Paper chromatography is one of the types of chromatography procedures which runs on a piece

of specialized paper. It is a planar chromatography system wherein a cellulose filter paper acts as
a stationary phase on which the separation of compounds occurs.

Principle of paper chromatography: The principle involved is partition chromatography wherein

the substances are distributed or partitioned between liquid phases. One phase is the water,
which is held in the pores of the filter paper used; and other is the mobile phase which moves
over the paper. The compounds in the mixture get separated due to differences in their affinity
towards water (in stationary phase) and mobile phase solvents during the movement of mobile
phase under the capillary action of pores in the paper.

The principle can also be adsorption chromatography between solid and liquid phases, wherein
the stationary phase is the solid surface of paper and the liquid phase is of mobile phase. But
most of the applications of paper chromatography work on the principle of partition
chromatography, i.e. partitioned between to liquid phases.

Applications - 1) For determination of amino acids.

2) In Pharma industry for determination of drugs, organic compounds etc.

3)For determination of organic compounds, biochemical compounds.

4) Polar and non polar compounds.

THIN LAYER CHROMATOGRAPHY

THIN LAYER CHROMATOG IS A TYPE OF PLANAR CHROMATOG ROUTINELY USED IN THE

LABORATORY FOR ANALYSIS OFphyto-chemicals, biochemistry, and so forth, to identify the
components in a compound mixture, like alkaloids, phospholipids, and amino acids.

Similar to other chromatographic methods, thin layer chromatography is also based on the
principle of separation.

The separation depends on the relative affinity of compounds towards stationary and the mobile
phase.

The compounds under the influence of the mobile phase (driven by capillary action) travel over
the surface of the stationary phase. During this movement, the compounds with higher affinity
to stationary phase travel slowly while the others travel faster. Thus, separation of components
in the mixture is achieved.

Once separation occurs, the individual components are visualized as spots at a respective level of
travel on the plate. Their nature or character are identified by means of suitable detection
techniques.

System Components

TLC system components consists of

TLC plates, preferably ready made with a stationary phase: These are stable and chemically inert
plates, where a thin layer of stationary phase is applied on its whole surface layer. The stationary
phase on the plates is of uniform thickness and is in a fine particle size.

TLC chamber. This is used for the development of TLC plate. The chamber maintains a uniform
environment inside for proper development of spots. It also prevents the evaporation of
solvents, and keeps the process dust free.

Mobile phase. This comprises of a solvent or solvent mixture The mobile phase used should be
particulate-free and of the highest purity for proper development of TLC spots. The solvents
recommended are chemically inert with the sample, a stationary phase.

A filter paper. This is moistened in the mobile phase, to be placed inside the chamber. This helps
develop a uniform rise in a mobile phase over the length of the stationary phase.

Applications
To check the purity of given samples.

Identification of compounds like acids, alcohols, proteins, alkaloids, amines, antibiotics, and
more.

To evaluate the reaction process by assessment of intermediates, reaction course, and so forth.

To purify samples, i.e for the purification process.

To keep a check on the performance of other separation processes.

Advantages

It is a simple process with a short development time.

It helps with the visualization of separated compound spots easily.

The method helps to identify the individual compounds.

It helps in isolating of most of the compounds.

The separation process is faster and the selectivity for compounds is higher (even small
differences in chemistry is enough for clear separation).

The purity standards of the given sample can be assessed easily.

It is a cheaper chromatographic technique.

CHROMATOGRAPHY - IT IS A TECHNIQUE EMPLOYED FOR THE SEPERATION OF COMPONENTS OF

A MIXTURE BY CONTINOUS DISTRIBUTION OF COMPONENTS OVER A STATIONARY PHASE AND A
MOBILE PHASE

COLUMN CHROMATOGRAPHY - The process of washing a compound through a column using a

solvent is known as elution. The solvent is sometimes known as the eluent.

COLUMN CHARACTERSTICS - FOR EFFICENT SEPERATION - LENGTH: DIAMETER = 100 :1

COLUMN MUST BE PACKED AS UNIFORMLY AS POSSIBLE TO PREVENT DISTORTION OF

CHROMATOGRAPHIC BOUNDARIES.

ELUTION TECHNIQUES- ISOCRATIC ELUTION - IN THIS PROCESS, SAME SOLVENT OR SOLVENT OF

SAME POLARITY IS USED THROUGHOUT

GRADIENT ELUTION - IN THIS SOLENT OF INCREASING POLARITIES ARE USED

FACTORS AFFECTING COLUMN CHROMAT- PRESSURE, TEMP, DIAMETER OF COLUMN, NATURE OF

SOLVENT, PARTICLE SIZE OF SOLVENT.
A packed column is a pressure vessel that has a packed section. Columns used in certain types of
chromatography consisting of a tube filled with packing material can also be called packed
columns and their structure has similarities to packed beds.

