Chromatography is essential for separating, purifying, and identifying bioactive compounds from crude plant or microbial extracts.
These techniques exploit differences in polarity, size, and other physicochemical properties.
1) Thin-Layer Chromatography (TLC)
Principle
Separates compounds based on migration rates on a coated plate (silica, alumina).
Application
Rapid, cost-effective screening of multiple components in crude extracts.
Rf value comparison with known standards.
2) Column Chromatography
Principle
Uses a stationary phase (e.g., silica gel) and a mobile phase (solvent) for separation.
Application
Scalable fractionation of plant extracts.
Pre-purification step before advanced techniques.
3) High-Performance Liquid Chromatography (HPLC)
Principle
High-pressure pumps drive solvents through a column with small particles for high-resolution separation.
Application
Quantitative analysis and quality control of phytochemicals.
Coupled with UV, fluorescence, or MS for improved identification (HPLC-UV, HPLC-FLD, LC-MS).
Standardization of herbal extracts in pharmaceuticals.
4) Gas Chromatography (GC)
Principle
Separates volatile compounds based on boiling points and stationary phase interactions.
Application
Analysis of essential oils and volatile compounds.
Commonly paired with MS (GC-MS) for compound identification.
5) Supercritical Fluid Chromatography (SFC)
Principle
Uses supercritical fluids (e.g., CO₂) as the mobile phase.
Application
Suitable for non-polar to moderately polar compounds.
Eco-friendly alternative to organic solvents.
6) Preparative Chromatography (Application of Chromatography)
Principle
Scaled-up column or HPLC for bulk purification.
Application
Produces milligram to gram quantities of pure compounds for pharmacological testing.
These chromatographic methods complement spectroscopic techniques, ensuring comprehensive compound identification and purification.