Re: What are two methods of identifying, rather than separating, an amino acid?

First of all, as far as I am aware, there is no straightforward way to 'calculate' Rf values. Second, published Rf values really aren't useful for identifying compounds; two different compounds can easily have the same or silimar Rf, and any particular compound will give a different Rf on a different chromatography system, unless the systems and conditions are very, very similar.

The easiest way to identify a compound using chromatography is to compare your unknown to known 'standard' (a pure sample a compound you buy from a company). You run your unknown through the system, and then run your standard. If the both the standard and unknown elute at the same Rf and the peaks (or dots) have the same shape, then the standard and unknown are probably the same thing. But this method has two weaknesses 1) you must know ahead of time what the unknown might be - if you have no idea what it is, you won't know what standards to try (and contrary to what you might have been taught, there are a lot more than 20 amino acids in biological systems) 2) It is possible that your peak contains two or more compounds that have not separated well.

The harder but more definitive way is to separate using chromatography, then analyze your unknown peak using mass spectrometry (see the Wikipedia article if you don't know what this is). Mass spectrometry instruments are more expensive, harder to operate and maintain and their data is harder to interpret, BUT they overcome both of the weaknesses of the other method.

Good luck! -Rafael

**Please note: The author is answering on his own behalf and his answer does not represent a statement from the USDA.

[Moderator's note: You can search our archives for answers that discuss mass spectrometry. --SM]