Column Chromatography is a preparative technique used to purify compounds depending on

their polarity or hydrophobicity. In column chromatography, a mixture of molecules is separated
based on their differentials partitioning between a mobile phase and a stationary phase.

Column chromatography in chemistry is a chromatography method used to purify individual

chemical compounds from mixtures of compounds. It is often used for preparative applications
on scales from micrograms up to kilograms. The main advantage of column chromatography is
the relatively low cost and disposability of the stationary phase used in the process. The latter
prevents cross-contamination and stationary phase degradation due to recycling.

The classical preparative chromatography column is a glass tube with a diameter from 5 mm to
50 mm and a height of 5 cm to 1 m with a tap and some kind of a filter (a glass frit or glass wool
plug – to prevent the loss of the stationary phase) at the bottom. Two methods are generally
used to prepare a column: the dry method and the wet method.

For the dry method, the column is first filled with dry stationary phase powder, followed by the
addition of mobile phase, which is flushed through the column until it is completely wet, and
from this point is never allowed to run dry.[1]

For the wet method, a slurry is prepared of the eluent with the stationary phase powder and
then carefully poured into the column. Care must be taken to avoid air bubbles. A solution of the
organic material is pipetted on top of the stationary phase. This layer is usually topped with a
small layer of sand or with cotton or glass wool to protect the shape of the organic layer from
the velocity of newly added eluent. Eluent is slowly passed through the column to advance the
organic material. Often a spherical eluent reservoir or an eluent-filled and stoppered separating
funnel is put on top of the column.

The individual components are retained by the stationary phase differently and separate from
each other while they are running at different speeds through the column with the eluent. At the
end of the column they elute one at a time. During the entire chromatography process the
eluent is collected in a series of fractions. Fractions can be collected automatically by means of
fraction collectors. The productivity of chromatography can be increased by running several
columns at a time. In this case multi stream collectors are used. The composition of the eluent
flow can be monitored and each fraction is analyzed for dissolved compounds, e.g. by analytical
chromatography, UV absorption spectra, or fluorescence. Colored compounds (or fluorescent
compounds with the aid of a UV lamp) can be seen through the glass wall as moving bands.

Gas chromatography (GC) is a common type of chromatography used in analytical chemistry for
separating and analyzing compounds that can be vaporized without decomposition. Typical uses
of GC include testing the purity of a particular substance, or separating the different components
of a mixture (the relative amounts of such components can also be determined). In some
situations, GC may help in identifying a compound. In preparative chromatography, GC can be
used to prepare pure compounds from a mixture.[1][2]

In gas chromatography, the mobile phase (or "moving phase") is a carrier gas, usually an inert
gas such as helium or an unreactive gas such as nitrogen. Helium remains the most commonly
used carrier gas in about 90% of instruments although hydrogen is preferred for improved
separations.[3] The stationary phase is a microscopic layer of liquid or polymer on an inert solid
support, inside a piece of glass or metal tubing called a column (an homage to the fractionating
column used in distillation). The instrument used to perform gas chromatography is called a gas
chromatograph (or "aerograph", "gas separator").

The gaseous compounds being analyzed interact with the walls of the column, which is coated
with a stationary phase. This causes each compound to elute at a different time, known as the
retention time of the compound. The comparison of retention times is what gives GC its
analytical usefulness.

Gas chromatography is in principle similar to column chromatography (as well as other forms of
chromatography, such as HPLC, TLC), but has several notable differences. First, the process of
separating the compounds in a mixture is carried out between a liquid stationary phase and a
gas mobile phase, whereas in column chromatography the stationary phase is a solid and the
mobile phase is a liquid. (Hence the full name of the procedure is "Gas–liquid chromatography",
referring to the mobile and stationary phases, respectively.) Second, the column through which
the gas phase passes is located in an oven where the temperature of the gas can be controlled,
whereas column chromatography (typically) has no such temperature control. Finally, the
concentration of a compound in the gas phase is solely a function of the vapor pressure of the
gas.[1]

Gas chromatography is also similar to fractional distillation, since both processes separate the
components of a mixture primarily based on boiling point (or vapor pressure) differences.
However, fractional distillation is typically used to separate components of a mixture on a large
scale, whereas GC can be used on a much smaller scale (i.e. microscale).[1]

Gas chromatography is also sometimes known as vapor-phase chromatography (VPC), or gas–

liquid partition chromatography (GLPC). These alternative names, as well as their respective
abbreviations, are frequently used in scientific literature. Strictly speaking, GLPC is the most
correct terminology, and is thus preferred by many authors.[1]

FACTORS AFFECTING - 1) LENGTH OF COLUMN, VAPOUR PRESSURE, The polarity of components

versus the polarity of stationary phase on column, Column temperature, Carrier gas flow
rate,Column length

High performance liquid chromatography (HPLC) is basically a highly improved form of column
liquid chromatography. Instead of a solvent being allowed to drip through a column under
gravity, it is forced through under high pressures of up to 400 atmospheres. That makes it much
faster. All chromatographic separations, including HPLC operate under the same basic principle;
separation of a sample into its constituent parts because of the difference in the relative
affinities of different molecules for the mobile phase and the stationary phase used in the
separation.

