Chromatography is a laboratory technique used to separate mixtures by distributing components between two phases. It was invented in 1903 by Russian botanist Mikhail Tswett to separate plant pigments. The basic terms include the stationary phase, mobile phase, elution, and retention factor. The main types are adsorption chromatography, partition chromatography, ion exchange chromatography, size exclusion chromatography, and affinity chromatography. These techniques have various forensic applications such as drug analysis, ink analysis, and determining if a deceased person consumed drugs or alcohol.

1. TOPIC:BASICS OF CHROMATOGRAPHY AND THEIR
FORENSIC APPLICATION
PRESENTED BY:
PALLAVI KUMARI
ID NO: 19MSFS001
M . Sc 1st SEMESTER 2019-21

2. CONTENTS:
 INTRODUCTION
 HISTORY
 BASIC TERMS
RELATED TO
CHROMATOGRAPHY
 TYPES AND THEIR
APPLICATIONS
 CONCLUSION

3. INTRODUCTION
 Chromatography comes from Greek Word
chroma : colour
graphein: writing
 What is chromatography?
Chromatography is a laboratory technique by which a
mixture is separated by distributing its components between
two immiscible phase.

4. HISTORY
 Chromatography was first invented by
a Russian Botanist Mikhail Tswett in
1903.
 He separate plant pigments such as
chlorophyll,xanthophyll and
carotenoids by using calcium
carbonate as stationary phase and
hydrocarbon as mobile phase.

5.  STATIONARY PHASE
 MOBILE PHASE
 ELUTION
 ELUTION VOLUME
 ELUTION TIME
 VOID VOLUME
 RETENTION FACTOR
 CHROMATOGRAM
 RESOLUTION
BASIC TERMS RELATED TO CHROMATOGRAPHY

6. TYPES OF CHROMATOGRAPHY
1. ADSORPTION CHROMATOGRAPHY
2. PARTITION CHROMATOGRAPHY
3. ION EXCHANGE CHROMATOGRAPHY
4. SIZE/MOLECULAR EXCLUSION CHROMATOGRAPHY
5. AFFINITY CHROMATOGRAPHY

7. 1.ADSORPTION CHROMATOGRAPHY
• DEFINITION: It is atype of chromatography which based on the
principle of adsorption. Here the separation is based on the
interaction of the absorbate with the adsorbent.
• Principle: It involves the analytical separation of a chemical mixture
based on the interaction of the absorbate with the adsorbent that
adsorbs different compounds at different rate
Adsorbent : silica gel,
alumina ,etc.
Mobile phase : either
liquid or gas

8. TYPES OF ADSORPTION CHROMATOGRAPHY
A. Adsorption column chromatography
Principle: Main principle of this technique is adsorption. Mixture of components
dissolved in the mobile phase is introduced in to the column and components
move depending upon their relative affinities.
The compounds which has more affinity towards stationary phase travels slow
and the compound which has lesser affinity towards stationary phase travels
faster.
Stationary phase: The stationary phase or adsorbent in this type is a solid. The most
commonly used is silica gel and alumina.
Mobile phase: The mobile phase or eluent is a solvent or a mixture of solvents.
History: Russian botanist Mikhail Tsvet invented column chromatography in
1906 as a means of studying plant pigments.

9. Instrumentation:
Applications:
Purification of tincture of alkaloids from pigments before determination of their alkaloidal
content.
Drug analysis, urine or other body samples ( high pH urine will elute first).
 Isolation and purification of vitamins and hormones.

10. B. Thin layer chromatography (TLC)
PRINCIPLE: Similar to other chromatographic method, TLC is also based on the principle of
separation. The separation depends on the relative affinity of compounds towards stationary
phase and mobile phase. High affinity travels slower and other travels faster.
HISTORY: TLC is discovered by A.A. Izmailov and M.S. Shraiber in 1938.
INSTRUMENTATION:
i. TLC plate
ii. TLC chamber
iii. Mobile phase
iv. A Filter paper

11. Applications of TLC:
To check the purity of given samples.
Identification of compounds like
acids,alcohols,proteins,alkaloids,amines, antibiotics and
more.
Used for identification of drugs, dyes ,inks, etc.
Comparison of drugs, dyes and inks.
Analysis of urine and blood.

12. C. Gas solid chromatography
PRINCIPLE: The basic principle of gas chromatography is that the resolution of
the constituents by partitioning them between the mobile phase and the stationary
phase inside the gas chromatography column.
HISTORY: The father of modern gas chromatography is John Porter Martin who
also developed the first liquid chromatograph in 1950.
INSTRUMENTATION:
i. Carrier gas
ii. Flow controller
iii. Sample injector
iv. Column
v. Oven
vi. Detector

13. Applications:
Gas chromatography is used to determined if a deceased person has taken any alcohols or drugs
prior to death as well as determining if they had been poisoned.
Separation and identification of volatile materials , plastics, natural, and synthetic polymers,
paints, and microbiological samples.
Analysis of drug products like antibiotics(penicillin), antiviral(amantidine), general anesthetics(
chloroform,ether), sedatives or hypnotics(barbiturates), etc.
In determining the levels of metabolites in body fluids like plasma, serum, urine, etc.

