absorbing water thereby rendering
the feces soft enough to pass out of the body readily and bulky enough to induce
defecation. Lignins absorb bile salts and cholesterol and thus play a role in lowering
cholesterol. Water-soluble fraction, on the other hand, slows down the gastric
emptying and also the rate of absorption of nutrients such as glucose. Consumption of
too much fiber is not beneficial, however. It may lead to mineral deficiency because
of excessive fecal excretion of electrolytes. The recommended average daily intakes
of dietary fiber for adult women and men are 12g and 17g respectively under the
condition of normal health.

44
 FOOD ANALYSIS

When the staple diet consists of refined materials such as sugar, flour, cakes, etc., the
person suffers from constipation because of dietary fiber deficiency. This problem is
common in developed countries. To overcome this problem, they use fiber
supplements such as those from wheat or oat bran in the diet. Dietary fiber
determination is usually very complicated. Several methods have been developed for
the determination of dietary fiber. Each method has its own scope and limitations.
Some of the methods used for dietary fiber determination are (i) Acid detergent fiber
(ADF) method, (ii) Neutral detergent fiber (NDF) methods, (iii) Enzyme-modified
neutral detergent fiber (ENDF) method, and (iv) Total dietary fiber (TDF) methods.
There are further variations in some of these methods.

The method given by Association of Official Analytical Chemists (AOAC, 1984) is

outlined as follows:

Defatted sample + blank  boiling  protease treatment (60°C)  amyloglucosidase

treatment (60°C)  ethanol saturation filter  washing and vacuum-drying of
residue  subtract ash, protein and blank  total dietary fiber.

Because of the complex nature of dietary fiber determination, crude fiber

determination is still widely used for routine purpose. Although crude fiber
determination is relatively simple crude fiber figure is indeed only a very rough
estimate. It represents only half to 1/8th of total dietary fiber in a given food!

Crude fiber is determination is a chemical method. An outline of the method is as follows:

Defatted sample (ground)  boiling in 1.25% H2SO4 for 30 min  filtration  washing
of residue with water to make it acid-free  boiling of residue in 1.25% NaOH for 30
min  washing of residue with water to make it alkali-free  washing of residue
with ethanol to remove any ethanol soluble materials  drying of residue 
weighing of residue  ashing of residue  subtracting ash from the weight of dry
residue  crude fiber.

Crude fiber is a very useful parameter in the analysis of food and feed. In particular,
crude fiber determination has following uses:

1. It serves as an index of feeding value of poultry and stock feeds. Seeds high
in crude fiber content are low in nutritional value
2. It is used for evaluating efficiency of milling and separating bran from the
starchy endosperm
3. It is useful in the chemical determination of succulence of fresh fruits and
vegetables. Overmature products have increased levels of crude fiber
4. It is used in the detection of adulteration of spices. If the crude fiber content
is higher than the normal value, adulteration is confirmed.
5. It can be used to evaluate the quality of tea. Tea containing matured parts of
the plant have high crude fiber content

45
 PROXIMATE ANALYSIS

PRINCIPLE

Determination of crude fiber involves recovery of ash-free residue after sequential

treatment of a solid sample (ground) with 1.25% sulfuric acid and 1.25% sodium
hydroxide each under standardized conditions. The ash that comes along with the
residue is removed by ashing in an ashless filter paper.

REQUIREMENTS

 H2SO4 (1.25g/100ml)  NaOH (1.25g/100ml)

 Buchner filter assembly  Whatman filter paper (rapid)
 Hot air over  Muffle furnace
 Phenolphthalein indicator6  Methyl orange indicator7
 Suction pump  Balance
 Beakers, crucible, etc  Heating arrangement
 Food sample  Silica crucible
 Desiccator  Linen cloth (~ 45 threads per inch)

PROCEDURE

1. Weigh 3 g of fat-free (preferably from crude fat determination), bone-dry sample

2. Prepare two 500-ml digestion flasks (conical or round bottom flask)
3. To one of the flasks, transfer 200ml of dilute (1.25g/100ml) H2SO4. Connect
a condenser and bring it to boil (Fig. IV-4)
4. To another flask, transfer 200ml of dilute (1.25g/100ml) NaOH. Connect a
condenser and bring to boil
5. Boil a liter of water in another flask or beaker. You will need this later on
6. Transfer the sample to the boiling H2SO4 solution. Bring it to boil immediately
and continue boiling for exactly 30 min from the time of boiling. Minimize
evaporation by continuously running the condenser. Rotate/shake briskly to
bring down any sample portion clinging to the internal sides of the flask
7. Filter through linen using Buchner set under light vacuum (see Fig. IV-5).
The transfer should be quantitative. Wash with hot water until acid-free (test
with methyl orange indicator)
8. Transfer the residue quantitatively to hot NaOH solution in the second flask.
You may use spatula to scrap off the residue quantitatively
9. Bring immediately to boil and continue boiling for 30 min as in step 6. Keep
an eye on the sample. Shake the flask intermittently to subdue the frothing
that occurs during boiling
10. Filter the digest through Buchner set as in step 7 but carry out with following
modifications: (i) place a piece of muslin cloth on the Buchner funnel (ii) over
this lining, snugly fit a tared, ashless filter paper. If the filtration becomes
difficult add hot, 10% solution of K2SO4 for facilitating filtration. In very
6
Dissolve 1g phenolphthalein powder in 50ml ethanol and dilute it to 100ml with distilled water
7
Dissolve 1g methyl orange indicator as for phenolphthalein

46
 FOOD ANALYSIS

persistent cases, or if the filtration is going to take a long time, add some
dilute, hot H2SO4 or HCl but do not make the condition neutral or acidic

Water out

Condenser

Water in

Fig. IV-4: Reflux unit for digestion

Buchner funnel
To vacuum

Side-armed flask

Rubber joint

Figure IV- 5: Filtration of digest

11. Wash repeatedly with hot water to make the residue NaOH-free (test the
filtrate with phenolphthalein indicator)
12. Dry the residue (along with the filter paper) at 100°C to bone-dryness and weigh
13. subtract the weight of the filter paper to obtain the weight of the residue
(contains crude fiber + some minerals)
14. Transfer the residue (along with the paper) to tared silica crucible and ignite
the content at 450-500°C in a muffle furnace until all the carbonaceous
materials are burnt out. This usually takes about 30 min. This step is done to
deduct the minerals present in the residue
15. Cool the crucible in desiccator and weigh for ash

47
 PROXIMATE ANALYSIS

CALCULATION

(Residue  Ash)g  (100  F)

% Crude fiber 8(wet basis) =
Sample(g)
(Residue  Ash)g  (100  F  M)
% Crude fiber (dry basis) =
Sample(g)

Where, M and F are moisture content (%) and crude fat (%) of the original sample.

4.3. DETERMINATION OF PROTEIN

4.3.1. PROTEIN DETERMINATION BY KJELDAHL NITROGEN METHOD

BACKGROUND

Protein determination in food analysis is a routine activity. Although it is primarily

concerned with the nutritional status of food or feed, it may also be used to assess
indirectly fruit part in wines, juices, etc. It may be used to differentiate synthetic and
artificial vinegar. In the flour confections, protein in flour is determined to assess its
suitability for noodles, breads and biscuits.

At the present time, all methods of determining the total protein content of foods are
empirical in nature. Isolation and direct weighing of protein would provide an
absolute method. Such a method is sometimes used in biochemical investigation but
is completely impractical for food analysis.

The Danish investigator Kjeldahl worked out in 1883 a method for determining
organic nitrogen in his studies on protein changes in grain used in the brewery
industry. Since the first publication of Kjeldahl, the method has undergone many
changes, the modifications made by Wilforth (1885), Gunning (1889), Van Slyke and
Hiller (1933), to name the least.

Today, Kjeldahl method is universally used for protein determination. Several

variations and modifications are available but all of them employ the amount of
nitrogen present in the sample to calculate indirectly the crude protein content. It is
assumed, in general, that protein contains 16% nitrogen, which means that each gram
of nitrogen determined reflects a protein content of 100÷16 = 6.25 g. The factor 6.25
has been worked out based on a number of studies on amino acid profile. In some
foods, other factors give more accurate result, for example, 6.8 for flour proteins, 5.71
for soybeans, 6.37 for milk, etc. During reporting the result, it is therefore customary
to mention the factor (usually 6.25) used in the calculation.

8
The sample has been assumed to be bone dry. If not make necessary correction in the formula

48
 FOOD ANALYSIS

The nitrogen content in the sample is obtained by first oxidizing the organic nitrogen
with conc H2SO4 into (NH4)2SO4 as under:

Nitrogenous organic compound + H2SO4  CO2 + H2O + (NH4)2SO4 + SO2

The oxidation, termed digestion, is carried out in a special heating chamber and,
depending on the particular protocol, it takes 10 min to several hours for the
completion. Digestion is the most difficult part of the operation and there is no
foolproof method for this. In practice, a small amount of catalyst mixture is added to
the sample to speed up the reaction. A very common digestion mixture contains
selenium dioxide (SeO2), potassium sulfate (K2SO4), and cupric sulfate
(CuSO4.5H2O). When carefully done, all the nitrogen of the sample is taken to be
converted to (NH4)2SO4. The nitrogen in (NH4)2SO4 is recovered as NH3 by steam
distilling the digest after decomposing the former with concentrated NaOH. The
distilled NH3 can be quantitatively determined by various means, including methods
that entail entrapment in standard boric acid and back titration.

Entrapment of NH3 in boric acid solution (the boric acid modification) is reportedly
very accurate and has the advantage that only one standard solution (of the titrating
acid) is required. Neither the amount nor the concentration of boric acid in the
receiving bottle has to be precise. The trapped NH3 is titrated with standard HCl and
nitrogen content back-calculated.

It has to be remembered at this point, the total nitrogen thus determined is not
necessarily from protein alone: nucleic acids and other nitrogenous compounds also
contribute to it. It is therefore appropriate to express the result in terms of crude protein.

PRINCIPLE

Nitrogen content estimated by the Kjeldahl method is based on the determination of

reduced nitrogen (NH2 and NH) present in the sample. The various nitrogenous
compounds are converted into ammonium sulfate by boiling with conc H2SO4. The
ammonium sulfate formed is decomposed with an alkali (NaOH), and the NH3
liberated is absorbed in excess of neutral boric acid solution and then titrated with
standard acid.

REQUIREMENTS

 Kjeldahl digestion and distillation set  Glassware for titration

 Mixed indicator solution9  2% boric acid10

9
Prepare 0.1% bromocresol green and 0.1% methyl red indicators in 95% ethanol separately. Mix 10ml
of bromocresol green with 2ml of the methyl red solution in a bottle provided with a dropper which will
deliver about 0.05ml per 4 drops. ALWAYS USE FRESHLY PREPARED INDICATOR
10
Dissolve 10g boric acid crystal in 500ml of boiling distilled water. After cooling, transfer the solution
to glass-stoppered bottle. It keeps indefinitely

49
 PROXIMATE ANALYSIS

 30% NaOH  0.01N HCl

 Digestion mixture (catalyst mixture)11  Sample (solid food sample)

Fume duct

Exhaust

Digestion flask
(Sample + catalyst
mixture + H2SO4)

Heater

Fig. IV-6: Digestion assembly for Kjeldahl nitrogen

PROCEDURE

1. Prepare samples as described in crude fat determination

2. Weigh 2g of sample12 and transfer quantitatively to a clean 250-ml Kjeldahl
digestion flask (see Fig. IV-6). Don’t spill the sample on the sides of neck
3. Add 2g catalyst mixture
4. Add 25ml conc H2SO4, a little at a time, swirling in between, to mix up the
sample
5. Swirl well to mix up the sample
6. Prepare blank (containing acid and catalyst only)
7. Digest the sample and the blank in the digestion assembly until pale-blue
color is obtained. It normally takes 3-5 hrs. Use water-jet vacuum all the
while to draw away the corrosive fumes of H2SO4. Swirl the digestion flask
(care!) gently (once in a while) to facilitate uniform digestion and bring down
sample portion clinging to the sides of the flask
8. Turn off the heat and let the flasks cool a little
9. Take out the flasks and cool them under running tap water
10. Transfer quantitatively the content to 100-ml volumetric flask (care!!). Use
small amounts of distilled water to complete the transfer. Bring down the heat
of reaction by cooling the flask repeatedly under running tap water
11. Adjust cooling and water addition such that the final volume of solution will
be exactly 100ml at room temperature
12. Mix thoroughly and set aside for distillation to be carried out later on

11
Mix 2.5g of powdered SeO2, 100g K2SO4 and 20g CuSO4..5H2O
12
For samples with high moisture contents, e.g., fruits and vegetables, drying must be done to
concentrate the protein. The minimum amount of wet sample needed is 1kg

50
 FOOD ANALYSIS

DISTILLATION

Rinsing of Kjeldahl distillation set

13. Set the assembly (see Fig. IV-7) and make all the necessary connections
14. Pour distilled water through the sample-introduction cock to fill the
distillation flask
15. Close the steam trap and the sample introduction cock (port)
16. Run the condenser
17. Generate stem by heating the generator with Bunsen burner
18. Carry out steam distillation until the vapor begins to condense and drip from
the condenser tip
19. Immerse the condenser tip in a beakerful of distilled water (~ 250ml)

Sample + NaOH

Line for venting

and filling water
water
out

Steam
Distillation trap
flask
Condenser

Steam
Water in generator

Fig. IV-7: Kjeldahl distillation set (modified Parnas-Wagner)

20. Remove the burner from the steam generator and allow the assembly to cool. As
the assembly cools, vacuum is created. This vacuum sucks in the distilled water
from the beaker. Allow the suction until water spills over to steam trap to nearly
full
21. Open the steam trap before water overflows back to the steam generator. As
the water begins to flow down from the condenser, further vacuum is created.
This sucks away the contents of the distillation flask to the last drop
22. Repeat steps 1721 for the next cycle of cleaning

51
 PROXIMATE ANALYSIS

Distillation of sample

23. Pipette out 5ml of 2% boric acid and 4 drops of mixed indicator into a clean
conical flask. Prepare 5-6 such sets
24. Fill a 25-ml burette (fine graduation) with 0.01N HCl. This will be needed for
online titration later on
25. Turn on the distillation assembly but leave the steam trap open
26. Immerse the condenser tip in the boric acid-indicator solution prepared in
step 23
27. Introduce from the sample introduction cock 5ml of blank digest, rinse the
funnel with 2-3ml portion of distilled water and then introduce 10ml of 30%
NaOH. Rinse with 2-3ml of distilled water and pinch close the cock
28. Close the steam trap
29. The steam now enters the distillation flask and stirs up the mixture. The
liberated ammonia finds its way through the condenser into the boric acid-
indicator trap. The boric acid solution changes from bluish purple to bluish
green as soon as it comes in contact with ammonia. 5 minutes after the boric
acid has changed its color, lower the conical flask such that the condenser tip
is 1 cm above the liquid. Wash the condenser tip with 1ml of distilled water.
Continue distillation for another minute and then remove the burner
30. Titrate the boric acid mixture in the flask with standard (0.01N) HCl until the
blue color just disappears
31. Clean the distillation set following steps 1721 between each addition of
sample
32. Repeat the distillation procedure for replicate samples

CALCULATION

(Sample titer  Blank titer)ml  N of HCl  14  100  100

Nitrogen (%, wet basis) 
Aliquot (ml)  Wt. of sample (g)  1000
100
Nitrogen  %, dry basis   Nitrogen  %, wet basis  
Dry matter  % 
Protein  %, dry basis   Nitrogen  %, dry basis   6.25

Note that the condensate can also be collected over a known volume of standard acid,
such as 0.1N H2SO4. The amount of ammonia (and therefore nitrogen) trapped can be
calculated from the decrease in the strength of the acid (determined by titrating with
standard alkali). A blank is also used. It must be noted that the volume of standard
acid used should be more than sufficient to trap ammonia.

52
 FOOD ANALYSIS

4.3.2. MACRO-KJELDAHL METHOD

REQUIREMENTS

 Sample (dry and powdered)  50% NaOH solution

 0.05 N HCl (see micro-Kjeldahl)  Boric acid (same as in micro-Kjeldahl)
 Mixed indicator (see micro-Kjeldahl)  Catalyst mixture (see micro-Kjeldahl)
 Digestion set  Distillation set
 Titration arrangement  Volumetric flask, and other glassware

Set up of the apparatus is shown in Fig. IV-8.

PROCEDURE

1. Prepare digest as in micro-Kjeldahl method

2. Make up the volume to 100ml with distilled water
3. Transfer 10ml of aliquot to a distillation flask
4. Add 400ml of ammonia-free distilled water
5. Add a large piece of granulated zinc in the flask (to prevent bumping)
6. Take 50ml boric acid in the receiving flask; add a few drops of mixed
indicator
7. Immerse the tip of the delivery tube (see Fig. IV-8) in the boric acid solution
8. Into the funnel of the distillation flask transfer 80ml of 50% NaOH solution
9. Open the tap, let the alkali drop into the flask, and close the tap
10. Heat the flask, and distil the ammonia into the boric acid solution
11. Stop distillation after about 300ml has distilled over
12. Open the tap and wash down the condenser and the delivery tube into the
receiving flask
13. Titrate the distillate with 0.05N HCl
14. Carry out blank determination in the same way without the sample

CALCULATION

(S  B)ml  N of HCl  14  100  Vol. made up (ml)

Nitrogen (%, wet basis) 
Wt. of sample (g)  Aliquot (ml)  1000
Protein (%, wet basis)  Nitrogen (%, wet basis)  6.25

Where, S = Sample titer, B = Blank titer

53
 PROXIMATE ANALYSIS

NaOH

Funnel

Water
out

Condenser

Digest

Water
in

Burner
Receiver

Boric acid

Fig. IV-8: Macro-Kjeldahl distillation set

4.3.3. DETERMINATION OF PROTEIN IN MILK BY FORMOL TITRATION

BACKGROUND

Formol titration (formol binding) was originally devised for the determination of proteins
in milk. The method has gained little popularity due to its low precision. However, this
method is probably the quickest method for the rapid survey of natural and processed
milk for protein content. This method can also be used to assess fruit part in soft drinks,
wines, etc., and may be applicable in differentiating synthetic and natural vinegar.

PRINCIPLE

The formol-binding method relies on the measurement of carboxylic group present in

the amino acids and proteins by titrating with NaOH. The amount of – COOH group
available for reaction with the alkali has been correlated with the actual protein
content (obtained by standard methods) of the milk sample.

Straightforward as it might seem, the stoichiometric reaction that occurs between the
alkali and –COOH is too week to perceive. This is due to the interference by amino
groups of proteins and amino acids. The titration end point can be made more
pronounced by masking/blocking the interference of primary amines and this is done
by reacting the latter with formaldehyde (methanal) at room temperature and neutral
pH to form dihydroxymethyl derivative, which is a tertiary amine.

54
 FOOD ANALYSIS

COO O COO
R CH 2HC R CH CH2OH
NH3 H N
CH2OH
Amino acid Formaldehyde Dihydroxymethyl derivative

The reaction may also lead to the formation of dimethylamine as:

COOH O COOH
R CH HC R CH
NH2 H N(CH3)2
Amino acid Formaldehyde Dimethyl derivative

The protein content and the formol titer (i.e., titer when the strength of NaOH is 0.1N)
are related thus:

F  titer (ml)
% Protein content =
Aliquot (ml)

The factor F has been worked out to be 17 for milk from Danish cows. It has to be
adjusted for other cows (by using standard methods, e.g., Kjeldahl method).

REQUIREMENTS

 Milk samples from 3 sources, ½ liter each  Titration arrangement

 Water bath  0.1N NaOH
 Saturated potassium oxalate13  0.0005% fuchsin solution
 2% phenolphthalein  40% formaldehyde

PROCEDURE

1. Heat milk in water bath in separate containers to 40°C and cool to 20°C
2. Into labeled, separate 250-ml conical flasks, add 2ml potassium oxalate, 50ml
milk and 0.5ml phenolphthalein
3. Stand the flasks for 2 min
4. Prepare control by mixing 50ml of milk, 2ml of fuchsin and 2ml of potassium
oxalate
5. Titrate (the sample mixture in step 2) with 0.1N NaOH to the same color as
the control
6. Add 10ml neutralized 40% formaldehyde and titrate again with 0.1N NaOH
to match the color of the control

13
Prepare in distilled water. Make sure that some crystals are still left undissolved

55
 PROXIMATE ANALYSIS

CALCULATION

17  formol titer (ml)

% Protein content =
Aliquot (ml)

Express the result as mean ± sample standard deviation

x  x 
x
n 1

Note: If the protein content obtained is unusually low, or if there is difficulty in observing the end point,
repeat the analysis by diluting the milk with water to a known volume (for example, 1+1).

4.3.4. DETERMINATION OF PROTEIN BY BIURET ASSAY

BACKGROUND

There are several methods available for the determination of proteins in a sample.
Because of the highly variable nature of proteins, however, no single method is
suitable in all cases. More sophisticated (but less routine) methods include: 1. Infrared
spectrophotometry, 2. Fluorimetry, 3. Polarography, and 4. Refractometry. For
routine tests, solid samples are usually tested by Kjeldahl nitrogen method. Liquid
samples can be tested by color reactions, such as:

1. Lowry (Folin-Ciocalteau) method

2. Coomassie Brilliant Blue Dye (Bradford) method
3. Silver Binding method
4. Turbidimetric method
5. Biuret method

Of the various protein analytical methods, the biuret reaction comes about the closest
to being most specific in that it requires at least a sequence of two or more peptides to
develop the diagnostic color. This is also a very rapid method of assay. Among the
limitations, the test lacks sensitivity if the protein concentration is below 1mg/ml.

Alkaline CuSO4 reacts with compounds containing two or more peptide bonds to give
violet-colored complex. The depth of the color is a measure of the number of peptide
bonds present in protein. The name of the test comes from the compound biuret,
which gives a typical positive reaction (Fig. IV-9). The reaction is not absolutely
specific for peptide bonds since any compound containing two carbonyl groups linked
through nitrogen or carbon atom will give a positive result.

56
 FOOD ANALYSIS

H H
CONH2 _ CON NOC
HN Cu
+2
OH HN Cu+2 NH
CONH2 CON NOC
Biuret molecule H H
Biuret complex
(Pink or violet)

Fig. IV-9: Biuret and biuret complex

O O
H H
R N N R
O _
NH Cu
+2
OH Cu+2
R
O O HN N
H O
HN

Protein residue Biuret complex

(Pink or violet)
Fig. IV-10: Mechanism of biuret action

The formation of color is due to probable formation of coordination complex of –NH of

the component amino acid with Cu++ (see Fig. IV-10 for the mechanism). Quantitative
analysis of protein can be done by measuring the absorbance of the color against a
standard at 540-560 nm. It may be noted, peptone produces a pink color in biuret assay.

PRINCIPLE

Biuret reagent (alkaline CuSO4) reacts with compounds containing two or more
peptide bonds to give a violet colored complex. The depth of the color is a measure of
the number of peptide bonds present in the protein. Dipeptides and most amino acids
(with the exception of histidine, serine and threonine) do not give this reaction. The
formation of color is probably due to the formation of complex between –NH group
of amino acid and copper ion (Cu++). The absorbance of the color of the reaction
mixture is measured at 540-560 nm in a colorimeter and compared with the standard
curve prepared by using standard protein solution.

REQUIREMENTS

 Protein standard14  Biuret reagent15

 Water bath or incubator  Colorimeter
 Pipettes and test tubes  Protein sample: fresh egg

14
Prepare 15mg/ml of analytical reagent grade albumin in water
15
Weigh 3g CuSO4.5H2O, 9g sod-pot-tartrate, and 5g KI. Dissolve them all in 0.2N NaOH to make 1
liter.

57
 PROXIMATE ANALYSIS

PROCEDURE

1. Weigh an egg
2. Break the egg, separate the contents and mix them well
3. Weigh 10g of the egg mixture and make up to 100ml with water. Mix well
4. Prepare test tubes as under:

Add sequentially in clean tubes

Albumin standard (ml) 0@ 0.2 0.4 0.6 0.8

Biuret (ml) 3 3 3 3 3
Water (ml) 12 11.8 11.6 11.4 11.2

Sample (ml) 0.2 0.4 0.6

Biuret (ml) 3 3 3
Water (ml) 11.8 11.6 11.4

5. Mix the contents in the tubes well. Warm the tubes at 37°C in water bath or
incubator for 10 min
6. Adjust the absorbance of blank at 540 or 560 nm in colorimeter at zero
7. Read the absorbance of the standard protein solution at 540 or 560 nm
8. Read the absorbance of the sample solutions
9. Prepare standard curve$ using data from step 7
10. Fit the absorbance you obtained in step 8 in the standard curve (Fig. IV-11) to
calculate the protein content. You may linearize the equation by least square
linear regression (as explained in vitamin C determination)

*
* Linear trendline

*
*
Protein content in the sample

Protein (mg)

Fig. IV-11: The standard curve

@
Meant for blank (i.e., with out protein)

58
 FOOD ANALYSIS

4.4. MOISTURE IN FOODS

BACKGROUND

Water is the most abundant compound present in most foods. Cellular material,
whether plant or animal, contains a significant amount of water. Green leafy
vegetables contain > 90 % water. Water content in relation to food material is
normally termed “moisture content”. Meat, milk and cereals contain 50-60, 87-88 and
11-13% moisture content respectively. Occasionally, a food such as oil will be dry;
but even crystallized substances which are relatively pure (such as sugar and salt),
contain small amounts of water adsorbed on the surfaces of the crystals.

Moisture content in food is often described by different terms, such as bound water,
free water, unfreezable water, Langmuir or monolayer water, capillary water, etc. It is
difficult to provide a rigid definition of these terms but the first two terms have been
explained here.

Bound water: It is generally understood as water that is unavailable as solvent and is

very difficult to remove by simple drying or dehydration method. Bound water is also
taken as water that remains unfrozen at some prescribed temperature below 0°C,
usually -20°C. However, this assumption is misleading because water can exhibit
freezing over a wide range of temperatures, depending on the presence of dissolved
solids, hydration effects of macromolecules and biological ultrastructures such as
membranes. A substantial amount of water may never freeze under given
experimental conditions. This water may be very difficult to remove, but still not
'bound'. Thus, bound water is simply the water whose rate of movement in the system
is so low that equilibrium cannot be achieved within the normal lifetime of food. It is
hypothesized that when foods enter the glassy state (freezing without crystallization)
the movement of water is so slow that it is effectively bound.

The unfreezable water accounts for about 8-10% of total water in foods

Free water: Free water exists as a dispersing medium for the colloids and as a
solvent for the crytalloids present. Free water can be determined by centrifugation,
pressing and heating or any other drying methods. The division between free and
bound moisture, however, is not very distinct because the difference between these
two forms of water is gradual rather than sharp.

The determination of moisture content in foods is one of the most frequently carried
out tests in food industries. The determination is essential for various reasons, for
example it may be essential to:

1. Ascertain the level of drying in dried foods

2. Check the efficiency of a given dryer or drying method
3. Ascertain microbiologically safe moisture level in cereal grains
4. Regulate moisture level of wheat during milling

59
 PROXIMATE ANALYSIS

5. Check whether a food conforms to the standards or not, e.g., in biscuits, flour, etc.
6. Aid in calculation of food constituents in terms of dry weight, and so on.

DETERMINATION OF MOISTURE CONTENT IN FOODS

Moisture content in food materials can be determined by a large number of methods.

The methods range from very simple to very sophisticated and this is mainly based on
the type and state of food sample. Some methods are suitable only for a limited range
of food types. There is no single method that can be applied to every type, state and
form of foods.

Basically, the methods available for the determination of moisture in foods can be
divided into following groups, namely, (i) Physical, and (ii) Chemical.

Physical method is more routinely used. Some of these methods are: Hot air oven
method, Infra Red method, Distillation with immiscible solvent (Dean and Stark
method), Hot plate method, Vacuum oven method, Freezing point, and Nuclear
magnetic resonance (NMR).

Chemical methods are less routinely used. Some of the tested methods but not
necessarily widely used or suitable for all food items are: (i) Karl Fischer method,
(ii) Reaction with calcium carbide, and (iii) Oxidation with potassium dichromate.

OVEN DRYING

This is by far the most widely used physical method for the determination of moisture
in a wide range of foods. The method is relatively simple. The sample is weighed and
heated in an insulated oven to constant weight. The difference in weight is the water
that has evaporated. The sample is usually weighed in a flat-bottomed, shallow dish
(made of material that will not react with food nor pick up moisture readily). The
oven must be thermostatically controlled and usually set at 100°C or 105°C. The size,
weight, etc., of the sample is very critical. To help fast and uniform drying, the
sample should be disintegrated into fine particles. Very often, an internal fan is also
fitted in the oven to circulate the hot air. This method is suitable for nuts, flour,
powders, meat and meat products, and most fruits and vegetables.

Many foods decompose to some degree if they are heated to 100°C. This is true, for
example, of all foods which contain fructose. It is necessary to dry them in a vacuum
oven where the temperature is maintained at lower figure (usually 60-70°C) and
pressure is reduce to less than 450mm of Hg to facilitate loss of moisture.

HOT PLATE

This method is usually used for fat and oil products such as cooking oil, vanaspati,
butter, ghee, etc. About 10g of sample is put in a tared, dry beaker and placed on a hot
plate. As the heating progresses, steam bubbles begin to appear and rise from the

60
 FOOD ANALYSIS

bottom. Soon after the foaming stops, slightly burnt smell accompanied by faint
brown fume becomes evident. The sample is immediately taken out, cooled in a
desiccator and weighed for loss in weight. The difference in weight represents the
amount of water present in the sample.

IMMISCIBLE SOLVENT DISTILLATION METHOD

Those foods which contain volatile compounds must be treated by another method.
None of the weight-loss methods are adequate to differentiate between loss of water
and loss of other volatile substance. The immiscible solvent distillation method can be
used for this purpose. The sample is placed in a flask which is connected with a reflux
condenser equipped with a distillate trap (see Fig. IV-12).

Water out

Condenser

Water in

Solvent Inclination
5
4
Trap
3
2
1 Water

Drain

Imiscible
Sample Solvent

Fig. IV-12: Immiscible solvent distillation method (Dean and Stark apparatus)

61
 PROXIMATE ANALYSIS

The sample is covered with a suitable solvent and the trap filled with the solvent. The
solvent must be immiscible with water so that as they distill separation of the two
liquids occur. One additional requirement of the solvent is that it should have a
boiling point slightly higher than that of water (that is, above 100°C).

Toluene is commonly used, although xylene and heptane are sometimes employed.
The flask is heated and the vapors of water and solvent are condensed by the
condenser and drop into the trap. The lighter solvent flows over to the flask, but water
is captured. It the trap is calibrated, the amount of water distilled out of the sample
can be read directly.

The method is suitable for the determination of moisture in items such as cardamom,
ginger, and aromatic plants.

INFRARED HEATING

Infrared moisture balance is an instrument for measuring moisture content of

materials that do not change their chemical structure while loosing water under
exposure to infrared radiation. The instrument comes in various shapes and sizes but
all contain graduated scale on wheels (or sometimes a direct readout meters). The
removal of moisture is by molecular vibration of water rather than the heat alone. It is
advised not to look at the light source with naked eyes. The greatest advantage of
infrared method is that the sample does not need weighing and cooling in desiccator.
The completion of the drying of sample can be easily ascertained by looking at the
pointer and scale provided along with the instrument. When moisture stops going out
(i.e., the weight remains constant) the pointer becomes stationary (does not move for
at least 5 min). The method is suitable for samples that rehydrate quickly. Samples as
diverse as flour, biscuits, and fruits and vegetables can be used for the determination.
Because the sample size the instrument can handle is usually very small (< 5g) the
accuracy may sometimes be affected. See Fig. IV-13 for a schematic drawing of the
instrument.

Movable cover
Filter glass

IR lamp Calibrated wheel and pointer

Plate and sample

Adjustment knob

Power switch

Fig. IV-13: Infrared moisture meter

62
 FOOD ANALYSIS

KARL FISCHER METHOD

This method is suitable for low moisture foods such as roasted coffee, oils/fats, and
sugar-rich products such as honey, sugar, etc. The method uses four-component
system, viz., pyridine, methanol, iodine, and SO2. Methanol and pyridine is used to
dissolve iodine and SO2. The reaction takes place in two steps. In all, for each mole of
water, 1 mole of iodine, 1 mole of SO2, 3 moles of pyridine and 1 mole of methanol
are used. The reaction sequence is:

C5 H 5 NI 2  C5 H 5 NSO 2  C5 H 5 N  H 2 O  C5 H 5 NHI  C5 H 5 NSO3

and
C5 H 5 NSO3  CH 3OH  C5 H 5 N(H)SO 4 CH 3

In practice, iodine is used in excess during the reaction. The residual iodine is later
determined by iodometric titration (with sodium thiosulfate) and back-calculated to
get the mole of iodine used in the reaction. The amount of iodine used in the reaction
is a measure of moisture content in the sample.

Another quick method of moisture determination is based on reaction of water with

calcium carbide to produce acetylene and calculating the moisture content, either
from loss in weight or from an increase in the pressure so produced. The procedure
has been used for quick determination of moisture in fresh and frozen sweet corn.

A method based on oxidation of potassium dichromate (K2Cr2O7) and

electromagnetic titration with ferrous sulfate (FeSO4) has been reported for the
determination of moisture in fresh and frozen fruits and vegetables.

4.4.1. MOISTURE CONTENT BY IR-MOISTURE METER

BACKGROUND AND PRINCIPLE

Already discussed elsewhere.

