Column Chromatography, TLC, Paper Chromatography & Electrophoresis
This unit introduces the fundamental chromatographic and electrophoretic separation techniques. Column chromatography (adsorption and partition modes), Thin Layer Chromatography (TLC — the workhorse of pharmaceutical labs for identification and purity testing), Paper Chromatography (an early planar technique), and Electrophoresis (separation based on charge — paper, gel, and capillary formats). These techniques form the foundation for understanding advanced methods in later units.
Syllabus & Topics
- 1Introduction to Chromatography: Chromatography: separation technique based on differential distribution of components between a STATIONARY phase and a MOBILE phase. Invented by Tswett (1906) — separated plant pigments on a CaCO₃ column (chroma = color, graphein = to write). Classification by mechanism: (1) Adsorption: components adsorbed on solid surface with different affinities. (2) Partition: components partition between two immiscible liquid phases. (3) Ion exchange: based on ionic interactions. (4) Size exclusion/gel: separation by molecular size. (5) Affinity: specific biological interactions. Classification by format: Column (packed bed), Planar (TLC, paper), Instrumental (HPLC, GC).
- 2Adsorption Column Chromatography: Stationary phase: solid adsorbent packed in a glass column. Common adsorbents (in order of activity): Alumina (Al₂O₃) > Charcoal > Silica gel > Florisil > MgO > CaCO₃. Silica gel and Alumina most widely used. Mobile phase: liquid solvent or solvent mixture — elutes components based on their adsorption affinity. Eluotropic series (increasing eluting power): Hexane < CCl₄ < CHCl₃ < Acetone < Ethanol < Methanol < Water. Methodology: (1) Pack column with adsorbent (slurry or dry packing). (2) Apply sample at top. (3) Elute with mobile phase (isocratic or gradient). (4) Collect fractions. (5) Analyze fractions (UV, TLC). Advantages: preparative scale, simple, versatile. Disadvantages: slow, large solvent volumes, poor resolution compared to HPLC.
- 3Partition Column Chromatography: Stationary phase: liquid coated on an inert solid support. Components partition between stationary liquid and mobile liquid based on their partition coefficients (K = Cs/Cm). Normal phase: stationary phase is POLAR (water on silica) → non-polar mobile phase → polar compounds retained longer. Reverse phase: stationary phase is NON-POLAR (C₁₈ bonded silica) → polar mobile phase (water/methanol/acetonitrile) → non-polar compounds retained longer. RPLC is the basis of modern HPLC. Craig Counter-Current Distribution: early partition technique using multiple extraction steps between two immiscible solvents → components distributed based on partition coefficient → separation.
- 4Thin Layer Chromatography (TLC): Planar chromatography: stationary phase = thin layer of adsorbent (silica gel G, alumina) coated on glass/aluminum/plastic plate (~0.25 mm thick). Principle: components travel different distances on the plate depending on their affinity for stationary phase vs mobile phase. Rf (Retardation Factor) = distance traveled by solute / distance traveled by solvent front. Rf is characteristic for a compound in a given system (0 < Rf < 1). Rf depends on: adsorbent, mobile phase, layer thickness, temperature. Methodology: (1) Prepare/activate plate (110°C, 30 min for silica gel). (2) Apply samples as spots using capillary (~1 cm from bottom). (3) Develop in chamber with mobile phase (ascending development). (4) Mark solvent front immediately. (5) Dry plate. (6) Visualize spots.
- 5TLC – Visualization & Applications: Visualization methods: (1) UV light: 254 nm (fluorescent plates — spots appear as dark zones on green background) and 366 nm (fluorescent compounds glow). (2) Iodine chamber: iodine vapor stains organic compounds as brown spots (reversible). (3) Spray reagents: Ninhydrin (amino acids — purple), Dragendorff’s (alkaloids — orange), Anisaldehyde-H₂SO₄ (sugars, terpenes). (4) Charring: spray with H₂SO₄ → heat → organic compounds char (brown/black). Advantages: rapid (20-30 min), inexpensive, simple, multiple samples simultaneously, minimal sample needed, semi-quantitative (densitometry). Disadvantages: qualitative/semi-quantitative only (unless densitometer used), limited resolution vs HPLC, open system (evaporation). Applications: identity testing (compare Rf with standard), purity checking, monitoring reaction progress, selecting HPLC mobile phase, separation of herbal constituents.
