Applications of Chromatography 1

APPLICATIONS OF CHROMATOGRAPHY
Chromatography is a process used to separate mixtures. The word chromatography is derived
from the Greek words "khroma" and "graphein" meaning "color" and "to write" or "to
represent". The chromatography technique is first discovered by Russian Biologists, Dr
Michael Tswett in 1906 for the separation of colored plant pigment on a column of alumina.
Now-a-days various types of chromatography are used to separate almost any given mixture
whether colored or colorless into its component.
Chromatography may be regarded as an analytical technique employed for the purification
and separation of organic and inorganic substances. It is also found useful for the
fractionation of complex mixture, separation of closely related compounds, such as isomers
and in the isolation of unstable substances.
Although there are several different types of chromatography, in each case a substance is
placed onto or into a medium and a solvent is passed through the test substance. In
chromatography science, the solvent is called "the mobile phase" or "the carrier fluid" and the
medium is called "the stationary phase".
Types of Chromatography:
There are three ways to classify chromatography. They area) Classification of chromatography according to mobile phase:
1. Liquid chromatography: mobile phase is a liquid. (Liquid-Liquid
Chromatography, Liquid-Solid Chromatography).
2. Gas chromatography: mobile phase is a gas. (Gas-Solid Chromatography, GasLiquid Chromatography)
b) Classification according to the packing of the stationary phase:
1. Thin layer chromatography (TLC): the stationary phase is a thin layer supported
on glass, plastic or aluminum plates.
2. Paper chromatography (PC): the stationary phase is a thin film of liquid supported
on an inert support.
3. Column chromatography (CC): stationary phase is packed in a glass column.

Applications of Chromatography 2

c) Classification according to the force of separation:

1. Adsorption chromatography.
2. Partition chromatography.
3. Ion exchange chromatography.
4. Gel filtration chromatography.
5. Affinity chromatography.
Table 01: Different Types of chromatography
Mode or type

Stationary phase

Mobile phase

Mechanism

Adsorption

Solid that attracts the Liquid or gas

Solutes move at different rates

Chromatography

solutes

according to the forces of

attraction to the stationary phase.

Partition

Thin film of liquid

Liquid or gas

Solutes equilibrate between the 2

Chromatography

formed on the

phases according to their

surface of a solid

partition coefficients

inert support
Ion Exchange

Solid resin that

Liquid containing Solute ions of charge opposite to

Chromatography

carries fixed ions & electrolytes

the fixed ions are attracted to the

mobile counter-ions

resin by electrostatic forces &

of opposite charge

replace the mobile counter-ions.

attached by covalent
bonds
Molecular

Porous gel with no

Exclusion

attractive action on

Chromatography

solute molecules

Liquid

Molecules separate according to

their size:
1. Smaller molecules enter the
pores of the gel, and need a
larger volume of eluent.

Applications of Chromatography 3

2. Larger molecules pass through

the column at a faster rate.

Affinity

Solid on which

Liquid or gas

Special kind of solute molecules

Chromatography

specific molecules

interact with those immobilized

are immobilized

on the stationary phase

Applications of Chromatography:
Chromatographic methods will separate ionic species, inorganic or organic, and molecular
species ranging in size from the lightest and smallest, helium and hydrogen, to particulate
matter such as single cells. No single configuration will accomplish this, however. Little preknowledge of the constituents of a mixture is required. At its best, chromatography will
separate several hundreds of components of unknown identity and unknown concentrations,
leaving the components unchanged. Amounts in the picogram or parts per billion ranges can
be detected with some detectors.
The solutes can range from polar to nonpolar i.e., water-soluble to hydrocarbon-soluble.
Substances of low critical temperature or low molecular weight, such as the gases at
laboratory conditions showing dispersive or London intermolecular forces only, are separated
with molecular sieves or gas-solid techniques. Gas-liquid chromatography is applicable to
species with high critical temperatures and normal boiling points as high as 400 C.
Substances that are solids at normal laboratory conditions with molecular weights below1000
are best separated with liquid-solid or liquid-liquid systems. Lower members of the molecular
weight scale range are amenable to separations. Methods are involved at molecular weights
above 1,000. Field-flow fractionation extends the size range to colloids and microscopic
particles.
General uses of chromatography in our real life are:
Pharmaceutical Company determine amount of each chemical found in new product.
Hospital detect blood or alcohol levels in a patients blood stream.