REQUIREMENTS

 Wheat flour  IR moisture meter

PROCEDURE

1. Prepare sample as usual (by quartering, discussed in fat determination)

2. Take out the aluminum pan (that receives the sample) from the moisture meter
and clean it with a piece of muslin cloth

63
 PROXIMATE ANALYSIS

3. Put back the pan, close the lid of the instrument and turn on the lamp. Do not
look at the lamp with naked eyes. Leave the instrument for 15 min to let the
pan dry
4. With the help of the adjustment knobs attached to the instrument, coincide the
red indicator needle (attached), 100 units of the circular scale (attached) and the
reference mark
5. Leave the instrument (with lamp on) for further 5 min. If the pointer needle
deviates during this period, make adjustment as in step 4 again
6. After everything is stabilized, turn off the lamp, take out the pan and allow it to
cool a little. Spread carefully onto the pan about 2 g of wheat flour
7. Put the pan (with the sample) back into the instrument. Do not turn on the lamp
(it will affect your eyes if you look at it with naked eyes). You will notice now
that the needle has run away
8. Turn the circular scale with the adjustment knob towards the zero direction.
The needle should appear as you continue to turn on the knob. In case the
needle doesn’t appear even after you have reached the zero mark, it is clear that
your sample size has to be reduced. Reduce the sample size by taking out small
portions until the needle appears. Carefully adjust the weight of the sample to
coincide reference mark, pointer needle, and zero. If the needle appears well
before you reach the zero mark you will need to add some flour and adjust to
coincide as before
9. After every thing has been made ready, turn on the lamp. Soon after you turn
on the lamp, you will notice movement in the red pointer needle
10. Bring the needle to the reference mark every 2-3 minutes by turning the
circular scale. Do this until the needle stops running away. Wait for another 5
min to be sure that the needle is stagnant (stable)
11. Make the final adjustment and read the mark on the circular scale at the point
of coincidence. The reading directly gives the percentage of moisture in the
sample
12. The direct percentage can be read off from the instrument only if you load the
sample to zero mark. If you do not reach the zero mark, note down the initial
units on the circular scale. This will be your initial load. Make necessary
calculation as given below:

CALCULATION

(Final  Initial) reading  100

% Moisture 
100  Final reading

4.4.2. MOISTURE CONTENT BY HOT-AIR OVEN METHOD

PRINCIPLE

The sample is weighed and heated in an insulated oven to constant weight. The
difference in weight is the water that has evaporated. The sample is usually weighed in
a flat-bottomed, shallow dish (made of material that will not react with food nor pick up

64
 FOOD ANALYSIS

moisture readily). The oven must be thermostatically controlled and usually set at
100°C or 105°C. The size, weight, etc., of the sample is very critical. To help fast and
uniform drying, the sample should be disintegrated into fine particles. Very often, an
internal fan is also fitted in the oven to circulate the hot air. This method is suitable for
nuts, flour, powders, meat and meat products, and most fruits and vegetables.

REQUIREMENTS

 Sample (flour, biscuit, rice, etc)  Petri dish or aluminum dish (~ 25 g cap)
 Weighing arrangement  Desiccator
(electrical or electronic balance,  Hot air oven
±1mg)

PROCEDURE

1. Prepare the sample by grinding, slicing or blending, depending on the type of

food
2. Take three Petri dishes or aluminum dishes that have been previously cleaned
and dried
3. Weigh about 10g sample in each dish by difference
4. Place the samples in hot air oven previously set at 103±2°C (for samples not
containing fructose or volatile materials)
5. Note the decrease in weight of the plate every hour until the two consecutive
weights differ only by ± 5mg. Before each weighing, cool the dish in a
desiccator

CALCULATION

Initial wt.  Final wt.

Moisture content, %  ×100
Initial wt.

4.4.3. MOISTURE CONTENT BY HOT PLATE METHOD

PRINCIPLE

This method is usually used for fat and oil products such as cooking oil, vanaspati,
butter, ghee, etc. A weighed amount of the sample is heated in a beaker on a hot plate
to the incipient faint brown smoking. The difference in weight of the sample
expressed in percentage gives the moisture content.

REQUIREMENTS

 Fat, oil or butter sample  Beaker, 50ml cap

 Hot plate  Desiccator
 Electronic or electrical balance  Glass rod (~ 5cm long)

65
 PROXIMATE ANALYSIS

PROCEDURE

1. Take a clean, 50ml-beaker with a glass rod stirrer

2. Weigh about 10g of fat sample in the beaker by difference
3. Place the beaker (along with the glass rod) on a hot plate and heat it at around
120°C
4. After the water begins to rise as fine bubbles, stir the contents intermittently
to facilitate faster escape of water. Stirring will also avoid the splattering (and
consequent loss) of the sample
5. Immediately the rising bubbles die away, be attentive to observe the incipient
smoking
6. As soon as the incipient smoking is observed, take out the beaker and cool it
in a desiccator. You may also notice light brown fume issuing from the hot
sample
7. Weigh the desiccator and calculate the moisture content in %

CALCULATION

Initial wt.  Final wt.

Moisture content, %  ×100
Initial wt.

4.4.4. MOISTURE CONTENT BY SOLVENT DISTILLATION METHOD

PRINCIPLE

For those foods which contain volatile compounds, none of the weight-loss methods
are adequate to differentiate between loss of water and loss of other volatile
substance. The immiscible solvent distillation method can be used for this purpose.

This method involves the reflux distillation of the food with an immiscible solvent
having a higher boiling point and a lower specific gravity than water, e.g., toluene,
heptane or xylene. The refluxed water settles as the solvent floats in a graduated tube,
in which it can be measured by volume. The volatile oils which also distil over
remain mixed with the solvent and are not measured. The equipment used for this
method of moisture determination is called Dean and Stark apparatus (Fig. IV-12) or
Bidwell-Sterling tube.

The method is suitable for the determination of moisture in items such as cardamom,
ginger, and aromatic plants which contain significant amounts of volatile principles.
These volatile principles normally get lost when subjected to vacuum oven and hot-
air oven methods, thereby introducing error in the determination.

66
 FOOD ANALYSIS

REQUIREMENTS

 Sample (fresh ginger, cardamom, etc)  Heating mantle (150°C range)

 Dean and Stark apparatus (250ml cap)  Cutting knife or grater
 Solvent (Toluene)  Weighing arrangement
 Cleaning reagent (saturated conc  Burette brush
H2SO4-K2Cr2O7 solution)

PROCEDURE

1. Clean the condenser and trap assembly of Dean and Stark apparatus with
cleaning agent (take instructor’s help)
2. Grate the ginger into coarse pieces
3. Weigh the sample which will give 2-5ml of water and transfer it to the boiler
flask. If bumping is likely to occur during boiling, add some dry sand at the
bottom of the flask before adding the sample
4. Add sufficient toluene to completely cover the sample
5. Connect the flask to the side arm of the condenser and trap assembly as in
Fig. IV-12
6. Pour toluene through the condenser until the trap (collecting tube) is filled
7. Run the condenser and start heating the boiler flask
8. Distil slowly at about 2 drops/sec until most of the water has distilled over
9. Increase the distillation rate to 4 drops/sec until no more water is distilled
10. Wash down the condenser with a small amount of toluene. If water droplets
adhere to the condenser, push them down with a burette brush saturated with
toluene
11. Continue distillation until no more water distils over and repeat the washing
process
12. Discontinue heating and allow the trap to attain room temperature
13. Read the volume of water distilled and, assuming its specific gravity to be
1.0, calculate the percentage of water present in the sample

CALCULATION

Volume of water (ml)

Moisture content, %  ×100
Wt. of sample (g)

4.5. DETERMINATION OF ASH IN FOOD SAMPLES

BACKGROUND

Ash is the inorganic residue from the incineration of organic matter. The amount and
composition of ash in food depends on the method of ashing used and the nature and
type of food. Ash represents all the minerals that do not volatilize at ashing
temperature. Ash content of food item is determined for following reasons:

67
 PROXIMATE ANALYSIS

1. Acid-insoluble ash is used to detect the presence of adulterants (sand, dirt,

etc.) in spices
2. To determine the efficiency of wheat-flour milling (test the thoroughness of
the separation of bran and germ from the rest of the kernel)
3. Water-soluble ash is a useful index of the fruit content of jelly and fruit
preserves
4. Total ash is a useful parameter in distinguishing fruit vinegar from synthetic
vinegar

Ash and mineral contents in most food items are determined by first destroying the
organic matter. The destruction can be carried out by two main methods, viz., (i) dry
ashing, and (ii) wet ashing (also called wet oxidation or wet digestion), the former
being more widely used.

The choice of the procedure depends on nature of the organic material, the nature of
any inorganic constituent present, the metal to be determined and the sensitivity of the
method used for determination. A brief description of both the methods is given in the
following paragraphs:

DRY ASHING

This is applicable to most common minerals except mercury and arsenic. It is

particularly useful in the determination of acid-insoluble and water-soluble materials.

The sample is incinerated at temperatures ranging from 400-700°C (most commonly

around 550°C). The temperature range can be varied, depending on the objective of
the ashing. For instance, zinc and potassium will be lost if temperatures exceeding
480°C and 450°C (respectively) are used. Excessive heating may also make certain
metallic compounds insoluble (as in the case of tin).

Dry ashing is done in special crucibles. Porcelain and silica crucibles are widely used
because of their good weight constancy and relatively low price. They can withstand
up to 1200°C. They are easily cleanable with dilute HCl. However, they are
susceptible to alkali and crack from sudden temperature changes.

The forms of mineral constituents in ash differ considerably from their forms in the
original food. Thus, calcium oxalates are converted into carbonates, and upon further
ashing, to oxides.

Dry ashing is arguably rapid compared to wet ashing. The method requires less
attention and does not need a blank. One of the limitations of dry ashing is that certain
trace elements are absorbed by silica or porcelain crucibles. Foods with high
phosphorus to base ratio fuse to give a dark melt in which carbon particles are trapped
and will not burn. In spite of this draw back, dry ashing is still the most widely used
technique for the determination of total ash and most minerals because of its
simplicity and rapidity.

68
 FOOD ANALYSIS

WET ASHING

Wet ashing is the digestion of samples for the determination of trace elements and
metallic poison (e.g., lead and arsenic). Mixed acids are generally used for
decomposing the organic matter. The mixture of H2SO4 and HNO3 is recommended
by many workers but there are several variations available. The digestion is done in
special glass apparatus.

4.5.1. DETERMINATION OF TOTAL ASH IN SOLID FOOD SAMPLE BY DRY

ASHING

PRINCIPLE

The determination of total ash can be conveniently carried out by incinerating all the
organic matter of the food sample at 550°C.

REQUIREMENTS

 Food sample  Silica crucible

 Muffle furnace  Electronic balance
 Desiccator

PROCEDURE

1. Prepare silica crucible by washing thoroughly with water, 6N HCl, and

distilled water. Dry the crucible in oven at temperatures above 150°C for half
an hour. Cool in a desiccator and tare to the nearest 1mg
2. Prepare sample by grinding
3. Weigh in the crucible (prepared as above) 8-10 g of sample by difference
4. Char the sample over a low Bunsen flame to volatilize as much of the organic
matter (until no more smoke is given out by the material) as possible. To
speed up the charring process and simultaneously reduce the smoke level,
ignite the smoke with a Bunsen flame
5. Transfer the crucible to a temperature-controlled muffle furnace. Use long
tongs for the transfer
6. Keep the furnace at 300°C until the carbon has ceases to glow and then raise
the temperature to 500°C. Continue ashing for 3-4 hours
7. Turn off the furnace and allow the crucible to cool to around 200°C and see
whether any traces of black residue (indicator of incomplete ashing) remain
8. If the carbon (i.e., the black residue) has not completely gone, take out the
crucible, add 1-2ml of conc HNO3, evaporate to dryness, and ash again for an
hour
9. Turn off the furnace and slowly cool the crucible to around 200°C. Don’t take
out the crucible all of a sudden: it will crack because of high temperature
gradient
10. Cool the crucible in desiccator and weigh to the nearest 1mg
69
 PROXIMATE ANALYSIS

CALCULATION

Ash (g)  100

Total ash (as-is basis) 
Sample (g)
Ash (g)  100  100
Total ash (%, dry basis) 
Sample (g)  Dry matter (%)

Note: As-is basis implies wet basis.

70
CHAPTER V: SOME ULTIMATE ANALYSES

5.1. DETERMINATION OF ACID-INSOLUBLE ASH

PRINCIPLE

The acid-insoluble ash is an ignited residue obtained after treatment of total ash in
10% HCl and subsequent filtration. Acid-insoluble ash mainly consists of silica
compounds that are resistant to dissolution in 10% HCl.

REQUIREMENTS

 Ash sample  Whatman® filter paper

 10% HCl solution  Heating arrangement
 Muffle furnace  Filtration assembly

PROCEDURE

1. To the ash prepared by dry ashing, add 25ml of 10% HCl solution (care!)
2. Cover with watch glass and boil gently over a low flame for 5 min
3. Filter quantitatively through ash-less filter paper
4. Reserve the filtrate for analysis of individual minerals, e.g., iron, calcium, etc.
5. Wash the filter paper thoroughly with hot water. Pool the washings to the
reserve filtrate
6. Return the filter paper to the original crucible
7. Ignite the contents in the muffle furnace, cool, and weigh

CALCULATION

Acid-insoluble ash (g)×100

Acid-insoluble ash (%, wet basis) 
Sample (g)

Acid-insoluble ash (g)×100×100

Acid-insoluble ash (%, dry basis) 
Sample (g)×Dry matter (%)
 ULTIMATE ANALYSIS

5.2. DETERMINATION OF CALCIUM CONTENT BY VOLUMETRIC METHOD

PRINCIPLE

Calcium is precipitated as calcium oxalate. The precipitate is dissolved in hot dilute

H2SO4 and titrated with standard KMnO4. 1ml of 0.01N KMnO4 is equivalent to
0.2mg calcium.

REQUIREMENTS

 10% HCl solution  Ashless filter paper

 Glassware
 Heating arrangement
 Dilute acetic acid17
 Methyl red indicator
 Dilute sulfuric acid18
 Ash
 Titration arrangement
 Dilute ammonia16
 Saturated ammonium oxalate
 Silver nitrate
 Standard KMnO4 (0.01N)19

PROCEDURE

1. To the ash prepared by dry ashing, add 25ml of 10% HCl solution (care!)
2. Dissolve the acid soluble ash by heating the solution carefully for about 5 min
3. Filter quantitatively and collect the filtrate in a clean 100-ml volumetric flask
4. Discard the residue left in the filter paper
5. Make up the volume of the solution to 100ml with distilled water
6. Transfer 50ml of the solution to a clean, distilled-water-rinsed 250-ml beaker.
Add about 50ml of distilled water and stir with a glass rod
7. Add 10ml of saturated ammonium oxalate and 2 drops of methyl red
indicator
8. Make the solution slightly alkaline be adding dilute ammonia (to give faint
yellow color)
9. Make the solution slightly acidic with few drops of dilute acetic acid until the
color is faint pink (pH 5)

16
Ammonia:water=1:4
17
Acid:water=1:4
18
Acid:water=1:4
19
Dissolve 3.3g of dry KMnO4 in 200ml water, transfer to 1-liter flask and make up the volume. Weigh
0.25-0.3g of sod oxalate (take into account the purity factor also) and transfer to a 500-ml beaker with
about 250ml of dilute sulfuric acid (acid:water=5:95). Stir to dissolve the oxalates. Warm the solution to
60°C and titrate rapidly with the above prepared KMnO4 solution to the first permanent pink end point.
Obtain the normality by routine method as follows:
Wt. of sod-oxalate(g)  1000
Normality of KMnO 4 
ml of KMnO 4  67

72
 FOOD ANALYSIS

10. Heat the solution to boiling point

11. Allow the solution to stand at room temperature for at least 4 hours.
Alternatively, leave the solution overnight
12. Filter through Whatman No. 42 paper and wash with water until the filtrate is
oxalate-free. Since HCl has been used for preparing the original ash solution,
it is convenient to test for the absence of chloride using AgNO3 solution.
Discard the filtrate
13. Break the point of the filter paper with pointed glass rod and wash down the
precipitate quantitatively with hot, dilute H2SO4 into the beaker in which
calcium was precipitated
14. Wash with hot water
15. Transfer the solution quantitatively to 100-ml volumetric flask and make up
the volume with distilled water
16. Do not tamper with the filter paper in the funnel. You will need it later on
17. Pipette out 25ml portions of solution in 3 conical flasks. Warm all the flasks
to 80°C
18. Titrate the content of the flask with 0.01N KMnO4 to the first permanent pink
19. To one of the flasks, after titration to end point, drop the filter used earlier
and titrate once again rapidly to the pink end point. Note the volume of
KMnO4 consumed by the filter paper
20. Average the reading as shown below:

4  V1 +V2 +V3 
Titer   V4
3

Where, V1, V2, and V3 = titer of the flasks 1, 2 and 3 respectively

V4 = volume of KMnO4 consumed by filter paper

CALCULATION

Titer  0.2  VT (ml)  100*  100

Ca++ (mg/100g, dry basis) =
VE (ml)  Wt. of sample (g)  Dry matter (%)

Where, VT = total volume of ash solution; VE = volume taken for estimation;

* = volume (ml) made up (in the above case, 100ml)

5.3. DETERMINATION OF IRON IN FOODS BY COLORIMETRY

PRINCIPLE

Iron in foods is determined by converting all the iron into ferric form using oxidizing
agents like potassium persulfate (K2S2O8) or hydrogen peroxide (H2O2) and treating
thereafter with potassium thiocyanate (KSCN) to form the red ferric thiocyanate
which is measured colorimetrically at 480nm
73
 ULTIMATE ANALYSIS

REQUIREMENTS

 Ash solution  Colorimeter

 Con H2SO4  Pot thiocyanide (KSCN, 3N)20
 G.S. measuring cylinders (25-ml cap)  Saturated pot persulfate (K2S2O8)21
 Pipettes, flasks, etc  Standard iron solution (0.1mg/ml)22

PROCEDURE

1. Prepare ash solution as described for calcium determination

2. Make up the volume with distilled water to 100ml
3. Into three separate stoppered measuring cylinders, pipette the solution as
given below:

Tube
Reagents Blank Standard Sample
Standard iron (0.1mg/ml) (ml) 0.0 1.0 0.0
Sample ash solution (ml) 0.0 0.0 5.0
Distilled water (ml) 15.0 14.0 10.0
Conc H2SO4 (ml) 0.5 0.5 0.5
Saturated K2S2O8 (ml) 1.0 1.0 1.0
3N KSCN (ml) 2.0 2.0 2.0

4. Mix the tubes well

5. Measure absorbance at 480nm setting the blank at 100% transmittance

CALCULATION

Absorbance of mg iron/ml of Total volume of

Iron (mg/100 g, sample × standard solution × ash solution (ml) × 100×100
dry basis) = Absorbance of Aliquot of ash Wt. of sample
standard × solution (ml) × taken for ashing (g) × Dry matter (%)

5.4. POTASSIUM BY FLAME PHOTOMETRY (EMISSION)

PRINCIPLE

20
Dissolve 146g of reagent grade KSCN in water and dilute to 500ml. Filter, if needed. Add 20ml pure
acetone to improve the keeping quality
21
Shake 7-8g of reagent grade iron-free pot persulfate with 100ml of water in a glass-stoppered flask.
The undissolved excess settles to the bottom and compensates for loss by decomposition. Shake briefly
before using. Keep the reagent in the refrigerator
22
Dissolve 0.702g of reagent grade crystalline FeSO4(NH4)2SO4.6H2O in 100ml of water. Add 5ml of
conc H2SO4, warm slightly, and add conc KMnO4 solution drop by drop until one drop produces a
permanent color. Transfer to a 1-liter volumetric flask, rinse with water and make up the volume. This
solution contains 0.1mg of ferric iron/ml and is stable indefinitely

74
 FOOD ANALYSIS

Potassium solution is atomized in an oxy-hydrogen or oxyacetylene flame. The flame

excites atoms of potassium causing them to emit radiations at specific wavelengths.
The amount of radiation emitted is measured by the emission flame photometer
(768nm). Under standard conditions, the amount of emission is proportional to the
concentration of potassium in the sample solution.

REQUIREMENTS

 Flame photometer  Potassium standard23

 Pipettes, tubes, beakers, etc  Con HCl
 Ash solution  Distilled water

PROCEDURE

1. Prepare ash solution as in the determination of iron or calcium

2. Collect the filtrate quantitatively and make up the volume to 1 liter
3. Into measuring cylinders, pipette the solutions as given below:

Blank Standard (ppm) Sample replicate

Reagents and sample
25 50 75 100 125 R1 R2 R3 R4
Standard, 1000ppm, (ml) 0.00 1.25 2.50 3.75 5.00 6.25 0.00 0.00 0.00 0.00
conc HCl (ml) 1.25 1.25 1.25 1.25 1.25 1.25 1.00 0.75 0.50 0.25
Ash solution from step 2 (ml) 0.00 0.00 0.00 0.00 0.00 0.00 10.00 20.00 30.00 40.00
Distilled water (ml) To make a total of 50 ml
Note: The volume of HCl has been calculated supposing that 25ml of it was used in the preparation
of ash solution.

4. Operate the flame photometer as per manual. The general outline of any
emission-type flame photometer is: maintain air/fuel ratio and pressure; ignite
the flame and wait for some time to warm up everything, rinse the sample
injection tube by sucking in distilled water and passing to the flame; etc.
5. Select the filter for potassium (you will find it attached) and calibrate the
photometer using blank and standards according to the operation manual of the
given instrument. Rinse with distilled water each time a new standard is used
6. Test the sample solutions, starting from the lowest concentration (highest
dilution, that is) and take the reading. Rinse the sample injection tube with
distilled water each time a new concentration is used
7. Carry out calculation according to the make of the instrument. If the
instrument is provided with a direct read-out meter, you will only need to
take the reading and calculate the concentration by including the fold of
dilution you have made in the original sample. In case only transmittance or
absorbance is available you will need to prepare a standard curve. Either way,
you will have to report the result in terms of dry basis

23
Dissolve 1.909g of analytical grade KCl in distilled water to make 1 liter (1,000 ppm of potassium)

75
 ULTIMATE ANALYSIS

Determination of sodium utilizes the similar technique. AR-grade NaCl is used for
preparing the standard.

5.5. DETERMINATION OF L-ASCORBIC ACID (VITAMIN C)

BACKGROUND

L-ascorbic acid is a water-soluble vitamin. It is present in nearly all fruits and vegetables.
It is synthesized by all higher plants. Certain molds are known to synthesize this vitamin.
Among animals, guinea pig, primates, and man are unable to synthesize this vitamin. In
humans, this inability is due to the lack of L-gulono oxidase, an enzyme needed for the
synthesis of this vitamin. The biosynthetic pathway (simplified) is given in Fig. V-1. The
amounts of vitamin C in some common fruits and vegetables (in mg/100ml juice) are:
amala, 600; lemon, 39; orange juice, 64; tomato, 29; and cabbage, 55.

D-glucose D-glucuronic acid L-gulonic acid L-gulonolactone

L-gulono oxidase

2-keto-L-gulonolactone

L-ascorbic acid

Fig. V-1: Biosynthetic pathway of ascorbic acid

Vitamin C has many biochemical functions. It takes part in tissue collagen formation,
tyrosine metabolism, electron transport in microsomal fraction, anti-aging, and folic
acid activation. Deficiency of ascorbic acid leads to scurvy, degenerative changes in
the cartilages and bone matrices, and defective formation of collagen fibers of
connective tissue. The recommended daily intake is about 40-60mg/day.

Chemically, ascorbic acid is a sugar acid, a -lactone of hexonic acid (see Fig. V-2).
The acid is a powerful reducing agent, giving up two hydrogen atoms to become
oxidized to L-dehydroascorbic acid. It is readily oxidized in the presence of metal
ions like Cu++, Fe++, Sn++, etc. The oxidation of ascorbic acid in body is reversible
(Fig. V-2): the presence of –SH group in cysteine, glutathione, etc., is responsible for
this reversible oxidation. Hydrolysis of dehydroascorbic acid, however, is an
irreversible reaction, leading to the formation of 2,3-diketo-L-gulonic acid.

Vitamin C can be determined by two main methods: (i) titrimetric, and (ii) colorimetric.
The most satisfactory titrimetric methods involve (a) reduction of 2,6-dichlorophenol
indophenol dye by ascorbic acid, and (b) reduction of dehydroascorbic acid with 2,4-
dinitrophenyl hydrazine.

5.5.1. THE 2,6-DICHLOROPHENOL INDOPHENOL TITRATION METHOD

76
 FOOD ANALYSIS

The 2,6-dichlorophenol indophenol visual titration method is the most widely used
routine method. This method can also be used in conjunction with a colorimeter. The
titrimetric method is of value for analyzing samples containing relatively high amounts
of ascorbic acid. As such, the dye is not absolutely specific as it is easily reduced also
by oxidizing impurities such as Fe++, Cu++, Sn++, etc., present in the sample.

CH2OH -2H CH2OH

HOCH O
oxidation HOCH O
O O

OH OH reduction O O
L-ascorbic acid +2H L-dehydroascorbic acid

Fig. V-2: Reversible oxidation of L-ascorbic acid

Ascorbic acid is unstable at neutral or alkaline pH. Its vitamin property is destroyed
by exposure to air or oxygen, light, and heat. It is stable at low pH. During analysis,
the sample as well as the standard is therefore prepared in acidic solution to give a pH
of 1-3.5. The commonly used acids are metaphosphoric acid (HPO3), oxalic acid, or
in some cases, acetic acid. The low pH not only stabilizes the vitamin but also makes
the reaction of dye and ascorbic acid more specific. Additionally, HPO3 and oxalic
acid have the ability to chelate the interfering metal ions.

PRINCIPLE

2,6-dichlorophenol indophenol, which is blue in alkaline solution, is reduced by

ascorbic acid to colorless form. The reaction is quantitative and practically specific
for ascorbic acid in solutions in the pH range 1 to 3.5. At this pH range the interfering
substances react more slowly, and a quantitative relation can be obtained (Fig. V-3).

CH2OH CH2OH
HOCH O HOCH O
O O

OH OH O O
L-ascorbic acid L-dehydroascorbic acid
Cl Cl
HO N O HO N OH
Cl Cl H
2,6-dichlorophenol indophenol (blue) Leuco dye (colorless)

Fig. V-3: Color reaction of ascorbic acid with dye

REQUIREMENTS

77
 ULTIMATE ANALYSIS

 Dye solution 24  Metaphosphoric acid (3%, aqueous) 25

 Ascorbic acid standard 26
 Sodium carbonate
 Fruit or vegetable sample
 Pipette, 5ml and 10ml cap
 Funnel
 Electronic balance, 0.1mg precision
 Sample
 Grinder or mortar and pestle
 Burette, 25ml cap
 Volumetric flask, 100ml cap
 Muslin cloth, centrifuge or cotton wad

PROCEDURE

1. Standardize the dye:

 Take 5.0ml of standard ascorbic acid solution and add to it 5ml of 3%
HPO3
 Fill a microburette (or a graduated pipette) with dye
 Titrate the dye solution to pink color, which should persist for 15 sec
 Determine the dye factor using the formula:
mg of ascorbic acid
Dye factor 
ml of dye
2. Prepare and test the sample:
 Take 10-20 g (or 10-20ml) of sample, the quantity being adjusted
according to the expected ascorbic acid content of the sample
 Grind well with 3% HPO3 and make the final volume to 100ml with the
same acid
 Mix well and centrifuge or filter to get a clear solution.
 Take 2-10ml of extract and titrate with the dye to pink end point. The color
must persist for at least 15 sec. Adjust the aliquot so that the titer is between
3 and 5ml
 Carry out titration in triplicate
_____________________________
Note: If samples containing SO2 is to be tested, add to the extract 1ml of 40% formaldehyde
followed by 0.1ml conc HCl. Stand it for 10 min before taking it for titration, OR Add 2ml
acetone to the aliquot before titration. SO2, sulfite or thiosulfate interferes with the test by
reducing the dye. Addition of acetone or formaldehyde eliminates the interference by
forming condensation products, for example, acetone bisulfite.

CALCULATION

24
Dissolve 50mg of sod-salt of 2,6-dichlorophenol indophenol dye in 150ml hot, glass-distilled water
containing 42mg Na2CO3. Cool and dilute with glass-distilled water to 200ml. Store in refrigerator and
standardize each time you use. It is stable for 3-7 days under refrigeration
25
Dissolve completely 30g metaphosphoric acid (HPO3) in glass-distilled water. Don’t store too long, as
it will gradually change to phosphoric acid
26
Weigh accurately 100mg of pure L-ascorbic acid and dissolve in 3% HPO3 solution to make 100ml.
Serially dilute it in 3% HPO3 to give a final concentration of 0.1mg/ml

78
 FOOD ANALYSIS

Titer  Dye factor  Vol. made up  100

mg% ascorbic acid 
ml of aliquot  ml (or gram) of sample taken

PRECAUTIONS

1. Use only glass-distilled water

2. Be careful to dissolve Na2CO3 and the dye completely
3. Use fresh reagents only

5.5.2. THE 2,4-DINITRO PHENYLHYDRAZINE METHOD

This method measures both ascorbic acid (AA) and dehydroascorbic acid (DAA)
contents. Vitamin C containing foods contain both AA and DAA, the proportion
being dependent on the sample type. For example, apple contains above 90% of the
ascorbic acid in DAA form as compared to around 10% in the case of lemon. DAA
has 80-100% of AA activity and can revert to AA under suitable condition.

PRINCIPLE

L-ascorbic acid (AA) in the sample is oxidized to dehydroascorbic acid (DAA) by

2,6-dichlorophenol indophenol. The resultant DAA is reacted with 2,4-
dinitrophenylhydrazine (DNPH) to from osazone crystals which are dissolved in
concentrated sulfuric acid to give an orange-red color solution. The concentration of
the color is measured at 520nm against standard L-ascorbic acid to obtain amount of
total ascorbic acid (TAA) in terms of DAA. A parallel sample without dye treatment
gives the amount of DAA originally present in the sample. The difference of TAA
and DAA therefore gives the L-ascorbic acid content in the sample.

REQUIREMENTS

 Sample: apple  2% Thiourea solution27

 9N Sulfuric acid28  Stock AA solution, 1mg/ml29
 85% Sulfuric acid30  Routine glasswares
 2,4-dinitrophenylhydrazine (DNPH)31  Spectrophotometer
 2,6-dichlorophenol indophenol,  Working AA standard solution,
200g/ml32 100g/ml33

27
Dissolve 1g thiourea in 5% HPO3 and make up to 50ml with water
28
Dilute 25ml conc. H2SO4 to 100ml with water
29
Dissolve 50mg AA in 5% HPO3 solution and make up to 50ml with HPO3
30
Add 177ml of conc H2SO4 (sp. grav. 1.84) in 23ml water with agitation
31
Dissolve 0.5g DNPH in 25ml 9N H2SO4. Store in an amber-colored bottle. Filter before use
32
Dissolve 10mg of the dye in 50ml water. Store in refrigerator and standardize daily
33
Dilute 5ml of stock solution to 50ml with HPO3 solution

79
 ULTIMATE ANALYSIS

 5% Metaphosphoric acid (HPO3)34  Electronic balance: ± 0.1mg

 Cotton wad

PROCEDURE

Prepare sample (see outline in Fig. V-5, you will find sample preparation for lemon also).

1. Split it into quarters and quickly weigh out 10g

2. Mix the weighed amount with a small amount of 5% HPO3 and grind it into
paste
3. Transfer the paste to a volumetric flask and make up to 50ml with 5% HPO3
4. Filter the mixture through a cotton wad and then through fine filter. Use 2ml
of the filtrate for analysis

Prepare standard graph

5. Label six 20-ml test tubes properly as shown below (T denotes tube)
6. Add standard ascorbic acid and other reactants in the sequence:

Tube label
Reactants T1 T2 T3 T4 T5 T6 Remarks
5% HPO3 2.0 1.6 1.2 0.8 0.4 0
Working AA solution, ml 0 0.4 0.8 1.2 1.6 2.0 Mixed
Dye, ml 1.0 1.0 1.0 1.0 1.0 1.0 Mixed
Thiourea, ml 2.0 2.0 2.0 2.0 2.0 2.0 Mixed
DNPH, ml 1.0 1.0 1.0 1.0 1.0 1.0 Mixed, covered,
incubated
Total, ml 6.0 6.0 6.0 6.0 6.0 6.0

7. Cap the tubes with aluminum foil and incubated at 50°C for 70 minutes, with
frequent stirring.
8. Take out the tubes and cool them in ice bath
9. Into each tube in the ice bath, add 4ml of 85% H2SO4 to dissolve the red
osazone crystals
10. Shake the tubes slowly to ensure thorough mixing
11. Take out the tubes and stand for 30 min
12. Read the absorbance is a spectrophotometer at 520nm by setting
transmittance to 100% for Tube 1
13. Note down the observation in tabulated form as follows:

Tube label ml of working g of AA Absorbance at 

34
Dissolve 25g HPO3 in water and dilute to 500ml

80
 FOOD ANALYSIS

standard = 520nm
Tube 1 0 0
Tube 2 0.4 40
Tube 3 0.8 80
Tube 4 1.2 120
Tube 5 1.6 160
Tube 6 2 200

14. Plot the standard graph of absorbance versus ascorbic acid and linearize the
relation as shown in the typical curve (Fig. V-4). The slope can be obtained
by linear regression that has the following formula: y = mx + c (Fig. V-5).

