An introduction to chromatography and its pplication in research

1. INTRODUCTION AND APPLICTIONS OF CHROMATOGRAPHY
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Table of Contents
1. Introduction…………………………………………………………………………………………………………………….1
1.1. Definition………………………………………………………………………………………………………………….1
2. Principle Of Chromatography ………………………………………………………………………………………….2
3. Commonly Used Terms In Chromatography……………………………………………………………………..2
4. Classification Of Chromatography…………………………………………………………………………………….3
5. Types Of Chromatography……………………………………………………………………………………………….4
5.1. Adsorption Chromatography…………………………………………………………………………………....4
5.2. Partition Chromatography…………………………………………………………………………………………6
6. Other Types Of Chromatography…………………………………………………………………………………..…7
7. Quantification Of Chromatogram…………………………………………………………………………………….7
8. Visualization Of The Spots Of Chromatogram………………………………………………………………..…8
9. Applications Of Chromatography……………………………………………………………………………………..8
10. References……………………………………………………………………………………………………………………….9

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1. Introduction:
Chromatography, literally "color writing", was first employed by Russian-Italian scientist
Mikhail Tsvet in 1900, primarily for the separation of plant pigments such as chlorophyll,
carotenes, and xanthophylls.
The word chromatography is derived from two Greek words
Chroma ….……..color
Graphos ………..writing
1.1. Definition:
Chromatography may be defined as
'A method of separating a mixture of components into individual components through
equilibrium distribution between two phases’.
OR
‘A technique by which a mixture is separated into its components on the basis of relative
ability of each component to be moved along/through a stationary phase by mobile phase’
One of these phases is a mobile phase and the other is a stationary phase. Porous medium
through which the mobile phase migrates is called the support.
The technique of chromatography is based on the differences in the rate at which the
components of a mixture move through a porous medium (called stationary phase) under
the influence of some solvent or gas (called moving/mobile phase).
Chromatography is a nondestructive procedure for resolving a multi-component mixture
of minor or major constituents into its individual fractions. It can be applied both for both
qualitative and quantitative studies as it is a separation technique.
2. Principle of Chromatography:
The principle of chromatography is like-dissolve-like or like-prefer-like. The basis of all forms
of chromatography is the partition or distribution coefficient ‘K’ which describes the way in
which a compound distributes itself between two immiscible phases. The Partition
coefficient; is defined as the molar concentration of analyte in the stationary phase divided
by the molar concentration of the analyte in the mobile phase
𝐾 =
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑐𝑜𝑚𝑝𝑜𝑛𝑒𝑛𝑡 𝑖𝑛 𝑠𝑡𝑎𝑡𝑖𝑜𝑛𝑎𝑟𝑦 𝑝ℎ𝑎𝑠𝑒
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑐𝑜𝑚𝑝𝑜𝑛𝑒𝑛𝑡 𝑖𝑛 𝑚𝑜𝑏𝑖𝑙𝑒 𝑝ℎ𝑎𝑠𝑒
The distribution of analytes between phases can be described simply. An analyte is in
equilibrium between the two phases;
Amobile Astationary

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3. Commonly used terms in chromatography:
 The analyte is the substance to be separated during chromatography.
 Analytical chromatography is used to determine the existence and the concentration of
analyte(s) in a sample.
 A chromatogram is the visual output of the chromatograph. In the case of an ideal
separation, different peaks or patterns on the chromatogram represent different
components of the separated mixture.

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 A chromatograph is an equipment that enables the separation e.g. gas chromatographic
or liquid chromatographic separation.
 The eluate is the mobile phase leaving the column.
 The eluent is the solvent that carries the analyte.
 An eluotropic series is a list of solvents ranked according to their eluting power.
 Elution is the process of extracting a substance that is adsorbed to another by washing it
with a solvent.
 An immobilized phase is a stationary phase that is immobilized on the support particles,
or on the inner wall of the column tubing.
 The mobile phase is the phase that moves over the stationary phase. It may be a liquid
(LC) or a gas (GC). The mobile phase moves through the stationary phase where the
sample interacts with the stationary phase and is separated.
 The retention time is the time required for the mobile phase to sweep a component from
the stationary phase.
 The retention volume is the volume of the mobile phase required to sweep a component
through stationary phase.
 The sample is the matter analyzed in chromatography. It may consist of a single
component or it may be a mixture of components. When the sample is treated, the phase
or the phases containing the analytes of interest is/are referred to as the sample whereas
everything else separated from the sample before or during analysis is referred to as
waste.
 The solute refers to the sample components in partition chromatography.
 The solvent refers to any substance capable of solubilizing another substance, and
especially the liquid mobile phase in liquid chromatography.
 The stationary phase is the substance fixed in place for the chromatography procedure.
It may be solid, gel or a liquid. e.g ; silica, alumina, cellulose
 The detector refers to the instrument used for qualitative and quantitative detection of
analytes after separation.
 Rf value or Retention factor (Rf) is defined as the ratio of the distance traveled by the
center of a spot (solute) to the distance traveled by the solvent front (solvent)
𝑅𝑒𝑡𝑒𝑛𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 =
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡
= 𝑅𝑓
4. Classification of chromatography:
The chromatography is either preparative or analytical
o Analytical ……….. to determine the chemical composition of a sample
o Preparative ………. used to purify and collect one or more components of a sample

