Chromatography Forum

Hi,
I'm doing development of HPLC methods for proteins. I usually collect abs data at 214/254/280 nm. I would like to report that the A280/A254 ratio can be used to distinguish between protein and non-protein peaks (as is quite common knowledge I guess). For instance if the ratio is ~2 the chance of having protein is relatively high and if the ratio is ~0.5 the peak might be DNA or something else other than protein.

Does anybody know a good reference on this?

Best regards ABV

The protein part is not correct. The common ratio in this context is A214/A280 and is usually between 10 - 15 depending on the number of aromatic amino acids there are in the protein in question.
Best regards

Thanks danko.
Do you have some kind of reference on that?

Non other than what I’ve saved on the virtual hard drive in my head.
I’ve learned it for maaaaaaaany yers ago and used it maaaaaaaaany times since.

Simple google search showed this hit: http://www.pnas.org/content/89/16/7380.full.pdf
And you’ll probably find tons of the kind by searching the net.

Best Regards