Where, y = absorbance and x = concentration of ascorbic acid

Determination of DAA and TAA in samples

15. Label properly five 20-ml test tubes (similar to those for standard graph)
16. Prepare assay mixture as follows:

Tube label
Reactants Blank Apple DAA Apple TAA Remarks
5% HPO3, ml 2.0 1.0 1.0
Sample, ml 0.0 2.0 2.0 Mixed
Dye, ml 1.0 0.0 1.0 Mixed
Thiourea, ml 2.0 2.0 2.0 Mixed
DNPH, ml 1.0 1.0 1.0 Mixed, covered,
Total, ml 6.0 6.0 6.0 incubated

17. Follow steps used for preparing standard graph, e.g., incubation, cooling in
ice bath, addition of H2SO4, etc.
18. Record the absorbance of the samples at 520nm and calculate the ascorbic
acid concentration using the linearized equation obtained from the standard
graph as follows

Sample Apple
Tube Blank DAA TAA
Absorbance, 520nm 0  
AA, g*/2ml aliquot 0  
AA, mg/100g (ml) sample** 0  

* AA = ascorbic acid (DAA or TAA); values calculated using trendline equation, y = mx +c

** Values calculated taking into account the dilution factor. For apple and lemon juice, each 2ml filtrate
aliquot represents 0.4g and 0.2ml of original sample respectively

19. Present the data as follows:

81
 ULTIMATE ANALYSIS

Sample, Species  DAA TAA AA

Apple, mg/100g   

Note that AA = TAA – DAA

n  xy    x   y    x    y     x   xy 
2

m and c 
n x 2    x  n x2    x 
2 2

0.6

0.5 *
*
*
0.4
*
0.3
*
*
0.2 Trendline: y = mx + c
*
*
0.1 *
*
0
0 20 40 60 80 100 120 140 160 180 200
L-ascorbic acid, g

Fig. V-4: Plot of Absorbance versus L-ascorbic acid for standard curve

apple lemon

splitting into quarters splitting into quarters

10g squeezing
grinding 5% HPO3
5ml juice
paste
50ml solution
50ml solution
coarse filtration
coarse filtration
fine filtration
fine filtration
2ml
2ml

osazone development osazone development

analysis analysis

Fig. V-5: Outline of sample preparation (for lemon and apple)

5.6. DETERMINATION OF REDUCING SUGARS

82
 FOOD ANALYSIS

BACKGROUND

Reducing sugars are an important group of carbohydrates. Their ability to act as a

reducing agent is due to free or potentially free aldehyde or ketone functions present
in the molecule. The reducing properties of these carbohydrates are usually observed
by their ability to reduce metal hydroxides like Cu(OH)2, Ag(OH)3, and Bi(OH)3 to
CuO, Bi (free), and Ag (free) respectively. The reducing sugar is in turn oxidized,
fragmented, and polymerized. As an example, the aldehyde group of an aldohexose is
readily oxidized to aldonic acid (monocarboxylic) at neutral pH with mild oxidizing
agents. In the presence of strong oxidizing agents such as HNO3, both aldehyde and
primary alcohol function will be oxidized to aldaric acid (dicarboxylic). Under the
influence of enzymes or reductants (e.g., NaBH4) reducing sugars can also yield
respective sugar alcohols (see Fig. V-6). The quantification of higher carbohydrates is
usually carried out by first hydrolyzing them into simple sugars, for example, the
reducing sugars.

mild oxidation
Glucose Gluconic acid
NaBH4
strong oxidation
Sorbitol

Glucaric acid

Fig. V-6: Fate of glucose during oxidation and reduction

All monosaccharides and a few disaccharides show reducing property. Lactose,

maltose, cellobiose, melibiose, etc., are some disaccharides that exhibit reducing
property because of the fact that they have potentially free aldehyde groups. Sucrose
cannot act as reducing sugar because the aldehyde and ketone groups are utilized in
bond formation. Similar is the case with polymeric carbohydrates like starch.

Reducing sugars can be quantitatively determined using a range of physical, chemical

and enzymatic methods. Many of the methods are not used on routine basis, as they
are more sophisticated. Lane and Eynon Method is a chemical (titrimetric) method
that has gained wide acceptance. Another widely used titrimetric method for all starch
and starch hydrolysates is the Luff-Schoorl method. This is an iodometric method in
which excess copper present in the Luff solution is allowed to react with iodine. Of
the instrumental methods, Nelson and Somogyi Method and Dinitrosalicylic acid
(DNS) Method are probably the most suitable when the sugar content of the sample is
very low. Recently, instrumental method that utilizes 3-Methyl-2-benzothiazolinone-
hydrazone (MBTH) to react with the sugar aldehyde has been described.

5.6.1. NELSON SOMOGYI METHOD

83
 ULTIMATE ANALYSIS

PRINCIPLE

The Nelson and Somogyi Method is one of the classical and widely used methods for
the quantitative determination of reducing sugars. The sugar sample is heated with an
alkaline solution of copper tartrate and cuprous oxide is produced, which reacts with
arsenomolybdate to give molybdenum blue. The intense blue color is then measured
in the photoelectric colorimeter. Sodium sulfate is included in the reaction mixture to
minimize the entry of atmospheric oxygen into the solution, which would otherwise
cause reoxidation of cuprous oxide. The blue color obtained is compared with a set of
standards in a photoelectric colorimeter at 620 nm. The calibration curve prepared
depends to some extent on the sugar being estimated, so the method is not suitable for
the determination of a complex mixture of reducing sugars.

If protein is suspected in the sample solution, deproteinization must be done before

the actual test. Deproteinization is usually done with zinc sulfate and barium
hydroxide: the technique gives a clear, protein-free, neutral solution.

REQUIREMENTS

 Arsenomolybdate reagent35  Water bath/Heating arrangement

 Barium hydroxide (0.3N)36  Test tubes/pipettes
 Alkaline copper tartrate37  Sample
 5% Zinc sulfate (in water)  Marbles
 Standard glucose solution (50g/ml)38  Laboratory centrifuge
 Electronic balance: 0.1mg sensitivity  Colorimeter (photoelectric)

PROCEDURE

1. Deproteinize the sample solution. If the sample is solid, dissolve it in water to

make a standard volume to give approximately 5-50g/ml
 Take 1ml sample in test tube, add 15ml water and mix thoroughly
 Add 2ml of Ba(OH)2 solution followed by 2ml of ZnSO4 solution

35
Dissolve 2.5g ammonium molybdate in 45ml water. Add 2.5ml conc H2SO4 and mix well. Then add
0.3g disodium hydrogen arsenate dissolved in 25ml water. Mix well and heat at 55°C for 30 min (or
incubate the solution at 37°C overnight). Use only the clear fraction of the solution for analysis
36
Dissolve 2.5g of Ba(OH)2 in water to make 100ml
37
Alkaline copper tartrate:
Solution A: Dissolve 2.5g anhydrous Na2CO3, 2.5g Na-K-tartrate and 20g anhydrous sod-sulfate in
80ml water and make up to 100ml
Solution B: Dissolve 15g CuSO4.5H2O in a small volume of water. Add 1 drop of conc H2SO4 and
make up the volume to 100ml with water
Mix 4ml of solution B and 96ml of solution A just before use
38
Prepare stock solution of 1% in distilled water. Prepare working standard of 50μg/ml in distilled
water by serial dilution. Don’t use old standards, as it is subject to microbial degradation.

84
 FOOD ANALYSIS

 Shake thoroughly and centrifuge a suitable amount. If centrifugation

is not possible filter the sample through a fine filter paper to collect
about 5ml of filtrate
 Take out 1ml of the supernatant (or filtrate), add 4ml distilled water
and mix thoroughly
 The total dilution will be 100 fold39 (based on the volume of liquid
sample)
2. Take out several clean test tubes (uniform size)and label them properly to
identify sample, blank, and standard
3. Pipette out into the test tubes reagents, standards, and samples (without
contamination, using separate pipettes) as under:

Reagent Blank (ml) Standard (ml) Sample (ml)

Standard (50g/ml) 0 0.2 0.4 0.8 1.6 0.1 0.2
Distilled water 2.0 1.8 1.6 1.2 0.4 1.9 1.8
Alkaline copper tartrate 1.0 1.0 1.0 1.0 1.0 1.0 1.0
Final volume 3.0 3.0 3.0 3.0 3.0 3.0 3.0

4. Mix well
5. Place a glass marble on top of each tube to minimize evaporation
6. Place the tube in boiling water bath for 10 min. Agitate the tubes several
times in between to allow uniform heating and mixing (and therefore uniform
reaction)
7. Cool the tubes to room temperature
8. Add 1ml of arsenomolybdate in each tube
9. Shake well until the gas evolution ceases
10. Add 10ml of water in each tube
11. Shake well and read the absorbance at 620nm after setting 100%
transmittance for blank as follows (T = Tube; Rep = replicate):

Blank Standard Sample

Solution
T1 T2 T3 T4 Rep 1 Rep 2
Sugar content, g 0 10 20 40 80  
Absorbance 0      

12. Construct the standard curve of absorbance against sugar content as shown in
Fig. V-7. Compute the trendline from the slope. See also colorimetric
determination of vitamin C (L-ascorbic acid) for least square linear regression

39
Remember the fold of dilution

85
 ULTIMATE ANALYSIS

0.6

0.5
*
0.4

0.3
Trendline: y = mx + c
0.2 *
Slope = m
0.1 *
*
0
0 10 20 30 40 50 60 70 80 90
Reducing sugar, g

Fig. V-7: Plot of Absorbance vs sugar concentration

CALCULATION

Calculate the reducing sugar content in the aliquot from the trendline equation (see
colorimetric determination of ascorbic acid) and back-calculate the % of reducing
sugar (m/v) as glucose anhydrous.

5.6.2. DINITROSALICYLIC ACID (DNS) METHOD

PRINCIPLE

This is an alternative to Nelson-Somogyi Method. It is simple, sensitive, and

adaptable during handling of large number of samples at a time. Under alkaline
condition, 3,5-dinitrosalicylic acid (DNS) solution is reduced by reducing sugars to 3-
amino-5-nitrosalicylic acid (Fig. V-8). The intensity of the dark-red color formed as a
result of the reaction is measured (against a standard) at 510 nm. The reagent is also
used for the test of amylase activity. The chemistry of reaction is complicated since
the standard curve does not always go through the origin and different sugars yield
different colors. The method is therefore unsuitable for the determination of a
complex mixture of reducing sugars.

COOH COOH
1 1
6
OH reduction 6
OH
2 + Glucose 2 + Gluconic acid
O2N 5 3 NO2 O2N 5 3 NH2
4 4

3,5-dinitrosalicylic acid 3-nitro-5-aminosalicylic acid

(yellow) (orange)

Fig. V-8: Principle of DNS test

86
 FOOD ANALYSIS

REQUIREMENTS

 DNS reagent40  Colorimeter

 Test tubes (uniform shape and size)
 Glass marbles
 Water bath
 Sample solution (prepared as in
Nelson-Somogyi method, sugar
content = 25-200mg/ml)
 NaOH, 2 M
 Pipettes: 0.01-10ml cap.
 Glucose standard (0.1mg/ml)
 Rochelle salt: 40% aqueous
solution of pot-sod-tartrate

PROCEDURE

1. Pipette out 0.5-3.0ml of prepared sample solution in labeled test tubes and
equalize the volume to 3ml with distilled water
2. Add 3ml of DNS reagent to each tube
3. Heat the contents in a boiling water bath for 5 min. Keep the tubes covered
with glass marbles
4. When the contents of the tubes are still warm, add 1ml of 40% Rochelle salt
solution
5. Mix, add 10ml distilled water and mix again
6. Cool and read the absorbance of the dark-red (sometimes orange-red) color at
510nm
7. Run a series of standards using glucose (0-500g) and plot a graph of
absorbance versus reducing sugar content (g)

CALCULATION

Calculate the % of reducing sugar as usual (method described for Nelson-Somogyi

method)

5.6.3. LANE AND EYNON METHOD

This method is based on copper reduction and utilizes copper-containing Fehling’s

solution to oxidize the reducing sugar. It is routinely used for the determination of
reducing sugars in foods. The test is suitable for foods with more than 0.2% reducing
sugar. It is can also used for the determination of sucrose and starch contents after
hydrolyzing them into reducing sugars by chemical or enzymatic means.

40
Dissolve by stirring 1g of DNS, 200mg crystalline phenol and 50mg sod sulfite in 100ml 1% NaOH.
Store at 4C

87
 ULTIMATE ANALYSIS

PRINCIPLE

Reducing sugar reduces the copper in Fehling’s solution to red, insoluble cuprous
oxide. The sugar content in a food is estimated by determining the volume of the
unknown sugar solution required to completely reduce a measured volume of
standardized Fehling’s solution. Normally, the impurities in the sample are
flocculated out by suitable clarifying agents, such as Carrez solution.

REQUIREMENTS

 Fehling’s solution41  Sample

 Methylene blue: 1% (aqueous)
 Carrez solution I42
 Carrez solution II43
 Electronic balance: 1mg sensitivity
 Phenolphthalein indicator
 Dilute NaOH: about 2N
 Filtration arrangement
 Pipette: 1ml cap
 Burette: 50ml cap
 Conical flask: 250ml cap
 Bunsen burner
 Dextrose standard: 2.5mg/ml of
dextrose anhydrous (or monohydrate)

PROCEDURE

Preparation of sample

1. Take 25g or 25ml of sample

2. If the sample is in solid form, solubilize it in adequate amount of water
3. Neutralize with dilute NaOH if the solution is acidic (use phenolphthalein
indicator)
4. Add 10ml of Carrez I and shake for 1 min
5. Add 10ml of Carrez II and shake for 1min
6. Transfer quantitatively to a volumetric flask and make up the volume with
water to give approximately 2.5mg reducing sugar per ml
7. Filter the solution through filter paper to get a clear extract. If the extract is
not clear (which is rare) repeat the clarification with more Carrez solution
8. Collect adequate amount of filtrate for the test (25 to 50ml)

41
Mix Fehling A and Fehling B in equal volumes to give a predetermined volume (i.e., not more than the
volume needed for test). Use only fresh mixture and discard the leftover
Fehling A: Dissolve 69.28g CuSO4.5H2O in water, dilute to 1,000ml and, if necessary, filter
through Whatman filter No. 4
Fehling B: Dissolve 346g of Rochelle salt (potassium sodium tartrate, KNaC4H4O6.4H2O) and
100g NaOH in water and make up to 1,000ml
42
Carrez I: Dissolve 21.9g Zn(CH3COO)2.2H2O and 3g glacial acetic acid to make 100ml with water
43
Carrez II: Dissolve 10.6g pot-ferricyanide, K4[Fe(CN)6].3H2O, in water to make 100ml

88
 FOOD ANALYSIS

Standardization of Fehling’s solution (trial)

9. Mix equal volumes of Fehling A and Fehling B (e.g., 50ml each)

10. Accurately pipette out 10ml of mixed solution into a 250ml conical flask
11. Add 50ml water
12. Take the standard dextrose in a burette
13. Heat the Fehling’s solution to boiling condition (this is necessary to avoid
oxidizing environment in the flask)
14. Rapidly titrate with standard dextrose until the color of Fehling’s solution
almost vanishes
15. Boil again for a minute and add 3 drops of methylene blue. The solution
should turn blue again
16. Titrate again with dextrose solution until the blue color vanishes. You will
also see at this time brick red precipitate
17. Note the titer and take it as trial titer

Standardization of Fehling’s solution (actual)

18. Take 10ml of Fehling’s mixture and add water 50ml water
19. Add standard dextrose in the Fehling’s solution (without heating) to represent
about 80% of the trial titer
20. Boil the mixture for 2 min and add 3 drops of methylene blue indicator
21. Without removing the flask from the burner titrate with standard dextrose to
colorless end point
22. Note the actual titer
23. Calculate the Fehling factor (quantity of reducing sugar in grams represented
by 10ml of Fehling’s solution) as follows:

 True titer × mg reducing sugar/ml 

Fehling Factor =   g per 10ml Fehling
 1000 

Titration of sample filtrate

24. Perform steps 9 to 22 using sample filtrate in place of standard dextrose.

Avoid contamination: use only clean glass wares

CALCULATION

Fehling factor × Dilution ×100

% Reducing sugar =
Aliqout titer ×Sample wt (g) or volume (ml)

Note: In place of Carrez solutions, you can use clarifying agents mentioned in Nelson Somogyi Method
(see 5.6.1). You may use 5ml each of Ba(OH)2 and ZnSO4 for every 10ml of sample.

89
 ULTIMATE ANALYSIS

5.7. DETERMINATION OF STARCH BY HYDROLYSIS

Starch content can be determined by a range of physicochemical and enzymatic

methods. Most of the methods are based on the reducing property of the glucose
(obtained after hydrolysis) and hence the test of reducing sugars can be readily
applied. Some errors are introduced due to increased molecular weight as the glucose
changes from residue form to free form during hydrolysis. This can be corrected by
using suitable conversion factor. For starch, the conversion factor can be derived as
follows:

General formula of starch is n(C6H12O6)  (n1)H2O where n = number of glucose

residues.

Upon hydrolysis, the above starch gives rise to n(C6H12O6) glucose molecules. The ratio
of molecular weights of starch to free glucoses formed due to hydrolysis is given by:

n(C6 H12 O 6 )  (n  1)H 2 O 180n  (n  1)18 162n  18

= =
n(C6 H12 O 6 ) 180n 180n

For n = 100, 500, 1000 and 5000, the ratios are 0.901, 0.9002, 0.9001 and 0.90002
respectively. Thus, the reducing sugar content can be multiplied by 0.9 to back-
calculate the starch content.

5.7.1. LANE AND EYNON METHOD OF STARCH DETERMINATION

PRINCIPLE

After the sugars present in the sample are leached out, starch is hydrolyzed using acid
or enzyme and estimated as glucose with standardized Fehling’s solution.

REQUIREMENTS

 Fehling’s solution A and B: see  Carrez solution I and II: see

reducing sugar determination reducing sugar determination
 Centrifuge  Concentrated HCl
 Ethyl alcohol: 50% and 95% alcohol  Strong NaOH: about 5N
 Alpha naphthol reagent: 10% alcoholic  Phenolphthalein indicator
 Concentrated sulfuric acid  Grinder or mortar-pestle
 Heating arrangement  Volumetric flasks
 Weighing arrangement  Filtration arrangement
 Dextrose standard: see reducing sugar  Conical flask: 250ml and 500ml
determination by Lane and Eynon
method

90
 FOOD ANALYSIS

PROCEDURE

1. Take 10g of sample (e.g., cereals)

2. Grind to very fine powder in mortar-pestle or grinder
3. Add a little water and heat to 60°C
4. Allow to stand for half an hour to obtain a solution of starch
5. Add about 50ml of 95% alcohol and mix well
6. Centrifuge till the precipitate settles at the bottom
7. Filter and wash the residue with 50% alcohol in small batches (20ml each
time) until the filtrate gives no test for sugars
8. Test the presence of sugars using -naphthol: to a few ml of the filtrate in a
small narrow test tube, add 2 drops of -naphthol. Allow 1ml of pure conc
H2SO4 to flow slowly down the side of the test tube so as to form a layer
beneath the aqueous solution. If sugars are present, a red ring will appear
within a few seconds at the junction of the two layers
9. Transfer the residue to 500ml conical flask with about 100ml of water
10. Add 20ml of concentrated HCl and place in a boiling water bath for 2.5 hrs.
Invert a funnel on the neck of the flask to prevent evaporation
11. Cool, and neutralize with NaOH using phenolphthalein indicator
12. Make up the volume to 250ml with water (or some suitable volume)
13. Take 10ml of the extract and follow steps 4 to 24 of Lane and Eynon titration.
You may make up the volume to 250ml after addition of Carrez solutions
14. Multiply the reducing sugar content with 0.9 to obtain starch content. Note
that the conversion factor is based on glucose anhydrous. If glucose
monohydrate has been used for preparing standard solution, the factor will be
0.818.
15. Do not forget the dilution factor you have used

5.8. DETERMINATION OF ACIDITY AND pH OF FOOD

Food materials contain a wide variety of acids, either native or added, but mostly
organic. Citrus foods have citric acid as the dominant acid while milk and milk
products have lactic acid as the dominant acid.

Acidity and pH are very important physicochemical properties of food materials. In a

technological sense, pH has to do with spoilage, stability, processing requirement, and
functionality of the food product while acidity has to do with the sensory aspects
(affects palatability and flavor). Preparation of products ranging anything from jam-
jelly to wine and enzymes all require measurement and manipulation of pH at some
point. From the processing aspect, foods are generally categorized as high-acid and
low-acid foods although the distinguishing characteristic in them is the pH rather than
acidity. High-acid foods exhibit some self-protective effects and therefore do not
require as extensive heat processing as for low-acid foods. In a physiological sense,
acidity and pH can be related to stages of maturity of fruits and vegetables. pH is a
measure of active acidity and is given by negative logarithm of hydrogen ion
concentration as follows:

91
 ULTIMATE ANALYSIS

1
pH = ; Where [H+] = hydrogen ion concentration in mole/ liter
log10  H  +

Increasing acidity decreases the pH but the relationship is not truly linear in the case
of acidity in foods because organic acids show weak dissociation. It is therefore easy
to see that at some point the pH ceases to decrease regardless of further increase in
the acid content. The measurement of pH is carried out by using pH indicators
(available as solution or paper strip) or by electronic pH meters. In very sensitive and
accurately calibrated pH meters, a precision of up to ± 0.01units can be obtained.

Foods with pH values equal to or less than 4.6 are generally called high-acid foods
and those below pH 4.6 low-acid foods, although some writers prefer to further
subcategorize foods into acid foods and medium-acid foods. The pH 4.6 is called the
’magic’ pH because Clostridium botulinum (a very dangerous, anaerobic food-
poisoning bacteria) do not grow at or below this pH. Thus, the knowledge of pH helps
a lot in food processing and food safety also. A useful categorization of foods based
on pH is given in Fig. V-9.

Normal ranges of pH and acidity of some food materials are as follows:

Food pH Acidity
Ripe tomato 3-4 0.4-0.5% as citric acid
Fresh milk 6.6-6.8 0.14-0.18% as lactic acid
Wine 2.8-3.8 0.7-0.8% as tartaric acid
Grape juice 3.0-3.5 0.5-0.15% as tartaric acid

Strongly acidic Strongly basic

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
pH values
* Most fruits * Most vegetables
* Bananas
-Apples -Beans
* Dates
-Berries -Beets
* Melons
-Cherries -Carrots
* Persimmons
-Citrus -Corn
* Papayas
-Peaches -Cucumbers
* Pineapples
-Plums -Onions
* Tomatoes
* Pickled vegetables -Peppers
* Most barbecue sauces -Potatoes
* Yogurt * Meat and poultry
* Vinegar * Milk
* Most cheeses
HIGH-ACID FOODS LOW-ACID FOODS

4.6
(pH value dividing high-acid foods and low-acid foods)

Fig. V-9: Classification of food based on pH

92
 FOOD ANALYSIS

Routine measurement of acidity in foods is done by titrimetric method in which the

food sample is titrated with a standard alkali and expressed as the percentage of
dominant acid, for example, citric acid in the case of fruits and their juices. The acidity
determined in this way is therefore called ‘titrable’ acidity or ‘total’ acidity. The term
‘total’ implies that both ‘volatile’ and ‘fixed’ acidity have been accounted for.

5.8.1. DETERMINATION OF ACIDITY IN (I) TOMATO, (II) MILK

PRINCIPLE

The determination of acidity in milk, tomato, as well as in most foods involves

preparation of aqueous suspension or solution of the food and titration with standard
NaOH using phenolphthalein indicator. The result is expressed as percentage of
dominant acid.

REQUIREMENTS

 Tomato sample  Pipettes (10ml cap)

 Milk sample  Burettes (50ml cap)
 Sodium hydroxide (0.1N)  Volumetric flask (100ml cap)
 Weighing arrangement  Beaker (50ml cap)
 Knife  Conical flask (100ml cap)
 Blender or mortar and pestle  Linen cloth
 Phenolphthalein (1% in 50% alcohol)

PROCEDURE FOR TOMATO SAMPLE

1. Cut tomato and blend it in an electric blender. Alternatively, you can use
mortar and pestle
2. Weigh 10g of the mixture in a beaker by difference
3. Transfer quantitatively to a 100ml-volumetric flask and make up the volume
with water without allowing the sample to froth
4. Mix well by shaking and inverting for a number of times
5. Strain away the residue in a piece of coarse linen and reserve the filtrate
6. Pipette out 10ml filtrate in a conical flask and titrate with 0.1N NaOH using 2
drops of phenolphthalein indicator to a persistent (up to 20 s) pink end point
7. Calculate the acidity in terms of citric acid anhydrous as follows:

Titer  N of NaOH  Vol.* made up (ml)  64  100

Acid, % 
Aliqout (ml)  Wt. or vol. of sample take (ml or g)  1000

Where, 64 = equivalent weight of citric acid anhydrous; * This does not apply if the
sample is directly taken for titration (i.e., without dilution as in the case of prepared
juices).

93
 ULTIMATE ANALYSIS

If the sample is deeply colored and there is difficulty in determining the end point,
add some more neutral distilled water just around the end point and titrate again. Do
not take into account the extra volume of water added.

PROCEDURE FOR MILK

1. Pipette out 10ml of milk in a conical flask

2. Add about 10ml distilled water and mix well
3. Rinse the pipette by drawing in and releasing a small amount of the diluted
milk (from step 2)
4. Titrate with 0.1N NaOH using 2 drops of phenolphthalein indicator to a
persistent pink end point
5. Calculate the acidity in terms of lactic acid anhydrous as follows:

Titer  N of NaOH  90  100

Acid, % 
Aliqout (ml)  1000

Note: If the normality of NaOH is exactly 0.1 N and an aliquot of 10ml has been taken, then
acidity (%) = 0.09 × Titer (ml).

5.8.2. DETERMINATION OF pH OF TOMATO AND MILK

PRINCIPLE

When pH meter is inserted in the food solution or juice an electrical circuit is

established due to protons (H+). The instrument is calibrated to directly reflect the
negative logarithm of hydrogen ion concentration in mole per liter. When pH
indicators are used, the constituent chemicals respond to hydrogen ion concentration
by changing their color that corresponds to certain pH unit.

REQUIREMENTS

 Tomato sample  Beaker

 Milk sample  Distilled water
 Mechanical press or juice extractor  Buffer solution or tablets (for pH
(hand-held type) 4.0 and 7.0)
 pH meter (for more accuracy) or pH
indicator (for rough estimate)

PROCEDURE FOR TOMATO

1. Cut the tomato sample and extract the juice with a press or extractor in a
beaker
2. If you are using a pH meter, read the instructions in the operation manual
carefully. If you are using pH indicators, look for the color guide

94
 FOOD ANALYSIS

3. For the pH meter, bring the temperature of tomato juice to the one mentioned
in the operation manual
4. Calibrate the pH meter at pH 4.0 and 7.0 using standard buffers
5. Rinse the pH meter several times in distilled water to remove buffer
6. Dip the pH meter in the juice and take the reading (digital or read-out meter)
according to the type of pH meter
7. Wash the pH meter several times in distilled water to remove the sample
8. Store the pH meter following the direction given in the operation manual
9. If a pH indicator paper is to be used, dip a small piece of pH paper into the
sample and match the color with the color guide
10. If pH indicator solution is to be used, take about 5ml sample in a 10ml test
tube, add 2 drops of indicator solution and match the color with the color
guide

PROCEDURE FOR MILK

1. Follow the guidelines given in the pH meter

2. Calibrate the pH meter with buffers of pH 4.0 and 7.0
3. Wash the pH meter with distilled water several times to remove buffer
4. Maintain the temperature of the milk
5. Dip the pH meter in the milk and take the reading (digital or read-out meter)
according to the type of pH meter
6. Wash the pH meter several times in distilled water to remove the sample
7. Store the pH meter following the direction given in the operation manual
8. If a pH indicator paper is to be used, dip a small piece of pH paper into the
sample and match the color with the color guide
9. If pH indicator solution is to be used, take about 5ml sample in a 10ml test
tube, add 2 drops of indicator solution and match the color with the color
guide

95
CHAPTER VI: DETERMINATION OF NATURAL PIGMENTS AND
RELATED COMPOUNDS

Natural pigments are a group of substances present in animal and vegetable cells that
impart color due to selective absorption of certain wavelengths of light and reflection
of the rest. Pigments do not account for structural color, which is the result of
reflection or iridescence in some multilayer structures. Some of the natural pigments
include chlorophyll, hemoglobin, anthocyanins carotenoids, and xanthophylls.

6.1. DETERMINATION OF CHLOROPHYLL

BACKGROUND

Chlorophylls are found in virtually all photosynthetic organisms. They occur in several
distinct forms: chlorophylls a and b are the major types found in higher plants and
green algae; chlorophylls c and d are found, often with a, in different algae; chlorophyll
e is a rare type found in some golden algae; and bacterio-chlorophyll occurs in certain
bacteria. In green plants chlorophyll occurs in chloroplasts, approximately in the ratio
3a:1b.

The determination of chlorophylls has great importance in food technology as well as

other disciplines. In food technology, chlorophyll is related to esthetic quality of fruits
and vegetables. It is also extensively used as a parameter in studying maturation and
ripening of fruits and vegetables. In aquatic ecology, chlorophyll determination is
used for biomonitoring.

Chlorophylls are very sensitive pigments. They are readily degraded by oxygen and
acid. Any activity (e.g., grinding) that frees the intracellular chlorophyllase enzyme
also leads to rapid destruction of chlorophylls. This warrants great care in sample
preparation.

The quantitative analysis of chlorophyll is universally carried out by spectro-

photometric method. Depending on the purpose of analysis and the nature of sample,
some variations, particularly with respect to absorbance wavelength and extraction
solvent, are also observed. Different investigators have used wavelengths of 600, 630,
642.5, 645, 663, 660, 665 and 750nm. The solvents commonly used are 80% acetone,
acetone-methanol (90% acetone), or 100% acetone. The centrifugation speed ranges
from 2,500 to 5,000 rpm.
 FOOD ANALYSIS

PRINCIPLE

Chlorophyll is extracted in 80% acetone and the absorption at 663 and 645nm are
read in a spectrophotometer. Using the absorption coefficients, the amount of
chlorophyll is calculated using the empirical formula:

V
Chl a, mg/g tissue = 12.7  A663   2.69  A645  
1000  W

V
Chl b, mg/g tissue = 22.9  A645   4.68  A663  
1000  W

Total chlorophyll, mg/g tissue = Chl a + Chl b (calculated above)

Where A = absorbance at specific wavelengths; V = final volume of chlorophyll

extract; W = fresh weight of tissue extracted

REQUIREMENTS

 Green vegetable sample  Spectrophotometer

 Centrifuge  Weighing arrangement
 Refrigerator  Measuring cylinder, 100ml
 Volumetric flask, 50ml  Beakers
 Knife, mortar-pestle, etc  80% acetone44

PROCEDURE

1. Cut the sample into small pieces (or thin slices)

2. Take 1.0g of sample and grind it to a fine pulp in mortar and pestle with
about 10ml of 80% acetone
3. Centrifuge the pulp at 5000 rpm for 5 min
4. Transfer the green supernatant to a 50-ml volumetric flask
5. Scrap the sediment in the centrifuge tube and grind it again in the same
mortar and pestle with a small amount of 80% acetone to extract the residual
chlorine
6. Centrifuge the mixture as done earlier and pool the extract in the 50-ml
volumetric flask (containing the previous supernatant)
7. Repeat the extraction until no perceptible green color in the residue
8. Add 80% acetone to the pooled extract to make up the volume (50ml, that is)
9. Stand the extract in refrigerator for 10 min to lower the temperature its
temperature

44
Prepared by adding water in pure acetone

97
 PIGMENTS AND RELATED COMPOUNDS

10. Read the absorbance of the extracts in spectrophotometer at 663 and 645 nm
using 80% acetone as the blank

CALCULATION

Calculate chlorophyll a and chlorophyll b in mg/g tissue using the formula given in
the principle.

6.2. DETERMINATION OF CAROTENE BY SOLVENT PARTITION METHOD

Carotene is a generic name for a group of carotenoid compounds and is represented

by -carotene, -carotene, γ-carotene and lycopene. Carotenes are highly unsaturated
hydrocarbons containing isoprene units. These unsaturated isoprene units are
responsible for the characteristic yellow, orange and orange-red color in
photosynthetic plants. Carotenes are precursors of vitamin A.

PRINCIPLE

Pigments (carotenoids and chlorophylls) in the sample are extracted in diacetone

alcohol and the carotenes transferred to petroleum ether (xanthophylls have limited
solubility in ether but are mostly soluble in alcohol). Chlorophylls are saponified with
methanolic KOH, which is removed from the mixture by washing with water. The
amount of carotene in the ether extract is then determined by spectrophotometric
method at 450 nm against -carotene standard. It has also been reported that a
0.036% potassium dichromate solution is equivalent to 0.00206mg carotene per ml.

