Quantitative question

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

5 posts Page 1 of 1
When monitoring peaks at 215 or 220nm (peptide bond) and comparing 2 different proteins. If both are loaded at the same conc (1g/l, same volume injection), but differ in molecular weight by 2, would you expect the peak area/height to be the same or since one has double the molar mass would it give twice the area at 220nm?

Thanks
Nope! Any molecule including peptides and proteins can have a dramatically different response to a detector. This can be especially true for a protein that has only a small but significant change (perhaps position) in an amino acid. Therefore, for each molecule I would develop a best fit (probably linear) curve of concentration versus detector response. In other words 'PROVE YOUR ASSUMPTION'!
resolution1 wrote:
would you expect the peak area/height to be the same or since one has double the molar mass would it give twice the area at 220nm?


There is no relationship between absorption of UV light and molar mass.

You can however assume that, for the same molar concentration, a molecule with a larger amount functional groups like alcohols, phenyls, halogens, amines, double/triple bonds,.... will have a higher UV-Vis absorption in general (200-800nm).
^ until you have variations in folding for proteins... folding differences can compound the vagaries of molar absorptivity too.
Thanks,
DR
Image
It is impossible just from scratch answer such questions if analytes aren't simple homologues. F.e. even benzene, benzyl alcohol, benzaldehyde and benzoic acid have similar in general but completely different UV spectra. There is quantum-chemical software to answer such questions in days of computer work.
Best regards,
Dmitriy Perlow
5 posts Page 1 of 1

Who is online

In total there are 7 users online :: 1 registered, 0 hidden and 6 guests (based on users active over the past 5 minutes)
Most users ever online was 188 on Wed Nov 02, 2016 2:58 am

Users browsing this forum: Google [Bot] and 6 guests