Applications of Chromatography 4

Law Enforcement to compare a sample found at a crime scene to samples from

suspects.
Environmental Agency determine the level of pollutants in the water supply.
Manufacturing Plant to purify a chemical needed to make a product.
Biotechnology industry establishing the purity or concentration of compounds in
biotechnological research.
Biological application Chromatography has many applications in biology. It is used to
separate and identify amino acids, carbohydrates, fatty acids, and other natural
substances. Environmental testing laboratories use chromatography to identify trace
quantities of contaminants such as PCBs in waste oil and pesticides such as DDT in
groundwater. It is also used to test drinking water and test air quality. Pharmaceutical
companies use chromatography to prepare quantities of extremely pure materials. The
food industry uses chromatography to detect contaminants such as aflatoxin.
For example we can consider the use of chromatography in Forensic Toxicology.
Forensic toxicology is the application of toxicology for the purpose of law.
Forensic Chemistry and Toxicology generally concerns the detection and characterization
of poisons or toxins exhibiting adverse physiological effects. Laboratory methods used in
chemical toxicological analysis cover a wide range and may be broadly classified as
follows: (1) physical tests (2) crystal tests (3) chemical spot tests (4) spectrophotometric
tests (5) chromatographic tests.
Toxicology is the study of substances that are harmful to human beings. Forensic
Toxicologists have the responsibility of detecting and identifying the presence of drugs
and poisons in fluids, tissues and organs. Their services are not just required in crime
laboratories and medical examiner's offices; they reach into hospital laboratories where
the identification of an overdose can mean the difference between life and death. The
work of a toxicologist generally falls into three main categories:
1. Routine testing for alcohol in blood or urine samples following a "breathalyzer" test.
However with the advent of new technology much of the testing can be carried out by
police with fewer cases being passed on to the forensic laboratory. Like Gas

Applications of Chromatography 5

chromatography was, and still is, used to identify exactly how much alcohol is present
in the blood or urine of a suspect. Because it is very accurate, results can be used as
evidence in a court of law.
2. Identification of drugs such as heroin, cocaine, cannabis, etc. It is common to perform
preliminary color or microcrystalline tests before using chromatography to identify a
substance as chromatography yields somewhat inconclusive results. However, both
thin layer and gas chromatography in conjunction with the preliminary tests is well
suited for drug analysis.
3. Detection of drugs and poisons in body fluids, tissues and organs. This area of
forensic toxicology involves the analysis of organs, tissues and body fluids in such
circumstances as sudden deaths and suspected poisonings. A post mortem is
performed by a pathologist who sends specimens of various body tissues and fluids to
the forensic toxicologist for examination. Many techniques are used in this area of
work including chromatography
Depend on the mechanisms different types of chromatography are specified in uses of
different actions. Applications according to the mechanism are given bellow:
Applications of Paper Chromatography:
1. Paper chromatography has widely been used for quantitative analysis of Inorganic,
organic and biochemical interest.
2. Paper chromatography is ideally suited for rapid analysis of reaction mixture and so it
is versatile tool in the hand of organic chemists.
3. Paper chromatography has been successfully used for characterizing and isolating the
following organic compounds.

Acids,
Alcohols,
Glycols,
Alkaloids,
Amines,

Amino acids
Proteins and peptides,

Antibiotics etc.

4. Paper chromatography has also been used in the analysis of mixture of sugars.
5. It can be used to detect traces of pollutants in water food or in soil.

Applications of Chromatography 6

6. It can be used for the identification of compounds in drugs, in biochemical

preparation and in natural products. It can be used for checking the purity of samples.
Applications of Gel-filtration Chromatography
1. Analytical applications
Analytical group separation
Analytical fractionation
Determination of molecular masses: Determination of M. wt. of peptides, proteins
& polysaccharides
2. Preparative applications
Preparative fractionation
Preparative group separations
Separation of mixture of mono-and polysaccharides.
Separation of amino acids from peptides & proteins.
Separation of proteins of different molecular weights.
Separation of mucopolysaccharides & soluble RNA.
Separation of myoglobin & haemoglobin.
Separation of alkaloids & purification of enzymes.
3. Biochemical applications: In general, Gel-filtration chromatography which is also
known as Size Exclusion chromatography is considered a low resolution
chromatography as it does not discern similar species very well, and is therefore often
reserved for the final "polishing" step of purification. The technique can determine the
quaternary structure of purified proteins that have slow exchange times, since it can
be carried out under native solution conditions, preserving macromolecular
interactions.

Applications of Chromatography 7

Size Exclusion chromatography can also assay protein tertiary structure, as it

measures the hydrodynamic volume (not molecular weight), allowing folded and
unfolded versions of the same protein to be distinguished. For example, the apparent
hydrodynamic radius of a typical protein domain might be 14 and 36 for the
folded and unfolded forms, respectively. Size Exclusion chromatography allows the
separation of these two forms, as the folded form will elute much later due to its
smaller size.
4. Polymer synthesis: Gel-filtration chromatography or Size Exclusion chromatography
can be used as a measure of both the size and the polydispersity of a synthesized
polymer, that is, the ability to be able to find the distribution of the sizes of polymer
molecules. If standards of a known size are run previously, then a calibration curve
can be created to determine the sizes of polymer molecules of interest in the solvent
chosen for analysis. In alternative fashion, techniques such as light scattering and/or
viscometry can be used online with Size Exclusion chromatography to yield absolute
molecular weights that do not rely on calibration with standards of known molecular
weight. Due to the difference in size of two polymers with identical molecular
weights, the absolute determination methods are, in general, more desirable. A typical
Size Exclusion chromatography system can quickly (in about half an hour) give
polymer chemists information on the size and polydispersity of the sample. The
preparative Size Exclusion chromatography can be used for polymer fractionation on
an analytical scale.
Applications of Thin Layer Chromatography
1. As a check on process: It has been used for checking of the other separation
procedures and purification processes.
2. In Organic Chemistry:
The main use of Thin Layer Chromatography is isolation and separation of
individual components of a mixture.
The main reasons for popularity of Thin Layer Chromatography as an analytical and
preparation methods are:
It can be used for most of chemical compounds.