REQUIREMENTS

 Sample, e.g., ripe mango  Whatman filter paper (rapid filter)

 Methanolic KOH45  Blender or mortar and pestle
 Petroleum ether46  Spectrophotometer
 Anhydrous sodium sulfate  Buchner funnel with vacuum set
 Separating funnel  Measuring cylinder
 Volumetric flask  Beakers
 Weighing arrangement  -carotene standard

PROCEDURE

Extraction

1. Take 5g sample
2. Add 62.5ml of diacetone alcohol
45
Take 2.5g KOH and dissolve in 12.5ml methanol
46
Boiling range = 60-80°C

98
 FOOD ANALYSIS

3. Blend in a blender for 5 min, or use a mortar and pestle for preparing the
paste
4. Filter through Buchner funnel set under water jet vacuum
5. Wash the residue, if required, with diacetone alcohol until washings are
colorless

Separation

6. Transfer the filtrate in a separating funnel and add 25ml of petroleum ether
7. Shake well and allow the phases to separate. The upper phase (petroleum
ether) contains carotenes while the lower phase contains other carotenoids
and some residual carotenes
8. Draw off the lower layer (diacetone alcohol) to a second separating funnel.
To this, add 25ml of petroleum ether (to extract the residual carotenes)
9. Shake well and draw the lower layer to discard
10. Pool the upper layer with the ether extract in the first separating funnel

Purification

11. Add 12.5ml of diacetone alcohol to the combined ether extracts and shake at
least 30 sec
12. Allow the layers to separate and discard the lower layer (diacetone alcohol)
13. Add 12.5ml of methanolic KOH and shake for 1 min
14. Allow phases to separate and discard the lower layer
15. Add 50ml water to the petroleum ether extracts in the separating funnel
16. Shake for 1 min, allow the phases to separate, and discard the lower layer
(water with soap)
17. Add a pinch or two of anhydrous sodium sulfate to the extract and shake well.
This is done to break the emulsion. If the emulsion is not broken, add more
sodium sulfate and shake again
18. Filter the extract through Whatman filter paper and collect the filtrate in a
100-ml volumetric flask
19. Pour some more petroleum ether to complete the washing (the total volume
of the extract should not exceed 100ml)
20. Make up the volume to 100ml with petroleum ether
21. Shake well and measure the absorbance of the sample at 450 nm against -
carotene standard

Standard solution

22. Take 5mg of -carotene

23. Dissolve in 50ml petroleum ether. This is the stock solution
24. Dilute the stock solution (in petroleum ether) as needed for preparing the
standard curve and read in spectrophotometer at 450nm. It is good to adjust
the maximum concentration of the standard and the sample by dilution so that
the absorbances are less than 0.7 in the spectrophotometer

99
 PIGMENTS AND RELATED COMPOUNDS

CALCULATION

Using the value obtained from the curve, calculate the carotene in the sample by using
the formula:

Concentration from the curve  Final vol. (ml)  dilution

 g carotene per gram 
Wt. of sample (g)

6.3. TANNINS

BACKGROUND

Tannins are astringent, bitter-tasting plant polyphenols that bind and precipitate
proteins. The term tannin refers to the source of tannins used in tanning animal hides
into leather; however, the term is applied to any large polyphenolic compound
containing sufficient hydroxyls and other suitable groups (such as carboxyls) to form
strong complexes with proteins and other macromolecules. Tannins have molecular
weights ranging from 500 to over 20,000.

Tannins are usually divided into hydrolyzable tannins and condensed tannins
(proanthocyanidins). At the center of a hydrolyzable tannin molecule, there is a
polyol carbohydrate (usually D-glucose). The hydroxyl groups of the carbohydrate
are partially or totally esterified with phenolic groups such as gallic acid (in
gallotannins) or ellagic acid (in ellagitannins). Hydrolyzable tannins are hydrolyzed
by weak acids or weak bases to produce carbohydrate and phenolic acids. Condensed
tannins, also known as proanthocyanidins, are polymers of 2 to 50 (or more)
flavonoid units that are joined by carbon-carbon bonds, which are not susceptible to
being cleaved by hydrolysis. While hydrolyzable tannins and most condensed tannins
are water soluble, some very large condensed tannins are insoluble.

Tannins may be employed medicinally in antidiarrheal, hemostatic, and

antihemorrhoidal compounds. Also, they produce different colors with ferric chloride
(either blue, blue black, or green to greenish black) according to the type of tannin.
Examples of gallotannins are the esters of tannic acid (C76H52O46) with glucose, found
in the leaves and bark of many plant species.

Estimation of tannins is carried out either by volumetric method or colorimetric

method.

6.3.1. VOLUMETRIC DETERMINATION OF TANNINS

PRINCIPLE

Tannins may be estimated by determining their oxidizability by potassium

permanganate solution. The interference of oxidizable non-tannin materials (that

100
 FOOD ANALYSIS

consume KMnO4) is taken in to account by carrying out a separate titration in which

the sample is treated with gelatin-acid sodium chloride solution. 1ml of 0.1N KMnO4
= 0.0042g of tannin (gallotannic acid).

REQUIREMENTS

 Sample (extract or juice)  Water bath

 Indigo carmine solution47
 Gelatin solutions48
 Acid sodium chloride solution 49
 0.04N KMnO4 (see calcium
determination)
 Heating arrangement
 Filtration unit
 Large porcelain dish
 Titration arrangement
 Filter aid (kaolin or kiesselguhr

PROCEDURE

1. Take 10-20ml of juice (containing about 0.01g tannins) in a porcelain dish. If

the sample is in solid form, boil 5g of sample (crushed) in 400ml of water for
30 min, cool, make up the volume to 500ml with water, shake well, and filter
2. Add 20ml of indigo carmine solution and about 500-750ml of water
3. Add potassium permanganate from a burette, 1ml at a time with vigorous
stirring, until the color becomes light green
4. Add potassium permanganate drop-wise until the color changes to bright
yellow or to a faint pink at the rim
5. Note the volume (ml) of KMnO4 used (say, A ml; this titer is due to total
tannin-like materials)
6. To 50ml of the clear filtered juice in a 250-ml flask, add 25ml gelatin solution
and make up the volume with acid sodium chloride solution
7. Transfer to a conical flask, add a little filter aid, shake for 15 min and filter
8. To 50ml of filtrate (≡ 10ml juice), add 20ml of the indigo carmine solution
9. Add about 500-750ml of water
10. Titrate with KMnO4 solution as before
11. Note the titer (say, B ml; this titer is due to non-tannin materials)

CALCULATION

% Tannin (A  B )  g of tannin per ml of KMnO 4 solution


(as gallotanninc acid) Vol. of sample taken (ml)

47
Dissolve 1.5g of indigo carmine (free from indigo blue) in 1 lit of water containing 50ml of conc
H2SO4
48
Soak 25g gelatin for 1hr in saturated sodium chloride solution, heat until gelatin dissolves, cool and
dilute with saturated NaCl solution to 1 liter
49
To 975ml of saturated NaCl solution, add 25ml of conc H2SO4

101
 PIGMENTS AND RELATED COMPOUNDS

Note that the gram of tannin per ml of KMnO4 solution in the present case is
(0.00424/100) = 0.000168

6.3.2. COLORIMETRIC DETERMINATION TANNINS

PRINCIPLE

Colorimetric estimation of tannins is based on the measurement of the blue color

formed by the reduction of phosphotungstomolybdic acid by tannin-like compounds
in alkaline condition. The measurement is done at 760nm.

REQUIREMENTS

 Folin-Denis reagent 50  Colorimeter

 Tannic acid standard solution51  Volumetric flaks, 100ml
 Saturated sodium carbonate solution52  Heating arrangement
 Filtration unit  Weighing arrangement
 Sample (solid53 or liquid)

PROCEDURE

1. Pipette 0-10ml aliquots of the standard tannic acid solution into 100-ml
volumetric flasks containing 75ml water
2. Pipette the sample (filtrate) containing not more than 0.1mg of tannic acid
into a separate 100-ml flask (use another pipette to avoid contamination)
3. Add 5ml of Folin-Denis reagent and 10ml of Na2CO3 solution into each of
volumetric flasks
4. Make up the volume to 100ml with water
5. Mix well and stand for 30 min
6. Measure the absorbances of the standard tannic acid solutions at 760 nm by
setting zero absorbance for flask containing 0ml of standard tannic acid
7. Measure the absorbance of the sample at 760 nm
8. Plot the graph of absorbance versus mg (not mg/ml) of tannic acid and find
out the amount (in mg) of tannic acid in the sample
9. Back calculate to express as tannic acid in %

50
To 750ml of water, add 100g of sodium tungstate (Na2WO4.2H2O), 20g of phosphomolybdic acid and
50ml of 85% phosphoric acid (H3PO4). Reflux the mixture for 2hr, cool to 25°C and dilute to 1000ml
with water.
51
Dissolve 100mg of tannic acid in 1 liter of water. Prepare fresh solution for each determination (1ml
= 0.1mg of tannic acid).
52
To 100ml of water, add 35g of anhydrous Na2CO3, dissolve at 70-80°C and cool overnight. Decant the
clear liquid before use.
53
If the sample is in solid form, boil 5g of sample (crushed) in 400ml of water for 30 min, cool, make up
the volume to 500ml with water, shake well, and filter

102
CHAPTER VII: FOOD ADDITIVES

In its broadest sense, a food additive is any substance added to food. Legally, the term
refers to “any substance the intended use of which results or may reasonably be
expected to result  directly or indirectly  in its becoming a component or otherwise
affecting the characteristics of any food”. This definition includes any substance used
in the production, processing, treatment, packaging, transportation, or storage of food.
The major categories of food additives are colors, flavors, preservatives, texture
improvers, fat substitutes, antioxidants, and nutritional additives.

7.1. ARTIFICIAL COLORANTS BY THIN LAYER CHROMATOGRAPHY

BACKGROUND

Color plays a major role in determining the appeal of most foods and we often use
color as an index of freshness and wholesomeness. Unfortunately, color may change
during processing, storage, or preparation in ways that detract from the appeal of the
food. Some foods (e.g. cola drinks and gelatins) are colorless unless a colorant is
added while other foods may be made more appealing by enhancing or changing the
natural color. Thus controlling, changing, and/or stabilizing the color of foods is a
major objective for food scientists and technologists.

There are 9 certified synthetic colors that are allowed for coloring foods. These colors
are assigned name and number, such as FD&C Blue No. 1, FD&C Yellow No.5, etc.
FD&C stands for Food, Drug and Cosmetic Act of U.S.A. that regulates colorants for
foods and drugs. Most of these colorants are sodium salts of sulfonic acids.

Food colorants are added to foods at low concentrations. Consequently, it is often

necessary to extract and concentrate the colorants in order to obtain sufficient
amounts for analysis. In many cases, a procedure for separating and concentrating
substances in foods can be developed from a knowledge of the properties imparted by
various functional groups in the molecules of interest.

Synthetic colors are routinely identified by Thin Layer Chromatography (TLC). TLC
is a form of adsorption chromatography. Separation is accomplished by a differential
adsorption of components in the sample on a stationary phase. Samples and standards
are applied at the bottom of a plate coated with silica gel. The plate is then placed
vertically in a chamber containing a small amount of solvent in the bottom, taking
 FOOD ADDITIVES

care that the solvent does not reach up to the sample. As the solvent system moves up
the plate by capillary action and moves through the sample, the components of the
sample which are soluble in the solvent are carried upward with the solvent. Since
some components are adsorbed more strongly than others, they begin to separate.
More polar compounds adsorb more strongly and remain nearer the origin. Less polar
compounds adsorb only weakly and thus spend more time in the moving solvent, thus
migrating faster. When the solvent front reaches the top of the plate, the plate is
removed and examined. By comparing the colors and migration distances of the
samples and standards, it is often possible to definitively identify components present
in the samples.

The positions of compounds on a TLC plate are often described by the Rf value.

d compound
Rf 
dsolvent

Where,

dcompound = distance traveled by the compound,

dsolvent = distance traveled by the solvent front (see Fig VII-1 for clarity)

If identical solvents and stationary phases are used, the Rf value for a particular
compound in that system should remain constant.

PRINCIPLE

All certified synthetic colorants remain ionized at pHs found in most foods. This ionic
character makes them water soluble and provides a means for separating them from
other components in the food.

The colorant is first electrostatically bound to wool utilizing the negative and positive
charges of the dye and the wool protein respectively. The mechanism can be
illustrated as:

Dye moleculeSO3+H3NWool protein.

The dye is later released into an aqueous solution by boiling in alkaline medium (the
electrostatic bond is disrupted by deprotonation of amino groups of wool proteins)
and subjected to thin layer chromatography. The identity of the dye is established by
comparing the Rf (retention factor) values of the sample and the standard.

104
 FOOD ANALYSIS

REQUIREMENTS

 Beakers (50 and 200ml)  25m blunt needle syringes

 Graduated cylinders (10 and 50ml)  Hair dryer
 Boiling beads (glass beads)  Developing tank
 Pipettes  FD&C color 54
 Oven set at 95°C  White knitting wool yarn55
 Hot plate  5N acetic acid
 Precoated silica gel plastic plates56  0.5N ammonium hydroxide
 n-butanol + methylethylketone +  Sample (soft drink)
NH4OH + H2O = 5 + 3 + 1 + 1

PROCEDURE

1. Transfer a 50ml aliquot of the soft drink to a 100-ml beaker and acidify with
1ml of 5N acetic acid
2. Drop a 20cm strip of white knitting wool (purified in advance by the
instructor) into each acidified sample.
3. Add boiling beads and boil the mixtures until the wool has adsorbed as much
color as possible, and cool
4. Wash the wool with cold water and transfer it to a small beaker
5. Add boiling beads and about 10ml of 0.5N ammonia
6. Boil gently until the color is released into solution
7. After the color is released, discard the wool and put the solution in a 95°C
oven until it reaches a state of near dryness. Alternatively, the water can be
evaporated on a hot plate. (If you choose to use the hot plates, use caution.
Hot solution may spatter out of the beaker.) Before spotting, add 1ml
isopropanol.

Separation and identification of the extracted colors

1. Spot 10-20μl of each FD&C colorant and your extracts on silica gel plates.
Spots should be at least 2cm from the bottom of the plate and no more than
0.5cm in diameter
2. Dry the spots by gently heating with a hair dryer
3. Wash the syringe with 5 rinses of 95% ethanol followed by 5 rinses of
distilled water between samples and when finished

54
Prepare stock solutions of FD&C Red No. 3 and 40, Blue No. 1 and 2, Green No. 3, Yellow No. 5 and
6 in water at the rate of 0.1mg/100ml. Dilute the stock 1:10 in methanol
55
Purified in advance by boiling in 0.01N NaOH and then boiling in water
56
Plates should be activated by heating for 4 hrs at 50°C prior to use. However, always chek the
manufacturer’s instruction before using pre-coated plates. When precoated plates are not available
plates can also be prepared in the laboratory as follows: mix 50g silica gel (without binder), 50ml of
0.6% starch solution and 80ml of 1.25% disodium EDTA. Make a slurry and spread on glass plates
(20cm20cm) to give 0.5mm thickness. Allow the plates to air dry and they dry at 120°C for 120 min.

105
 FOOD ADDITIVES

lid

coated plate

syringe for
introducing sample
solvent
sample spot chamber
base line
1 2 3 4 5
1 2 3 4 5
standard colorant solvent

dsolvent movement 4
5
dsample of spot 1

Fig. VII-1: Outline for Thin Layer Chromatography

4. The food colorants can be spotted directly (5 μl) without dilution

5. Use a maximum of 9 spots per plate. When all of the samples and standards
have been spotted on the plate, transfer it to a developing tank containing the
mobile phase [isopropanol/conc. ammonia (4:1, v/v)]
6. Allow the plates to develop until the solvent front is within 2 to 4 centimeters
of the top of the plate.
7. Calculate Rf values for all spots and compare known FD&C standards with
unknowns in food products for tentative identification

7.2. DETERMINATION OF SULFUR DIOXIDE

BACKGROUND

Sulfur dioxide (SO2) is used as a preservative in foods such as raisins, squashes,

wines, pickles, chutneys, jams/jellies/marmalades, etc. SO2 is a reactive molecule and
can disrupt microbial metabolism in a number of ways. As a reducing agent, it can
break disulfide linkages in proteins and interfere with redox processes. It can also
form addition compound with pyrimidine bases in nucleic acids, sugars and a host of
key metabolite intermediates. One disadvantageous consequence or this reactivity is
its ability to destroy the vitamin thiamine in foods. SO2 is reactive against bacteria,
yeasts and molds, although some molds are more resistant. Gram-negative bacteria
are most susceptible to SO2.The levels of SO2 permitted in different foods are as
follows:

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 FOOD ANALYSIS

Item Level (ppm)

Fruit juice and concentrates  350
Soft drinks and RTS (natural)  70
Squash  350
Raisins  2, 000
Fruit chutney and pickles  100
Jam/Jelly/Marmalade  40

SO2 readily dissolves in water to establish a pH-dependent equilibrium:

SO 2(gas)  SO 2  H 2 O  H 2SO3  H   HSO3  2H +  SO32

Sulfurous acid (H2SO3) is a dibasic acid with pKa values of 1.86 and 6.91. The
unionized forms of SO2, which can readily penetrate the cell, have the greatest
antimicrobial activity. It has been reported that they are between 100 and 1,000 times
more active than the bisulfite anion (HSO3). Since the unionized forms predominate
at low pH values, it follows that SO2 is used to best effect in acidic foods. At neutral
pH, SO2 is present as a mixture of the relatively inactive bisulfite and sulfite (SO32–)
ions, although salts of these anions prove the most convenient way of handling the
preservative in the food industry. The most common form of salt used is potassium
metabisulfite (KMS, K2S2O5).

7.2.1. DETERMINATION OF SO2 IN FOODS

SO2 added to foods as preservative may exist as undissociated sulfurous acid H2SO3,
as free bisulfite HSO3¯, as free SO32–, and/or as combined SO2 in the form of hydroxy
sulfonate. The available methods of determination are designed to measure the free
and total SO2. Free SO2 is estimated by direct titration with iodine solution. In the
estimation of total SO2, the combined SO2 is liberated by:

1. Treatment with excess alkali at room temperature, subsequent acidification to

prevent recombination, and titration with iodine, or
2. By distillation from acid solution and titration with standard NaOH

THE DISTILLATION METHOD FOR TOTAL SO2

This method is relatively involved in that it requires special apparatus and

considerable care in operation. In principle, the sample is acidified and the evolved
SO2 is swept with CO2 or nitrogen into cold H2O2, where the sulfurous acid is
oxidized to H2SO4. The latter is determined by titration with standard NaOH.

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 FOOD ADDITIVES

FREE AND TOTAL SO2 BY MODIFIED RIPPER TITRATION METHOD

PRINCIPLE

This is a relatively simple and fast method for the determination of total and free SO2
in liquid samples. The method utilizes the ability of SO2 and its species to reduce
iodine:

SO2 + I2 + 2H2O = 4H+ + SO42– + 2I ‾

SO32– + I2 + H2O = 2H+ + SO42– + 2I ‾

HSO3– + I2 + H2O = 3H+ + SO42– + 2I ‾

The color of iodine vanishes as long as SO2 (or its species) or similar reducing
agent is present. After reduction has completed, the excess iodine reacts with starch
indicator to give a blue colored end point.

Determination of free SO2

For the determination of free SO2, the sample is acidified with H2SO4 to reduce the
rate of dissociation of bound SO2 (H2SO4 supplies excess protons) and make the
process selective for free SO2 only. Oxygen is expelled from the solution (to maintain
a reducing environment) by adding Na2CO3 (to generate CO2) and titrated rapidly
with standard iodine in the presence of starch indicator.

Allowance has to be made for reducing substances (other than SO2) that produce a
similar effect on iodine. This can be done by subtracting the titer value due alone to
non-SO2 reducing substances. In practice, the same amount of sample is taken and
treated with a small amount of formalin (40% formaldehyde) to bind SO2 selectively
and titration carried out as usual. The difference in the titer values of the ‘sample’ and
the ‘blank’ can now be attributed to free SO2. The calculation is done using following
relation:

1ml of 0.02N iodine solution = 0.64mg SO2

Determination of total SO2

For the determination of total SO2, the sample is first treated with NaOH solution to
liberate the bound SO2, HCl added in slight excess, and titrated with iodine as for free
SO2. Reducing substances other than SO2 has to taken into account here also. To the
aliquot that has been treated with NaOH and HCl as described in the preceding
paragraph, formalin is added and titration carried out as usual. The difference in the
‘sample’ and the ‘blank’ titer can be attributed to total SO2. The difference of total
and free SO2 gives the amount of bound SO2.

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 FOOD ANALYSIS

REQUIREMENTS

 Sample (squash)  Dilute H2SO457

 Na2CO3  Standard iodine (0.02N)58
 Formalin (40% formaldehyde)  Dilute HCl (≈ 5N)
 Dilute NaOH (≈ 5N)  Titration arrangement
 Starch indicator (1% aqueous)

PROCEDURE (FOR FREE SO2)

1. Take 50ml sample (dilute with water, if needed)

2. Add 5ml dilute H2SO4
3. Expel air by adding 0.5g Na2CO3 to the sample
4. Mix by gentle swirling (do not beat in air)
5. Titrate rapidly with 0.02N iodine solution using starch indicator (to a blue-
colored end point). Let the 1st titer be A ml
6. Take another flask and carry out steps 1  4
7. Add to the mixture 10ml of formalin, mix, and stand for 10 min
8. Titrate rapidly with 0.02N iodine solution as usual to a permanent blue color.
Let the 2nd titer be B ml

CALCULATION

(First titer  Second titer)ml  0.64  1000

Free SO 2 (ppm) 
Wt. of sample (g)
(A  B)  0.64  1000
Or, Free SO 2 (ppm) 
Wt. of sample (g)

If the strength of iodine is different, say N, multiply the above expression by N/0.02

PROCEDURE (FOR TOTAL SO2)

1. Take 50ml aliquot each in two 250-ml flasks

2. Add 5ml of 5N NaOH in both of them and mix gently (without beating in air)
3. Allow to stand for 20 min
4. Into both the samples, add 7ml of 5N HCl and stir immediately to avoid local
concentration
5. Add 1ml starch indicator and titrate immediately with 0.02N iodine solution
to a definite dark blue color. Let the 1st titer value be C ml

57
Three parts water and one part conc H2SO4
58
Dissolve 2.7g of pure resublimed iodine in a solution containing 4.8g of KI in 200ml water and dilute
to 1 liter. Standardize the solution by titrating with standard sod thiosulfate using starch indicator. The
reagent is not very stable it should be restandardized before using it on another date.

109
 FOOD ADDITIVES

6. To another sample in the flask, add 10ml formalin, stir and stand for 10 min
7. Add 1ml starch and titrate rapidly with iodine solution to a dark blue end
point. Let the 2nd titer be D ml

CALCULATION

(First titer  Second titer)ml  0.64  1000

Total SO 2 (ppm) 
Wt. of sample (g)
(C  D)  0.64  1000
Or, Total SO 2 (ppm) 
Wt. of sample (g)

If the strength of iodine is different, say N, multiply the above expression by N/0.02

Combined SO2 = Total SO2 – Free SO2

PRECAUTION

In the determination of total SO2, the titration should be very rapid. Slow titration will
result in the reaction of a small part of the sulfite due to decomposition of sulfite-
formaldehyde complex. A small portion of the SO2 released from the combined form
may also revert to its original form, thereby producing a low titer.

7.2.2. DETERMINATION OF BENZOIC ACID/SODIUM BENZOATE IN FOOD

INTRODUCTION

Benzoic acid as a preservative acts principally in the undissociated form. Since it is a

relatively strong acid (pKa = 4.19) it is effective only in acid foods. As a consequence,
its practical use is to inhibit the growth of spoilage yeasts and molds. Activity against
bacteria has been reported but the organisms show greater variability in their
sensitivity.

Inhibition by benzoic acid appears multifactorial. The ability of the undissociated

molecule to interfere with membrane energetics and function appears to be of prime
importance since growth inhibition has be shown to parallel closely the inhibition of
amino acid uptake in the whole cells and membrane vesicles. Some inhibition may
also result from benzoic acid once it is inside the cell as a number of key enzyme
activities have also been shown to be adversely affected.

Benzoic acid or its salt, viz., Na-benzoate, is used in a number of foods as

preservative, for example: in canned fruit juice and concentrates (≤600 ppm); tomato
puree and paste (≤250 ppm); tomato ketchup (≤750 ppm); soy sauce (≤750 ppm);
jam, jelly and marmalade (≤200 ppm); squash and fruit syrup (≤600 ppm); fruit
chutney and pickles in brine (≤250 ppm); and soft drinks and RTS, natural ≤150 ppm.

110
 FOOD ANALYSIS

Sample preparation for the determination of benzoic acid

Benzoic acid in food is first converted to water-soluble sodium benzoate and back
again to benzoic acid for the titration. In general, the sample is mixed thoroughly and
ground. It is neutralized with NaOH in the presence of excess NaCl. The sample is
stood at least for 2 hrs with intermittent shaking to mix up the contents. The whole is
then filtered through Whatman no. 4.

Manipulation may be essential according to the type of product tested. If fat is

suspected in the sample, additional dose of NaOH is given to the filtrate before
proceeding for the determination. If alcohol or vinegar is present, the filtrate needs to
be evaporated to less than half the original volume.

PRINCIPLE

In NaCl solution of sample, the benzoic acid present is converted into water-soluble
sodium benzoate by the addition of NaOH. When the sodium benzoate solution is
acidified with excess HCl, water-insoluble benzoic acid is formed which is extracted
with chloroform. The chloroform is removed by evaporation and the residue
containing benzoic acid is dissolved in alcohol and then titrated with standard NaOH.

REQUIREMENTS

 Sample (at least 500g)  10% NaOH

 0.05N NaOH
 Dilute HCl59
 Water bath
 Whatman no. 4 filter paper
 Neutral alcohol60
 NaCl powder and saturated solution
 Chloroform
 Titration arrangement
 Separating funnels, 500ml cap

PROCEDURE

1. Prepare sample as follows:

 Ketchup: To 100g of sample, add 15g NaCl and transfer to 500-ml volumetric
flask rinsing with ~ 150ml of saturated salt solution. Add NaOH to make the
mixture alkaline to litmus paper and make up the volume with saturated salt
solution. Allow to stand for at least 2 hours (with frequent shaking) and filter
through Whatman filter
 Jams/jellies/marmalades and preserves: Mix 100-150g of sample with
~300ml of saturated NaCl solution. Add 15g NaCl and make alkaline (with
NaOH) to litmus paper. Transfer to a 500-ml volumetric flask and dilute to

59
One part conc HCl and 3 parts water
60
Neutralize the alcohol (in the presence of phenolphthalein indicator) with 0.1N NaOH to faint pink

111
 FOOD ADDITIVES

mark with saturated salt solution. Allow to stand for at least 2 hours, shaking
frequently, centrifuge (if necessary) and filter
 Cider and alcohol containing products: Make 250ml of the sample alkaline to
litmus paper with 10% NaOH solution and evaporate on steam bath to
~100ml. Transfer the sample to a 250-ml volumetric flask, add 30g NaCl, and
shake until dissolved. Make up the volume with saturated salt solution, allow
to stand for at least 2 hours, shaking frequently, and filter

2. Pipette out 100ml of the filtrate into a 500-ml separating funnel

3. Neutralize to litmus with dilute HCl and add 5ml HCl in excess
4. Extract with chloroform using successive portions of 70, 50, 40, and 30ml.
Avoid formation of emulsion
5. Pool the extract in a flask
6. Recover most of the chloroform by distillation
7. Evaporate the remaining chloroform in a water bath until only a few drops
remain
8. Leave the residue in a desiccator overnight for drying
9. Dissolve the residue in 50ml of neutral alcohol and titrate with 0.05N NaOH
using phenolphthalein indicator

Following relation exists between NaOH and sodium benzoate:

1ml of 0.05N NaOH = 0.0072g of anhydrous sodium benzoate

CALCULATION

Titer (ml)  N of NaOH  144  V (ml)  1000

Anhydrous Na-benzoate61, ppm =
Aliquot (ml)  Wt. of sample (g)

Where, V = volume made up

61
To estimate as benzoic acid, use 122 in place of 144 in the above expression

112
CHAPTER VIII: GENERAL TEST OF ALCOHOLIC BEVERAGES

Alcoholic beverages such as distilled products, wines and beers are generally
analyzed for alcohol content, acidity, pH, color, methanol, esters and higher alcohols.
Determination of ethanol content and methanol content will be described here.
Ethanol content can be determined by different methods (physical and chemical). For
routine testing, alcohol is determined by:

1. Specific gravity bottle or hydrometers

2. Alcoholmeter
3. Zeiss immersion refractometer.

8.1. ETHANOL CONTENT BY SPECIFIC GRAVITY METHOD

PRINCIPLE

The alcohol content of the distillate is inversely proportional to the specific gravity in
the range 0.7981 (for 100% alcohol) and 0.9992 (for 0% alcohol) at 15.56°C. The
specific gravity can be determined using hydrometer or specific gravity bottle and the
value read against standard density-alcohol chart. This chart also contains values for a
range of temperatures likely to be encountered in the laboratory.

REQUIREMENTS

 Sample (500ml), alcohol content > 2%  ~ 1N NaOH

 Distillation unit, 500ml cap  Heating mantle
 Specific gravity bottle, 50ml cap  Weighing arrangement, ±1mg
 Distilled water  Thermometer
 Volumetric flask, 100ml  Boiling beads
 Phenolphthalein indicator  Measuring cylinder, 200ml

PROCEDURE

Preparation of specific gravity bottle (Fig. VIII-1)

1. Clean the bottle and the stopper thoroughly with detergent and then with
cleaning agent (H2SO4-K2Cr2O7 mixture, care!)
 ALCOHOLIC BEVERAGES

2. Wash thoroughly again with distilled water

3. Dry the bottle (keep it horizontal to prevent condensation of moisture from
the neck) in hot air oven at ~ 120°C for 1hr
4. Take out the bottle and allow it to cool sufficiently. If vapor condensate are is
observed, dry the bottle again (until completely dry)
5. Weigh the dry bottle and note it down (say, We)

Preparation of sample

6. Measure out 150ml of sample in a volumetric flask and transfer it to

distillation flask
7. Add few drops of phenolphthalein and neutralize the sample with ~ 1N NaOH
8. Add some boiling beads
9. Start the distillation unit (Fig. VIII-1), controlling the temperature to avoid
vigorous boiling. Do not forget to run the condenser
10. Collect nearly 100ml of the distillate in a conical flask. You can easily
determine the completion of the distillation by looking at the condenser.
Characteristic water vapor condensates appear on walls of the condenser
when the alcohol has completely distilled off
11. Dismantle the distillation unit
12. Rinse the internal of the condenser with 1-2ml of distilled water (use wash
bottle) and pool the washings to the distillate
13. Cool the distillate to room temperature (say, T°C)
14. Transfer the distillate to 100ml volumetric flask. Complete the transfer by
rinsing the original flask (that contained the distillate) with distilled water
15. Make up the volume to 100ml and mix well
16. Slowly and gently fill the specific gravity bottle with the distillate (without
spilling), slightly above the base of the neck. Gently insert the capillary
stopper to ensure that the capillary is filled to the top. If the alcohol
overflows, quickly and completely soak it with a blotting paper. If the
capillary is not filled, take out the stopper and add few more drops of
distillate in the bottle and repeat the insertion of stopper until the capillary is
completely filled
17. Wipe the entire surface of the bottle and the stopper with dry filter paper (do
not hold in the hand for long, as this will cause temperature gradient)
18. Note the weight of the bottle + distillate (say, Wd)
19. Empty the bottle back to the flask
20. Empty the capillary by blowing out the contents
21. Rinse the inside of the bottle repeatedly (~ 5 times) with 10-20ml portions of
distilled water
22. Fill the bottle with distilled water (as previously done)
23. Insert the stopper (as previously done)
24. Dry the surface of the bottle (as previously done)
25. Note down the weight of bottle + distilled water (say, Ww)

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 FOOD ANALYSIS

plug
capillary

50cc

specific gravity bottle

Water out

Condenser

Alcoholic
broth
Water in

Heat
Receiver
Distillation assembly

Fig. VIII-1: Apparatus needed for alcohol determination

26. Calculate the specific gravity from the relation:

Wt. of a given vol. of distillate at TC Wd  We

Sp. gravity = =
Wt. of the sample vol. of distilled water at T C Ww  We

27. Look up the standard table to find out the corresponding alcohol content. A
full version of the table for 30°C is given in the Appendix.

8.2. DETERMINATION OF METHANOL BY COLORIMETRIC METHOD

Minute amounts of methanol are naturally present in spirits. Significant quantities are
found especially in low-grade brandies. Methanol is toxic to the consumer and as
little as 30ml can cause death. The toxic effect is due to the oxidation by cellular
alcohol dehydrogenase and consequent formation of formaldehyde (which is a toxic
protein denaturant). During fermentation, methanol is formed by the de-esterification
of methoxylated pectin present in the fermentation medium. The enzyme responsible
for this hydrolysis is called pectin methyl esterase and is produced by contaminants,
particularly molds.

115
 ALCOHOLIC BEVERAGES

PRINCIPLE

Methanol is separated from most of the ethanol by fractional distillation and oxidized
by acid permanganate to formaldehyde, which reacts with chromotropic acid to form
a red color. The concentration of methanol is determined colorimetrically by
measuring the absorbance of the red color at 575 nm against known standard.

REQUIREMENTS

 KMnO4 solution62  Weighing arrangement

 Sodium salt of chromotropic acid63  Pipette, 5ml (graduated)
 Sample distillate  Measuring cylinder, 25ml
 Methanol anhydrous  Ice water bath
 Absolute alcohol  Hot water bath
 Volumetric flask, 100ml  Conc sulfuric acid
 NaHSO3  Colorimeter

PROCEDURE

1. Prepare sample as follows:

Dilute or adjust sample (distillate) to total alcohol concentration of 5-6%. If
the sample is in the undistilled form, distil 50ml sample in ordinary
distillation unit and collect 40ml of distillate. Dilute to 50ml with water.
Adjust the alcohol content to 5-6% by adding water and/or ethanol and note
the volume formed.
2. Prepare methanol standard: Make 0.025% methanol solution in 5.5% ethanol.
3. Prepare reagent blank: Dilute ethanol with water to give 5.5% solution
4. Pipette 2ml KMnO4 solution into three 50-ml volumetric flasks
5. Label the flasks ‘blank’, ‘sample’, and standard
6. Chill the flasks in ice bath
7. Add 1ml of chilled sample (prepared as above) to the ‘sample’ flask
8. Add 1ml of methanol standard (prepared as above) to the ‘standard’ flask
9. Add 1ml of blank solution (prepared as above) to the ‘blank’ flask
10. Stand all the three flasks in ice bath for 30 min
11. Decolorize the contents of the flasks with little dry NHSO3
12. Add 1ml of chromotropic acid solution in each of the flasks
13. Add 15ml of H2SO4 slowly with swirling (in each of the flasks)
14. Place the flasks in hot (60-75°C) bath for 15 min
15. Cool, make up the volume (50ml) with water, mix well, and allow it to come
to room temperature
16. Read absorbance at 575nm using blank first

62
Dissolve 3.0g of KMnO4 and 15.0ml H3PO4 in 100ml water. Prepare monthly.
63
5% aqueous solution of sodium 1,8-dihydroxynaphthalene-3,6-disulfonate. Filter if not clear. Prepare
weekly. Either acid or salt may be used.

