Greetings
I am trying to find out what is the origin of the 220nm absorbance peak of free amino acids? It is usually attributed to peptide bond but I find it also in single aa...
Yoram
All amino acids absorb at 200-220 nm due to the C=O group. Additionally, based on the solvent and concentration of the sample, hydrogen bonding may be present increasing the observed nm.
If you are using an aromatic amino acid (Tyr, Phe, Trp), it absorbs UV with two different peaks. The peak at ~270-280 nm is due to the conjugation system of the aromatic ring and the absorbance peak at lower wavelengths is attributed to carboxylic acid moiety of the amino acid.
Tryptophan
And many thanks! So, if I see decrease in the 220nm absorbance and broadening of the the absorbance ~260nm this will suggest change in the carboxylic side? I Oxidation?
Yoram
I fully agree with Dr.Eirinaios I. Vrettos.
Therefore, I have chosen the 210 nm for protein determination; i.e., amino acid elutes in the small molecule region and proteins elute at the exclusion limit portion in SEC. Then, HPLC-Surf-SEC protein determination method is the most reliable and quantitative protein-determination method nowadays (please see file; HPLC-Surf-SEC protein determination method).
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