Chromatography Forum

I am running chloramphenicol using colume Hypersil gold,50x2.1,5u.
25% ACN(0.1% formic acid):water (0.1% formic acid) at flow rate of 200ul\min. I am gettting at huge peak at .88-.90 min. what could be that. I get the peak when I use Methanol instead of ACN. and also i am not getting stable base line too..method is so simple i am not able to figure out why.. I get my chp at 2.5 min with ACN and 3.9 min with MeOH..please advise

As always, more details would help:
- what is your sample matrix?
- what is your sample diluent?
- what injection volume?
- what kind of detector (and what conditions, e.g., wavelength, sensitivity range, etc.)

In the absence of specific information, all I can say is the following: the dead time for that size column / flow rate is about 0.5 minute, so your huge peak is almost unretained (k' < 1). It is very likely the "solvent front" caused by your injection disturbing the equilibrium of the system.

If that's the case, the situation can be improved by dissolving the sample in the mobile phase and/or keeping the injection volume small. The latter is especially important with a small column such as you are using.

I am using PDA detector at 254nm. My std( Chloroamphenicol at 50ppm/ml conc.) is dissolved in 100% ACN. But I did make sample in 25:75 ACN:water( my mobile phase) still get that huge peak. I am only injecting 5-10 ul.

A spectrum of the peak may tell you whether it is air, goo, or still a system peak.