116
 FOOD ANALYSIS

CALCULATION

 Absorbance of sample 
Methanol content, % =    0.025  F
 Absorbance of standard 

Where, F = dilution factor of sample

Example:

Sample was diluted 25 times; absorbance of sample = 0.421; Absorbance of standard

methanol = 0.368. Then, methanol = (0.421/0.368)0.02525 = 0.715%.

 If the color of the sample is too intense (absorbance greater than 1.0), dilute
with H2SO4-alcohol blank (as prepared above). Not more than 3-fold dilution
is permitted, as the ratio of chromotropic acid to formaldehyde is too low if
the dilution is greater.
 If the absorbance of the blank is greater than 0.05, purify the reagents as
follows:

Dissolve 10g of chromotropic acid or its salt in 25ml water (Add 2ml H2SO4
to aqueous solution of salt to convert it to free acid). Add 50ml methanol,
heat just to boiling point and filter. Add 100ml isopropanol to precipitate free
chromotropic acid (add more isopropanol to increase yield of purified acid).

8.3. ETHANOL CONTENT BY OXIDATION METHOD

Although determination of alcohol content by pyknometric method is the reference

method deviation may magnify if errors occur in measurements and cleaning of
apparatus. A simultaneous use of oxidation method is therefore desirable in many
cases. The latter method is less prone to error magnification when carefully carried
out. The method assumes that oxidizable substances other than ethanol are present
only in negligible amounts in the distillate. Since this condition cannot be generally
achieved by routine distillation process, some errors (though insignificant) do exist.

PRINCIPLE

The method entails separation of ethanol by distillation followed by oxidation by

potassium dichromate in sulfuric acid medium and determination of excess
dichromate by ferrous ammonium sulfate in the presence of ferrous-1,10
phenanthroline as indicator.

117
 ALCOHOLIC BEVERAGES

REQUIREMENTS

 Conc H2SO464  KMnO4 solution65

 Dilute H2SO466
 Ca(OH)2 suspension68
 K2Cr2O7 solution70
 Distillation set, 500ml cap
 Pipettes: 5, 10, and 20ml cap
 Conical flask: 250ml cap, stoppered
 (NH4)2FeSO4.6H2O solution67
 Fe-1,10 phenanthroline solution69
 Heating mantle
 Volumetric flask, 100ml cap
 Burette, 50ml cap
 Balance, 0.01g sensitivity

1. Prepare sample for the determination. Mechanical blending is needed if the

sample is solid or viscous. For liquid samples, uniform mixing is sufficient
2. Weigh to the nearest 0.01g sufficient quantity of sample (in case of liquids,
volume) so that the quantity of ethanol collected in 100ml of distillate is less
than 1g. Dilute the test portion with about 50ml water and transfer
quantitatively to the flask of the distillation set
3. Rinse the vessel used to take the test portion with not more than 120ml water
and transfer it to the flask
4. Make the product slightly alkaline (pH 8±0.2) with calcium hydroxide
suspension shaken before use
5. Add glass beads or porcelain to control the rate of boiling
6. Pour 10ml water in a 100-ml volumetric flask and immerse the outlet tip of
the condenser in the liquid (water)
7. Distil the diluted test portion (previously made alkaline) in such a way that
the distillate, when it reaches the volumetric flask, is at a relatively low
temperature (15-20°C)
8. Collect about 80-85ml of the distillate
9. Stop distillation, avoid back suction of the distillate and rinse the condenser
and tip with a few ml of water.
10. Shake the volumetric flask to mix the contents. If necessary, immerse the
flask in cold water at 15-20°C of a few minutes
11. Dilute the contents of the volumetric flask to the mark with water and shake

64
Density = 1.84g/ml
65
Weigh 1.372g of KMnO4 and dissolve in water to make 1 liter. 10.0ml of this solution is equivalent to
1ml of ammonium ferrous sulfate solution (prepared in this test)
66
Acid + Water = 1+1
67
Dissolve 170.2g of (NH4)2FeSO4.6H2O in 500ml water. Add 20ml of conc sulfuric acid and dilute to 1
liter with water. Stabilize the solution by adding some aluminum chips. 2.0ml of this solution is
equivalent to 1ml of K2Cr2O7 solution (prepared in this test)
68
Obtain the suspension by shaking 110-112g CaO in 1 liter water
69
Dissolve 0.695g of ferrous sulfate heptahydrate (FeSO4.7H2O) in 100ml water. Add 1.485g of 1,10
phenanthroline monohydrate and heat to aid solution. This solution keeps well.
70
Weigh 42.572g of pure K2Cr2O7 and dissolve in water to make 1 liter. 1.0ml of this solution is
equivalent to 0.01g ethanol

118
 FOOD ANALYSIS

12. Pour 20ml (V1) of potassium dichromate solution accurately measured and
20ml of dilute sulfuric acid into a 250-ml conical flask with stopper and
shake
13. Add 10ml (V0) of distillate accurately measured. Stopper the flask,
moistening the stopper with a drop of sulfuric acid
14. Shake the flask and wait for at least 30 min, shaking the flask from time to
time. The resultant mixture should in no case assume the green coloration of
the chromium cation as this would indicate that the ethanol content of the test
portion was too high. If this occurs, recommence the oxidation using smaller
portion of the distillate. If the test portion has too little ethanol, a smaller
amount of potassium dichromate solution may be used. Take account of any
such changes in calculations
15. Titrate the excess dichromate, using the ammonium ferrous sulfate solution.
The excess of dichromate should be at least equal to 20% of the quantity used
for the blank test. Shake the flask after each addition
16. When the color changes to greenish blue, add 4 drops of ferrous 1,10
phenanthroline solution. Continue the addition of ammonium ferrous sulfate
solution until the color of the medium changes from green blue to brown
17. If, for some reasons, the end point is passed, return to it precisely by adding
potassium permanganate solution. Deduct from the volume of ammonium
ferrous sulfate used, one-tenth of the volume of potassium permanganate
solution added. Let V2 be the volume remaining after this deduction which
represents the exact volume of ammonium ferrous sulfate equivalent to the
excess potassium dichromate
18. Carry out a blank test under the same conditions as for the titration replacing
the volume (V0) of the distillate by the same volume of distilled water. Let V3
be the volume of ammonium ferrous sulfate solution used

CALCULATION

For solid products:

V3  V2 100 100
Ethanol content, % weight  0.01V1   
V3 V0 m

Where,

m = weight in g of the test sample

V0 = volume in ml of distillate taken for the titration
V1 = volume in ml of potassium dichromate solution used for the oxidation
V2 = volume in ml of ammonium ferrous sulfate solution used for the back
titration of the dichromate
V3 = volume in ml of ammonium ferrous sulfate solution used in blank test

119
 ALCOHOLIC BEVERAGES

Liquid products:

V3  V2 100 100
Ethanol content, % weight  0.01V1   
V3 V0 V4

Where V0, V1, V2, V3 have the same meanings as above and V4 = volume in ml of the
test portion.

Note: In case of products having essential oils the distillate is turbid with drops of essential oil floating
on the surface. The method has to be modified as follows:

Collect the distillate in a 100ml volumetric flask and allow it to stand for 2 hrs. Dilute
to the mark with water, the interface between the two phases (essential oil and water)
being at the level of the mark. Allow to stand for a further 1 to 2 hrs. Discard the
small quantity of essential oil collected on the surface either by suction with a fine
pipette or by filtration through paper in a covered funnel.

Transfer the still turbid filtrate to a 150-ml flask and add 10g of polystyrene granules
(granule size 1-2mm). Shake the stoppered flask for 15 min and then filter the mixture
through gauge in a covered funnel. The liquid should then have become clear and
have lost its odor completely. Proceed with the determination on this liquid.

120
CHAPTER IX: ANALYSIS OF FATS AND OILS

Fats and oils are a heterogeneous group of predominantly hydrophobic compounds.

The distinction between fats and oils does not have a chemical basis. Those fats/oils
that remain liquid at normal (ambient) temperature are generally taken as oil and
those that remain solid, fats. Analysis of fats and oils is carried out for various
reasons, viz.:

1. Shelf life study (how long the item will remain without deterioration in
quality under a given set of conditions)
2. Functional quality (e.g., suitability for use in biscuits, bakery, hydrogenation,
etc.)
3. Sensory quality (e.g., rancidity)
4. Nutritional quality (e.g., melting point, polyunsaturated fatty acids)
5. As an aid in controlling production operation (e.g., control of hydrogenation,
recovery of oil in mills)
6. Conformance to regulatory standards (e.g., with respect to free fatty acids,
saponification value, peroxide value, moisture)
7. Detection of adulteration (e.g., contamination with mineral oil and argemone
oil, adulteration of dairy ghee with vegetable ghee)
8. Advanced research (e.g., determination of fatty acid profile)

Some of the routine tests carried out on fats and oils are as follows:

1. Acid value/Free fatty acid (FFA)

2. Saponification value, SV (also termed Saponification number)
3. Iodine value, IV (Also termed Iodine number)
4. Unsaponifiable matter
5. Refractive index
6. Melting point (for solid and semisolid items)
7. Moisture content
8. General tests for adulteration, such as Hexabromide test for the presence of
linseed oil, Halphen test for the presence of cottonseed oil, Baudouin test for
the presence of vegetable ghee in dairy ghee, Bellier turbidity test for the
presence of ground nut oil, etc.

Some of the special tests used for particular fats and oils are:
 FATS AND OILS

1. Crismer test for rapeseed and mustard oil

2. Reichert-Meissl, Polenske and Kirschner values for dairy ghee
3. Polybromide test for linolenic oils such as linseed oil

Some of the important physicochemical characteristics of common fats and oils are:

Fat/oil Ref. index at Saponification value, Iodine Unsaponifiable

40°C mg KOH/g oil value, Wij’s matter, %
Soybean oil 1.466-1.470 189-195 120-143 1.5
Mustard oil 1.461-1.469 170-184 92-125 1.5
Maize oil 1.465-1.468 187-195 103-128 2.8
Sunflower oil 1.467-1.469 188-194 110-143 1.5

9.1. DETERMINATION OF FREE FATTY ACIDS (FFA) AND ACID VALUE

BACKGROUND

For the most part, natural fats and oils are in the triglyceride form when freshly
extracted from the source. With prolonged storage, however, the triglycerides begin
to break down giving rise to free fatty acids (FFA). This hydrolysis is brought about
by a variety of agents: presence of moisture in the oil, elevated temperature and, most
important of all, lipases (enzyme) coming from the source or contaminating
microorganisms. Consequently, the neutral oil becomes a mixture of triglycerides,
diglycerides, monoglycerides, free fatty acids and glycerol. Some fats/oils are
relatively stable but others, such as crude rice-bran oil, are notoriously susceptible to
hydrolysis. Whichever the oil, presence of excess free fatty acids is a sure indicator to
unnatural state of oil. The presence free fatty acid in large excess, though not a health
hazard is undesirable for several reasons; some of them are:

1. The oil is no longer the same as the virgin oil

2. The oil tends to smoke during deep-frying
3. The oil is susceptible to rancidity
4. The product prepared from such oil turns rancid very soon

Rancid oils markedly lower the esthetic value of oil. Such oils also bring about health
problems. In connection with the afore-mentioned points, regulating bodies have set
mandatory standards for edible oils, for example, in Nepal:

Mandatory standards of selected fats/oils

Fat/oil FFA (as % oleic acid)

Vanaspati ≤ 0.5
Refined oil ≤ 0.25
Mustard/Rapeseed oil ≤ 3.0

122
 FOOD ANALYSIS

The operational definitions of Acid Value and FFA are:

Acid Value: Number of milligram of KOH needed to neutralize the FFA present in
1 g of oil
FFA : Percentage of free fatty acids present in oil. It is calculated using an
assumed average molecular weight of fatty acids, usually 282 (oleic
acid)

PRINCIPLE

Free fatty acids are readily soluble in rectified spirit or absolute alcohol. A suitable
amount of oil is therefore mixed with neutralized rectified spirit to extract free fatty
acids and the amount of the latter calculated by titrating with standard NaOH or KOH
using phenolphthalein indicator. To facilitate extraction, the mixture may be warmed
to about 70C and swirled vigorously. Calculation for both acid value and FFA can be
carried as follows:

REQUIREMENTS

 Neutral alcohol (95%, v/v)71  Titration arrangement

 Phenolphthalein indicator (1%, alcoholic)  Weighing arrangement
 0.1N NaOH  Hot plate

PROCEDURE

1. Weigh out 10 g of fat/oil in a 250-ml conical flask (by difference)

2. Add 50ml of neutral alcohol
3. Add a drop or two of phenolphthalein indicator
4. Swirl the contents and place flask on the hot plate
5. Warm the mixture to about 70C. Swirl well
6. Titrate warm with 0.1N NaOH to persistent pink color
7. In case of doubt, tilt the mixture to allow separation of alcohol and fat
fractions. Observe the color of the alcohol fraction for persistent pink color
8. Carry out titration in triplicate

CALCULATION

ml of alkali  N of alkali  28.2

% FFA  (as oleic acid)
Wt. of sample (g)

71
Neutralize the acidity in the with 0.1N NaOH using phenolphthalein indicator

123
 FATS AND OILS

ml of alkali  N of alkali  56.1

Acid value 
Wt. of sample (g)

9.2. DETERMINATION OF SAPONIFICATION VALUE OF FAT/OIL

BACKGROUND

Saponification value of fat/oil is a very valuable test for the determination of

adulteration. The test is a rough measure of the average molecular weight of fatty
acids in the oil and related thus:

561.0937 100  P 
M   12.683
Saponification value

Where M =Average molecular weight, P = percentage of unsaponifiable matter

Since the oil from a given source has a remarkably constant saponification value any
deviation found in the test is an indication to adulteration. Some of the common
examples of edible oils and their saponification values are:

Soybean oil: 189 – 195 Butter oil (ghee): 210 – 230

Rapeseed oil: 168 – 181

The test merits considerable attention in that successful testing is more of an art.
There are at least two titrimetric methods for the determination of saponification
value. A relatively easy method utilizes double indicator, viz., phenolphthalein and
bromophenol blue.

When fat is boiled with an excess of alcoholic KOH, the glycerides irreversibly
hydrolyze, giving rise to glycerol and fatty soap (Fig. IX-1). The alkali consumed for
this is a measure of saponification value, and is defined as the number of milligram of
KOH needed to saponify one gram of oil or fat.

CH2OCO-R1 CH2OH R1-COOK

R2-COOCH + 3KOH HOCH + R2-COOK
CH2OCO-R3 CH2OH R3-COOK
Mixed triglyceride Glycerol Potassium soap

Fig. IX-1: Saponification of triglyceride

The hydrolysis is limited to glycerides, waxes and phosphatides. Sterols,

hydrocarbons, pigments, etc., although lipids, do not react with KOH under the above

124
 FOOD ANALYSIS

condition and they contribute to what is known as unsaponifiable matter. A

recapitulative presentation of the above-mentioned points is given in Fig. IX-2.

PRINCIPLE

When the oil is saponified with a slight excess of alcoholic KOH, the reaction results
in potassium soaps, glycerol and unreacted KOH. The free KOH can be determined
by titrating with 0.5N HCl using phenolphthalein as an indicator. The KOH in the
form of soap is determined by further titrating with 0.5N HCl using bromophenol blue
indicator solution as the indicator. Bromophenol blue changes from blue to permanent
greenish yellow upon complete breakdown of the soap. The amount of HCl consumed
is back calculated to reflect the milligrams of KOH consumed by one gram of fat/oil
during the saponification.

CRUDE FAT
Fats KOH Potassium salts + Glycerol
KOH
Waxes Potassium salts + Alcohol
KOH
Phosphatides Potassium salts + Glycerol + K3PO4 + Amine
Non-fat Sterols
KOH
Hydrocarbon No reaction
Pigments (Unsaponifiable matter)

Fig. IX-2: Saponifiable- and unsaponifiable matter in oil

REQUIREMENTS

 Oil sample  Conical flask with condenser

 KOH pellets (pure)  Heating arrangement (hot plate or water bath)
 Standard HCl (0.5N)  Titration arrangement
 Rectified spirit or absolute  Indicators: bromophenol blue (1%, alcoholic)
alcohol (aldehyde-free) and phenolphthalein (1%, alcoholic)

PROCEDURE

1. Melt the sample (if not already liquid) and filter warm. Ensure that the sample
is free from moisture and impurities
2. Weigh accurately by difference about 2g of sample in a 250-ml conical flask
3. Add about 500mg KOH pellet, a small amount of rectified spirit ( 10ml), and
emulsify by swirling briefly
4. Add about 30ml rectified spirit and gently reflux the whole until the oil
becomes transparent (this usually takes 25 min)
5. Add some rectified spirit (if the volume decreases) and continue refluxing till
completely saponified. The oil-alcohol mixture appears transparent at this
stage

125
 FATS AND OILS

6. Slightly cool the flask and add a drop or two of phenolphthalein indicator.
Intense red color indicates the presence of excess KOH. If the color does not
change, repeat the whole process using more KOH (e.g. 600-800mg)
7. Add a drop or two of distilled water. If a milky color develops, the sample
contains significant amounts of unsaponifiable matter or is contaminated with
mineral oil
8. add more water (about 50ml) and mix well
9. Neutralize the excess KOH with 0.5N HCl. The pink color should just
disappear
10. Add a drop of bromophenol blue indicator and swirl. It should give a blue
color
11. Note the reading on the burette (containing the standard HCl) and titrate till a
permanent greenish-yellow color appears. If, during titration, fat-like globules
suddenly appear, warm the flask a little and continue titration to the end point
12. Note the volume of 0.5N HCl consumed (the second reading, that is) and
calculate the saponification value

CALCULATION

ml of HCl  N of HCl  56.1

Saponification value 
Wt. of sample (g)

9.3. DETERMINATION OF IODINE VALUE OF FAT/OIL BY WIJ'S METHOD

BACKGROUND

Probably no analytical test method in all of oleochemistry has had the universal
widespread use as the measurement of unsaturation in fats and oils by iodine value
determination. The first to use this concept was Von Hubl in 1884. Since 1898 great
many innovations have been made, continuing until recently.

Iodine Value is the number of grams of iodine absorbed per 100 g of oil or fat, when
determined using Wij'’ solution. The test is a measure of unsaturation of a given fat or
oil. Since the degree of unsaturation is more or less characteristic to oil source, the
test is routinely used for the determination of adulteration by other types of oils.
Iodine values of some common edible oils are:

Soybean oil: 120-143

Rapeseed oil: 94-120
Butter oil: 26-38

The test is of tremendous value in vanaspati (hydrogenated oil) plants. It is routinely

used for monitoring the degree of hydrogenation. Iodine value is also used to
calculate the amount of hydrogen used or wasted in vanaspati plants. In general, a
drop in 1 unit of iodine value means to the vanaspati manufacturer that 0.075kg of

126
 FOOD ANALYSIS

hydrogen has been added to every 1000 kg oil. There are several methods for
measuring the iodine value of fats and oils. Some of the variations and /or equivalent
methods are: Hanus method, Bromine Value method, Rosenmund-Kuhnhenn method,
etc. There are some difference vis-à-vis reagent preparation in Wij’s method also.

PRINCIPLE

Halogens add across the double bonds of unsaturated fatty acids to form addition
compounds. Iodine monochloride (ICl) is allowed to react with the fat in the dark.
The amount of iodine consumed is then determined by titrating the iodine released
(after adding KI) with standard thiosulfate and comparing with blank in which the fat
is omitted. The reaction occurring in the test can be shown in Fig. IX-3.

CH CH + ICl CH CH
Unsaturated Iodine I Cl
portion of fat monochloride Addition
compound

ICl + KI KCl + I2
Residual Added after Molecular
titration iodine

Na2S2O3 + I2 2NaI + 2Na2S4O6

Na-thiosulfate Na-tetrathionate

Fig. IX-3: Reaction scheme during iodine value determination

The reaction mixture is kept in dark and the titration carried out as quickly as possible
since halogens are oxidized in the light. The amount of Wij’s reagent used in this test
should be more (usually by 150%) than shown by the stoichiometry.

REQUIREMENTS

 Carbon tetrachloride  Soluble starch (1%)72

 Potassium iodide (10%, aqueous)  Iodine value flasks
 Standard Na-thiosulfate (0.1N)73  Pipette: 25- and 5ml (graduated)

72
Dissolve 1g of reagent grade starch in hot water. Transfer the clear fraction into another container.
Use only fresh solution, as it is subject to microbial degradation
73
Dissolve 25g AR grade Na2S2O3.7H2O in distilled water to make 1000ml. Mix the solution thoroughly,
allow to stand for a few days, and then siphon off the clear liquid. Standardize the solution with AR
grade potassium dichromate (K2Cr2O7). Weigh 0.20 to 0.23g of K2Cr2O7 (dried for 2 hrs at 105°C).
Transfer to a 250-ml beaker using 150ml of water. Add 2g of KI and mix. Add 20ml of 1N HCl, swirl,
and allow to stand for 10 min. start titrating with Na2S2O3 solution from the burette, adding about
80% of the required amount. Add 1ml of starch indicator and complete the titration to a point where
the solution changes from blue-green to light green. Calculate the strength of Na-thiosulfate as follows
(footnote):

127
 FATS AND OILS

 Wij’s solution74  Measuring cylinder, 25ml

 Electronic balance (± 1mg )  Burette: 50ml, 2 sets
 Oil or fat sample

PROCEDURE

1. Weigh accurately by difference suitable quantity of oil using the formula:

(20.3 ÷ expected iodine value) grams, in to a clean, dry 250-ml IV flask (see
Fig. IX-4)
2. Add 10ml of CCl4 and allow oil to dissolve
3. Add accurately 20ml of Wij’s solution. Swirl once and close the flask with the
stopper. The stopper may be moistened with minimum of 10% KI solution
4. Stand the flask at 15-20C for 30 min in dark
5. Add 15ml of 10% KI solution, followed by 100ml distilled water
6. Titrate with 0.1N Na2S2O3 using starch indicator towards the end of the
titration (The mixture turns straw color near the end point. Add two drops of
starch solution. The mixture immediately turns dark blue. Continue the
titration until the blue color just disappears)
7. Carry out a blank test upon the same quantities of reagents, omitting the oil, at
the same time and under the same conditions. The excess of reagent remaining
for titration in the test must be 150% of the reagent absorbed

CALCULATION

Iodine value 
 Blank titer  Sample titer  ml  N of Na 2S2O3  12.69
Wt. of sample (g)

Stopper

Fig. IX-4: Wij’s IV flask

Wt . of pot - dichromate  1000

N of sod-thiosulfate 
ml of sod - thiosulfate  49.037
Add a pinch of Na2CO3 and 1ml of chloroform to preserve it from microbial degradation
74
Dissolve 8g iodine trichloride in 150ml glacial acetic acid and mix with 9g iodine dissolved in 350ml
glacial acetic acid. The strength of Wij’s solution, as determined by titrating with Na-thiosulfate, should not
be less than 0.2N. Store the reagent in a colored bottle in dark. The solution is stable for about 30 day.

128
 FOOD ANALYSIS

PRECAUTIONS

1. The Wij’s solution should be  0.2 N

2. The reagent should be used in excess (150% of the amount absorbed by
fat/oil)
3. Use only freshly prepared starch solution

Note: Wij’s solution can be prepared by other methods also, viz., (i) using iodine monochloride, and
(ii) using chlorine gas and resublimed iodine. The latter method is described here.

Preparation of Wij’s solution by chlorination

Before anything else, prepare standard sod-thiosulfate, conc sulfuric acid, 10% KI
solution and starch indicator. Assemble pipettes, burettes and other glassware needed
for iodometric titration.

 Take 13g resublimed iodine in a 1-liter beaker

 Add 200ml glacial acetic acid and dissolve by gentle heating (along with
stirring). Iodine dissolves very slowly and the complete dissolution can be
carried out in stages by using small portions of glacial acetic acid
 Transfer the dissolved portion to 1-liter volumetric flask
 Add more glacial acetic acid (~ 200ml) to the undissolved iodine in the
beaker and heat gently (as previously done) to affect dissolution
 Transfer the dissolved portion to the volumetric flask (to pool the solution)
again
 Carry out this operation until iodine is completely dissolved. However, do not
exceed the total volume of 1000ml. If some space is available, make up the
volume to 1000ml by glacial acetic acid. Mix the solution well. Take out
about 25ml solution and set aside (as a reserve) in a separate flask (you will
need this later)
 Transfer the bulk iodine solution in a Woulfe bottle and assemble the parts as
in Fig. IX-5
 Generate chlorine75 and pass through the iodine solution to form iodine
monochloride
 Continue passing the chlorine until the characteristic color of free iodine is
discharged (solution suddenly lightens because of free chlorine)
 Stop passing chlorine and test the Wij’s solution for dismantle the assembly
for chlorination
 Add small amounts of iodine solution (reserved earlier) until the free chlorine
has been destroyed (the color again darkens). A slight excess of iodine does

75
Chlorine is generated in the laboratory by reacting KMnO4 and con. HCl:
2KMnO4 + 16HCl  2KCl + 2MnCl2 + 8H2O + 5Cl2
The amounts of HCl and KMnO4 needed for chlorination are not very large. However some amounts of
chlorine go waste during chlorination. Besides, sufficient amounts of chlorine must be produced to force
itself through the solution. To take this into account, use about 5-10g of KMnO4 and 50-100ml of conc HCl.

129
 FATS AND OILS

little or no harm but excess chlorine must be avoided. Typically, the

iodine/chlorine ratio should be 1.1± 0.1 and this can be ascertained by
determining iodine content and total halogen content as follows:

Iodine content:

o Take 150ml of Chlorine-saturated water in a 500-ml conical flask and

add some glass beads
o Add 5ml of Wij’s solution
o Mix, and heat to boiling for 10 min
o Cool and add 30ml of 2% H2SO4
o Add 15ml of 15% fresh KI solution
o Titrate with 0.1N sod-thiosulfate to starch end point
o Note the titer (say A)

Total halogen content:

o Take 150ml of recently boiled, cooled water in a 500-ml conical flask

o Add 15ml of 15% KI solution
o Pipette 20ml Wij’s solution
o Titrate immediately with 0.1N sod-thiosulfate to starch end point
o Note the titer (say B)
o Iodine/Chlorine ratio as follows:

2A
I/Cl  , the Wij’s solution thus prepared should consume
 3B  2 A
approximately double the amount of 0.1N sod-thiosulfate

Delivery tube Conc. HCl

Small vent to release

excess pressure

KMnO4
Iodine solution

Chlorine bubbles

Fig. IX-5: Preparation of Wij’s solution

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 FOOD ANALYSIS

9.4. DETERMINATION OF PEROXIDE VALUE

BACKGROUND

Peroxide value (PV) is a very sensitive indicator of the early stages of oxidative
deterioration of fats and oils. PV therefore provides a means of predicting the risk of
the development of flavor rancidity. There are numerous analytical procedures for the
measurement of peroxide value. In all cases the results and accuracy of the test
depend on the experimental conditions, as the method is highly empirical. The most
common methods are those based on the iodometric titration originally reported by
Lea and Wheeler, which measure the iodine produced from potassium iodide by the
peroxides present in the oil. It has been contended that the two principal sources of
error in these methods are the absorption of iodine at unsaturated bonds in the fatty
acids on the one hand, and on the other, the liberation of iodine from potassium iodide
by oxygen present in the solution to be titrated. Other types of error which can arise
include variation in weight of the sample, the type and grade of solvent used,
variation in the reaction conditions such as time and temperature, and the constitution
and reactivity of the peroxides present in the oil.

Other methods have been recommended for peroxide value determination and these
include a colorimetric method based on the oxidation of ferrous to ferric ion and the
determination of the latter as ferric thiocyanate; a variation in the iodometric method
reported by Swoboda and Lea, in which the liberated iodine is converted into a blue
starch iodine complex; and the Sully method, in which the mixture is boiled.

Peroxide value of an oil or fat is the amount of peroxides present and expressed as
milli-equivalents of peroxide per 1,000g of sample.

PRINCIPLE

When a rancid fat or oil sample is treated with potassium iodide after dissolving in an
appropriate solvent, peroxides present in the fat liberate iodine. The test is a
volumetric one where I2, formed from KI in the presence of peroxide is determined by
titrating with sodium thiosulfate and conducting a blank determination.

Now, milliequivalent peroxide = milliequivalent thiosulfate at the equivalence point

Again, milliequivalent = (strength × volume), when volume is in milliliter

Therefore, PV = milliequivalent thiosulfate / kg sample

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 FATS AND OILS

REQUIREMENTS

 Oil or fat sample  Iodine flasks: 250ml cap

 Acetic acid-chloroform solvent76  Burette: 25-50ml cap
 Saturated potassium iodide77  Pipette: 25ml cap
 0.01N and 0.1N sod-thiosulfate (see  0.5% starch indicator (see
determination of Iodine Value) determination of Iodine Value)
 Measuring cylinder: 25ml cap  Weighing arrangement

PROCEDURE

1. Weigh accurately (by difference) 5g of fat or oil sample in the Iodine flask
2. Add 25ml of solvent and displace the air with CO2
3. Add 1ml of KI solution, stopper the flask, and allow it to stand for 1min (with
gentle shaking)
4. Add 35ml of distilled water and a few drops of starch indicator. Appearance
of blue color on addition of starch indicates presence of free iodine
5. Titrate the liberated iodine with 0.01N or 0.1N sod-thiosulfate until the blue
color just vanishes
6. Carry out a blank determination simultaneously (omitting oil)
7. Calculate Peroxide value using following equation:

N  (VS  VB )  1000
PV (meq/kg) 
Wt. of sample (g)

Where,

N = normality of sod-thiosulfate, VS = sod-thiosulfate consumed by sample

(ml), and VB = sod-thiosulfate consumed by blank (ml).

9.5. MELTING POINT OF FAT BY OPEN-TUBE CAPILLARY METHOD

BACKGROUND

Fats do not melt sharply because they contain different types of fatty acids with
different melting points. Melting point of fat increases with an increase in the degree
of saturation and chain length of fatty acid. Unsaturated bonds produce kinks in the
fatty acid chain and therefore allow very loose molecular packing. This facilitates
faster slipping away of molecules, thereby resulting in low melting point. Melting
point also depends on isomeric forms and polymorphism in fatty acids. Trans isomers
of fatty acids (that usually form during hydrogenation process) have higher melting
point because the chains are less kinked.

76
2 volumes of acetic acid and 1 volume of chloroform
77
4 parts of pure KI in 3 parts of distilled water. Keep in brown bottle

132
 FOOD ANALYSIS

Polymorphism in fats and oils refers to existence of more than one crystalline form of
fatty acid or glyceride. Three such polymorphic forms, viz., alpha (), beta prime (’)
and beta () have been identified. Polymorphism results from different patterns of
molecular packing in fat crystals. A comparison of the three polymorphs is given
below:

Polymorph Crystal size Melting point Stability

Alpha () 5m Lowest Least stable
Beta () 20-50m Highest Most stable
Beta prime (’) 1-2m Intermediate Intermediate

The stability of fat is related to the polymorphic form and the associated melting
point. The melting points of -, ’-, and  forms of tristearin are 55°C, 64°C, and
73°C, respectively. Polymorphic transformations occur from  to ’ to  and are
irreversible. When fat is cooled rapidly the  polymorph is produced, which is
usually quickly converted to the ’ form. These polymorphic forms also affect the
appearance and texture of fat. ’ form gives a smooth texture whereas  form results
in a very coarse granules. It is therefore very important to control the balance of
polymorphic forms in the production of fat and fatty foods like margarine (needs ’
form), ghee (needs  form), etc.

Because of the reasons described above, melting point as such is not very reliable for
establishing identity of the fat and oil. However, it is extensively used in controlling
process operation (e.g., hydrogenation), quality control, and determining suitability of
fat for a particular purpose. The methods used for the determination of melting point
vary considerably. A typical method used in vanaspati manufacture is the open-tube
capillary method. The melting point is therefore defined by the specific conditions of
the method by which it is determined.

PRINCIPLE

The temperature at which the oil or fat softens or becomes sufficiently fluid to slip or
run as determined by the open-tube capillary-slip method.

REQUIREMENTS

 Capillary tubes78  Beaker or Thiele tube

 Thermometer (0.2°C sensitivity)  Heat source (gas burner or spirit lamp)

78
Thin-walled with uniform bore capillary glass tubes opn at both ends with following dimensions:
Length = 50-60mm
Inside diameter = 0.8-1.1mm
Outside diameter1.2-1.5mm

133
 FATS AND OILS

PROCEDURE

1. Melt the sample and filter it through a filter paper to remove any impurities
and last traces of moisture
2. Make sure that the sample is absolutely dry
3. Mix the sample thoroughly
4. Introduce a capillary tube into the molten sample, so that a column of the
sample, about 10mm long, is sucked into the tube
5. Chill the tube containing the sample immediately by touching the tube,
against a piece of ice until the fat solidifies
6. Place the tube in a small beaker and hold it for one hour either in a
refrigerator or in water maintained at 4-10°C

Thermometer

Capillary Rubber band

Heat source
Fat
Theile tube

Chilled water

Fig. IX-6: Arrangement for melting point determination of fat

7. Remove the tube and attach with a rubber band to the thermometer bulb, so
that the lower end of the capillary tube and the thermometer bulb are at the
same level
8. Take water at 10°C in the Thiele tube and immerse the thermometer with the
capillary tube containing the sample of fat (see Fig. IX-6)
9. Gradually increase the temperature by heating at the side-tube of the Thiele
tube at the rate of 2°C pen min, till the temperature reaches 25°C, and
thereafter at the rate of 0.5°C per min
10. Note the temperature of the water when the sample column begins to rise in
the capillary tube
11. Report the average of two such separate determinations as the melting point,
provided that the readings do not differ by more than 0.5°C

9.6. TESTS FOR THE ADULTERATION OF FATS AND OILS

Physicochemical properties of fats and oils are often used to identify them. Usually,
more than one property is measured so that the identification can be made with some
assurance since natural fats and oils vary somewhat in their properties. A few special
134
 FOOD ANALYSIS

tests are now available for the unequivocal determination of adulteration in fats and
oils. Some of these tests are described next.

