The default settings of the FPLC UV detector are for wavelengths 215, 255, 280 and 495 nm. I know that the 280 nm is the most important since it directly measures protein via the aromatic ring in some aminoacids. What are the other wavelengths for and for diagnostic purpose what would it tell you (for example, more peptides would mean proteolitic lysis)?
215 nm (absorption wavelength for peptide bond) can be used to detect a peptide deficient in tryptophan, tyrosine and phenylalanine or all the proteolytic digestion products of a protein which otherwise will not be detectable with 280 nm as some fragments may be deficient in the aromatic amino acids.
BTW: Phenylalanine shows only a very low absorbance at 280 nm. Therefore, it can be neglected in this context in nearly all cases.
255 nm is I think for phenylalanine and when the 280 nm filter of our Akta prime FPLC system got damaged, the other one available was 254 which we used for tracking protein purifications.
495 nm I am guessing may be used as a reference wavelength, same as in HPLC. I am waiting for a response on this
255 nm is useful for monitoring nucleic acids. If the 255 nm absorbance is higher than the 280 nm absorbance, it is probably because the peak contains a lot of nucleic acids relative to protein.
Aside from the reasonable possibility of using 495 nm absorbance as a reference, as Mangal Singh suggested, it might also be useful for detecting proteins with visible chromophores, like heme-containing proteins and fluorescent proteins.
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