Site author Richard Steane
The BioTopics website gives access to interactive resource material, developed to support the learning and teaching of Biology at a variety of levels.

Chromatography of amino acids

Amino acids have no colour. Therefore all of these procedures need to be carried out "blind", and the results will be seen when a revealing agent (ninhydrin) is sprayed on the resulting chromatogram.

You are provided with a number of solutions of amino acids, and solution X (a mixture of 2 amino acids).  Do not use the same pipette for more than one liquid.

Chromatography paper must not be touched with the hands (at the bottom, at least). Use plastic/rubber gloves, and work on a clean surface (e.g. inside page of pad of paper).

Cut a suitable length of chromatography paper (slightly longer than the glass chamber) and mark it with a pencil line about 1.5 cm from the bottom. Again using a pencil, put 3 marks on the line forming crosses, the outer ones labelled with (3 letter codes for) the amino acids you are going to use, and X in the middle. Put your name at the top.

Using 3 different pipettes, place a drop of each amino acid, and the mixture X, at the appropriate positions on the line. If you have time, gently warm the paper and repeat the spotting process (on the same positions) to raise the concentration of the amino acids, but ensure that the paper is completely dry before proceeding.

Partially fold the paper in half lengthways, to remove its tendency to curl up.

In a fume cupboard or a secluded (well ventilated?)  area of the lab :
Using a funnel, pour a small amount of chromatography solvent (butanol/ethanoic acid) into the glass chamber (to about 1 cm depth). Place on the lid to allow the atmosphere to become saturated with vapour. Leave the chamber on the bench in its final position, so that it does not splash up; bring the paper to meet it.

Line up the paper with the (outside of the) chamber, and either fold over the top or cut it so that it will fit. The solvent should touch the lower part of the paper but not cover the drops on the line.

Attach the paper to the lid, and then place the lid on. The paper should not touch the sides of the chamber.

The solvent should gradually rise up the paper, passing the line and heading upwards.

After about 3 hours, the liquid should have risen about three-quarters of the height of the paper. If the solvent nears the top of the paper, proceed immediately to the next stage.

Remove the paper from the apparatus, and use a pencil to mark the position of the solvent front. Up to 5 pieces of chromatography paper can be placed across a clean A4 sheet of paper, stapled at the top of the chromatograms.

Place the chromatograms into an oven at about 45 C to dry.

Either in a fume cupboard or outside on a still day, spray ninhydrin evenly over the paper. Care: ninhydrin is dissolved in an inflammable solvent.

Return the chromatograms to the oven to develop the colour. Spots should be visible as purplish smears on the paper.

The image of the spots may be enhanced if the sheet is photocopied on a darker than normal setting.

Calculation of Rf values

Measure the distance from the start line to the solvent front and to the front of each spot.

For each spot, calculate the Rf value (Rf means relative to front):

       distance moved by spot      
distance moved by solvent front

Compare the values you obtain with reference Rf values. Different solvents and different types or makes of chromatogaphy papers will give slightly different results.

One or both of the spots from solution X may be at the same level as another (known) amino acid alongside it. This should assist in identification.

Amino acid Rf value
alanine 0.38
arginine 0.20
asparagine 0.5
aspartic acid 0.24
cysteine 0.4
glutamine 0.13
glutamic acid 0.30
glycine 0.26
histidine 0.11
isoleucine 0.72
leucine 0.73
lysine 0.14
methionine 0.55
phenylalanine 0.68
not a true amino acid - shows up as yellow
serine 0.27
threonine 0.35
tryptophan 0.66
tyrosine 0.45
valine 0.61

Also on this site:
More info about amino acids including links to Jsmol versions
3-D views of amino acid molecules using Jsmol

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