9.6.1. REICHERT-MEISSL, POLENSKE, AND KIRSCHNER VALUE

BACKGROUND

These tests are widely used for identification and test of adulteration of butter. The
tests are based on the quantitative measurement of low molecular weight fatty acids
(C4-C14) that are predominant in butter. Although the RM value varies for butter with
season, nutrition, and time in the lactational cycle of the cow, it is usually between 24
and 34, higher than other edible oils.

The definitions of the terms are:

Reichert-Meissl value: It is the number of milliliters of 0.1N NaOH required to

neutralize the steam-volatile, water-soluble fatty acids distilled from 5g sample of fat
under precise conditions specified in the method. This test measures the quantity of
C4 and C6 fatty acids.

Polenske value: It is the number of milliliters of 0.1N NaOH required to neutralize

the steam-volatile, water-insoluble fatty acids distilled from 5g sample of fat under
precise conditions specified in the method. This test measures the quantity of C8 to
C14 fatty acids.

Kirschner value: It is a measure of steam-volatile, water-soluble fatty acids forming

water-soluble silver salts (which is the property of butyric acid). In recent years, this
analysis is not usually done.

PRINCIPLE

Steam-volatile fatty acids can be collected by saponification and steam-distillation of

oil. Reichert Meissl value can be determined by titrating the steam condensate (that
contains water-soluble fatty acids) with0.1N NaOH. Polenske value can be
determined by eluting the fatty acids adhering on the condenser with neutral ethanol
and titrating with 0.1N NaOH. Determination of Kirschner value involves
neutralization of the water-soluble fatty acids with barium hydroxide, preparation of
their silver salts, separation of the water-soluble butyric acid salt by filtration,
liberation of butyric acid by acidification, separation by steam distillation, and
quantification by titrating again with barium hydroxide.

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 FATS AND OILS

REQUIREMENTS

 Fat sample  90% neutral ethyl alcohol (v/v)

 Glycerol  0.05 N barium hydroxide
 50% NaOH  Finely powdered silver sulfate
 Pumice powder: 1.4-2.0mm in diameter  Titration arrangement
 Dil. H2SO4 (25ml H2SO4 + 1000ml H2O)  Weighing arrangement
 RM-Polenske-Kirschner apparatus79  Phenolphthalein indicator
 0.1N NaOH (not to be used, if Kirschner  Heating arrangement
value is to be determined)

PROCEDURE

1. Melt the fat sample if solid but do not heat above 50°C
2. Weigh 5±0.01g of fat sample into a Polenske flask
3. Add 20g of glycerol and 2ml of 50% NaOH solution from a burette which is
protected from CO2 pick up. Wet the tip of the burette before adding alkali to
free it of carbonate deposit and reject the first 0.5ml of NaOH
4. Heat the mixture over a low flame with wire gauze until the liquid becomes
clear and the fat has saponified. Do not overheat at this stage which causes
discoloration
5. When all the fat has saponified, cover the flask with a watch glass, and allow
to cool
6. Add 93ml of boiling distilled water which is free of CO2 and mix. The
solution must be completely clear at this stage and pale yellow in color. If the
solution is not clear which indicates incomplete saponification, or if it is
darker which indicates overheating, repeat the procedure with a fresh sample.
An old sample of oil or fat may behave similarly
7. Add 0.1g of pumice powder and 50ml of dilute H2SO4
8. Connect to the distillation apparatus (Fig. IX-7)
9. Warm the mixture until any insoluble material which may be present melts
10. Increase the heat and distil 110ml of solution in 19 to 21 min. The distillation
is considered to begin when the first drop forms in the shill-head
11. Stop heating soon after 110ml has distilled over, and replace the graduated
flask by a measuring cylinder to collect drippings from the condenser
12. Close the graduated flask with the stopper. Do not mix
13. Place the flask in a water bath at 15°C for 10 min and ensure that the 110ml
graduation is below the water level
14. Mix and filter through a 9cm Whatman No. 4 paper. Reject the first 2-3ml of
the filtrate and collect the rest in a dry flask
15. Wash the condenser, still head and the 110ml graduated flask with three lots
of 15ml distilled water passing each washing through the measuring cylinder,
100ml flask and stopper

79
Apparatus consisting of flat-bottom boiling flask, still head (10.7cm wide and 18cm high), condenser (52cm
long with 30cm cooling length and 7cm entry tube) and a receiver (with graduations at 100ml and 110ml)

136
 FOOD ANALYSIS

20 78 37

110ml

100ml

Fig. IX-7: Reichert-Meissl distillation apparatus

16. Filter through the same filter paper ensuring that all insoluble matter is
transferred to the paper. Discard the filtrate. Do not mix with filtrate of the
distillate got in the previous step. The filtrate should be free from water
insoluble fatty acids
17. Pipette out 100ml of the filtrate to a dry 250ml conical flask and titrate with
0.1N NaOH using phenolphthalein as indicator
18. Calculate RM value as follows:

RM value = (Sample titer – Blank titer)ml × N of NaOH × 11

The factor 11 has been obtained as follows:

137
 FATS AND OILS

Total volume collected (ml) 110

= = 11
Aliqout taken (ml) × Required N of NaOH (i.e., 0.1) 100× 0.1

19. Dissolve the insoluble fatty acids by three washings of the condenser, the
measuring cylinder, the 110ml flask with stopper and the filter paper
containing the main bulk with 3 similar washings as before using 15ml
portions of neutral ethanol
20. Collect the alcoholic washings (45ml) in a clean dry flask and titrate with
0.1N NaOH using phenolphthalein indicator
21. Carry our a blank determination similarly
22. Calculate Polenske value as follows:

Polenske value = (Sample titer – Blank titer)ml ×N of NaOH ×10

RM and Polenske values are affected by low barometric pressures which occur at
high altitudes. Under such conditions, correct the readings as follows:

(Observed RM  10) log 760

Corrected RM value = +10
log p

Observed value × (760  45)

Corrected Polenske value =
p  45

Where, p = barometric pressure in mm of Hg at the place of determination

23. For Kirschner value, proceed as in RM value determination but replace 0.1N
NaOH with 0.05N Ba(OH)2 for titration
24. After determination of RM value, add 0.5g of finely powdered silver sulfate
to the solution
25. Keep in a dark place for 1hr with intermittent shaking
26. Filter through a dry Whatman No. 4 paper
27. Add 35ml of cold, CO2-free distilled water, 10ml of dilute H2SO4 and 0.1g of
pumice powder
28. Connect to the distillation apparatus, and distil 110ml in 19-21 min
29. Cool the distillate at 15°C for 10 min, mix and filter through 9cm Whatman
No. 4 filter paper as before
30. Titrate 100ml of the filtrate as in RM value using 0.05N barium hydroxide
31. Carry out a blank determination similarly
32. Calculate Kirschner value as follows:

100 +  Tr - Ta 121
Kirschner value =  Tk - Tb  
10000

138
 FOOD ANALYSIS

Where,

Tk and Tb = sample and blank titer respectively in Kirschner value

determination

Tr and Tb = sample and blank titer respectively in the RM value determination

9.6.2. BAUDOUIN TEST

This test is useful in the detection of adulteration of dairy ghee with vanaspati ghee.
The test is based on the color reaction between sesamolin (a compound present in
sesame oil) and furfural In Nepal, use of sesame oil in vanaspati is mandatory. Dairy
ghee containing sesamolin gives a positive Baudouin test, thereby indicating the
presence of vanaspati ghee.

PRINCIPLE

The development of pink color with furfural solution in the presence of hydrochloric
acid indicates the presence of sesame oil. The color is produced on account of
reaction with sesamolin present in sesame oil.

REQUIREMENTS

 Stoppered test tube/measuring cylinder  Furfural solution80

 Conc Hydrochloric acid  Oil sample

PROCEDURE

1. Take 5ml of the oil or melted fat in a 25-ml measuring cylinder (or test tube)
provided with a glass stopper
2. Add 5ml of conc. hydrochloric acid
3. Add 0.4ml of furfural solution
4. Insert the glass stopper and shake vigorously for two minutes
5. Let is stand and allow the mixture to separate. The development of pink or
red color in the lower acid layer indicates presence of sesame oil
6. Confirm by adding 5ml of water and shaking again. If the color in acid layer
persists, sesame oil is present; if the color disappears it is absent

9.6.3. HEXABROMIDE TEST

This test is of importance for detecting adulteration of edible oil with linseed oil
(which is inedible). The test is based on the formation of insoluble polybromides

80
2% furfural, freshly distilled in ethyl alcohol

139
 FATS AND OILS

when unsaturated fatty acids are brominated. Di- and tetrabromides that result from
oleic and linoleic acids are soluble and therefore do not interfere with this visual test.

PRINCIPLE

The formation of a precipitate of hexabromide when the oil in chloroform is treated

with bromine and then with alcohol and ether in cold condition indicates the presence
of linseed oil.

REQUIREMENTS

 Boiling tubes  Ice water bath

 Chloroform  Ethyl alcohol
 Liquid bromine  Diethyl ether

PROCEDURE

1. Pipette 1ml of oil into a boiling tube (wide-mouthed, 100ml cap)

2. All 5ml chloroform and about 1ml of bromine drop-wise till the mixture
becomes deep red in color
3. Cool the test tube in an ice water-bath
4. Add about 1.5ml of rectified spirit drop-wise while shaking the mixture until
the precipitate which was first formed just dissolves
5. Add 10ml diethyl ether
6. Mix the contents and place the tube within the ice water-bath for 20 min
7. Appearance of precipitates indicates the presence of linseed oil

The sensitivity of this test is about 1% if linseed oil in other oils. The test has some
limitations. It is not suitable for test in mahua oils. Besides, marine oils, which
contain polyunsaturated fatty acids, also give insoluble polybromide precipitate.

9.5.4. THE PRESENCE OF ANIMAL FAT BY MICROSCOPIC EXAMINATION

Animal body fats such as beef tallow and lard have been shown to contain trisaturated
glycerides. On crystallization these glycerides exhibit a characteristic crystalline
appearance when viewed under microscope. The procedure recommended by
Williams Sutton for the microscopy of fat crystals have been suitably modified and
given.

REQUIREMENTS

 Fat sample  Test tubes  Microscope

 Ice water-bath  Filtration unit
 Ethyl alcohol  Glycerin

140
 FOOD ANALYSIS

PROCEDURE

1. Take about 2g of melted fat samples in test tubes

2. Add 10ml of diethyl ether and mix
3. Plug the tubes with cotton and allow them to stand for 30 min in ice water or
24 hrs at 20ºC (slow crystallization gives bigger crystals). In certain cases it is
preferable to first crystallize with a stronger solution of fat from a mixture of
ether and ethyl alcohol (1:1). In such cases separate the crystals by filtration
and recrystallized in ether
4. Place the crystals on a drop of glycerin previously taken on a microscopic
slide
5. Cover the crystals immediately with cover glass
6. Examine the crystals under 160 and finally 400 magnifications. The typical
appearance of beef tallow crystallized into characteristic fan like tufts, the
ends of which are more or less pointed can be seen. Lard crystals are chisel-
shaped. Hydrogenated fats deposit smaller size crystals. The size and shape of
the crystals depend upon the strength of solution, amount of fat taken and the
time allowed for crystallization

9.6.5. PRESENCE OF ARGEMONE OIL

Argemone (Argemone maxicana L.), yellow poppy, is a wild herb, which grows in
mustard field and bears capsules full of brown black seeds. Because of its
resemblance with black mustard, it is often used as an adulterant. The oil is reported
to cause glaucoma, dropsy and sometimes total blindness due to the presence of
alkaloids namely, sanguinarine and dihydrosanguinarine.

PRINCIPLE

The hydrochloric acid extract of the oil sample containing argemone oil when
subjected to TLC for separation of alkaloid gives fluorescent spot under UV light.

 Standard argemone oil extract  UV chamber (long wave, 366nm)

 Pear-shaped flask
 Hot water bath
 Separating funnel, 50ml cap.
 Glass beaker, 10ml cap
 Aqueous NaOH, 20%
 TLC plates coated with silica gel-
G or precoated ready made plates
cut to suitable size
 Solvent mixture (mobile phase)
 Diethyl ether
 Conc. HCl, sp. grav. = 1.19
 Chloroform:Acetic acid:Water =
70:20:10 (v/v)
 Chloroform:Acetic acid (90:10, v/v)
mixture

PROCEDURE

1. Take 10ml sample in a separating funnel and dissolve in 15ml Diethyl ether

141
 FATS AND OILS

2. Add 5ml conc HCl and shake vigorously for 2-3 minutes. Allow to separate.
Contents of the separatory funnel may be heated cautiously over the vent of
heating water bath for some time for quick separation
3. Transfer the acid layer to a 25ml beaker
4. Place the beaker into a boiling water bath and evaporate till dryness
5. Dissolve the residue obtained after evaporation of hydrochloric acid in 1ml of
a mixture of chloroform and acetic acid (9:1)
6. Spot on TLC plate with the help of spotting capillary. Spot side by side
standard Argemone oil extract (0.1 % in ether)
7. Develop the plate in (a) Butanol:Acetic acid:water; or (b) Hexane:Acetone
mixture
8. Allow the solvent front to move up a distance of 10cm
9. Allow the plate to dry
10. Place the plate under UV light in the visualization chamber
11. Bright yellow or orange yellow fluorescent spots having Rf similar to the
standard argemone oil will confirm presence of argemone oil. The spot gives
blue florescence under UV-light if plate is sprayed with 20% aqueous sodium
hydroxide solution

The method is very sensitive and can detect argemone oil up to 50 ppm level.

9.6.6. KRIES TEST FOR RANCIDITY IN FATS/OILS

Kries test is a very rapid test for the assessment of rancidity in fats and oils. It can be
carried out quantitatively as well as quantitatively. The qualitative method involves
vigorous mixing of 5ml of oil with 5ml of 0.1% phloroglucinol solution (in diethyl
ether), adding 5ml of conc HCl and observing for pink color as the evidence of
incipient rancidity.

PROCEDURE FOR QUANTITATIVE METHOD

1. Shake 5ml of oil and 5ml chloroform in a stoppered test tube

2. Add 10ml of a 30% solution of trichloroacetic acid (in glacial acetic acid)
3. Add 1ml of 1% solution of phloroglucinol (in glacial acetic acid)
4. Incubate the test tube at 45ºC for 15 min
5. Add 4ml of ethanol and immediately measure the absorbance at 545nm.

Absorbance values below 0.15 indicate no rancidity. Absorbance values greater than
0.2 denote incipient rancidity, and absorbance values around 1.0 show that the sample
is highly rancid.

142
CHAPTER X: ANALYSIS OF WATER

The single most important requirement for drinking water and water for food
industries is the potability. Potable water implies water that is fit for drinking
(esthetically satisfactory, does not bring about health hazard). For food industries,
however, potability of water does not necessarily connote suitability. Thus, suitable
water for industries implies water that is potable and has defined physicochemical
properties to satisfy a particular processing requirement. It is very difficult to
prescribe an overall quality of water because there are so many specialized
requirements. In other words, a given set of water characteristics that is objectionable
to one use may not necessarily be detrimental to another.

Water analysis is done utilizing parameters signifying the quality of water. The two
main parameters used for this are (i) physicochemical parameter and (ii) microbiological
parameter. Physicochemical parameter includes characteristics like taste, color, turbidity,
pH, hardness, alkalinity, dissolved solids, iron, arsenic, chlorides, nitrites, ammonia, etc.
while microbiological parameter includes counts for total coliform, sulfate-reducing
clostridia, and aerobic bacteria.

10.1. TOTAL DISSOLVED SOLIDS

BACKGROUND

Total dissolved solids (TDS) of natural water consist mainly of carbonates,

bicarbonates, sulfates, phosphates, and nitrates of calcium, sodium, potassium, iron,
manganese, etc. TDS is an important characteristic for drinking water and other water
quality standards.

Increased levels of TDS reduce palatability of water. Concentrations above

3000mg/liter produce distress in cattle and livestock. In industries, the use of water with
high TDS may lead to scaling in boilers, corrosion, and degraded quality of product.

PRINCIPLE

TDS is determined as the dried residue left after evaporation of filtered water sample.
Since the temperature range used routinely to dry the residue is seldom sufficient to
completely drive off the water of hydration attributed to salts like CaSO4 and MgSO4,
the result may be erroneous. In expressing the result, it is therefore customary to state
 WATER

the temperature at which the residue was dried. It is also customary to express the
result to the nearest 5mg/liter.

REQUIREMENTS

 Hot air oven  Desiccator

 Fiberglass filter assembly  Volumetric flask, 100ml
 Electronic balance  Beaker, 200ml

PROCEDURE

1. Measure 100ml (or some suitable volume) of water in a volumetric flask

2. Filter the water through fiberglass filter paper into a tared 200-ml beaker
3. Evaporate the water on a hot plate to dryness. Do no use temperature above 98°C
4. Dry the residue at 103-105°C in an oven for 1 hour
5. Cool the residue in a desiccator and weigh the residue by difference

CALCULATION

Wt. of residue (mg)  1000

TDS (mg/liter) 
Vol. of water (ml)

10.2. TOTAL HARDNESS IN WATER

BACKGROUND

The hardness of water is entirely due to the salts of calcium and magnesium. Hardness
is expressed by convention as mg/liter of CaCO3. Occasionally, presence of free CO2,
large excess of NaCl and carbonates of polyvalent metals like Zn, Fe, Al, etc., may add
to hardness. The factors for different hardness producing cations are: Fe = 1.792,
Sr = 1.142, Al = 5.564, Zn = 1.531, Mn = 1.822, Ca = 2.498, and Mg = 4.116.

Hardness is a property of water which prevents lather formation with soaps and
increases boiling point of water. Bicarbonate- and carbonate salts of cations produce
what is known as temporary hardness. Temporary hardness can be removed simply by
boiling the water. On the other hand, permanent hardness is mainly due to sulfates and
chlorides of metals, and as the term implies, cannot be removed by boiling the water.

Hardness has no known adverse effects on health. Hard water is undesirable in boilers:
it forms scales in the tubes and impedes heat transfer. Hard water is unsuitable also for
domestic uses such as cleaning, washing and laundering. In the beverage industries,
where water forms the main bulk of the final product, hardness is a determinant to the
quality of the beverage. Breweries need water of hardness around 300mg/liter (but is
subject to variation). Water used for canning peas, beans and lentils should have zero
hardness (hardness contributes to stiffening/toughening of the product).

144
 FOOD ANALYSIS

PRINCIPLE

Calcium and magnesium form a complex of wine-red color with Eriochrome Black T
at pH of 10.0 ± 0.1. Ethylene diamine tetra-acetic acid (EDTA) has a strong affinity
towards Ca++ and Mg++ and if the sample is titrated with EDTA the former complex is
broken down to form a new soluble complex. The indicator acquires its original blue
color after it is stripped of the cations. The sharpness of the color increases with
increasing pH (10.0 ± 0.1). A limit of 5 min is set for the duration of titration in order
to minimize the tendency towards CaCO3 precipitation.

REQUIREMENTS

 EDTA solution (0.01M)81  Ammonia buffer82

 Eriochrome Black T indicator83  Titration arrangement
 Water sample (at least 1 liter)

PROCEDURE

1. Take 50ml (or some suitable volume) of sample in a 250-ml conical flask
2. Add 1ml ammonia buffer
3. Add 100-200mg of Eriochrome indicator. The solution turns wine-red in the
presence of hardness. Otherwise it will remain sky blue to deep blue
(depending on the amount of indicator used)
4. Titrate the content rapidly against 0.01M EDTA to a blue end point

CALCULATION

ml of EDTA  1000
Hardness (mg/liter) as CaCO3 
Vol. of water (ml)

10.3. CALCIUM IN WATER

BACKGROUND

Calcium is one of the most abundant mineral substances of the natural waters.
Calcium as such has no hazardous effect on human health (up to 1800mg/liter). High
calcium content in water is disadvantageous in household and industrial use. It
suppresses lather formation; produces scales in boilers; if sulfate is present, inhibits
malt fermentation; and with chlorine, inhibits growth of yeasts.

81
Dissolve 3.723g disodium EDTA in distilled water to make 1 liter (1ml of 0.01M EDTA = 1mg of CaCO3
82
Solution A: Dissolve 16.9g NH4Cl in 143ml of conc NH4OH. Solution B: Dissolve 1.179g of disodium EDTA
and 0.79g MgSO4.7H2O in 50ml water. Mix solutions A and B and dilute to 250ml with distilled water
83
Mix Eriochrome Black T with 100g reagent grade NaCl and grind. Store it in a bottle

145
 WATER

PRINCIPLE

Murexide indicator (ammonium purpurate) is a purple-colored reagent that can

selectively react with calcium at a high pH to form a pink-colored complex. When
this mixture is titrated with EDTA, the complex is broken down. EDTA has the
property of binding magnesium also but because of the high pH the latter is
precipitated as Mg(OH)2.

REQUIREMENTS

 Reagents for hardness determination  Sodium hydroxide (~1N)

 Murexide indicator84  Titration arrangement

PROCEDURE

1. Take 50ml (or some suitable volume) of sample in a 250-ml conical flask
2. Add 2ml of 1N NaOH in the sample
3. Add 100-200mg murexide indicator. A pink color develops if calcium is
present. Otherwise, a purple color is seen
4. Titrate rapidly with 0.01M EDTA to a purple end point. A blank consisting of
distilled water can also be used for comparing the color of the end point

CALCULATION

ml of EDTA  1000
Ca ++ , mg/liter 
ml of sample  2.498

If the strength of EDTA is xM instead of 0.01M, multiply the above expression by

(x/0.01)

10.4. MAGNESIUM IN WATER

PRINCIPLE

The value of Mg++ can be obtained by subtracting the value of calcium (obtained
using ammonium purpurate) from total (Ca++ + Mg++) obtained by manipulating the
numerical value of total hardness.

CALCULATION

Assuming that same volume of water was used for the determination of total hardness
as well as calcium, following relation can be worked out:

84
Mix 0.2g of ammonium purpurate with 10g of reagent grade NaCl and grind

146
 FOOD ANALYSIS

ml of EDTA (for total hardness  for calcium)  1000

Mg ++ , mg/liter 
ml of sample  4.116

10.5. ALKALINITY IN WATER

BACKGROUND

Alkalinity of the water is its capacity to neutralize a strong acid and is characterized
by the presence of all hydroxyl ions capable of combining with the hydrogen ion.
Alkalinity in natural waters is due to the presence of free hydroxyl ions, bicarbonates,
carbonates, phosphates, nitrates, etc.

Alkalinity in itself is not harmful to human beings, still water supplies with alkalinity
of <100mg/liter are desirable for domestic use. The alkalinity value is useful in
calculating the dose of alum and biocides in water. Alkalinity-producing substances,
such as sodium bicarbonate, are added to check corrosion in soft water supplies.
Alkalinity measurements are also important in controlling water and wastewater
treatment processes. The ratio of alkalinity to alkaline earth metals is a good
parameter of suitability of irrigation water.

PRINCIPLE

The total alkalinity can be estimated by titrating the sample with a strong acid (HCl or
H2SO4), to pH between 4.2 and 5.4 with methyl orange indicator. The
phenolphthalein alkalinity is estimated by titrating the water sample with a strong
acid to pH of 8.3 using phenolphthalein indicator.

REQUIREMENTS

 0.1N HCl  CO2-free distilled water

 Phenolphthalein indicator  Methyl orange indicator
 Titration arrangement

PROCEDURE

1. Take 100ml (or some suitable volume) of sample in a 250-ml conical flask
2. Add 2 drops of phenolphthalein indicator. If the color does not change, the
phenolphthalein alkalinity (PA) is nil. Determine the total alkalinity
following description given in step 4.
3. If the color changes to pink after the addition of phenolphthalein, there is PA.
Titrate with 0.1N HCl until the color disappears. Note the volume of HCl
consumed, say X ml


See hardness factors for different cations given elsewhere

147
 WATER

4. Now, add 2-3 drops of methyl orange indicator to the same sample (i.e.,
sample from step 2 or 3) and continue titration with HCl to a pink end point.
Note the volume of HCl consumed for this second phase of titration, say Y ml

CALCULATION

X  N of HCl  1000  50
Phenolphthalein alkalinity (mg/liter) as CaCO3 
ml of sample

( X  Y )  N of HCl  1000  50
Total alkalinity (mg/liter) as CaCO3 
ml of sample

10.6. CHLORIDES IN WATER

BACKGROUND

Chlorides occur naturally in all types of waters. The most important source of
chloride in the water is the discharge of domestic sewage. Excess chloride levels in
water are therefore an indicator to pollution. Chloride is harmless to man up to
1500mg/liter but imparts salty or brackish taste at 250-500mg/liter. Chloride corrodes
concrete structures by extracting calcium in the form of calcide. MgCl2-water
generates HCl upon heating, which is corrosive. This creates problems in boilers.

PRINCIPLE

Chlorides are determined by Mohr’s method. When water containing chlorides is

titrated with silver nitrate using potassium chromate as an (internal) indicator), silver
chloride is quantitatively precipitated, and at the end point a reddish brown silver
chromate is formed.

REQUIREMENTS

 Titration arrangement  Silver nitrate (0.02N)85

 Potassium chromate indicator (5% solution86

PROCEDURE

1. Take 50ml of sample in a conical flask and add 2ml of pot-chromate indicator

85
Dissolve 3.4g of analytical reagent grade silver nitrate in distilled water to make 1 liter. Keep the
solution in dark bottle
86
Dissolve 5g K2CrO4 in 50ml of distilled water. Add AgNO3 solution until a definite red precipitate is
formed. Allow to stand for 12 hours, filter, and make up the volume to 100ml

148
 FOOD ANALYSIS

2. Titrate the contents against 0.02N AgNO3 solution until a persistent red tinge
appears. Carry out a blank titration using distilled water. This will also serve
as a control color for the end point

CALCULATION

(Sample titer  Blank titer)  N of AgNO3  1000  35.5

Chloride (mg/liter) 
ml of sample

10.7. IRON BY 1,10-PHENONTHROLINE METHOD

BACKGROUND

All kinds of natural waters have some amounts of iron. Iron in water is not of much
concern form health hazard point of view but is considered as a nuisance if present in
excessive amounts. Iron in excess of 0.3g/liter causes staining of clothes and utensil.
Water with high concentrations of iron is not suitable for processing food and
beverage. In tea and coffee, iron present in water reacts with tannins of the
commodity to form iron tannate, a black inky compound. Coffee becomes unpalatable
at iron levels more than 1mg/liter. Iron in higher concentrations may also cause
vomiting. The limits of iron in drinking water are based on esthetic and taste
considerations rather that the physiological effects.

PRINCIPLE

All iron is converted into ferrous state by boiling with HCl and hydroxylamine
hydrochloride (NH2OH.HCl). The reduced iron chelates with 1,10-phenonthroline at
pH 3.2-3.3 to form an orange-red complex, which is measured colorimetrically at
510nm.

Strong oxidizing agents like cyanides, nitrite, polyphosphates, chromium, etc.,

interfere with the iron determination. The boiling of sample with an acid, coupled by
the addition of hydroxylamine hydrochloride removes this interference.

REQUIREMENTS

 Conc HCl  Hydroxylamine-HCl solution87

88
 Ammonium acetate buffer  1,10-phenonthroline solution89
90
 Stock iron solution (200mg/liter)  Working standard iron (10mg/liter)91

87
Dissolve 10g of NH2OH.HCl in distilled water to make 100ml
88
Dissolve 250g of ammonium acetate in 150ml distilled water and add 700ml glacial acetic acid
89
Dissolve 200mg of 1,10-phenonthroline monohydrate in 100ml of distilled water by thorough stirring
and heating up to 80°C (should not be boiled)

149
 WATER

PROCEDURE

1. Take 50ml (or an aliquot containing not more than 4mg iron/liter) in a 200-ml
conical flask
2. Add 2ml conc HCl and 1ml hydroxylamine hydrochloride
3. Boil the contents to half the volume to dissolve all the iron
4. Cool, and add 10ml ammonium acetate buffer and 2ml of 1,10-
phenonthroline reagent
5. Make up the volume to 100ml, and after 10 min, take the absorbance in a
spectrophotometer at 510 nm
6. Prepare standard curve in the range 1 to 4mg iron/liter using various dilutions
of standard solution. Follow steps 2 5
7. Calculate the iron content from the standard curve☻

10.8. MICROBIOLOGICAL ANALYSIS OF WATER

BACKGROUND

The microbiological examination of water enjoys a special status in pollution studies

as it is a direct measurement of sanitary status of water. The microbiological
examinations are routinely conducted to ensure the safety routinely conducted to
ensure the safety of potable water, monitor the water quality for recreational,
industrial and agricultural uses and also to evaluate prospective resources for drinking
purposes.

Methods of examining water bacteriologically are set forth in the book of “Standard
Methods for the Examination of Water and Wastewater”, prepared and published
jointly by the American Public Health Association (APHA), the American Water
Association, and the Water Pollution Control Federation. The Environmental
Protection Agency (EPA) has also developed a manual for this purpose.

Routine microbiological test of water consists of:

 Plate count to determine the total number of bacteria

 Test to reveal the presence of coliform bacteria

90
Add some conc H2SO4 in 50ml of distilled water. Dissolve in this solution 1.404g of ferrous ammonium
sulfate [Fe(NH4)2SO4.6H2O]. Add 0.1N KMnO4 slowly until a persistent pink color appears. Dilute to
1000ml
91
Dilute stock solution 20 times in distilled water
☻
If the sample contains less than 1mg iron/liter, mix standard iron solution of strength 4mg iron/liter
and the sample in the ratio 1:1 (i.e., 25ml each). Follow steps 2  5 and compare with the reading for
standard containing 2mg iron/liter. If the absorbance for the standard =A and that of the mixture = B,
in the above condition, iron content of the sample in mg/liter = 2(B-1)/A

150
 FOOD ANALYSIS

It is essential that strict attention be given to the following details when water samples
are collected for bacteriological analysis:

 The sample must be collected in a sterile bottle

 The sample must be representative of the supply from which it is taken
 Contamination of the sample must be avoided during and after sampling
 The sample must be tested as promptly as possible after collection
 If there is delay in examination of the sample it should be stored at a
temperature between 0 and 10°C

10.8.1. THE STANDARD PLATE COUNT OF WATER SAMPLE

BACKGROUND

The Standard Plate Count (SPC), also called Aerobic Plate Count (APC), provides the
density of aerobic and facultative bacteria in water sample. The test is useful in
warning about the excessive microbial load in any water and also in judging the
efficiency of water treatment in removing bacteria. A good water may be expected to
have SPC value of less than 100/ ml.

The SPC value actually represents only a fraction of the true number because many
bacteria fail to grow in the given medium: the medium simple cannot support the
growth of bacteria with diverse cultural and environmental requirements. No great
error is introduced by this failure, though. For, in general, the normal flora of water
are not of much sanitary significance.

PRINCIPLE

Colony counts are performed after plating the aliquots of the water sample in Plate
Count Agar (PCA) or Tryptone Glucose Yeast Extract Agar (TGYA) at 35 (or 37)°C
and 20 (or 22)°C and the result expressed as colony forming units per milliliter
(cfu/ml)

REQUIREMENTS

 Water sample  PCA medium92

 Sterile pipettes (1-ml cap)  Sterile Petri plates
 Dilution blanks (9-ml tubes, 2)  General facilities existing in
microbiology lab

92
The medium is commercially available. It can also be prepared as follows:
Take 2.5g yeast extract, 5g tryptone, 1g glucose, 1g skim milk powder (SMP) and, 12-18g agar. Dissolve
the ingredients in 1000ml water by boiling. Adjust pH to 6.9±0.1 with dilute NaOH or HCl and sterilize
at 121°C for 15 min.

151
 WATER

PROCEDURE

1. Label 4 Petri plates with “control”, “1”, “10-1”, and “10-2” to represent
(respectively) control, no dilution, and 10-fold dilution, and 100-fold dilution
2. Make two more sets for the triplicate analysis. You can omit the “control”
this time
3. Shake the sample at least 25 times to obtain uniform distribution of
microorganisms
4. Transfer aseptically (see Fig. X-1 and Fig. X-2) with a single pipette 1ml
each of water sample to the plates labeled “1” and another 1ml to a 9-ml
dilution blank
5. Mix the inoculated blank thoroughly by rotating the tube between the palms,
followed by a couple of quick jerk. Don’t worry if the cotton plug soaks up
some water
6. Take another sterile pipette and transfer 1ml each to the plates labeled “10-1”
and another 1ml to yet another 9-ml dilution blank
7. Do as in step 4
8. Take another sterile pipette and transfer 1ml each to the plates labeled “10-2”
9. Melt the medium thoroughly and cool it to ≈ 50°C
10. Pour the medium aseptically to all the Petri plates at the rate of 20ml per plate
11. Mix the contents thoroughly by swiftly moving the plates in the shape of “8”
for 10 times (don’t spill the medium in the process)
12. Allow the agar to harden, invert the plates and incubate them at 37°C for 48
hours
13. Count the plates showing 30-300 colonies. If the total number of colonies
given by undiluted sample is less than 30, record the result disregarding the
above rule. Register the spreading (but non-overlapping) colony as a single
colony

Sterile
Sterile zone
zone

cotton
plug
Fig. X- 1:Aseptic inoculation Fig. X- 2: Aseptic pipetting

CALCULATION

cfu/ml  Average number of colonies  fold of dilution

152
 FOOD ANALYSIS

10.8.2. THE PRESUMPTIVE COLIFORM TEST BY MEMBRANE FILTRATION

BACKGROUND

Polluted water is a hazard to health. The most important pollution that results in
water-borne diseases is the fecal pollution. Unfortunately, while not all fecal bacteria
are pathogenic, the few that are so are the hardest to determine by routine methods.
The search for organisms of fecal origin that would be easy to determine and also
reliably indicate the possibility of fecal pollution (and therefore the presence of
pathogens) led to the concept of “indicator organisms”. For routine purpose, the most
widely accepted indicator organisms are the coliforms, particularly Escherichia coli.

