History, Introduction, General Principles, Terminology, Classifications of Chromatography and Applications.

1. Dr. BASAVARAJAIAH S. M.
Assistant Professor and Coordinator
P.G. Department of Chemistry
Vijaya College
Bangalore-560 004
CHROMATOGRAPHY
(PART-I)

2. History
Introduction
GENERAL principleS
Terminology
Classification
Applications
CONTENTS:

3. HISTORY
M. Tswett,
(1872-1919)
Russian, Botanist
The first chemist to develop the chromatography
was a Russian botanist-M. S. Tswett.
He developed this useful technique in 1903 to
separate plant pigments under gravity using a
calcium carbonate column.
Tswett also coined the term ‘chromatography’,
which comes from the words chroma (Greek) i.e.
color and graph.
In fact, this technique can be used to distinguish
between two compounds that are quite similar in
molecular mass or charge; however, this requires an
appropriate combination of materials and operating
conditions.

4. Historical Developments in Chromatography
Investigator(s) Year Contribution
Karrer, Kuhn, and
Strain
1930-1932
Used activated lime, alumina and
magnesia absorbents.
Holmes and Adams 1935
Synthesized synthetic organic ion
exchange resins.
Reichstein 1938
Introduced the liquid or flowing
chromatogram
Izmailov and
Schraiber
1938
Discussed the use of a thin layer of
unbound alumina
spread on a glass plate.
Brown 1939
First use of circular paper
chromatography.
Martin and Synge 1941
Introduced column partition
chromatography.
Consden, Gordon,
and Martin
1944
First described paper partition
chromatography.

5. Investigator(s) Year Contribution
Boyd, Tompkins, et al 1947
Ion-exchange chromatography
applied
to various analytical problems.
M. Lederer and
Linstead
1949
Applied paper chromatography to
inorganic compounds.
Kirchner 1951
Introduced thin-layer
chromatography
James and Martin 1952 Developed gas chromatography.
Sober and Peterson 1956
Prepared first ion-exchange
celluloses
Porath and Flodin 1959
Introduced cross-linked dextran for
molecular sieving.
J. C. Moore 1964
Gel permeation chromatography
developed as a practical method

6. Introduction
Chromatography is an efficient analytical technique used
for the separation, identification, and analysis of various
components of a mixture.
The sample components often vary in physical and/ or
chemical properties, and this forms the very basis of their
separation through chromatography.
The compound that is separated during chromatography
is called analyte.
Chromatography was long back recognized as a powerful
separation technique that can separate the components
from a mixture with great precision.

7. All types of chromatographic systems involve two phases-
a stationary phase and a mobile phase.
The sample mixture to be analyzed is applied and allowed
to adhere to a stationary material known as the stationary
phase or adsorbent.
The stationary phase is usually a bonded phase that is covalently
bound or immobilized to the support particles or to the inside wall
of the column tubing.
The mobile phase is the phase which flow through
the stationary phase in a definite direction.
It is also called the carrier fluid or eluent as it
mobilizes and elutes the analyte out of the stationary phase.

9. GENERAL PRINCIPLES
Chromatographic separation involves a dynamic and rapid
equilibrium of molecules between the two phase’s i.e. stationary
phase and mobile phase.
Analyte molecules may exist in the free state i.e. absolutely
dissolved in the liquid or dispersed in the gaseous mobile
phase, or in bound state, i.e. adsorbed on the surface of the
solid stationary phase.
The equilibrium between the free and absorbed states
depends on following factors-
Polarity and size of the molecule
Polarity of the stationary phase
Polarity of the solvent

10. TERMINOLOGY
Eluotropic series: An eluotropic series is listing of various
compounds in order of eluting power for a given adsorbent.
Eluent: The eluent or eluant is the "carrier" portion of the
mobile phase. It moves the analytes through the chromatograph.
Eluate: The eluate is the analyte material that emerges from
the chromatograph. It specifically includes both the analytes and
solutes passing through the column, while the eluent is only the
carrier.
Elution time and elution volume: The "elution time" of a
solute is the time between the start of the separation and the
time at which the solute elutes. In the same way, the elution
volume is the volume of eluent required to cause elution.

