[dekalina]

First of all, I'm sorry for my English. I hope that I write in the appropriate section.

I make an analysis of a mixture of four antibiotics: chloramphenicol, thiamfenikol, neomycin and erythromycin (mobile phase ACN and water). Every time I get a double peak from tiamfenikol. I am sure that it doesn't combine with the peak from other antibiotics. Does anyone know why this is happening? Or where can I find the answer to this question?
I tried to analyze tiamfenikol alone and I get a double peak too.

[Arkcon]

First of all, I'm sorry for my English. I hope that I write in the appropriate section.

You've picked the correct sub-forum.

I make an analysis of a mixture of four antibiotics: chloramphenicol, thiamfenikol, neomycin and erythromycin (mobile phase ACN and water).

Hrm.  Are you getting good results in general with this mobile phase?  Its a simple one, which is best to avoid complications, but may adversely impact peak shape, and the reproduciblity of your area.  Consider attaching a sample chromatogram.

Every time I get a double peak from tiamfenikol. I am sure that it doesn't combine with the peak from other antibiotics. Does anyone know why this is happening? Or where can I find the answer to this question?
I tried to analyze tiamfenikol alone and I get a double peak too.

Thank you for evaluating the standard.  There are many possibilities for the cause of your problem.  You column may be dying, having lost its separation ability, developed a void, or channels.  Yes, splitting only happens with thiamphenicol peak.  But are the others widening, or otherwise changing shape?  Are the other peaks later in the separation?  That may hide their splitting.

Are your samples and standards pure?  Can you purchase another, fresher standard?  If partially degraded, or if the substance is naturally impure, you may get two peaks.

I can't easily find the pKa of this substance.  However, you have no pH buffer in your mobile phase, so if this molecule isn't pH neutral, then you could be evaluating two substances on column, one charged, and one not.  Although usually, the double peaks usually have poor shape in this case.

If you have two perfect peaks from one pure substance, you most likely have a channel or void in the column.