7.
Affinity Chromatography
Principle
Based on specific biological interactions between a ligand and its target molecule.
Examples of interactions:
Antigen–Antibody
Enzyme–Substrate
Receptor–Ligand
Components
Stationary phase: Matrix with immobilized ligand
Mobile phase: Buffer containing sample
Steps
1. Sample loading
2. Binding of specific target
3. Washing of unbound materials
4. Elution of target molecule
Applications
Purification of antibodies
Enzyme isolation
DNA/RNA purification
Forensic DNA sample preparation
8. Size Exclusion Chromatography (SEC)
Also called:
Gel Filtration (aqueous systems)
Gel Permeation Chromatography (organic systems)
Principle
Separation based on molecular size (hydrodynamic volume).
Stationary phase: Porous gel beads
Mobile phase: Liquid
Mechanism
Large molecules → Cannot enter pores → Elute first
Small molecules → Enter pores → Elute later
No chemical interaction is involved.
Applications
Protein molecular weight determination
Polymer analysis
Blood protein separation
9. Types of Chromatography (Broad
Overview)
Technique Mobile Phase Stationary Phase Mechanism
TLC Liquid Solid Adsorption
Paper Chromatography Liquid Liquid Partition
HPLC Liquid Solid/Liquid Partition/Adsorption
GC Gas Liquid/Solid Partition/Adsorption
Ion Exchange Liquid Charged Resin Ionic interaction
Affinity Liquid Ligand-bound matrix Biological binding
SEC Liquid Porous beads Size exclusion
10. Forensic Applications of
Chromatography
Chromatography plays a vital role in forensic science due to its high sensitivity and
specificity.
1. Drug Analysis
Detection of narcotics
Identification of synthetic drugs
Toxicological screening
2. Poison Detection
Identification of pesticides
Heavy metal analysis
Detection of toxic compounds in viscera
3. Explosive Analysis
Detection of TNT, RDX
Residue analysis
4. Ink and Document Examination
Separation of ink components
Detection of forged documents
5. Alcohol Determination
Blood alcohol analysis using GC
6. DNA and Protein Analysis
Purification of biomolecules
Forensic serology
7. Environmental Forensics
Soil and water contamination analysis
Oil spill identification
11. Advantages of Chromatography
High sensitivity
High selectivity
Rapid analysis
Small sample requirement
Quantitative and qualitative analysis
12. Limitations
Expensive instrumentation (HPLC, GC)
Skilled operation required
Maintenance cost
Sample preparation sometimes complex
13. Short Notes (Exam-Oriented Points)
Why is chromatography important?
Because it allows separation, identification, and quantification of complex mixtures with high
accuracy.
What factors affect separation?
Polarity
Molecular size
Charge
Temperature
Flow rate
Nature of stationary phase
14. Conclusion
Chromatography is a versatile and powerful analytical tool based on the differential
interaction of components between stationary and mobile phases. Depending on the
mechanism—adsorption, partition, ion exchange, affinity, or size exclusion—different types
of chromatography are used for various analytical purposes. In forensic science,
chromatography is indispensable for drug detection, toxicological investigations, document
examination, and environmental analysis.
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