HPLC Method help

[KarenJ]

We are developing an HPLC method for measuring PMCol in a mixture of ethanol, propylene glycol, and ascorbic acid buffer. We are using a Prevail C18 column and our mobile phase is 75% ACN/25% MeOH. We often see a second small peak come off right next to the main peak (not quite baseline resolved). We haven't been able to separate the two peaks from each other although they will move together relative to other peaks when we vary the mobile phase ratio. The relative height of the small peak varies from day-to-day but not within runs. One day it was gone altogether. When we vary the injection volume, the relative heights do not change nor does the separation between them. Any ideas?

[HW Mueller]

What happens if you collect portions of those peaks and reinject?

[KarenJ]

Thanks for your response. We aren't set up for collecting fractions so we haven't tried that.

[HW Mueller]

Where does your waste go???

[Johneg]

Are you sure that it is another compound and not just the primary peak being split as a result of voids in the system.

[Daren]

Is it there when you make a blank (0 uL) injection?

The things I would check are needle wash contaminant or vial cap contaminant. when it is there try making 3-4 blank (0 uL ) injections in a row and see if it goes away, if it does than you have some type of contamination with your injections probably one of the two mentioned.

Daren