11. Elution: Elution is the process of extracting one material from another by
washing with a solvent; as in washing of loaded ion-exchange resins to remove
captured ions.
Gradient Elution: The process by which the strength and
composition of the eluent is increased during the chromatographic
run thereby reducing analysis time.
Isocratic: Chromatographic conditions in which a constant
composition eluent is used.
Dead Volume: A measure of solvent accessible volume between
injector and detector after the space occupied by the column packing
material has been subtracted.
 Detection or Visualizing agents: The agent or chemical which help
or aid in the detection of spot of the analyte. Ex: UV light, I2 vapor,
Ninhydrin, H2SO4, 2,4-DNP.

12. Capacity Factor (k’): A factor which measures sample retention (tR)
independently of eluent flow rate or column length.
Column Efficiency (N): A term used to express the width of a peak
produced by a column. Efficiency is measured in terms of the number
of plates, a parameter which is inversely related to the square of the
peak width.
Overload: A saturation of the stationary phase by the solute which is
evidenced by band broadening, tailing and flat edged chromatographic
peaks.
Retention factor: The retention factor of a particular material is the
ratio of the distance the spot moved above the origin to the distance the
solvent front moved above the origin.
Retention Time: The elapsed time between sample injection and the
appearance of the chromatographic peak apex.

13. Resolution: A measure of the separation of two adjacent peaks. The
higher the resolution value the greater the separation.
Theoretical Plate: Measure of column efficiency. Length of column
relating to this concept is called height equivalent to a theoretical plate
(HETP).
Void: The formation of a space, usually at the head of the column,
caused by a settling or dissolution of the packing. A void in the column
leads to decreased efficiency and loss of resolution.
Void Volume (V0): The total volume of eluent in the column, the
remainder being taken up by packing material. Can be determined by
injecting an unretained substance.
Degassing: The practice of removing dissolved gases in the eluent. It
can be achieved by helium sparging, applying vacuum to the eluent,
ultrasonification or heating.

14. CLASSIFICATION OF CHROMATOGRAPHY TECHNIQUES
The classification of Chromatography is based on several
criteria, listed as follows:
Purpose of the chromatography experiment
Geometry of stationary phase and support used
Physical states of stationary phase and mobile phases
Principle of separation used
Polarity of stationary phase and mobile phase used

15. The Types of Chromatography Classified on
Each Basis are:-
I. Basedon purpose of chromatographyexperiment
a. Preparative chromatography: For this purpose, a large
amount of sample may be applied and the separated
compounds are collected for further use.
b. Analytical chromatography: The sample size applied to the
chromatographic system is very small the sample eluted from the
column is often disposed of.

16. II. Basedon the shape of stationaryphase and support:
a.Planar Chromatography: In this type the stationary
phase is planar and the development of chromatogram is
two-dimensional. In this chromatography, the mobile phase
moves through the stationary phase by capillary action
and/or by gravity. For example Paper chromatography &
Thin-Layer Chromatography (TLC).
b. Column chromatography: In this type, the stationary
phase is filled in a tube or column and the separation is
achieved based on different retention times of analytes as
they move through the column with the help of the mobile
phase. Examples: HPLC & GC.

17. III. Basedon the physical stateof stationaryphase and mobile phases:
a. Solid-Liquid Chromatography: In which stationary phase is solid
and the mobile phase is liquid.
b. Liquid-Liquid Chromatography: In this type of chromatography,
both the phases i.e. stationary phase and mobile phase are liquid but of
different polarities.
c. Gas-Solid Chromatography: In this type, the stationary phase
which is an active solid adsorbent in powdered form, is filled in a tube.
An inert gas like helium is used as the mobile phase or carrier gas.
d. Gas-Liquid Chromatography: Carrier gas (inert) is used as a mobile
phase and stationary phase comprises a nonvolatile liquid coated as a
thin layer on inactive solid support or the inside walls of the capillary
tube. The compounds are separated according to their partition-
coefficients.

18. IV. Basedon the principle of separation
a. Adsorption Chromatography: In adsorption
chromatography, the stationary phase is a solid material
and the mobile phase is either a liquid (solid-liquid
chromatography) or a gas (gas-solid chromatography). The
sample compounds are adsorbed on the solid stationary
phase through various interactions like covalent bonding
and electrostatic attraction. The basis of separation is the
difference in solubility of molecules in the mobile phase by
virtue of their polarity.
b. Partition Chromatography: In partition
chromatography, the stationary phase is a liquid supported
on an inert solid, while the mobile phase is either a liquid
(liquid-liquid chromatography) or gas (gas-liquid
chromatography). The basis of separation is the partitioning
of the solubility of the compounds between the liquid
stationary phase and the liquid or gaseous mobile phase.

