Chromatography Forum

Hi,

I am seeing some sample solvent / MP effects that are causing my first analyte peak to split. I was wondering how high of a % organic you can start a gradient? My sample solvent is ~35% MeOH, while my starting MP conditions are around 12% MeOH.

For RP columns 3-5% organic modifier should be in MP. If you have a big difference in organic modifier % in MP and sample that can disturb the Equilibrium on the column and you can get split Peaks. Try to use MP also in your sample, if possible.

There is no general rule about how much the mobile phase and the sample solvent can be different in %organic without causing any troubles. It depends on many variables apart from the different %organic, including the injection volume, column volume, flow rate, retention factor of the analytes and even the system volume of the HPLC itself. If you use the initial mobile phase as solvent, as suggested by Gerhard, then you're safe. If you want to use more organic in the solvent, well, you have to try if it works...but you should be aware that this weakens the robustness of your method. Even if it looks good once, you might be just on the edge and if you for example try to run that method on a different HPLC, early peaks suddenly might look much worse.

So maybe im mis-understanding what you guys are saying. My sample is plasma that is precipitated out with 100% MeOH in a 3/4 ratio, the votexed and spun down. Water and an derivatizing agent are added to a amount of the supernatant. The final sample is then injected into the HPLC system. Like i said before, my first peak splits after around 200 injections but the second shows no change. I tryed replacing the MeOH with H2O on my aqueous standards and the first peak looks amazing. How can i get my gradient to allow for such a high organic sample being injected into it?