Show Menu

TLC:Separation of Amino Acids Cheat Sheet by

Thin Layer Chromatography: Separation and Identification of Amino Acids


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


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
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.

Materials Required

Plant Material
Green Mung Beans (Vigna radiata) extract.
{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
Glass Plates for TLC / Readymade TLC plate.
TLC chamber.
Capillary tubes.
Reagent spray bottle.
Spreader for silica gel.
Conical flasks,
Conical flasks
Measuring Cylinder
Weighing Balance


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

Observ­ation Table

**Std AA/ Sample
Distance Travelled By Compound (cm)
Distance Travelled by Solvent (cm)
Rf value
Std AA 1
Std AA 2
Sample Band 1
Sample Band 2
Sample Band 3
Std AA -Standard Amino acid
Sample Band 1- Separated Amino acid in Plant sample numbered accord­ingly as Band 1, Band 2 etc


Calculate and record the Rf values for each amino acid to identify them in the mixture.
Perform the experiment and fill in the Rf values and the identified amino acids.

Figure TLC Chroma­togram of Amino Acids

MET- Methio­nine; TRY-Ty­rosine; PA- Phenyl alanine, VAL-Valine
MIX- Mixure (Sample)
M1-Mixture Band 1
M2- Mixure Band 2


No comments yet. Add yours below!

Add a Comment

Your Comment

Please enter your name.

    Please enter your email address

      Please enter your Comment.

          Related Cheat Sheets

          Micrometry Technique Cheat Sheet
          Study of Peroxisomes (Catalase activity) Cheat Sheet

          More Cheat Sheets by UmeshJagtap

          Viruses Demystified Cheat Sheet
          Isolation of Plant Genomic DNA Cheat Sheet
          Understanding Question Terms: Bloom's Taxonomy Cheat Sheet