Chromatogram analysis

This chromatogram was run over a period of around 40 minutes, and the separation was not particularly good. The differing sizes and colour densities of the spots further complicates the analysis. A better separation could have been achieved by running a bigger chromatogram over a longer time span, possibly using a different solvent combination. However, I am reasonably satisfied with the conclusion that the mixture contained glycine, phenylalanine and tyrosine.

You may be concerned that the heights of the spots do not match exactly. However, a more careful examination of the chromatogram shows that the solvent did not rise evenly. This is often the case in this experiment, particularly if there is any overlap of the edges of the paper. This causes the solvent to rise more rapidly at this point. Given that the solvent has risen slightly further at the mixture position, we would expect the spots to be slightly higher here. This is the case. This effect is removed if we calculate the R_f values. Try to do this for the chromatogram above and check your answers here.

The shade of the spots also gives a clue to their identity, although this is complicated by the relative depth of the colour in the two positions. Control over the exact size of each of the spots and the exact quantity of solution deposited is quite difficult.

Students often find that their chromatogram has a large purple band across it. This is caused by the spots being placed too low on the paper or by careless handling of the beaker. This causes the amino acids to leach out into the chromatography solvent and rise up with it. Often purple fingerprints will give the careless culprit away!