Separation Techniques

Separation & Chromatographic Methods in Pharmacognosy

A. Fractional Techniques

  1. Fractional Crystallization

  • Principle: Separation based on solubility differences. On cooling, less soluble compounds crystallize first.

  • Use: Purify natural compounds (e.g., crystalline products from plant extracts).

  1. Fractional Distillation

  • Principle: Separation based on boiling points. Uses repeated vaporization & condensation in a column.

  • Use: Isolation of volatile oils and compounds with close boiling points.

  1. Fractional Liberation (for alkaloids)

  • Principle: Acidify → convert alkaloids into salts → extract with organic solvent → adjust pH stepwise → alkaloids precipitate depending on basicity.

  • Use: Purify different alkaloids from plants.

B. Chromatography Types

1. Adsorption Chromatography

  • Principle: Separation based on adsorption to a solid surface (silica/alumina).

  • Use: Separate phytochemicals (oils, terpenes, amino acids, alkaloids, vitamins, steroids).

2. Partition Chromatography

  • Principle: Separation based on solubility in two immiscible liquids.

  • Use: For alkaloids, flavonoids.

3. Ion-Exchange Chromatography

  • Principle: Separation based on charge interactions with ion-exchange resin.

  • Use: For amino acids, peptides, alkaloids.

4. Electrophoresis (Electrochromatography)

  • Principle: Separation by charge + size under an electric field.

  • Use: Proteins, nucleic acids.

5. Gel Filtration (Size-Exclusion)

  • Principle: Separation by molecular size using porous beads (large molecules come out first).

  • Use: Polysaccharides, proteins.

C. Chromatographic Methods

  1. Column Chromatography

  • Principle: Stationary phase in column, solvent passes through, separation occurs.

  • Use: Purify alkaloids, flavonoids (large amounts).

  1. Thin Layer Chromatography (TLC)

  • Principle: Silica layer on plate, solvent moves upward, separation by adsorption & solubility.

  • Use: Quick test, analysis of plant extracts, detect drugs, pesticides, amino acids.

  1. Paper Chromatography

  • Principle: Uses filter paper, separation by solubility.

  • Use: Plant pigments (chlorophyll, anthocyanins).

  1. Gas Chromatography (GC)

  • Principle: Sample vaporized, separated by volatility & interaction with stationary phase.

  • Use: Essential oils, volatile plant compounds.

  1. High-Performance Liquid Chromatography (HPLC)

  • Principle: High pressure forces solvent through finely packed column for better separation.

  • Use: Quantification & purification of plant compounds (small to large biomolecules).

👉 Quick Mnemonics

  • Fractional Techniques: Crystallization (solubility), Distillation (boiling point), Liberation (alkaloids).

  • Chromatography Types:

    • Adsorption (surface), Partition (solubility), Ion-Exchange (charge), Electrophoresis (charge+size), Gel Filtration (size).

  • Methods: Column, TLC, Paper, GC, HPLC.