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Separation of amino acids using thin layer chromatography - page 1
Keywords: Separation of amino acids, thin layer chromatography
By riya on 22/02/2012
Level: Bachelor Honours Degree (BA, BEng, BSc etc)
Page Number: 1 of 3 pages: 1 2 3
ANGLIA RUSKIN UNIVERSITY
DEPARTMENT OF LIFE SCIENCES
Introduction to Biochemistry & Molecular Biology (EK115017S)
Separation of amino acids using thin layer chromatography
Introduction
Thin layer chromatography (TLC) is a convenient and rapid small scale technique for the separation of various compounds including sugars, lipids, nucleotides and amino acids. The principle underlying the separation process depends on the material constituting the thin layer and the basis of the separation can be partition and/or adsorption, ion-exchange or even gel filtration.
In this experiment, the separation is carried out using a thin layer of silica gel and both partition and adsorption phenomena contribute to the separation.
In partition chromatography solutes partition themselves between a so-called “stationary” aqueous phase, which becomes associated with the particles of the gel as the solvent (containing water) moves through the previously dehydrated gel, and a “mobile” organic phase which does not associate with the gel. Clearly, compounds which are more soluble in the organic phase than in the aqueous phase move through the gel more rapidly than those which are more soluble in the aqueous phase.
In adsorption chromatography, compounds which have a high affinity for the gel are adsorbed more strongly than those which have a low affinity for the gel. Since the solutes are dissolved in, and carried along with, the solvent, these differences in adsorption contribute to a differential movement of the solutes through the gel.
The aims of this practical are to familiarize you with the technique of thin layer chromatography and to identify the amino acids in two unknown mixtures.
Materials and Reagents
TLC plates
These are 20 x 20 mm commercially prepared aluminium plates with a coating of silica
gel G at a uniform thickness of 0.2 mm. The plates are activated prior to use by desiccation overnight. N.B. Handle these plates with care throughout. Take care not to release silica dust from the plates as this is HARMFUL.
Samples
(All are provided as 0.2% solutions in 10% aqueous propan-2-ol. (Caution! This is an IRRITANT - avoid breathing the vapour).
Unknown amino acid mixtures (M1 and M2).
Reference amino acids:- aspartic acid (asp)
glycine (gly)
leucine (leu)
lysine HCl (lys)
phenylalanine (phe)
proline (pro)
tryptophan (trp)
Solvent system
(N.B. FLAMMABLE, HARMFUL, IRRITANT, CORROSIVE. Avoid breathing the vapour and avoid contact with skin and eyes). Kept in fume cupboard.
Butan-1-ol : glacial acetic acid : water (3 : 1 : 1 by volume).
This is freshly prepared.
Detection reagent
(N.B. FLAMMABLE, HARMFUL, IRRITANT. Avoid breathing the vapour and prevent contact with the eyes).
Ninhydrin (0.2% w/v in acetone)
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