Chromatography Forum

My hplc condition is 20mmol KH2PO4 (PH=3.2):acetonitrile=96.3:3.7 ,which is operated by two pumps.when i add a new batch of mobile phase ,the retention of my object would change a little and during the running time ,it changed as well.My column is C18,250*4.6,5,intex.because the condition is used to detect the serum samples by hplc-uv,a little change in retention will bring me into troubles.In my opinion,the chromatograhy method may be not rough enough,but my question is that whether the column stable enough in such conditon.thanks a lot!

You know, it would have made more sense to simply continue your previous thread on this topic:
viewtopic.php?t=13033&highlight=

That said, most C18 columns should be reasonably stable at pH 3.2. Depending on your sample prep, garbage from the serum may be accumulating and changing the column chemistry. If that's the case, a better sample cleanup is the best remedy. The use of a guard cartridge can also help, but you may have to change it frequently.

I really appreciate your reply!thanks very much!

I am also confused that my condition which contains only 3.7% acetonitrile,is it one of the cause of unstable method?

If it only takes 3.7 % ACN to elute your analyte from a C18 column, maybe it’s not the right technique for this application.
The above could also (to some degree) explain the retention instability you’re experiencing.

Best Regards

thanks a lot