Coliforms include all aerobic and facultatively anaerobic, Gram-negative, non-

sporulating rods which ferment lactose to produce acid and gas within 48 hours at
35°C. The classical examples of coliforms are Escherichia coli and Enterobacter
aerogenes. Other examples include the genera Citrobacter and Klebsiella.

PRINCIPLE

The presumptive coliform test is the first step in the standard test of water fork the
presence of coliforms. The standard test consists of three sequential tests, viz.,
(i) presumptive, (ii) confirmed, and (iii) completed test. Several selective and
differential media (as also the methods) are available for the presumptive test. The
membrane filter technique is a very rapid technique compared to the classical
Multiple Tube Fermentation. The membrane filter method consists of filtration of a
measured volume of water through a sterile membrane filter, in situ growth of the
“residue” bacteria on a selective-cum-differential medium, and direct counting of the
typical colonies.

REQUIREMENTS

 Membrane filter assembly (Fig. X-3 and X-4)  Water sample

 Endo broth93  Sterile Petri plate
 Sterile pipette  Autoclave
 Forceps

PROCEDURE

1. Sterilize the different parts of the membrane filter unit (you may skip the
filter receiving unit) in an autoclave at 15 psig for 15 min
2. Place a sterile Millipore filter membrane on the filter holder with a forceps

93
Dissolve in 1000ml water: 3.5g KH2PO4, 10g peptone, 10g lactose, 0.5g basic fuchsim, and 2.5g sod
sulfite. Sterilize in autoclave.

153
 WATER

3. Put the adsorbent pad (that comes along with the filter membrane) on a sterile
Petri plate. Place 2ml of Endo broth on the pad and keep the plate covered
until needed
4. Place the funnel over the filter holder (that contains the filter membrane). Join
the two with a clamp

Funnel Forceps
100
Clamp 50
100
Clamp 50

Cork
Filter
Porous membrane
filter holder

To vacuum

Receiving flask Receiving flask

Fig. X- 3: Detailed view of membrane Fig. X- 4: Assembled view of membrane
filter filter

5. Insert the above unit in the side-armed receiving flask (that is connected to
vacuum pump)
6. Pour 100ml of water sample in the funnel and affect rapid filtration with the
attached vacuum pump
7. Detach the funnel from the filter holder
8. With sterile forceps, take out the filter membrane and place it on the pad
previously impregnated width Endo broth (step 2)
9. Observe the colonies with pink to red rose color showing distinguishable
green metallic “sheen”
10. Report the finding as coliform/100ml water

154
CHAPTER XI: ANALYSIS OF COMMON SALT

11.1. SALT CONTENT BY POTASSIUM DICHROMATE METHOD

PRINCIPLE

Silver nitrate reacts with sodium chloride to form silver chloride and sodium nitrate
quantitatively. The end point is determined with an internal indicator such as
potassium chromate.

REQUIREMENTS

 Silver nitrate (0.1N), prepared and standardized as usual

 Potassium chromate indicator (5% solution in distilled water)

PROCEDURE

1. Weigh 2g of salt sample

2. Dissolve in distilled water to make 250ml in a volumetric flask
3. Pipette out 25ml of the sample solution in a clean conical flask
4. Add 1ml of potassium chromate indicator
5. Keep silver nitrate solution in a burette
6. Titrate with silver nitrate until the appearance of a permanent reddish tinge
7. Note the volume of silver nitrate consumed. This is called sample titer
8. Carry out a blank determination along side as follows: take 25ml of distilled
water (the same water used for preparing the sample solution), add 1ml of
potassium chromate, and titrate with silver nitrate to the similar permanent red
tinge. Note the volume silver nitrate consumed. This volume is called blank titer.

CALCULATION

(Sample titer  Blank titer)  N of silver nitrate  5.845

% NaCl (wet basis) 
gram of sample in the aliquot

Note: This method is generally used for the determination of chloride content in water, rather
than NaCl. This method is also used for determining salt in ketchup and similar products.
 SALT

11.2. IODINE CONTENT IN COMMON SALT

Iodine is a natural element in top soil. It is absorbed by and utilized by plants,

including food grains, vegetables which we consume. Loss of iodine occurs from the
loss of top soil by erosion. In Nepal due to erosion of soil there is a lack of iodine in
most foods included in the normal diet and iodine deficiency has long been endemic.
Most peoples living in isolated place far from the sea are suffering from IDD.

Iodine is needed in very small amounts but essential for normal growth and
development of animals and plant. It is essential for the synthesis of thyroid hormones
and in a state of iodine deficiency, thyroid hormones are synthesized inadequately
resulting in decreased metabolism of key nutrients affecting overall development.

For endemic goiter areas each person should get daily supplement of 150μg of iodine.
When goitrogenic factors occur it may be necessary to increase the supplement to 300
to 400μg

The average daily consumption of salt per person may be taken as 10g (2 to 20g in
range). Therefore, in order to ensure a daily intake of 150μg of iodine, the minimum
level of iodine supplement is 25 ppm. In fact the iodization level should not be less
than 50 ppm so as to compensate for the loss which takes place between salt
iodization plant and the consumer.

PRINCIPLE

The test is essentially an iodometric test. The sample is treated to sulfuric acid to
liberate the iodine present in it. The liberated iodine is reacted with standard sodium
thiosulfate and the stoichiometric relation used to calculate the iodine content. It can
be shown that 1ml of 0.005N sodium thiosulfate ≡ 0.001058g of iodine

REQUIREMENTS

 Starch indicator (1%): Dissolve 1g starch in 100ml of hot distilled water. Use
fresh
 10% potassium iodide solution: Dissolve 10g of potassium iodide in distilled
water to make 100ml. No need to use a volumetric flask
 Sulfuric acid (about 2N): In a 100ml volumetric flask take some distilled
water. Measure out 5.56ml of concentrated sulfuric acid and to the flask
slowly. Mix well
 Potassium iodate solution (0.005N), prepare as follows:
 Dry a small amount of potassium iodate at 120°C in hot-air oven for 1 hr.
 Cool the reagent in desiccator.
 Quickly weigh (0.35675 ÷ purity factor)g of potassium iodate and dissolve
in distilled water in a 100-ml volumetric flask.
 Take out 50ml of this stock solution in distilled water to 1000ml in a
volumetric flask
156
 FOOD ANALYSIS

 Sodium thiosulfate solution (0.005N), prepare as follows:

 Weigh 2.5g of sodium thiosulfate pentahydrate and dissolve in distilled
water in a volumetric flask to make 100ml.
 Take out 50ml of this stock solution and dilute it to 1000ml in distilled
water in a volumetric flask.
 Standardize the sodium thiosulfate solution as follows:
o Place sodium thiosulfate solution in a burette
o Take 25ml of potassium iodate solution in a conical flask
o Add 2ml of sulfuric acid (2N) and shake to mix
o Add 5ml of potassium iodide (10% solution) and shake to mix
o Titrate with sodium thiosulfate until the color becomes pale yellow
o Add 1ml of starch solution. The mixture turns deep purple due to free
iodine
o Continue titration until the purple color completely disappears
o Note the volume of sodium thiosulfate consumed
o Calculate the normality of sodium thiosulfate as follows:

N of pot. iodate  ml of pot. iodate

Normality of sod. thiosulfate 
ml of sod. thiosulfate

PROCEDURE FOR IODINE DETERMINATION (See Fig. XI-1)

1. Weigh 10g of salt sample

2. Transfer the sample to a clean conical flask with 50ml of distilled water
3. Add 2ml of sulfuric acid (2N)
4. Add 5ml of potassium iodide (10% solution) and shake to mix. The color of
the solution turns deep yellow
5. Cover the conical flask with watch glass and keep it in dark for 10 minutes
6. Take the sodium thiosulfate solution (standard prepared above) in burette
7. Titrate the salt solution with sodium thiosulfate until the color turns pale
yellow
8. Add 1ml starch solution to the mixture. The color of the mixture should turn
purple
9. Continue titration (this time, add sodium thiosulfate drop by drop) until the
deep blue color completely disappears
10. Note down the volume of sodium thiosulfate solution needed for the titration
11. Calculate the iodine content (in ppm) using the formula given below:

R  100  1000  0.127  N

Iodine (ppm, wet basis)   R  N  2116.67
6

Where, R = burette reading (ml), N = Normality of sodium thiosulfate

157
 SALT

2 10g salt

3 50ml distilled water

1
4 2ml H2SO4, 2N
11

12

24 5 5ml pot. iodide (10% solution)

25

Deep yellow color

250ml
Sod. thiosulfate
soution (0.005N) 6 Covering

7
Stand for 10 min

Titrate to pale yellow color

1ml starch

8 9

Titrate to purple color Titrate until blue color disappears

Fig. XI-1: Outline of method for iodine determination in salt

Note: The numbers in the circle represents the sequence of operation

11.3. TEST OF MAGNESIUM AND CALCIUM

PRINCIPLE

Eriochrome Black-T is blue in color. When it combines with calcium and/or

magnesium, it forms a wine red complex. Calcium and magnesium can also form
complex with EDTA stoichiometrically at a pH of 10.1±0.1. In fact, the EDTA can
strip the magnesium and calcium from the wine red complex to produce free
(uncomplexed) Eriochrome Black-T. Thus, at the end point, the color of mixture
returns to pure blue.

Murexide (ammonium purpurate) is purple in color. At pH around 13, it combines

with calcium preferentially to form a wine red complex. The magnesium, even if
present, is converted to magnesium hydroxide, thus making it unavailable for the
complexing reaction. When EDTA is added, it strips calcium from murexide
complex. The color of the solution will return to purple color at the end point.

158
 FOOD ANALYSIS

REQUIREMENTS

 Eriochrome black-T indicator94  Standard calcium solution95

 Standard EDTA solution96  Ammonia buffer solution97
 10% NaOH  Murexide indicator98
 Weighing arrangement  Glasswares (flasks, burette, pipette, etc)
 Sample 

PROCEDURE FOR CALCIUM AND MAGNESIUM CONTENT

1. Weigh 2g of salt sample and dissolve in distilled to make 100ml in a

volumetric flask
2. Pipette out 50ml of the above solution in a conical flask
3. Add 10ml of buffer solution
4. Add 5 drops of Eriochrome Black T indicator
5. The color of the sample should be wine red if magnesium is present
6. Titrate with EDTA until a pure blue color is obtained
7. Note the volume of EDTA consumed. Let this be B ml
8. Take in another conical flask 50ml of the sample solution again
9. Add 5ml of sodium hydroxide solution (prepared above)
10. Add 0.2g of murexide indicator. Alternatively, add 100mg of calcein
indicator
11. Titrate with EDTA to a definite end point. The color will be changed from
wine red color to purple color if murexide indicator is used.
12. Note the volume of EDTA consumed. Let this be C ml
13. Calculate the calcium and magnesium as follows:

A  C  1000
% Calcium as Ca ++ (dry basis) 
W  Dry matter

94
Dissolve 0.1g in 20ml of methanol or rectified spirit. Prepare fresh every week
95
Dry calcium carbonate at 120°C, cool, weigh 1.0g, dissolve in minimum quantity of dilute HCl (1+1)
dropwise, and make up to 1 liter. 1ml of this solution is equivalent to 0.4008mg of calcium (as Ca)
96
Dissolve 3.72g of disodium salt of EDTA in distilled water and make to 1000ml. Standardize against
standard calcium solution as follows: Take 25ml of standard calcium solution in a conical flask, add
25ml of water, 10ml of buffer solution, 5 drops of the Eriochrome Black T indicator, and titrate
against EDTA to a blue end point. Calculate the calcium equivalent of 1ml of EDTA (that is, gram of
calcium per ml of EDTA), Let this be A
97
Ammonia buffer solution: Dissolve 67.5g of ammonium chloride in a mixture of 570ml of ammonia
(sp. grav. 0.90) and 250ml of distilled water. Separately, dissolve a mixture of 0.931g of disodium salt
of EDTA dihydrate and 0.616g of magnesium sulfate heptahydrate in about 50ml of distilled water.
Mix the two solutions and make up to 1 liter
98
Grind 0.2g of murexide with 10g of sodium chloride until the mixture is homogenous. Alternatively,
calcein indicator can also be prepared as follows: Mix 0.1g of calcein and 0.06g of thymophthalein
with 10g K potassium chloride

159
 SALT

6068  A( B  C )
% Magnesium as Mg ++ (dry basis) 
W  Dry matter

Where, W = gram of salt in the 50ml sample solution taken for titration. (In the
present case, W = 1g).

160
BIBLIOGRAPHY

Jayaram, J. (1992). Laboratory Manual in Biochemistry. Wiley Eastern Ltd

KC J.B. and Rai B.K. (2000). Experiments in Basic Food Microbiology. Ekta
Publishers, Nepal
Das, D. (1993). Biochemistry. 8th edn. Academic Publishers, Calcutta
Conn, E. and Stumph, P.K. (1972). Outlines of Biochemistry. 3rd edn. John Wiley
and Sons Inc
Plummer, D.T. (1988). An Introduction to Practical Biochemistry. 3rd edn
Ranganna, S. (1986). Handbook of Analysis and Quality Control of Fruit and
Vegetable Products. 2nd edn. Tata McGraw-Hill Publ. co. India
Sadasivam, S. and Manickam A. (1992). Biochemical Methods in Agricultural
Sciences
Williams, K.A. (1966). Oils, Fats and Fatty Foods: Their Practical Examination. J&A
Churchill Limited
Aneja, K.R. (1996). Experiments in Microbiology, Plant Pathology and Tissue
Culture. 2nd edn. Wishwa Prakashan, India
AOAC (2005). Association of Official Analytical Chemists
Anonymous (2005). Manual of Methods of Analysis of Foods: Oils and Fats,
Directorate General of Health Services, Ministry of Health and Family
Welfare, Govt. of India, New Delhi
Trivedy, R.K. and Goel, P.K. (1984). Chemical and Biological Methods for Water
Pollution Studies, Environmental Publications, India
APPENDICES

APPENDIX 1

EQUIVALENT AND FORMULA WEIGHTS

Chemical Molecular formula Formula wt Equivalent wt
Citric acid, anhydrous C6H8O7 192 64
Citric acid, monohydrate C6H8O7.H2O 210 70
Hydrochloric acid HCl 36.5 36.5
Lactic acid C3H6O3 90 90
Malic acid C4H6O5 134 67
Oxalic acid dihydrate C2H2O4.2H2O 126 63
Phosphoric acid H3PO4 97.98 32.66
Sulfuric acid H2SO4 98.08 49.04
Tartaric acid C4H6O6 150 75

Chemical Molecular formula Formula wt Equivalent wt

Aluminum hydroxide Al(OH)3 77.98 26
Ammonium hydroxide NH4OH 35 35
Barium hydroxide Ba(OH)2 171.36 85.68
Calcium carbonate CaCO3 100.08 50.04
Calcium hydroxide Ca(OH)2 74.08 37.04
Potassium carbonate K2CO3 138 69
Potassium hydroxide KOH 56.1 56.1
Sodium bicarbonate NaHCO3 84 84
Sodium carbonate Na2CO3 106 53
Sodium hydroxide NaOH 40 40

Chemical Molecular formula Formula wt Equivalent wt

Ferric chloride FeCl3 162.5 54.2
Ferrous amm. sulfate.6H2O Fe(NH4)2(SO4)2.6H2O 392.14 196.07
Ferrous sulfate FeSO4 152 152
Potassium chloride KCl 74.6 74.6
Potassium dichromate K2Cr2O7 294.222 49.037
Potassium iodate KIO3 214.02 35.67
Potassium oxalate K2C2O4 166.22 83.11
Potassium permanganate KMnO4 158 31.6
 FOOD ANALYSIS

Chemical Molecular formula Formula wt Equivalent wt

Silver nitrate AgNO3 169.87 169.87
Sodium chloride NaCl 58.5 58.5
Sodium oxalate Na2C2O4 134 67
Sodium thiosulfate Na2S2O3 158 158
Sod. Thiosulfate.5H2O Na2S2O3.5H2O 248 248
Zinc sulfate ZnSO4 161.446 80.723

Preparation of acids and bases of approximate strength

Substance Specific Normality % m/m ml to be taken to make

gravity, g/ml 1L of 1N (approx
solution)
Hydrochloric acid 1.18 11.3 35 89
1.16 10.33 32 98.2
Nitric acid 1.42 16.0 70 63
Sulfuric acid 1.84 36 96 28
Phosphoric acid 1.69 41.1 85 23
Acetic acid 1.05 17.4 99.5 58
Ammonium 0.90 14.3 27 71
hydroxide

163
 APPENDIX

APPENDIX 2

INTERNATIONAL ATOMIC WEIGHTS (based on 12C = 12)

Element Atomic weight Element Atomic weight
Actinium (Ac) 227.00 Mercury (Hg) 200.59
Aluminum (Al) 26.9815 Molybdenum (Mo) 95.94
Americium (Am) 243.00 Neodymium (Nd) 144.24
Antimony (Sb) 121.75 Neon (Ne) 20.183
Argon (Ar) 39.948 Neptunium (Np) 273.00
Arsenic (As) 74.9216 Nickel (Ni) 58.71
Astatine (At) 210.00 Niobium (Nb) 92.906
Barium (Ba) 137.34 Nitrogen (N) 14.0067
Berkelium (Bk) 247.00 Nobelium (No) 255.0
Beryllium (Be) 9.0122 Osmium (Os) 190.2
Bismuth (Bi) 208.980 Oxygen (O) 15.9994
Boron (B) 10.811 Palladium (Pd) 106.4
Bromine (Br) 79.909 Phosphorus (P) 30.9738
Cadmium (Cd) 112.40 Platinum (Pt) 195.09
Calcium (Ca) 40.08 Plutonium (Pu) 244.00
Californium (Cf) 251.00 Polonium (Po) 209.00
Carbon (C) 12.01115 Potassium (K) 39.102
Cerium (Ce) 140.12 Praseodymium (Pr) 140.907
Cesium (Cs) 132.905 Promethium (Pm) 145.00
Chlorine (Cl) 35.453 Protactinium (Pa) 231.00
Chromium (Cr) 51.996 Radium (Ra) 266.00
Cobalt (Co) 58.9332 Radon (Rn) 222.00
Copper (Cu) 63.54 Rhenium (Re) 186.2
Curium (Cm) 247.00 Rhodium (Rh) 102.905
Dysprosium (Dy) 162.50 Rubidium (Rb) 85.47
Einsteinium (Es) 254.00 Ruthenium (Ru) 101.07
Erbium (Er) 167.26 Samarium (Sm) 150.35
Europium (Eu) 151.96 Scandium (Sc) 44.956
Fermium (Fr) 257.00 Selenium (Se) 78.96
Fluorine (F) 18.9984 Silicon (Si) 28.086
Francium (Fr) 223.00 Silver (Ag) 107.870
Gadolinium (Gd) 157.25 Sodium (Na) 22.9898
Gallium (Ga) 69.72 Strontium (Se) 87.62
Germanium (Ge) 72.59 Sulfur (S) 32.064
Gold (Au) 196.967 Tantalum (Ta) 180.948
Hafnium (Hf) 178.49 Technetium (Tc) 97.00
Helium (He) 4.0026 Tellurium (Te) 127.60
Holmium (Ho) 164.930 Terbium (Tb) 158.924
Hydrogen (H) 1.00797 Thallium (Tl) 204.37
Indium (In) 114.82 Thorium (Th) 232.038
Iodine (I) 126.9044 Thulium ™ 163.934
Iridium (Fr) 192.2 Tin (Sn) 118.69

164
 FOOD ANALYSIS

Element Atomic weight Element Atomic weight

Iron (Fe) 55.847 Titanium (Ti) 47.90
Krypton (Kr) 83.80 Tungsten (W) 183.85
Lanthanum (La) 138.91 Uranium (U) 238.03
Lawrencium (Lw) 256.00 Vanadium (V) 50.9415
Lead (Pb) 207.19 Xenon (Xe) 131.30
Lithium (Li) 6.9417 Ytterbium (Yb) 173.04
Lutetium (Lu) 174.967 Yttrium (Y) 88.905
Magnesium (Mg) 24.312 Zinc (Zn) 65.37
Manganese (Mn) 54.9380 Zirconium (Zr) 91.22
Mendelevium (Md) 258.00

165
 APPENDIX

APPENDIX 3

ALCOHOL CONTENT AND SPECIFIC GRAVITY OF ALCOHOLIC SOLUTION

100
95

90
85
80

75

70
Alcohol content (% v/v) at 15.56°C

65
60
55

50
45
40

35
30

25
20
15

10
5
0
0.7839 0.8039 0.8239 0.8439 0.8639 0.8839 0.9039 0.9239 0.9439 0.9639 0.9839
Specific gravity (30°C)

Graphical representation for a rough estimate of alcohol content

Specific gravity versus alcohol content (tabular form)

Note: The specific gravity has been measured at 30°C and the corresponding alcohol content is
expressed as % vol/vol at 15.56°C (60°F).

S. G. A. C. S. G. A. C. S. G. A. C. S. G. A. C. S. G. A. C. S. G. A. C. S. G. A. C.
1.0000 0.00 0.9690 23.77 0.9380 43.98 0.9070 59.00 0.8760 71.85 0.8450 83.17 0.8140 92.84
0.9995 0.33 0.9685 24.16 0.9375 44.25 0.9065 59.21 0.8755 72.05 0.8445 83.34 0.8135 92.98
0.9990 0.66 0.9680 24.54 0.9370 44.52 0.9060 59.43 0.8750 72.24 0.8440 83.51 0.8130 93.12
0.9985 0.99 0.9675 24.93 0.9365 44.79 0.9055 59.65 0.8745 72.43 0.8435 83.68 0.8125 93.26
0.9980 1.32 0.9670 25.32 0.9360 45.06 0.9050 59.87 0.8740 72.63 0.8430 83.85 0.8120 93.40
0.9975 1.66 0.9665 25.70 0.9355 45.32 0.9045 60.09 0.8735 72.82 0.8425 84.02 0.8115 93.53
0.9970 2.00 0.9660 26.09 0.9350 45.58 0.9040 60.31 0.8730 73.01 0.8420 84.18 0.8110 93.67
0.9965 2.34 0.9655 26.47 0.9345 45.85 0.9035 60.53 0.8725 73.20 0.8415 84.35 0.8105 93.80
0.9960 2.68 0.9650 26.85 0.9340 46.11 0.9030 60.75 0.8720 73.39 0.8410 84.52 0.8100 93.94
0.9955 3.02 0.9645 27.22 0.9335 46.37 0.9025 60.97 0.8715 73.58 0.8405 84.69 0.8095 94.07
0.9950 3.37 0.9640 27.59 0.9330 46.63 0.9020 61.18 0.8710 73.77 0.8400 84.85 0.8090 94.20
0.9945 3.72 0.9635 27.96 0.9325 46.89 0.9015 61.40 0.8705 73.97 0.8395 85.02 0.8085 94.33
0.9940 4.06 0.9630 28.33 0.9320 47.15 0.9010 61.61 0.8700 74.16 0.8390 85.18 0.8080 94.47

166
 FOOD ANALYSIS

S. G. A. C. S. G. A. C. S. G. A. C. S. G. A. C. S. G. A. C. S. G. A. C. S. G. A. C.
0.9935 4.42 0.9625 28.70 0.9315 47.40 0.9005 61.83 0.8695 74.31 0.8385 85.35 0.8075 94.60
0.9930 4.77 0.9620 29.07 0.9310 47.65 0.9000 62.04 0.8690 74.54 0.8380 85.51 0.8070 94.73
0.9925 5.12 0.9615 29.43 0.9305 47.91 0.8995 62.26 0.8685 74.73 0.8375 85.68 0.8065 94.86
0.9920 5.49 0.9610 29.80 0.9300 48.16 0.8990 62.47 0.8680 74.92 0.8370 85.84 0.8060 94.89
0.9915 5.85 0.9605 30.16 0.9295 48.41 0.8985 62.68 0.8675 75.10 0.8365 86.00 0.8055 95.11
0.9910 6.23 0.9600 30.51 0.9290 48.66 0.8980 62.89 0.8670 75.29 0.8360 86.16 0.8050 95.24
0.9905 6.60 0.9595 30.87 0.9285 48.91 0.8975 63.11 0.8665 75.48 0.8355 86.33 0.8045 95.37
0.9900 6.98 0.9590 31.22 0.9280 49.16 0.8970 63.32 0.8660 75.66 0.8350 86.49 0.8040 95.50
0.9895 7.36 0.9585 31.56 0.9275 49.41 0.8965 63.53 0.8655 75.85 0.8345 86.65 0.8035 95.62
0.9890 7.74 0.9580 31.91 0.9270 49.66 0.8960 63.74 0.8650 76.04 0.8340 86.81 0.8030 95.75
0.9885 8.12 0.9575 32.25 0.9265 49.91 0.8955 63.95 0.8645 76.22 0.8335 86.97 0.8025 95.87
0.9880 8.50 0.9570 32.58 0.9260 50.15 0.8950 64.16 0.8640 76.41 0.8330 87.13 0.8020 96.00
0.9875 8.89 0.9565 32.92 0.9255 50.40 0.8945 64.37 0.8635 76.59 0.8325 87.29 0.8015 96.12
0.9870 9.27 0.9560 33.25 0.9250 50.64 0.8940 64.58 0.8630 76.78 0.8320 87.45 0.8010 96.25
0.9865 9.72 0.9555 33.59 0.9245 50.88 0.8935 64.79 0.8625 76.96 0.8315 87.61 0.8005 96.37
0.9860 10.11 0.9550 33.92 0.9240 51.13 0.8930 65.00 0.8620 77.15 0.8310 87.77 0.8000 96.49
0.9855 10.50 0.9545 34.25 0.9235 51.37 0.8925 65.21 0.8615 77.33 0.8305 87.93 0.7995 96.61
0.9850 10.89 0.9540 34.57 0.9230 51.61 0.8920 65.42 0.8610 77.51 0.8300 88.08 0.7990 96.73
0.9845 11.29 0.9535 34.90 0.9225 51.85 0.8915 65.63 0.8605 77.69 0.8295 88.24 0.7985 96.85
0.9840 11.68 0.9530 35.22 0.9220 52.09 0.8910 65.83 0.8600 77.88 0.8290 88.40 0.7980 96.97
0.9835 12.07 0.9525 35.53 0.9215 52.33 0.8905 66.04 0.8595 78.06 0.8285 88.55 0.7975 97.09
0.9830 12.48 0.9520 35.85 0.9210 52.57 0.8900 66.25 0.8590 78.24 0.8280 88.71 0.7970 97.21
0.9825 12.88 0.9515 36.16 0.9205 52.81 0.8895 66.45 0.8585 78.42 0.8275 88.87 0.7965 97.32
0.9820 13.29 0.9510 36.47 0.9200 53.05 0.8890 66.66 0.8580 78.60 0.8270 89.02 0.7960 97.44
0.9815 13.70 0.9505 36.78 0.9195 53.28 0.8885 66.87 0.8575 78.78 0.8265 89.18 0.7955 97.56
0.9810 14.11 0.9500 37.09 0.9190 53.51 0.8880 67.07 0.8570 78.96 0.8260 89.33 0.7950 97.67
0.9805 14.51 0.9495 37.39 0.9185 53.75 0.8875 67.28 0.8565 79.14 0.8255 89.48 0.7945 97.78
0.9800 14.92 0.9490 37.70 0.9180 53.98 0.8870 67.48 0.8560 79.32 0.8250 89.64 0.7940 97.89
0.9795 15.34 0.9485 38.00 0.9175 54.21 0.8865 67.68 0.8555 79.49 0.8245 89.79 0.7935 98.01
0.9790 15.59 0.9480 38.30 0.9170 54.45 0.8860 67.89 0.8550 79.67 0.8240 89.94 0.7930 98.12
0.9785 16.00 0.9475 38.60 0.9165 54.68 0.8855 68.09 0.8545 79.85 0.8235 90.09 0.7925 98.23
0.9780 16.41 0.9470 38.90 0.9160 54.92 0.8850 68.29 0.8540 80.03 0.8230 90.24 0.7920 98.33
0.9775 16.83 0.9465 39.19 0.9155 55.15 0.8845 68.48 0.8535 80.02 0.8225 90.39 0.7915 98.42
0.9770 17.24 0.9460 39.49 0.9150 55.38 0.8840 68.69 0.8530 80.38 0.8220 90.54 0.7910 98.55
0.9765 17.65 0.9455 39.78 0.9145 55.61 0.8835 68.89 0.8525 80.56 0.8215 90.69 0.7905 98.63
0.9760 18.07 0.9450 40.07 0.9140 55.84 0.8830 69.09 0.8520 80.73 0.8210 90.84 0.7900 98.76
0.9755 18.48 0.9445 40.35 0.9135 56.07 0.8825 69.29 0.8515 80.91 0.8205 90.99 0.7895 98.87
0.9750 18.90 0.9440 40.64 0.9130 56.30 0.8820 69.49 0.8510 81.09 0.8200 91.13 0.7890 98.97
0.9745 19.32 0.9435 40.93 0.9125 56.53 0.8815 69.69 0.8505 81.26 0.8195 91.27 0.7885 99.07
0.9740 19.74 0.9430 41.21 0.9120 56.76 0.8810 69.89 0.8500 81.44 0.8190 91.42 0.7880 99.18
0.9735 20.15 0.9425 41.49 0.9115 56.99 0.8805 70.09 0.8495 81.61 0.8185 91.56 0.7875 99.28
0.9730 20.56 0.9420 41.77 0.9110 57.21 0.8800 70.29 0.8490 81.78 0.8180 91.71 0.7870 99.38
0.9725 20.97 0.9415 42.06 0.9105 57.44 0.8795 70.48 0.8485 81.96 0.8175 91.85 0.7865 99.48
0.9720 21.38 0.9410 42.33 0.9100 57.66 0.8790 70.68 0.8480 82.13 0.8170 92.00 0.7860 99.58

167
 APPENDIX

S. G. A. C. S. G. A. C. S. G. A. C. S. G. A. C. S. G. A. C. S. G. A. C. S. G. A. C.
0.9715 21.79 0.9405 42.61 0.9095 57.89 0.8785 70.88 0.8475 82.30 0.8165 92.14 0.7855 99.68
0.9710 22.19 0.9400 42.89 0.9090 58.11 0.8780 71.07 0.8470 82.48 0.8160 92.28 0.7850 99.78
0.9705 22.59 0.9395 43.16 0.9085 58.33 0.8775 71.27 0.8465 82.65 0.8155 92.42 0.7845 99.88
0.9700 22.98 0.9390 43.44 0.9080 58.55 0.8770 71.46 0.8460 82.82 0.8150 92.56 0.7840 99.98
0.9695 23.38 0.9385 43.71 0.9075 58.77 0.8765 71.66 0.8455 83.00 0.8145 92.70 0.7839 100.00

S.G. = specific gravity at 30°C, A.C. = alcohol content (% v/v) at 15.56°C (60°F)

168
 FOOD ANALYSIS

APPENDIX 4

NUTRIENT CONTENT OF NEPALESE FOODS, HMG-N (2043)

CEREAL AND CEREAL PRODUCTS

Common name Edible Moisture, Protein, Fat, Ash, Fiber, Carb, Energy, Ca, P, Fe,
part, % g g g g g g kcal mg mg mg
Bajra 84 12.4 11.6 5 2.3 1.2 67.5 361 42 296 5
Barley 100 12.5 11.5 1.3 1.2 3.9 69.6 336 26 215 3
Buck wheat 74 11.3 10.3 2.4 2.3 8.6 65.1 323 64 355 15.5
Foxtail millet 79 11.2 12.3 4.3 3.3 8 60.9 331 31 290 12.9
Jowar 100 11.9 10.4 1.9 1.6 1.6 72.6 349 25 222 5.8
Maize, dry 100 14.9 9.1 3.6 1.5 2.7 66.2 342 10 348 2
Maize flour, white 100 12 9.2 3.9 1.2 1.6 73.7 355 20 255 2.4
Maize flour, yellow 100 12 9.2 3.9 1.2 1.6 73.7 355 20 256 2.4
Maize, granular 100 12 9 3.4 1.1 1 74.5 362 17 223 1.8
Maize, tender 37 67.1 4.7 0.9 0.8 1.9 24.6 125 9 121 1.1
Oatmeal 100 10.7 13.6 7.6 1.8 3.5 62.8 374 50 380 3.8
French millet 59 11.9 12.5 1.1 1.9 2.2 70.4 341 14 206 5
Ragi (finger millet) 100 13.1 7.3 1.3 2.7 3.6 72 328 344 283 6.4
Rice, parboiled, 100 13.3 6.4 0.4 0.7 0.2 79 346 9 143 4
Rice, hand-pounded 100 13.3 7.5 1 0.9 0.6 76.7 346 10 190 3.2
Rice, raw, milled 100 13.7 6.8 0.5 0.6 0.2 78.2 345 10 160 3.1
Rice, flakes 100 12.2 6.6 1.2 2 0.7 77.3 346 20 238 20
Rice, puffed 100 14.7 7.5 0.1 3.8 0.3 73.6 325 23 150 6.6
Rice bran -- 11 13.5 16.2 6.6 4.3 48.4 393 67 1410 3.5
Semolina 100 -- 10.4 0.8 -- 0.2 74.8 348 16 102 1.6
Sorghum milled -- 14.3 7.6 2.4 1 0.6 74.7 357 17 196 3.6
Uwa, white 100 10.9 12.61 1.6 2.1 2.3 70.5 346 25 -- 4.12
Uwa, black 100 12.1 10.39 1.75 2.33 2.51 70.9 340 20.3 -- 7.48
Vermicelli 100 11.7 8.7 0.4 0.7 0.2 78.3 352 22 92 2
Wheat flour, whole 100 12.2 12.1 1.7 2.7 1.9 69.4 341 48 355 11.5
Wheat flour, refined 100 13.3 11 0.9 0.6 0.3 73.9 348 23 121 2.5
Wheat germ 100 5.2 29.2 7.4 3.5 1.4 53.3 397 40 846 6
Wheat bran -- 11.9 14.6 3 4.5 6.8 66 207 132 975 13.8
Biscuits, salty 100 4.5 6.6 32.4 1.9 -- 54.6 534 -- -- --
Biscuit, sweet 100 5.4 6.4 15.2 1.1 -- 71.9 450 -- -- --
Bread, brown 100 39 8.8 1.4 -- 1.2 49 244 18 -- 2.2
Bread, white 100 39 7.8 0.7 -- 0.2 51.9 245 11 -- 1.1
Papad 100 20.3 18.8 0.3 8.2 -- 52.4 288 80 300 17.2