19. c. Ion Exchange Chromatography: In this type of
chromatography the stationary phase is an ion exchange
resin on which the ionic sample components bind
electrostatically. The resin is either cationic or anionic. The
mobile phase is a buffer of predetermined pH. The buffer
serves to weaken the electrostatic interactions between the
analyte and the resin and elutes it out at a particular pH.
d. Molecular Exclusion Chromatography: It is also known
as gel permeation, gel filtration, or size exclusion
chromatography. The stationary phase is a porous gel with
a specific pore size. As the mobile phase passes through a
porous gel, larger solute molecules pass through the void
spaces while smaller molecules are entrapped in the pores
of gel beads. This allows the larger molecules to pass
through the column at a faster rate than the smaller ones,
and thus the compounds are separated based on their
molecular size.

20. e. Affinity Chromatography: It is a highly selective type of
chromatography that is based upon the specific interaction
between the analyte molecule and another compatible
molecule immobilized on a stationary phase. For example,
for the separation of antigens, an antibody may be
immobilized on a matrix that forms a stationary phase.
When a sample consisting of a mixture of proteins is passed
through the column, only the specific antigen is bound to
the antibody immobilized on the stationary phase. This
antigen can be extracted either by changing the ionic
strength / pH or through dialysis.

21. V. Basedon polarityof thestationaryphase andmobile phase used
a. Normal phase chromatography: In this type of chromatography, the
stationary phase is polar in nature, and the mobile phase is non-polar.
Therefore, while carrying out the elution, non-polar compounds are
eluted first. The polar compounds have a greater affinity to the polar
stationary phase, thus their mobility is slow in the system, therefore
are eluted later in the sequence. Ex: TLC, Paper chromatography etc
b. Reverse phase chromatography: This process is reverse to the
normal phase chromatography. The stationary phase is non-polar and
the mobile phase is polar in nature, therefore polar compounds are
eluted first and non-polar are eluted later. This technique is largely
used in the routine analysis for common polar compounds like drugs
and other pharmaceutics. Ex: HPLC, Gas chromatography.

22. APPLICATIONS
Chemical industry
In testing water samples and also checks air quality.
HPLC and GC are very much used for detecting various
contaminants such as polychlorinated biphenyl (PCBs) in
pesticides and oils.
In various life sciences applications
Pharmaceutical sector
To identify and analyze samples for the presence of trace
elements or chemicals.
Separation of compounds based on their molecular weight and
element composition.
Detects the unknown compounds and purity of mixture.
In drug development.

23. Environmental Applications
Detection of phenolic compounds in drinking water.
Bio-monitoring of pollutants.
Applications in Forensics
Quantification of drugs in biological samples.
Identification of steroids in blood, urine etc.
Forensic analysis of textile dyes.
Determination of cocaine and other drugs of abuse in
blood, urine etc.
In forensic pathology and crime scene testing like
analyzing blood and hair samples of crime place.

24. Applications in Clinical Tests
Urine analysis, antibiotics analysis in blood.
Analysis of bilirubin, biliverdin in hepatic disorders.
Detection of endogenous Neuropeptides in extracellular
fluid of brain etc.
Food and Flavour:
Measurement of Quality of soft drinks and water.
Sugar analysis in fruit juices.
Analysis of polycyclic compounds in vegetables.
Preservative analysis.

25. Applications in Clinical Tests
Urine analysis, antibiotics analysis in blood.
Analysis of bilirubin, biliverdin in hepatic disorders.
 Detection of endogenous Neuropeptides in extracellular fluid
of brain etc.
Molecular Biology Studies
Various hyphenated techniques in chromatography such as
EC-LC-MS are applied in the study of metabolomics and
proteomics along with nucleic acid research.
HPLC is used in Protein Separation like Insulin Purification,
Plasma Fractionation, and Enzyme Purification and also in
various departments like Fuel Industry, biotechnology, and
biochemical processes.

26. Thank you