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5. Types of chromatography:
5.1. Adsorption chromatography
5.2. Partition chromatography
5.1. Adsorption chromatography:
It uses a mobile phase or gaseous phase that is adsorbed
onto the surface of a stationary solid phase. The
equilibration between the mobile and stationary phase
accounts for the separation of different solutes. Following
are the chromatographic techniques that are included in
this category:
1. Thin Layer Chromatography (TLC)
2. Column Chromatography
3. Ion Exchange Chromatography
4. High Performance (pressure) Liquid Chromatography (HPLC)
5. Gel Permeation Chromatography
6. Gas Solid Chromatography (GSC)
1. Thin layer chromatography (TLC):
Thin layer chromatography is similar to paper chromatography, but the stationary
phase is a thin layer of a solid such as alumina or silica supported on an inert base
such as glass, aluminum foil or insoluble plastic. The mixture is ‘spotted’ at the
bottom of the TLC plate and allowed to dry. The plate is placed in a closed vessel
containing solvent (the mobile phase) so that the liquid level is below the spot. TLC
has advantages over paper
chromatography in that its results
are more reproducible, and that
separations are very efficient
because of the much smaller
particle size of the stationary
phase.
TLC is also called as drop, strip,
spread layer, surface
chromatography and open column
chromatography.

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2. Column chromatography:
A solvent acts as the mobile phase while a finely divided solid surface acts as the
stationary phase. The stationary phase will adsorb the
components of the mixture to varying degrees. As the
solution containing the mixture passes over the
adsorbent, the components are distributed between the
solvent and adsorbent surface. This process may be
described by three-way equilibrium between the sample,
the solvent and the adsorbent. The solvent and sample
compete for positions on the solid adsorbent, the solvent
displacing the sample reversibly and continuously in the
direction of the solvent flow. Consequently, a weakly
adsorbed compound will spend more time in the solvent,
and will therefore be eluted first.
3. Ion exchange chromatography:
Ion exchange chromatography is used to remove
ions of one type from a mixture and replace them
by ions of another type.
The column is packed with porous beads of a resin
that will exchange either cations or anions. There
is one type of ion on the surface of the resin and
these are released when other ions are bound in
their place – e.g. a basic anion exchange resin
might remove nitrate ions (NO3–) from a solution
and replace them with hydroxide ions (OH–).
4. High Performance (pressure) Liquid Chromatography (HPLC):
HPLC involves a liquid sample being passed over a solid adsorbent material packed
into a column using a flow of liquid solvent under pressure.
o Normal Phase HPLC:
NP-HPLC uses a non-polar, non-aqueous mobile phase (e.g. Chloroform), the
analyte associates with and is retained by the polar stationary phase. Adsorption
strengths increase with increased analyte polarity.
o Reversed Phase HPLC:
RP-HPLC has a non-polar stationary phase and an aqueous, moderately polar
mobile phase. With such stationary phases, retention time is longer for molecules

7. INTRODUCTION AND APPLICTIONS OF CHROMATOGRAPHY
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which are less polar, while polar molecules elute more readily (early in the
analysis).
5. Gel Filtration or Gel Permeation Chromatography:
The separation of large molecules, often in biochemical situations, can be achieved
in a column which works on the basis of molecular exclusion. The mixture of solutes
is carried through the column by a solvent. The stationary phase (the gel) typically
consists of particles of a cross-linked polyamide which contains pores. Separation
occurs according to molecular size – the larger molecules passing through the column
fastest. Different gels are available that allow the separation of proteins with relative
masses. The greatest resolution is achieved by using very small gel particles, but the
flow rate through the column then becomes much slower.
5.2. Partition chromatography:
This form of chromatography is based on a thin
film formed on the surface of a solid support by a
liquid stationary phase. Solute equilibrates
between the mobile phase and the stationary
liquid.
a. Paper Chromatography
b. Gas Liquid Chromatography (GLC)
a. Paper chromatography:
Cellulose filter paper is often used as stationary phase in paper chromatography.
Since it is hydrophilic, it is usually covered with a thin film of water. That’s why it is
often called as liquid-liquid chromatography.
Principle:
The substances are distributed between liquid
phases. One phase is the water which present
in pores of filter paper and other phase is mobile
phase which moves on the paper. The separation of
mixture is due to different attraction force
towards stationary phase (water) and mobile phase
(solvents).
Types of paper chromatography:
The classification is based on the procedure of the development of chromatogram
on the paper. So there are five types of paper chromatography.
 Ascending chromatography