14. 2. PARTITION CHROMATOGRAPHY
PRINCIPLE: The separation of the components from the sample mixture is carried out by the
process of partition of the components between two phases (both are in liquid phase). The liquid
surface is immobilised by stationary phase which results in making it stationary phase. The
mobile phase moves from the stationary phase and components get separated. The separation
depends on different co-efficient.
HISTORY: This separation theory was introduced by Richard Laurence Millington and Archer
Martin in 1940s.
APPARATUS REQUIRED:
i. Chromatography jar
ii. Liquid impregnated paper (stationary phase)
iii. Capillary tube (to apply sample mixture)
iv. Mobile phase (chloroform, methanol, acetone, ethanol)

15. SOME MAIN TYPES OF PARTITION CHROMATOGRAPHY
A. Liquid liquid chromatography
This is a chromatographic technique where a sheet of blotting paper is used instead of
adsorption column. The components are separated based on their differential migration
velocities.
B.Gas liquid chromatography
This is a type of chromatography technique in which the separation of the mixture is done by
an inert gas along a tube. The tube is filled with with finely divided inert solid. The solid is
coated with non-volatile oil. The migration of each component occurs at a rate determined by
its solubility in oil as well as its vapour pressure.
C. Paper chromatography
PRINCIPLE: Differential partitioned co-efficient of the components between water on the solid
stationary phase and the mobile phase.
Solid support: Paper
Stationary phase: Whatman’s paper no.1 & 3, CM-cellulose, DEAE( diethylaminoethane)
Mobile phase: solvent system (ex: chloroform, benzene ,butanol, cyclohexane ,etc.)

16. INSTRUMENTATION/COMPONENTS REQUIRED
i. Solid support
ii. Mobile phase
iii. Stationary phase
iv. Chromatographic chamber
APPLICATIONS:
Separation between polar and non-polar compound.
To determine organic compounds , biochemicals in urine.
For determination of hormones and drugs.
Identification of inks.

17. 3) ION EXCHANGE CHROMATOGRAPHY
PRINCIPLE: Ion exchange chromatography separates
molecules based on their respective charged groups. It
retains analyte molecules on the column based on ionic
interactions.
HISTORY: It was first reported by Peterson and Sober in
1956. In modern form it was invented by Small, Stevens
and Bauman in 1975.
TYPES:
1). Cation exchange chromatography
2). Anion exchange chromatography

18. INSTRUMENTATION:
i. Pump,
ii. Injector,
iii. Column,
iv. Suppressor,
v. Detector,
vi. Recorder or data system.
APPLICATIONS:
 Determination and identification of chlorate, nitrite and nitrate in cases of explosives
and explosive residues were investigated.
 Analysis of amino acid mixtures.
 In treatment of water.
 In the analysis of products of hydrolysis of nucleic acids.

19. 4) AFFINITY CHROMATOGRAPHY
PRINCIPLE: Separation in chromatography depends
upon the reversal adsorption of biomolecules through
biospecific interactions on the ligands.
HISTORY: Discovered by Pedro and Wilcheck in
1968.
COMOPONENTS:
i. Matrix
ii. Spacer arm
iii. Ligand

20. To identify the biological compound binding to a specific sites.
It is essentially a sample purification technique, used primarily for biological molecules such as
proteins.
Removal of impurities or in purification process.
Nucleic acid purification.
Investigation of binding sites of enzymes.
In in vitro antigen-antibody reactions
APPLICATIONS:

21. 5) SIZE/MOLECULAR EXCLUSION CHROMATOGRAPHY
PRINCIPLE: The principle of size exclusion chromatography is that
particles of different sizes or different molecular weight elute from the
stationary phase at different rates.
HISTORY: In 1979, Yau and Kirkland published the first text on size
exclusion chromatography.
TYPES:
A. Gel permeation chromatography(GPC): In this type, a
hydrophobic column packing material is used and a non-
aqueous mobile phase (organic solvent) to measure the
molecular weight distribution of synthetic polymers.
B. Gel filtration chromatography(GFC): In this type, a hydrophilic
packing material and an aqueous mobile phase is used to
separate, fractionate, or measure the molecular weight
distribution of molecules soluble in water, such as
polysaccharides and proteins.

22. INSTRUMENTATION:
i. INJECTION VALVE
ii. PUMP
iii. COLUMN
iv. DETECTOR
v. RECORDER

23. APPLICATIONS:
 Purification of biological macromolecules like viruses, protein, enzymes, nucleic acids,
antibodies and polypeptides.
separation of low molecular weight components from mixture.
Separation of biological molecules in food specimens.

24. CONCLUSIONS
Chromatography is important because it is a versatile and
small quantities of a material can be separated with ease, it
is fast and accurate if the hardware is
maintained. Chromatography is one of the most common
techniques in analytical technology and needs only a few
micrograms of material in order to work.

25. THANK YOU