Types of HPLC

There are following variants of HPLC, depending upon the phase system (stationary) in the
process :

Normal Phase HPLC:

This method separates analytes on the basis of polarity. NP-HPLC uses polar stationary phase
and non-polar mobile phase. Therefore, the stationary phase is usually silica and typical mobile
phases are hexane, methylene chloride, chloroform, diethyl ether, and mixtures of these. Polar
samples are thus retained on the polar surface of the column packing longer than less polar
materials.

Reverse Phase HPLC:

The stationary phase is nonpolar (hydrophobic) in nature, while the mobile phase is a polar
liquid, such as mixtures of water and methanol or acetonitrile. It works on the principle of
hydrophobic interactions hence the more nonpolar the material is, the longer it will be retained.

Size-exclusion HPLC:

The column is filled with material having precisely controlled pore sizes, and the particles are
separated according to its their molecular size. Larger molecules are rapidly washed through the
column; smaller molecules penetrate inside the porous of the packing particles and elute later.

Ion-Exchange HPLC:

The stationary phase has an ionically charged surface of opposite charge to the sample ions. This
technique is used almost exclusively with ionic or ionizable samples. The stronger the charge on
the sample, the stronger it will be attracted to the ionic surface and thus, the longer it will take
to elute. The mobile phase is an aqueous buffer, where both pH and ionic strength are used to
control elution time.

Instrumentation of HPLC

High-performance-liquid-chromatography-hplc

As shown in the schematic diagram in Figure above, HPLC instrumentation includes a pump,
injector, column, detector and integrator or acquisition and display system. The heart of the
system is the column where separation occurs.

Solvent Resorvoir : Mobile phase contents are contained in a glass resorvoir. The mobile phase,
or solvent, in HPLC is usually a mixture of polar and non-polar liquid components whose
respective concentrations are varied depending on the composition of the sample.

Pump : A pump aspirates the mobile phase from the solvent resorvoir and forces it through the
system’s column and detecter. Depending on a number of factors including column dimensions,
particle size of the stationary phase, the flow rate and composition of the mobile phase,
operating pressures of up to 42000 kPa (about 6000 psi) can be generated.

Sample Injector : The injector can be a single injection or an automated injection system. An
injector for an HPLC system should provide injection of the liquid sample within the range of 0.1-
100 mL of volume with high reproducibility and under high pressure (up to 4000 psi).
Columns : Columns are usually made of polished stainless steel, are between 50 and 300 mm
long and have an internal diameter of between 2 and 5 mm. They are commonly filled with a
stationary phase with a particle size of 3–10 µm. Columns with internal diameters of less than 2
mm are often referred to as microbore columns. Ideally the temperature of the mobile phase
and the column should be kept constant during an analysis.

Detector : The HPLC detector, located at the end of the column detect the analytes as they elute
from the chromatographic column. Commonly used detectors are UV-spectroscopy,
fluorescence, mass-spectrometric and electrochemical detectors.

Data Collection Devices : Signals from the detector may be collected on chart recorders or
electronic integrators that vary in complexity and in their ability to process, store and reprocess
chromatographic data. The computer integrates the response of the detector to each
component and places it into a chromatograph that is easy to read and interpret.

Applications of HPLC

The information that can be obtained by HPLC includes resolution, identification and
quantification of a compound. It also aids in chemical separation and purification. The other
applications of HPLC include :

Pharmaceutical Applications

1. To control drug stability.

2. Tablet dissolution study of pharmaceutical dosages form.

3. Pharmaceutical quality control.

Environmental Applications

1. Detection of phenolic compounds in drinking water.

2. Bio-monitoring of pollutants.

Applications in Forensics

1. Quantification of drugs in biological samples.

2. Identification of steroids in blood, urine etc.

3. Forensic analysis of textile dyes.

4. Determination of cocaine and other drugs of abuse in blood, urine etc.

Food and Flavour

1. Measurement of Quality of soft drinks and water.

2. Sugar analysis in fruit juices.

3. Analysis of polycyclic compounds in vegetables.

4. Preservative analysis.

Applications in Clinical Tests

1. Urine analysis, antibiotics analysis in blood.

2. Analysis of bilirubin, biliverdin in hepatic disorders.

3. Detection of endogenous Neuropeptides in extracellular fluid of brain etc.