A Typical problem in column chemistry

[vijayak]

Hi,
I have a typical problem. I am analyzing proteins from fermentation supernatant and injecting around 5µl. I have connected a new column(ACE 300A 4.6X250mm;5µm) and injected my sample, i got gautian shape with lot of tailing.Then, i injected 200-300µl of the same sample supernatant in to the column and continued my regular injection volume.
After the Higher injection load to the column the usual injection samples is giving very good symmetrical peaks. can any one say what is really happening? Am i blocking any active sites by loading higher injection volume?.
Am i avoiding any secondary intreaction between solute and stationay phase?

If any one joins this discussion, it will be good to find out a answer for a new problem.

Thanks
vijayak

[Vlad Orlovsky]

part of your analyte sticks to silanol active sites and make the surface less sticky, you now have a positive charge on the surface. This kind of mimics what we have on our mixed-mode column when positively charged surface shields analyte from secondary interaction with silanols.