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 Descending chromatography
 Ascending- descending mode
 Radial mode
 Two dimensional chromatography
Mostly ascending type or radial type chromatography is used because they are easy
to handle and not time consuming. They give chromatogram with fast speed.
b. Gas chromatography (GC):
o Gas-liquid chromatography (GLC):
In GLC the mobile phase is a gas and stationary phase is a thin layer of a non-volatile liquid
bound to a solid support thus a partition process occurs. In such case small inert particles
such as
Diatomaceous earth is coated with the liquid so that a large surface area exists for the
solute to equilibrate with.
o Gas-solid chromatography (GSC):
GSC utilizes a solid adsorbent as the stationary phase while gas as a mobile phase and an
adsorption process takes place.
The separation method can be affected by the polarity of stationary phase, temperature,
carrier gas flow, length of column, material amount etc.
6. Other types of Chromatography:
i. Affinity Chromatography:
This is the most specific type of chromatography. It is based on the specific interaction
between two molecules. The one is solute molecule and a second molecule is immobilized
on a stationary phase. For example, the immobilized molecule can be an antibody which
interacts on the particular area of protein.
When a solute with mixture of proteins is passed
by immobilized molecule, only the particular
protein is reacted to this antibody. Thus the
antibody binds to the stationary phase. After this
interaction, the extraction of protein is
completed by altering its ionic strength or pH.
The packing material is called the affinity matrix.
It should be inert. ‘Agarose’ is used for this
purpose.
ii. Countercurrent Chromatography (CCC):
Countercurrent chromatography (CCC), the liquid-liquid chromatography, technique, is
much less widely known and used than solid liquid chromatography. This technique uses
two immiscible liquids to form the stationary and mobile phase of a chromatographic
system. In all CCC systems, the stationary phase is held and maintained by gravitational

9. INTRODUCTION AND APPLICTIONS OF CHROMATOGRAPHY
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force. All modern, commercially manufactured CCC instruments use high gravitational
force fields and are essentially centrifuges.
7. Quantification of Chromatogram:
The components elute from the column can be quantified by a detector and/or collected for
further analysis. An analytical instrument can be combined with a separation method for on-
line analysis. Examples of such "hyphenated techniques" include gas and liquid
chromatography with mass spectrometry (GC-MS and LC-MS), Fourier-transform infrared
spectroscopy (GC-FTIR), and diode-array UV-VIS absorption spectroscopy (HPLC-UV-VIS).
8. Visualization of the Spots of Chromatogram:
It can be done by following method,
o Use of fluorescent material like manganese with
activated zinc silicate
o Spray of Iodine vapors
o UV exposure
o Ninhydrin for identification of amino acids
9. Applications of chromatography:
o It is commonly used technique for the separation of molecule. For example, it is used to
remove pesticides and insecticides like DDT in the water and poly chlorinated biphenyls.
o It is widely used to determine the purity of a substance.
o In pharmaceutical companies, it is used for producing pure materials for medicines and
also for checking the contamination presence in medicines.
o It is used in pharmacy for detecting the chiral compounds (Enantiomers and optical
isomers).
o In the food industry, this technique is very useful for analyzing and the separation of
additives, proteins and amino acids etc.
o It used in forensic science for detecting the presence of drugs.
o Chromatography has been extensively used in the isolation, characterization and
determination of the 60 or so carotenoids at present known.
10. References
1. M. Younus, Ilmi Kitab Khana, 2011. Organic Spectroscopy And Chromatography, Chromatograph,
pg. 269, ed. 3rd
.
2. Scott, R. P. W. Introduction to Analytical Gas Chromatography; 2nd ed.; Marcel Dekker,
1998.
3. Jennings, W. G.; Mittlefehldt, E.; Stremple, P. Analytical Gas Chromatography; 2nd ed.;
Academic Press, 1997.
4. McNair, H. M.; Miller, J. M. Basic Gas Chromatography; Wiley, 1997.
5. Grant, D. W. Capillary Gas Chromatography; Wiley, 1996.
6. Fowlis, I. Gas Chromatography; 2nd ed.; Wiley, 1995.

10. INTRODUCTION AND APPLICTIONS OF CHROMATOGRAPHY
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7. Scott, R. P. W. Techniques and Practices of Chromatography; 2nd ed.; Marcel Dekker,
1995.
8. Grob, R. L. Modern Practice of Gas Chromatography; 3rd ed.; Wiley, 1995.
9. Baugh, P. E. Gas Chromatography: A Practical Approach; Oxford, 1994.
10. Hinshaw, J. V.; Ettre, L. S. Introduction to Open Tubular Column Gas Chromatography;
Advanstar, 1994.
11. Grob, K. Split and Splitless Injection in Capillary Gas Chromatography; 3rd ed.; Hüthig,
1993.
12. Hill, H. H.; McMinn, D. G. Detectors for Capillary Chromatography; Wiley, 1992.
13. Grob, K. On-Column Injection in Capillary Gas Chromatography; 2nd ed.; Hüthig, 1991.
14. Poole, C. F.; Poole, S. K. Chromatography Today; Elsevier, 1991.
15. Baars, B.; Schaller, H. Fehlersuche in der Gaschromatographie; VCH, 1994.
16. Kolb, B. Gaschromatographie in Bildern; Wiley-VCH, New York, 1999.
17. Kenndler, E.; Huber, J. F. K. In Analytiker Taschenbuch; Springer, 1989