TLC:Separation of Amino Acids Cheat Sheet

To separate and identify amino acids in a mixture using thin layer chroma­tog­raphy (TLC).

Chroma­tog­raphy
Chroma­tog­raphy is a technique used for separating compounds in a mixture based on differ­ences in their distri­bution between a stationary phase (like silica) and a mobile phase (solvent mixture).

Thin Layer Chroma­tog­raphy (TLC)
TLC separates compounds based on
solubi­lity,
intera­ction with the stationary phase, and
molecular size.
It's useful for qualit­ative and quanti­tative analysis.

Chroma­tog­raphic Separation of Amino Acids
Amino acids, having different R groups, interact variably with silica, affecting their movement on a TLC plate. Ninhydrin is used to visualize amino acids, forming purple spots on reaction.

Plant Material
Green Mung Beans (Vigna radiata) extract.
Reagents
{fa-sq­uar­e-o}} Standard solutions of individual amino acids (Known Concen­tra­tions).
Solvent mixture (butan­ol:­acetic acid:water in 12:3:5 ratio).
Ninhydrin reagent (2% in acetone).
Silica gel for TLC
Equipments
Glass Plates for TLC / Readymade TLC plate.
TLC chamber.
Capillary tubes.
Reagent spray bottle.
Spreader for silica gel.
Conical flasks,
beakers.
Conical flasks
Measuring Cylinder
Weighing Balance
Oven
Ruler

1. TLC Plate Prepar­ation:
Clean the glass plates thorou­ghly.
Prepare a slurry of silica gel with water (1:2) in a beaker.
Pour the silica gel slurry onto the glass plate and spread evenly using a spreader.
Allow the silica layer to dry and then activate it by heating in an oven at 110°C for 1 hour.
2. Solvent Prepar­ation: Pour solvent mixture into TLC chamber and let it saturate for 30 minutes.
3. Sample Applic­ation: Use capillary tubes to spot amino acid solutions onto the baseline (appro­xim­ately 2 cm from the bottom of the plate). Allow to dry.
4. Develo­pment: Place the plate in the TLC chamber ensuring the baseline is above the solvent. Let the solvent ascend to about 1 cm from the top.
5. Drying: Remove the plate and mark the solvent front with a pencil. Dry the plate under a hood.
6. Detection: Spray the dry plate with ninhydrin and dry in an oven at 105°C for 5 minutes.
7. Analysis: Measure the distances moved by the solute and solvent, and calculate Rf values.
Rf Value Calcul­ation:
Rf = Distance moved by solute / Distance moved by solvent X 100

Calculate and record the Rf values for each amino acid to identify them in the mixture.