Please help me if you can. If I do not use the mobile phase as the solvent for my sample, why do I get split peaks? Why is the interaction of the mobile phase with the sample as it flows through the column not enough to overcome the effects of the small volume (~20 uL) of solvent that the sample was in?

Thanks for any help

you haven't provided enough details about your problem to enable a response of much insight. You will note from previous forum postings that information rich questions generally get in depth answers.

If your split peaks are a result of the solvent used to prepare the sample, then the split occurs at the instant the sample hits the column. Part of the sample partitions between the mobile phase and the column (the part that got mixed with mobile phase) and part partitions between sample solvent and column (that part that didn't get mixed). If the sample solvent is stronger and not adequately diluted by mobil phase upon injection , then that portion of sample will "run ahead" of the sample that was sufficiently mixed with mobile phase. Once this part of the sample gets ahead of the rest, there is no means of rejoining it with the rest that is lagging behind. A split peak results at the head of the column and the peaks stay split as they traverse the column. Therefore, dilution of the sample solvent volume must occur nearly instantaneously upon injection to avoid the possibility of a split peak.

I rarely saw a split peak with a 10 uL injection and 2 mL/min flow, no matter what the sample was dissoved in. An exception to this case would be a highly buffered sample injected into a poorly buffered mobile phase. In this case, no reasonable amount of dilution will save the day.

There are other causes of split peaks that have nothing to do with sample solvent.

I recently had the same problem with OPA-derivatized glutathione in a mobile phase consisting of 15:85 ACN/0.05M H3PO4 on a C18 column. This derivative is a dicarboxylic acid, so my first idea was "ion-suppression" RP-HPLC. The derivatizing buffer was 0.4M pH 9.5 borate buffer. The peak split badly due to non-instantaneous mixing of the mobile phase with the borate in the sample. When I diluted the sample with water, and/or adjusted the pH to 4 using acetic acid, there was a great improvement in the peak shape. Unfortunately, the derivative was unstable in acid, so back to the drawing board. I finally came up with an ion pairing method that ran at pH 7 and was unaffected by the high pH buffer. It was still sensitive to ionic strength. The ionic strength could not be greater than that of the mobile phase, or distortion of the peak would result.

Thanks for the info.