Applications of Chromatography 8

It has high speed of separation.

Its selectivity is high.
The following are the various applications of Thin Layer chromatography in organic
chemistry:
For checking the purity samples as a purification process.
Examination of reactions.
For identifying organic compounds.
Thin Layer Chromatography has been successfully used for characterizing and
isolating the following organic compounds.
i. Acids

v. Amines

ii. Alcohols

vi. Amino acids, proteins and

iii. Glycols
iv. Alkaloids

peptides
vii. Antibiotics

Besides these, there are compounds like carbohydrates, carbonyl compounds, Dyes,
Hydrocarbons, lipids, nucleic acids, pesticides, natural pigments, pharmaceutical products,
phenols, steroids, terpenes, essential oils, vitamins, adhesives, explosives plasticizers etc.
which have been separated and characterized by Thin Layer Chromatography.
3. For separation of Inorganic Ions: Recently Thin Layer Chromatography has been used for separating cationic,
anionic, purely covalent species and also some organic derivatives of the metals.
4. Applications of Thin Layer Chromatography in quantitative analysis:
i. Spectrophotometric
Measurement
ii. Fluorimetric Method
iii. Visual comparison of spots

iv. Spectral reflectance

v. Spot densitometer
vi. Vapour phase chromatography

Applications of Chromatography 9

vii. Radioactive methods

viii. Volumetric analysis

Application of Column Chromatography:1. Analytical uses: For analytical purposes, column chromatography finds limited
applications. Vestergaard and Sayegh could separate seven urinary steroids within 5
hours which requires 36 hours on a normal column. They have used narrow Teflon
tubing packed with aluminum oxide or silica gel.
2. Separation of geometrical isomers: The separation of cis/trans isomer is based on the
steric factors. Isomers whose functional groups can approach the surface of the
adsorbent more easily are more strongly adsorbed.
3. Separation of Diastereomers.
4. Separation of tautomeric mixtures.
5. Separation of racemates.
Applications of Affinity chromatography:
1. Purify and concentrate a substance from a mixture into a buffering solution.
2. Reduce the amount of a substance in a mixture.
3. Discern what biological compounds bind to a particular substance, such as drugs.
4. Purify and concentrate an enzyme solution.
Applications of Ion Exchange Chromatograph:
1. Water softening: Removal of Ca2+, Mg2+ & other multivalent ions causing hardness
of water by filtration through a layer of strong cation resin.
2. Separation of electrolytes from non-electrolytes.
3. Neutralization: Cationic exchanger in [H+] can be used to neutralize alkali hydroxide
& anionic exchanger in [OH-] form to neutralize the acidity.
4. Water demineralization: Removal of cations & anions dissolved in water. Usually
carried by the two step technique in which two columns of strongly acid cation

Applications of Chromatography 10

exchanger in [H+] form & strongly basic anion exchanger in [OH-] form are used in
sequence.
5. Separation of carbohydrates & their derivatives:
Uronic acids separated on anion exchanger.
Sugars converted into ionized form by using borate& separated on strong anion
exchanger.
Hexosamines separated on strong cation exchanger.
Medical Applications of High Performance Liquid Chromatography:
The isolation and purification of compounds is of critical importance to medicinal
chemists discovering and developing drugs. In these activities it is extremely important
to be able to produce mg quantities of the target compounds, from a synthetic reaction
mixture. Then later on use the same separation technologies to provide gram and Kilo
quantities. Similarly, the technique can isolate, purify and concentrate suitable
quantities of low level impurities and metabolites to support compound identification
and further characterization.
One of the key advantages of high performance counter current chromatography is that
it scales easily and simply. This allows the medicinal chemist to focus on the valueadded part of the development process i.e. the chemistry, and alleviates the need to
waste time developing scaled-up chromatography techniques, as demand for the
quantity of compound increases.
A further important issue is sample solubility since this can affect the throughput to
produce a specified quantity of the compound of interest. Typically, this can become an
issue when the purification is performed in reverse phase (RP) which generates aqueous
fractions. These aqueous fractions are laborious to concentrate by evaporation and this
process can lead to degradation of the product.
As scale of production increases the volumes of solvents used and those of product
containing fractions also increase. This is particularly an issue where Reverse PhaseHigh Performance Liquid Chromatography produces large volumes of aqueous
fractions. High performance counter current chromatography can be used in normal

Applications of Chromatography 11

phase for the same separation which means that fractions can be collected in essentially
non-aqueous solvents which makes their processing simpler, faster and less energy
consuming.
High Performance Liquid Chromatography has found many applications in medicinal
chemistry, to which the Spectrum or Midi bench top products are normally found to be
the most suitable for the quantities of compound required.