Chromatography Forum

Hi all,
I was wondering what could be the impact if i use a wide pore Column (300A) for analysis of relatively smaller peptide (3kDa)? Will I be noticing inconsistencies with retention times? What else would I notice.

Thanks and regards,

Resolution will be lost using the wrong support type.

300A columns are for larger molecules (10K and above). Use a conventional pore size (~ 100A; 90 - 120A) for such small molecules.

I am aware that the peaks tend to become broader because of diffusion effects. But would it have effect on the RT?

Yes, of course it would. A column packed with 300A particles vs 120A (or std sized pores) is a completely different HPLC column. Different column phases would not, and should not, be expected to show the same results. Retention times are a result of: (1) The HPLC method used; (2) the exact column selected and (3) the specific HPLC system used (and how it is configured). *Change the column and you change everything.

Please ask your teacher for assistance with your project. These types of topics are well covered in the classic texts on the fundamentals of liquid chromatography and are required reading (lots of useful info).

Just to pile on here:

In general, surface area is inversely proportionate to pore diameter. Accordingly, peptides will elute earlier from a 300-Å pore column than from one with 100- or 120-Å pores. If their retention is still good enough, though, then there's no reason you couldn't try a 300-Å pore column for your application.