Chromatography Forum

Hi All,

I am trying to develop a SEC-HPLC method to separate some aggregate/high MW species in a peptide sample ( ~4000Da). If I use this isocratic conditions with 5 mM ABC buffer, I can see there is a peak at the front of the main peak. If I run a gradient with (a) 100% water, (b) 100mM ABC, is this against the SEC rules?

If runing the ABC in a grdiaent fashion improves the resoution of these 2 species and if I can use this method to detect these multimers in my samples without too much reliability on the MW of the 2 species is this method acceptable to FDA or any other regulatory agencies? Is there is anything wrong doing a gradient in a SEC-HPLC method?

Thanks in advance for your input and comments.

Ananda

One of the assumptions underlying SEC is that there are no "chemical" interactions between your analytes and the column packing. If you wish to obtain molecular weight information, all the molecules must "see" the same chemical environment during the separations (i.e., no gradients).

If all you want to do is assay different compounds, then there is no requirement that you do "pure" SEC. If a secondary interaction helps your resolution, then go for it -- just recognize that you are exploiting a hybrid mechanism, and don't try to deduce size information from the results.

A final note: one possibility is that the buffer ionic strength is affecting the aggregation rather than the separation chemistry per se.

Thank you very much for your reply. You are exactly right and I just want to use this assay as a qualitative method to detect aggreagtes and not for any mass or any other qualitative evaluations.

Thanks again!

Ananda