- 6Paper Chromatography: Stationary phase: water held in cellulose fibers of filter paper (Whatman No. 1 commonly used). Mobile phase: organic solvent (less polar). Mechanism: primarily partition (liquid-liquid). Development techniques: (1) Ascending: paper hangs in tank, mobile phase rises by capillary action (most common). (2) Descending: mobile phase flows down from trough at top (faster, better for less polar compounds). (3) Radial/circular: sample at center, solvent moves outward in circles. (4) Two-dimensional: develop in one direction → rotate 90° → develop with different solvent (better resolution for complex mixtures). Methodology: similar to TLC — apply spots, develop, dry, visualize. Advantages: very inexpensive, good for amino acids. Disadvantages: slow (6-12 hours), limited loading capacity, poor resolution vs TLC.
- 7Electrophoresis – Principle: Electrophoresis: migration of charged particles/molecules in an electric field. Electrophoretic mobility (μ) = v/E (velocity per unit electric field). μ = q / (6πηr) → mobility depends on: (1) Charge (q): more charge → more mobility. (2) Size/radius (r): smaller → more mobility. (3) Medium viscosity (η): less viscosity → more mobility. (4) Applied voltage (E): higher E → faster migration. (5) pH of buffer: affects ionization state of molecules (proteins: above pI → negative → migrate to anode; below pI → positive → migrate to cathode; at pI → no migration). Supporting media: paper, gel (agarose, polyacrylamide), capillary.
- 8Paper & Gel Electrophoresis: Paper electrophoresis: filter paper strip soaked in buffer → sample applied as a spot at center → voltage applied (100-300V) → ionic species migrate toward respective electrodes. Simple, used for serum protein separation (albumin, globulins). Gel electrophoresis: gel matrix provides molecular sieving effect → separation by BOTH charge and size. (1) Agarose gel: large pore size → for DNA/RNA separation (nucleic acids — uniform charge/mass ratio → separated by size only). (2) Polyacrylamide gel (PAGE): smaller pore → for protein separation. SDS-PAGE: SDS (sodium dodecyl sulfate) denatures proteins + coats them uniformly with negative charge → separation PURELY by molecular weight. Molecular weight determination by comparing migration with standard proteins. Visualization: Coomassie Blue staining (proteins), Ethidium bromide/UV (DNA).
- 9Capillary Electrophoresis (CE): Modern, high-resolution electrophoresis in narrow-bore fused silica capillary (25-75 μm ID, 30-100 cm length). Buffer-filled capillary + high voltage (10-30 kV) → separation in minutes. Electroosmotic flow (EOF): bulk flow of liquid from anode to cathode (surface silanol groups on capillary → negative charge → attracts cations → cation layer moves toward cathode dragging bulk solution). All species (cations, neutrals, anions) eventually reach cathode detector. Migration order: cations (own mobility + EOF) > neutrals (EOF only) > anions (own mobility opposes EOF, but EOF dominates). Detection: on-column UV, fluorescence, mass spectrometry. Advantages: extremely high efficiency (>100,000 plates), tiny sample volumes (nL), fast, versatile. Applications: protein/peptide analysis, chiral separations, drug analysis, DNA sequencing.
Learning Objectives
Exam Prep Questions
Q1. What does Rf value tell us?
Rf (Retardation Factor) = distance traveled by solute ÷ distance traveled by solvent. It reflects how strongly a compound interacts with the stationary vs mobile phase. High Rf (~0.8–0.9): compound has low affinity for stationary phase → moves with mobile phase (non-polar compound on silica with non-polar mobile phase). Low Rf (~0.1–0.2): compound strongly adsorbed on stationary phase → moves slowly. Rf is used for IDENTIFICATION (compare with standard) — same Rf in same system suggests same compound (but not conclusive alone). Ideal Rf for separation: 0.2–0.8.
Q2. Why is SDS added in SDS-PAGE?
Without SDS, proteins separate by BOTH charge AND size → complex, unpredictable pattern. SDS (sodium dodecyl sulfate) solves this: (1) Denatures proteins (unfolds native structure). (2) Binds uniformly (~1.4 g SDS per gram protein) along the polypeptide chain. (3) Masks the protein’s native charge with a UNIFORM NEGATIVE charge. Since all proteins now have the same charge-to-mass ratio, the ONLY difference is SIZE. The polyacrylamide gel acts as a molecular sieve → smaller proteins migrate faster → separation purely by molecular weight.
Q3. What is the difference between adsorption and partition chromatography?
Adsorption: solute molecules are adsorbed onto the SURFACE of a solid stationary phase (silica gel, alumina). Separation based on differences in adsorption affinity. Stationary phase = solid.
Partition: solute molecules DISSOLVE into (partition between) two immiscible liquid phases. Separation based on differences in partition coefficient (solubility). Stationary phase = liquid coated on solid support.
In practice, TLC on silica is primarily adsorption. HPLC on C₁₈ is primarily partition (reverse phase).