Values are per 100g edible portion

169
 APPENDIX

NUTRIENT CONTENT OF NEPALESE FOODS, HMG-N (2043)

VEGETABLES (NATIVE)
Common name Edible Moisture, Protein, Fat, g Minerals, Fiber, Carb, Energy, Ca,
part, % g g g g g kcal mg
Armale -- 93 2 traces 1.5 1.4 2.1 16.4 20.5
Asparagus -- 92.9 2.1 0.3 0.7 0.9 3.8 27 22.5
Bander bheti 51.48 56.2 2.4 0.2 0.7 3.1 27.3 320 45.1
Bandari sag -- 90 3.5 0.3 1.3 0.8 4.3 33 89
Bantarul -- 76.2 1.7 0 3.1 1.3 17.7 77 43
Betha sag -- 78 7.1 traces 3.6 2 8.8 64 400
Bhringraj -- 84.9 3.1 0.8 2.7 1.7 6.7 46 224
Chamsur jhar -- 86.6 3.6 0.6 2.3 1.2 5.7 43 382
Chinia -- 81.5 6.1 traces 2.2 1.5 8.7 59 --
Chitlang sag -- 87.5 2.4 0.2 2 1.1 6.7 38 105
Chutro -- 72 2.5 6.9 1 1.4 16.2 87 --
Damaiphal -- 85 0.5 0.3 0.8 1.7 11.5 51 60
Frase tarul -- 75 1.6 traces 0.7 1.5 21 91 --
Gande -- 82 2.1 0.3 2 2.5 11 55 --
Ghod tapre -- 79 3 0.2 1.5 2.6 13.6 68 20505
Ginare, dried -- 7.2 22 traces 21.1 12 37.7 238 2032.4
Githa 74 1.5 0.1 0.8 1.3 22 96 -- --
Guyenlo -- 68 4.6 0.7 1 7.8 17.9 96 70.2
Halhale sag -- 90 3.1 0.3 1.3 1.3 3.8 31 76.6
Jamun -- 87 4 traces 1.6 1.4 6 40 --
Kali kath -- 65.4 1.5 0.7 1.1 5.7 25.4 114 95.1
Kali mayal -- 69 0.7 traces 0.7 8.3 21.3 89 --
Kamal phal 100 81.7 1.8 0.3 1 4.7 10.3 51 21.1
Kane sag -- 91 2.3 0.1 1.6 1.7 3.2 23 86
Kavro -- 69 2.8 traces 0.8 1.6 25.8 114 --
Khaneo 100 85.1 1.4 0.3 1.3 6.1 5.6 31 180.7
Koiralo -- 84.3 1 3.4 2 0.8 8.1 54 75.9
Kukur diano -- 93 1.6 traces 0.6 0.8 2.8 17 20.1
Latte sag, green stem -- 78 6.4 traces 3 1.2 11.4 71 --
Latte sag, red stem -- 81 4.4 traces 2.9 2.4 9.3 48 --
Lude sag -- 83.9 4.7 0.2 2.8 1.1 7.1 49 406.1
Malsahare -- 80 3.3 0.05 0.9 1.6 14.1 70 --
Mayal -- 79.8 0.4 0.2 0.4 2.9 16.2 68 --
Neuro (niguro) -- 88 4.4 0.2 1.3 1.8 4.2 36 30
Phaphar sag -- 90 3.9 0.1 1.9 1 3.8 12 --
Pidale -- 93 2.2 0.1 1.5 0.8 2.4 19 --
Pudina -- 87 4.5 0.1 1.9 1.8 4.6 38 --
Rato tarul -- 74 1.9 traces 0.7 0.2 2.3 100 --
Sati bayer -- 52.5 2.8 0.1 2 10 32.2 142 170.5
Sisnu -- 81.7 6.9 0.5 4.2 1.4 5 53 981.3
Siplicane -- 84 6.3 0.2 1.9 1.8 5.8 50 196
Tarul githa -- 76 2.2 traces 0.9 1.8 19 85 --
Tarul munta -- 89 2.8 0.05 1.1 1.6 5 33 --
Theki phal -- 88.3 0.5 0.1 0.9 2.4 7.5 33 35.1
Thutne -- 91 1.5 traces 0.8 0.9 5.8 29 21.8
Timila, pakche -- 88 1.1 0.3 0.7 2.7 7.2 37 --
Timila, wakche -- 88 1 0.4 0.8 1.6 7.4 37 --
Vyakur -- 77 1.6 traces 0.6 -- -- -- --
Vyakur githa -- 80.7 2 0.05 0.9 1.8 14 66 --
Vyakur jhutre -- 78 1.5 traces 0.8 1.7 18 78 --
Values are per 100g edible portion

170
 FOOD ANALYSIS

NUTRIENT CONTENT OF NEPALESE FOODS, HMG-N (2043)

MEAT, FISH AND EGG

Common name Edible Moisture, Protein, Fat, Ash, Fiber, Carb, Energy, Ca, P, Fe,
part, % g g g g g g kcal mg mg mg
Buffalo meat -- 78.7 19.4 0.9 1 -- -- 86 3 189 --
Duck meat -- 72.3 21.6 4.8 1.2 -- 0.1 130 4 235 --
Egg, duck -- 71 13.5 13.5 1 -- 0.8 181 70 260 3
Egg, hen -- 73.7 13.3 13.3 1 -- -- 173 60 220 2.1
Field rat's meat -- 73.9 23.6 1 1.4 -- 0.1 104 30 242 --
Finch -- 68.8 26.6 3 1.7 -- -- 133 90 347 --
Goat meat -- 74.2 21.4 3.6 1.1 -- -- 118 12 193 --
Goat liver -- 76.3 20 3 1.3 -- -- 107 17 279 --
Mutton, muscle -- 71.5 18.5 13.3 1.3 -- -- 194 150 150 2.5
Pigeon -- 70.4 23.3 4.9 1.4 -- -- 137 12 290 --
Pork, muscle -- 77.4 18.7 4.4 1 -- -- 114 30 200 2.2
Snail, small -- 78.9 12.6 1 3.8 -- 3.7 74 1321 147 --
Snail, big -- 74.1 10.5 0.6 2.4 -- 12.4 97 870 116 --
Turtle -- 79.4 16.5 1.5 1.1 -- 1.5 86 7 162 --
Venison -- 75.3 21 0.6 1.2 -- 1.9 97 3 233 --
Chicken -- 66 20.2 12.6 1 -- -- 195 -- -- --
Bam fish -- 74.8 16.1 0.9 1.3 -- 6.9 100 330 240 0.8
Crab, small -- 65.3 11.2 9.8 4.6 -- 9.1 169 1606 253 --
Hilsa fish -- 53.7 21.8 19.4 2.2 -- 2.9 273 180 280 2.1
Katla fish -- 73.7 19.5 2.4 1.5 -- 2.9 111 530 235 0.9
Koi fish -- 70 14.8 8.8 2 -- 4.4 156 410 390 1.4
Mungri fish -- 78.5 15 1 1.3 -- 4.2 86 210 290 0.7
Prawn 45 77.4 19.1 1 1.7 -- 0.8 89 323 278 5.3
Rahu fish 78 76.7 16.7 1.4 0.9 -- 4.4 97 650 175 1
Singhi fish -- 68 22.8 0.6 1.7 -- 6.9 124 670 650 2.3
Tengra fish, fresh -- 70 19.2 6.4 2.1 -- 2.3 144 270 170 2

SUGAR AND SUGAR PRODUCTS

Product Edible Moist, Prot, Fat, Ash, Fiber, Carb, Energy, Ca, P, Fe,
part, % g g g g g g kcal mg mg mg
Cane sugar 100 0.4 0.1 0 0.1 0 99.4 398 12 1 --
Honey -- 20.6 0.3 0 0.2 -- 79.5 319 5 16 0.9
Sugarcane juice -- 90.2 0.1 0.2 0.4 -- 9.1 39 10 10 1.1
Jaggery (cane) -- 3.9 0.4 0.1 0.6 -- 95 383 80 40 11.4

SOME INDIGENOUS FOODS

Food Edible Moist, Prot, Fat, Ash, Fiber, Carb, Energy, Ca, P, mg Fe,
part, % g g g g g g kcal mg mg
Areca nut -- 31.3 4.9 4.4 1 11.2 47.2 249 50 130 1.5
Betel leaves -- 85.4 3.1 0.8 2.3 2.3 6.1 44 230 40 7
Coconut, tender -- 90.8 0.9 1.4 0.6 -- 6.3 41 10 30 0.9
Coconut water 100 93.8 1.4 0.1 0.3 0 4.4 24 24 10 0.1
Groundnut cake -- 7.2 10.9 7.4 2.5 3.2 38.8 386 213 548 --
Gundruk, mustard leaves -- 11.8 -- -- -- -- -- -- 2458 -- 94.3
Pumpkin seeds 70 8 24.3 47.2 4.7 0.2 15.6 584 50 830 5.5
Masyaura -- 9.1 21.2 4.1 -- -- -- -- 478.1 -- 44.9
Mushroom 88 88.5 4.6 0.8 1.4 0.4 4.3 43 6 110 1.5
Values are per 100g edible portion

171
 APPENDIX

NUTRIENT CONTENT OF NEPALESE FOODS, HMG-N (2043)

OTHER VEGETABLES, ROOTS AND TUBERS

Common name Edible Moist, Prot, Fat, Ash, Fiber, Carb, Energy, Ca, P, Fe,
part, % g g g g g g kcal mg mg mg
Agathi flower -- 92.9 1 0.5 0.4 0.8 4.4 26 9 5 --
Ashgourd 67 96.5 0.4 0.1 0.3 0.8 1.9 10 30 20 0.8
Bittergourd 97 92.5 1.6 0.2 0.8 0.8 4.2 25 20 70 1.8
Bottle gourd 86 96.1 0.2 0.1 0.5 0.6 2.5 12 20 10 0.7
Brinjal 91 92.7 1.4 0.3 0.3 1.3 4 24 18 47 0.9
Broad beans 88 85.4 4.5 0.1 0.8 2 7.2 48 50 64 1.4
Cauliflower 70 90.8 2.6 0.4 1 1.2 4 30 33 57 1.5
Celery stalks -- 93.5 0.8 0.1 0.9 1.2 3.5 18 30 38 4.8
Cho-cho marrow -- 92.5 0.7 0.1 0.4 0.6 5.7 27 140 30 0.6
Cluster beans -- 81 3.2 0.4 1.4 3.2 10.8 60 130 57 4.5
Colocasia stems 86 94 0.3 0.3 1.2 0.6 3.6 18 60 20 0.5
Cowpea pods -- 85.3 3.5 0.2 0.9 2 8.1 43 72 59 2.5
Cucumber 83 96.3 0.4 0.1 0.3 0.4 2.5 13 10 25 1.5
Bouble beans -- 73.8 8.3 0.3 1 4.3 12.3 85 40 140 2.3
Drumstick 83 86.9 2.5 0.1 2 4.8 3.7 26 30 110 5.3
Drumstick flowers -- 85.9 3.6 0.8 1.3 1.3 7.1 50 51 90 --
Field beans, tender 93 86.1 3.8 0.7 0.9 1.8 6.7 48 210 68 1.7
French beans 94 91.3 1.7 0.1 0.5 1.8 4.5 26 50 28 1.7
Giant chillies (capsicum) 94 91.3 1.7 0.1 0.5 1.8 4.5 26 50 28 1.7
Jack, tender -- 84 2.6 0.3 0.9 2.8 9.4 51 30 40 1.7
Karonda, fresh 98 91 1.1 2.9 0.6 1.5 2.9 42 21 28 --
Ol-k 74 92.7 1.1 0.2 0.7 1.5 3.8 21 20 35 0.4
Ladies finger 84 89.6 1.9 0.2 0.7 1.2 6.4 35 66 56 1.5
Lakooch, raw -- 89.4 1.6 1.2 1.1 2.8 13.9 73 67 25 --
Leeks -- 78.9 1.8 0.1 0.7 1.3 17.2 77 50 70 2.3
Lotus stem, dry 100 9.5 4.1 1.3 8.7 2.5 51.4 234 405 128 60.6
Mango, green 72 87.5 0.7 0.1 0.4 1.2 10.1 44 10 19 5.4
Onion stalks 100 87.6 0.9 0.2 0.8 1.6 8.9 41 50 50 7.4
Papaya, green -- 92 0.7 0.2 0.5 0.9 5.7 27 28 40 0.9
Parwar 95 92 2 0.3 0.5 3 2.2 20 30 40 1.7
Peas 53 72.1 7.2 0.1 0.8 4 15.9 93 20 139 1.5
Pink beans 94 86.8 3.1 0.4 0.6 2.1 7 44 54 70 1.5
Plantain flower 43 89.9 1.7 0.7 1.3 1.3 5.1 34 32 42 1.6
Plantain stem -- 88.3 0.5 0.1 0.6 0.8 9.7 4.2 10 10 1.1
Pumpkin 79 92.6 1.4 0.1 0.6 0.7 4.6 25 10 30 0.7
Pumpkin flowers -- 89.1 2.2 0.8 1.4 0.7 5.8 39 120 60 --
Rape plant, stem -- 91.4 3.1 0.1 1.4 -- 4 29 100 100 1.2
Red gram, tender 72 65.1 9.8 1 1 6.2 16.9 116 57 164 1.1
Ridge gourd 82 95.2 0.5 0.1 0.3 0.5 3.4 17 18 26 0.5
Sannhemp flowers -- 78.9 4.8 0.6 1.4 3.9 10.4 66 200 100 --
Silk cotton flowers -- 86.4 1.5 0.3 0.7 1.6 9.5 47 22 45 --
Snake gourd 98 94.6 0.5 0.3 0.5 0.8 3.3 18 26 20 0.3
Spinach stalks -- 93.4 0.9 0.1 1.8 -- 3.8 20 90 20 1.6
Sword beans 98 87.2 2.7 0.2 0.6 1.5 7.8 44 60 40 2
Tinda, tender 99 93.5 1.4 0.2 0.5 1 3.4 21 25 24 0.9
Tomato, green 98 93.1 1.9 0.1 0.6 0.7 3.6 23 20 36 1.8
Vegetable marrow 94 94.8 0.5 0.1 0.3 0.8 3.5 17 10 30 0.6
Water chestnut, dry -- 90.8 1.6 0.6 0.7 0.9 5.4 33 29 18 --
Water lily flower 35 85.1 0.4 0.2 1.4 1.1 11.8 51 25 10 1.1
Values are per 100g edible portion (continued….

172
 FOOD ANALYSIS

….continued) OTHER VEGETABLES, ROOTS AND TUBERS

Edible Moist, Prot, Fat, Ash, Fiber, Carb, Energy, Ca, P, Fe,
Common name part, % g g g g g g kcal mg mg mg
Beet root 85 87.7 1.7 0.1 0.8 0.9 8.8 43 18 55 1
Carrot 95 86 0.9 0.2 1.1 1.2 10.6 48 80 530 2.2
Colocasia -- 73.1 3 0.1 1.7 1 21.1 97 40 140 1.7
Garlic, dry 85 62 6 0.1 1 0.8 29.8 145 30 310 1.3
Ghartarul 100 58.61 4.07 0.07 2.06 2.56 32.6 147 69.8 -- 24.03
Githa 66 68.31 3.72 0.94 0.93 0.91 25.19 124 12.41 -- 0.95
Lotus root -- 85.9 1.7 0.1 0.2 0.8 11.3 53 21 74 0.4
Mango ginger 87 85 1.1 0.7 1.4 1.3 10.5 53 25 90 2.6
Onion big 95 86.6 1.2 0.1 0.4 0.6 11.1 50 47 50 0.7
Onion small -- 84.3 1.8 0.1 0.6 0.6 12.6 59 40 60 1.2
Potato 85 74.4 1.6 0.1 0.6 0.4 22.6 97 10 40 0.7
Potato, boliled -- 81 1.9 0.1 0.7 0.3 16.3 72 7 44 0.8
Potato chips, fried -- 4.2 3.6 4.38 2.5 0.9 45.9 562 18 74 1.6
Radish, pink 98 90.8 0.6 0.3 0.9 0.6 6.8 32 50 20 0.5
Radish rat-tailed -- 92.3 1.3 0.3 0.7 1.1 4.3 25 78 24 --
Radish, white 99 94.4 0.7 0.1 0.6 0.8 3.4 17 35 22 0.4
Rani bhyakur 78 72.72 2.29 0.16 1.41 0.7 22.71 101 24.71 -- 8.38
Sweet potato 97 68.5 1.2 0.3 1 0.8 28.2 120 46 50 0.8
Sweet potato, boiled -- 70.7 1 0.1 0.8 0.6 27.4 114 36 56 0.9
Turnip 65 91.6 0.5 0.2 0.6 0.9 6.2 29 30 40 0.4
Yam elephant -- 75.7 1.2 0.01 0.8 0.8 18.4 79 50 34 0.16
Yam, wild 89 70.4 2.5 0.3 1.4 1 24.4 110 20 74 1

LEGUMES, PULSES AND THEIR PRODUCTS

Edible Moist, Prot, Fat, Ash, Fiber, Carb, Energy, Ca, P, Fe,
Common name part, % g g g g g g kcal mg mg mg
Bengal gram (whole) 100 9.8 17.1 5.3 3 3.9 60.9 360 202 312 10.2
Bengal gram (chick pea) 100 9.9 20.8 5.6 2.7 1.2 59.8 372 56 331 9.1
Bengal gram, roasted 100 10.7 22.5 5.2 2.5 1 58.1 369 58 340 9.5
Black gram, whole 100 10.6 21 1.6 3.6 4.4 59 344 110 348 8.4
Black gram dal 100 10.9 24 1.4 3.2 0.9 59.6 347 154 385 9.1
Broad bean or horse bean 100 13.8 25 1.2 3.1 5.1 -- 328 104 397 4.2
Cow pea 97 13.4 24.1 1 3.2 3.8 54.5 323 77 414 5.9
Field bean, dry -- 9.6 24.9 0.8 3.2 1.4 60.1 347 60 433 2.7
Green gram, whole 100 10.4 24 1.3 3.5 4.1 56.7 334 124 326 7.3
Green gram (mung bean) 100 10.1 21.2 1.2 3.5 0.8 59.9 348 75 405 8.5
Horse gram, black 100 11.8 22 0.5 3.2 5.3 57.2 321 287 311 8.4
Lentil 100 12.4 25.1 0.7 2.1 0.7 59 343 69 293 4.8
Moth beans 100 10.8 23.6 1.1 3.5 4.5 56.5 330 202 230 9.5
Peas, dry 100 16 19.7 1.1 2.2 4.5 56.5 315 75 298 5.1
Peas, roasted 100 10.1 22.9 1.4 2.4 4.4 58.8 340 81 345 6.4
Rajmah (French bean) -- 12 22.9 1.3 3.2 -- 60.6 346 260 410 5.8
Red gram (pegion pea) 100 13.4 22.5 1.7 3.5 1.5 57.6 335 73 304 5.8
Soybean, grey 100 8.1 43.2 19.5 4.6 3.7 20.9 432 240 690 11.5
Soybean, black 100 12.5 33.3 15 4 4.3 30.9 395 213 509 9.5
Soybean, white 100 10.2 33.3 17.7 5 4.2 29.6 400 226 546 8.5
Khesari dal 100 10 28.2 0.6 2.3 2.3 56.6 345 90 317 6.3
Values are per 100g edible portion

173
 INDEX
INDEX
1,10-phenonthroline ............................ 149, 150
2,4-dinitro phenylhydrazine .......................... 79
2,6-dichlorophenol indophenol ......... 77, 78, 79
3,5-dinitrosalicylic acid ................................. 86
Absorbance ... 57, 58, 74, 76, 80, 81, 85, 87, 96-
99, 102, 116, 117, 142, 150
Accuracy ........................ 11, 30-33, 62, 94, 131
Acetone ........................... 42, 74, 78, 96, 97, 98
Acid value ................................................... 121
Acidity ..................... 8, 12, 27, 91-94, 113, 123
Adulteration ...........45, 121, 124, 126, 135, 139
Alcoholic beverages .................................... 113
Aldehyde ............................................... 83, 125
Alkali . 43, 45, 49, 52-54, 68, 93, 107, 124, 136
Alkalinity ............................................ 143, 147
Alternative hypothesis............ 14-17, 22, 23, 26
Amino groups........................................ 54, 104
Ammonia ... 52, 53, 72, 105, 106, 143, 145, 159
Ammonium purpurate ......................... 146, 158
Ammonium sulfate ........................ 49, 150, 166
Amyloglucosidase ......................................... 45
ANOVA ............................................ 27, 28, 29
Anthocyanins ................................................ 96
APHA.......................................................... 150
Argemone oil .............................. 121, 141, 142
Arsenomolybdate .................................... 84, 85
Ascorbic acid ..................................... 76-81, 86
Ash .............................. 26, 45-47, 67-69, 71-75
Barium hydroxide ................. 84, 135, 136, 138
Baudouin test ...................................... 121, 139
Bellier turbidity test .................................... 121
Benzoic acid ................................ 110, 111, 112
Biuret ............................................................ 57
Boric acid .......................................... 49, 52, 53
Bound water .................................................. 59
Bromophenol blue ....................... 124, 125, 126
Buchner filter ................................................ 46
Buffer ............................ 95, 145, 149, 150, 159
Butyric acid ................................................. 135
174
Calcium .. 60, 63, 68, 71-75, 101, 143-146, 148,
158, 159
Calibration .................................................... 30
Capillary ................ 59, 104, 114, 133, 134, 142
Carbohydrates ................................... 38, 39, 83
Carmine solution ......................................... 101
Carotenoids ....................................... 96, 98, 99
Carrez solution ........................................ 88, 90
Catalyst mixture ...................................... 49, 50
Cellulose ................................................. 43, 44
Chlorine ........................................ 97, 129, 145
Chloroform .......... 111, 112, 128, 132, 140, 142
Chromotropic acid .............................. 116, 117
Citric acid...................................... 4, 91, 92, 93
Coliform ...............................143, 150, 153, 154
Colorants ..................................... 103, 104, 106
Colorimeter .................. 57, 74, 84, 87, 102, 116
Completed test ............................................ 153
Condenser ..40, 41, 46, 51-53, 61, 67, 114, 125,
135, 136, 138
Confidence limit ......................... 18, 19, 20, 22
Confirmed test ...................................... 45, 153
Crismer test ................................................. 122
Crude fat ....................................................... 39
Crude fiber ................. 38, 41, 43, 44, 45, 46, 47
Dean and Stark .................................. 60, 66, 67
Degrees of freedom ................................. 12, 22
Dehydroascorbic acid........................ 76, 77, 79
Deprotonation ............................................. 104
Desiccator ......................40, 46, 65, 66, 69, 144
Diacetone alcohol ................................... 98, 99
Dietary fiber ...................................... 43, 44, 45
Dihydrosanguarine ...................................... 141
Dilution factor ................................. 81, 91, 117
Dinitrosalicylic acid ...................................... 83
Disaccharides ................................................ 83
Distillation .....49-54, 61, 66, 67, 107, 112, 114,
116, 135, 136, 138
Distillation .................................................... 60
EDTA........................... 105, 145, 146, 158, 159
Electronic balance ....................... 35, 80, 84, 88

Endo broth........................................... 153, 154
Enzymatic methods ................................. 83, 90
Eriochrome Black T ............................ 145, 159
Ethanol ....... 45, 46, 49, 105, 113, 116, 135, 142
Fatty acid............................................. 132, 133
FD&C standards.......................................... 106
Fehling’s solution.............................. 87, 88, 90
Flame photometer ......................................... 75
Food additive .............................................. 103
Formaldehyde ...54, 55, 78, 108, 109, 110, 115,
116, 117
Formol binding.............................................. 54
Free fatty acid ..................................... 121, 122
Free water ..................................................... 59
F-test ............................................................. 27
Furfural ....................................................... 139
Gallotannins ................................................ 100
Glucose ..............44, 83, 84, 86, 87, 90, 91, 100
Halogen content .......................................... 130
Hardness................................... 18, 19, 143-147
Hemicellulose ......................................... 43, 44
Hemoglobin................................................... 96
Hexabromide ............................................... 140
Hot air oven........................................... 60, 144
Hot plate .............60, 65, 66, 105, 123, 125, 144
Hydrometers................................................ 113
Hydroxylamine hydrochloride ............ 149, 150
Immiscible solvent .................................. 60, 66
Indicator organisms ..................................... 153
Infrared moisture balance .............................. 62
Iodine ... 1, 63, 83, 107-110, 126-129, 131, 132,
156-158
Iodine value......................... 121, 122, 126, 127
Iodometric titration ..................................... 131
Iron ............................. 71, 73-75, 143, 149, 150
Isopropanol ................................. 105, 106, 117
Karl Fischer method ...................................... 60
Kirschner value ................... 135, 136, 138, 139
Kjeldahl ................38, 48, 49, 50, 51, 53, 54, 56
Lactic acid ......................................... 91, 92, 94
Lane and Eynon ................................ 83, 90, 91
L-ascorbic acid .................................. 79, 82, 85
Level of significance . 14, 15, 17, 18, 20-23, 25-
28
Lignin ............................................................ 43
Linear regression ..................................... 81, 85
Linseed oil........................... 121, 122, 139, 140
Lycopene ....................................................... 98
Magnesium .................. 144, 145, 146, 158, 159
Margarine .................................................... 133
Melting point............................... 132, 133, 134
Melting point............................... 121, 132, 133
FOOD ANALYSIS
Membrane filter .................................. 153, 154
Metaphosphoric acid ............................... 77, 78
Methanol ................................63, 115, 116, 117
Methyl orange ....................................... 46, 147
Microscope ................................................. 140
Milk ................... 4, 6, 39, 48, 54, 55, 59, 91-95
Mineral.............................44, 68, 121, 126, 145
Mixed indicator ....................................... 52, 53
Moisture ...................................... 59, 60, 65, 66
Moisture content ................8, 20, 48, 59, 62, 63
Molybdenum blue ......................................... 84
Muffle furnace .................................. 47, 69, 71
Multiple Tube Fermentation ....................... 153
Murexide ..................................... 146, 158, 159
NaCl ..............101, 111, 112, 144-146, 155, 167
Natural pigments ........................................... 96
Nelson and Somogyi ............................... 83, 84
Normal curve .......................................... 13, 18
Normality ...........................33, 72, 94, 132, 157
Null hypothesis ................... 14-6, 20, 22, 23, 26
One-tailed test ................................... 16, 26, 27
Oxalate ...................................... 55, 72, 73, 167
Oxalic acid .............................34, 35, 36, 37, 77
PCA ............................................................ 151
Pearson Square.............................................. 34
Peroxide value..................................... 131, 132
Petroleum ether ........................... 39, 40, 98, 99
pH ... 5, 54, 72, 77, 83, 91, 92, 94, 95, 107, 113,
143, 145-147, 149, 158
Phenolphthalein ..... 35, 46, 88, 90, 93, 113, 123,
136, 147
Phosphotungstomolybdic acid .................... 102
Physiological effects ................................... 149
Polenske value .................................... 135, 138
Polymorphism ............................................. 132
Polyunsaturated fatty acids ................. 121, 140
Population ............ 5, 6, 7, 12, 13, 14, 16, 18, 23
Potable water .............................................. 143
Potassium.. .... 34, 49, 55, 60, 63, 68, 73, 75, 88,
98, 100, 101, 107, 125, 127, 131, 132, 143,
148, 155-157, 159
Potassium dichromate ..................... 63, 98, 127
Potassium thiocyanate ................................... 73
Precision ....................................................... 11
Preservatives ......................................... 34, 103
Presumptive test .......................................... 153
Primary amines ............................................. 54
Probability..........................7, 14, 15, 16, 18, 22
Protease ......................................................... 45
Protein ..... 12, 27, 38, 43, 45, 48, 49, 50, 54, 55,
56, 57, 58, 84, 104, 115
Proximate .................................................. 8, 38
Purity factor .................................................. 36
Pyridine ......................................................... 63
175
 INDEX
Rancidity ..................................................... 131
Reducing sugar............................. 38, 83, 86-91
Reflux.................................. 40, 41, 61, 66, 125
Refractometry ............................................... 56
Reichert-Meissl ........................... 122, 135, 137
Retention factor ........................................... 104
RM value...................................... 135, 137-139
Sampling ...........................................................
Sangunarine................................................. 141
Saponification value .................... 121, 122, 124
Screening ...................................................... 10
Scurvy ........................................................... 76
Sesame oil ................................................... 139
Sesamolin .................................................... 139
Significant values .................................... 17, 22
Silica compounds .......................................... 71
Silica crucibles .............................................. 68
Sodium sulfate ........................................ 98, 99
Sodium thiosulfate ................ 63, 131, 156, 157
Solvent front................................................ 104
Soxhlet .................................................... 40, 42
Specific gravity ........ 66, 67, 113-115, 170, 172
Spectrophotometer .............. 80, 97, 98, 99, 150
Standard deviation............ 12- 14, 18, 22-24, 56
Standard deviation.............................................
Standard error.............................. 13, 14, 18, 27
Standard Plate Count ................................... 151
Starch content.......................................... 90, 91
Starch indicator ............ 108, 109, 127-129, 132
Statistical hypothesis ................................ 13-16
176
Strength ... 33, 34, 36, 37, 52, 55, 109, 110, 127,
128, 131, 141, 146, 150, 167
Sulfur dioxide ............................................. 106
Sulfuric acid . 46, 72, 79, 90, 116, 129, 156, 157
t value ........................................................... 22
Tannic acid.......................................... 100, 102
Tannins ................................100, 101, 102, 149
TDS............................................................. 143
Tertiary amine ............................................... 54
TGYA ......................................................... 151
Thimble ......................................................... 40
Thin Layer Chromatography ............... 103, 106
Thymophthalein .......................................... 159
Transmittance ............................. 74, 76, 80, 85
Trendline .......................................... 81, 85, 86
Two-tailed test ............................ 16, 18, 20, 25
Types of errors .............................................. 15
Ultimate analysis........................................... 38
Unsaponifiable matter ......................... 121, 122
Unsaturation ................................................ 126
Vacuum oven ...................................... 8, 60, 66
Vanaspati .........................60, 65, 126, 133, 139
Variance ...........................12, 18, 19, 20, 21, 27
Vitamin A ..................................................... 98
Vitamin C...........................5, 12, 28, 58, 79, 85
Wij’s reagent ............................................... 127
Wool ................................................... 104, 105
Working solution .................................... 36, 37
Xanthophylls ................................................. 96
γ-carotene...................................................... 98
Prof. Jagat Bahadur K.C. is one of the leadin g Food
Scientists of Nepal. He was the founder tea cher of
Food Technology in Ne pal. He was the foun der
President of Nepal Food Scientists and Technologists
Association (NEFOSTA) a nd is also th e president of
NEFOSTA at present. He established NEFOSTA Gold
Medal and is asso ciated with a nu mber of social
organizations in Nepal. Recently, he completed a term
for the prest igious post of the Vice Chancellor of
Purwanchal University, Nepal.

Prof. K.C. passed his MSc. (Chemistry) from T.U. and

received Mahendra Vidya Bhusan. He passed is M.Sc.
(Food Technology) from U.K. also. He started hi s
teaching career as an assistant lecturer from 1968 and
has come a long way teaching subje cts as diverse as
Chemistry, Food Technology (Quality Control, Food
Analysis, Food Processing, etc.) and Fo od
Microbiology to graduate and post graduate levels. He
has over 2 dozens of research papers to his credit. He
also has 5 prestigious awards under his belt.
Popularizing science, especially Food Technology, he
says, is his motto of life.

Mr. Basanta Kumar Rai earned his B. Tech (Food)

and M. Tech (Food, Dairy as the major) from
Tribhuvan University. Pre sently, he is a lecture r of
Food Technology at Ce ntral Campus of Technology,
Tribhuvan University. He teaches Industrial
microbiology. Because of the interrelated nature of
subjects in the Food Te chnology course, his intere st
also lies in teaching subj ects such as Micro biology,
Biochemistry, Food Analysis, and Food Chemistry.
To this end, he has co-authored two books:

1. Practicals in basic biochemistry and in dustrial

microbiology
2. Experiments in basic food microbiology

Other books in line for publication are:

1. Essentials of Food Chemistry

2. Industrial Microbiology