1. INTRODUCTIONTO
CHROMATOGRAPHY
AND ITSAPPLICATIONS
Kalsoom Saleem
CMS # 8107
1

2. Introduction
Chromatography, literally "color writing",
was first employed by Russian-Italian
scientist MikhailTsvet 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
2

3. Definition
 ‘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’
 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
 Applied both for both qualitative and quantitative
studies
3

4. Principle
Like-dissolve-like or like-prefer-like.
The basis-partition or distribution coefficient ‘K’
which describes the way in which a compound
distributes itself between two immiscible phases.
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
4

6. 6

7. CommonlyUsed
Terms
7
Analyte
The substance to be separated during
chromatography
Analytical chromatography
Determines the existence and the
concentration of analyte(s) in a sample
Chromatogram
Visual output of the chromatograph

8. Chromatogram
8

9. CommonlyUsed
Terms
9
Chromatograph
An equipment that enables the separation e.g. gas
chromatographic or liquid chromatographic separation.
Eluate
Mobile phase
Eluent
The components obtained after the process.
Eluotropic series
List of solvents ranked according to their eluting power
Elution
Process of extracting a substance that is adsorbed on
another surface by moving it with solvent.

10. Commonly used
terms
Immobilized phase
Stationary phase that is immobilized on the support
particles, or on the inner wall of the column
Mobile phase
Phase that moves over the stationary phase. It may
be a liquid (LC) or a gas (GC).
Retention time
Time required for the mobile phase to sweep a
component from the stationary phase.
Sample
Matter analyzed in chromatography- single
component or multiple components
Solute
Sample components in partition chromatography.

11. Commonly
UsedTerms
11
Solvent
Any substance capable of solubilizing another substance, and
especially the liquid mobile phase in liquid chromatography
Stationary phase
Substance fixed in place for the chromatography procedure-
solid, gel or a liquid. e.g ; silica, alumina, cellulose
Detector
Instrument used for qualitative and quantitative detection of
analytes after separation
Rf value or Retention factor (Rf)
The ratio of the distance traveled by the center of a spot
(solute) to the distance traveled by the solvent front (solvent)

12. Classification
12
Analytical
• To determine the
chemical composition
of sample
Preparative
• Used to purify and
collect one or more
components of a
sample

13. Types
13
Adsorption
Partition

14. 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:
14
Techniques
Gas Solid
Chromatography
(GSC)
Gel Permeation
Chromatography
High
Performance
(pressure) Liquid
Chromatography
(HPLC)
Ion Exchange
Chromatography
Column
Chromatography
Thin Layer
Chromatography
(TLC)

15. Principle
15

16. Thin Layer
Chromatography
 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 is also called as drop, strip, spread layer, surface
chromatography and open column chromatography.
16

17. TLC

18. TLC PAPER

19. 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.

21. 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–).

22. Principle

23. High
Performance
(pressure)
Liquid
Chromatograp
hy (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.
Normal
Phase
HPLC
• 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.
Reversed
Phase
HPLC
• A non-polar stationary phase and an
aqueous, moderately polar mobile
phase.With such stationary phases,
retention time is longer for molecules
which are less polar, while polar
molecules elute more readily (early in
the analysis).

24. HPLCWorking

25. 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.

28. 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.
Paper
Chromatography
Gas Liquid
Chromatography
(GLC)

29. Principle

30. Gas
Chromatography
 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.
 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.

31. Principle

32. Working

33. 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).

34. Principle

35. Affinity chromatography

36. Detectors
 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)

37. Visualization of
Chromatogram
It can be done by following method,
Use of fluorescent material like
manganese with activated zinc silicate
Spray of Iodine vapors
UV exposure
Ninhydrin for identification of amino
acids

38. Applications
 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.
 It is widely used to determine the purity of a substance.
 In pharmaceutical companies, it is used for producing pure
materials for medicines and also for checking the contamination
presence in medicines.
 It is used in pharmacy for detecting the chiral compounds
(Enantiomers and optical isomers).
 In the food industry, this technique is very useful for analyzing
and the separation of additives, proteins and amino acids etc.
 It used in forensic science for detecting the presence of drugs.
 Chromatography has been extensively used in the isolation,
characterization and determination of the 60 or so carotenoids at
present known.

39. References
 M.Younus, Ilmi Kitab Khana, 2011. Organic Spectroscopy And Chromatography,
Chromatography, pg. 269, ed. 3rd.
 Scott, R. P.W. Introduction to Analytical Gas Chromatography; 2nd ed.; Marcel
Dekker, 1998.
 Jennings,W. G.; Mittlefehldt, E.; Stremple, P.Analytical Gas Chromatography;
2nd ed.; Academic Press, 1997.
 McNair, H. M.; Miller, J. M. Basic Gas Chromatography; Wiley, 1997.
 Grant, D.W. Capillary Gas Chromatography; Wiley, 1996.
 Fowlis, I. Gas Chromatography; 2nd ed.;Wiley, 1995.
 Scott, R. P.W.Techniques and Practices of Chromatography; 2nd ed.; Marcel
Dekker, 1995.
 Grob, R. L. Modern Practice of Gas Chromatography; 3rd ed.;Wiley, 1995.
 Baugh, P. E. Gas Chromatography: A Practical Approach; Oxford, 1994.
 Hinshaw, J.V.; Ettre, L. S. Introduction to OpenTubularColumnGas
Chromatography; Advanstar, 1994.
 Grob, K. Split and Splitless Injection in Capillary Gas Chromatography; 3rd ed.;
Hüthig, 1993.
 Hill, H. H.; McMinn, D. G. Detectors for Capillary Chromatography; Wiley, 1992.
 Grob, K. On-Column Injection in Capillary Gas Chromatography; 2nd ed.; Hüthig,
1991.
 Poole, C. F.; Poole, S. K. ChromatographyToday; Elsevier, 1991.
 Baars, B.; Schaller, H. Fehlersuche in der Gaschromatographie; VCH, 1994.
 Kolb, B. Gaschromatographie in Bildern;Wiley-VCH, NewYork, 1999.
 Kenndler, E.; Huber, J. F. K. In AnalytikerTaschenbuch; Springer, 1989