Chromatography Forum

Hello All,

I am trying to separate some proteins (15K daltons) using Vydac C4 column, I am using gradient of ACN and buffer (I tried ammonium formate and TFA), but have no separation-just overlapping humps. My column is 4.6x150 mm (300A pore size) and I am wondering if I have no separation because my conditions are wrong or my column (may be I need a different pore size or higher/lower hydrophobicity?). What is the general approach and initial steps in retention control? How can I increase efficiency of separation?

Thank you and have a good weekends

Hard to be specific without more details, but in general:

• - gradients for proteins should be significantly more shallow than gradients for smaller molecules (for 15k Da, something under 1%/min might be a good starting point)
  
  - reversed-phase is inherently a denaturing technique. If denaturation occurs during chromatography, then you can actually have a distribution of native, partially-denatured, and denatured conformers from each protein, leading to some really bizarre peak shapes. You'll get the best results if you denature your proteins before injection, and then run under conditions that ensure they stay denatured (higher temperature, TFA, surfactants, etc.)