Experiment 07Separation Biophysics • B.Sc. / M.Sc.
Separation and Identification of Amino Acids by Thin-Layer Chromatography (TLC)
🎯 Aim: To separate a mixture of standard amino acids using silica gel G thin-layer chromatography, visualize developed spots with ninhydrin, calculate retention factor ($R_f$) values, and identify amino acids present in an unknown sample.
🔬 Theoretical Principle & Partition Thermodynamics
Thin Layer Chromatography (TLC) operates on differential partition and adsorption chromatography between two phases:
- Stationary Phase: A thin ($0.25,text{mm}$) layer of microcrystalline silica gel ($text{SiO}_2cdot xtext{H}_2text{O}$) bound to an inert aluminum or glass plate with calcium sulfate binder (Gypsum, silica gel G). Highly polar silanol groups ($-text{Si}-text{OH}$) form hydrogen bonds with polar solutes.
- Mobile Phase: A non-polar/moderately polar solvent mixture, typically BAW (n-Butanol : Glacial Acetic Acid : Water in $4:1:1 text{v/v/v}$).
As the solvent ascends the plate by capillary action, amino acids with non-polar hydrophobic side chains (e.g., Leucine, Valine) partition preferentially into the organic mobile phase and migrate rapidly (high $R_f$). Highly polar or charged amino acids (e.g., Aspartic acid, Lysine) interact strongly with the stationary phase and migrate slowly (low $R_f$).
R_f = frac{text{Distance migrated by solute from origin (cm)}}{text{Distance migrated by solvent front from origin (cm)}}
Figure 7.1: Thin Layer Chromatography (TLC) of Amino Acids & Rf Determination
📊 Representative Amino Acid TLC Retention Table
| Amino Acid Spot | Chemical Nature of Side Chain | Migration Distance (cm) | Solvent Front (cm) | Calculated Rf Value | Ninhydrin Spot Color |
|---|---|---|---|---|---|
| Aspartic Acid (Asp) | Acidic, polar charged ($-text{CH}_2-text{COO}^-$) | 2.4 cm | 10.0 cm | 0.24 | Purple-Blue |
| Glycine (Gly) | Small, uncharged ($-text{H}$) | 4.0 cm | 10.0 cm | 0.40 | Red-Violet |
| Alanine (Ala) | Non-polar aliphatic ($-text{CH}_3$) | 4.8 cm | 10.0 cm | 0.48 | Violet |
| Leucine (Leu) | Bulky branched aliphatic ($-text{CH}_2text{CH(CH}_3)_2$) | 7.3 cm | 10.0 cm | 0.73 | Deep Violet |
| Proline (Pro) | Cyclic secondary imino group | 4.3 cm | 10.0 cm | 0.43 | Bright Yellow |
💬 Viva-Voce Questions & Model Answers
Q1: Why is chamber saturation mandatory before running TLC plates?
Chamber saturation (lining the tank with filter paper soaked in developing solvent for 30 minutes) creates an atmosphere saturated with solvent vapor. If the chamber is unsaturated, solvent continuously evaporates from the rising edge of the TLC plate, creating non-linear solvent movement, edge effects (“smiling bands”), and falsely elevated $R_f$ values.
Q2: Why must the sample spot origin line be drawn above the solvent level in the developing chamber?
If the sample spot is submerged beneath the liquid surface, the concentrated amino acid spots immediately dissolve directly into the reservoir solvent pool instead of ascending the plate via capillary partition.
Q3: Why can an Rf value never exceed 1.0?
The solvent front represents the maximum thermodynamic boundary of liquid movement driven by capillarity. Solutes can only migrate as fast as the mobile phase carrier solvent; thus, solute distance is always $le$ solvent distance ($R_f le 1.0$).
