Chromatography Forum

Dear everyone,
May I kindly request you to help me.I have very low background in HPLC.Last night,I run the sequence on HPLC machine overnight.The sequence include 25 samples (retention time 35 mins) and after that sequence for column cleaning. Eventhough I command post shut down,the pump didn't close and continue for cleaning. In the morning,when I check my column (C18,Hypersil gold),the peak shapes become broadening and can't get the normal sharp peak.Does it my column packing degrade or not?Now, I worry for the column.
Could you please some suggestions for this case.I am a Master degree student and I can't buy new column.May I request your kind help.Thank you.

Reverse, then wash the column with 95% Methanol, 95% Acetonitrile, and finally 50/50 Methanol/Water and your mobile phase. It seems likely your column has gone dry.

Broad peaks are also indication of post column dead volume. Minimize connections to detector from column and be sure the capillary diameter isnt too large comared to other connections.
HPLC chemist gave good advice on reversing column with organic %B flush. Use low flow rate for a few hours, monitor baseline, if it goes erratic than flat, thats a good sign

Broad peaks are also indication of post column dead volume.

That would be true if the plumbing had been changed, but that's not the situation here. A void space at the *head* of the column is, unfortunately, a possibility.

If the suggested wash of the column doesn't help, the next thing to try would be to reverse-flush the column (just turn it around and run it in the backward direction).

If *that* doesn't help, the next step would be to disassemble the inlet fitting and change the frit and/or repack any void. In an industrial lab, this would almost never be cost effective (unless you are in the worst-case situation of having no replacement column available!). For a graduate student, however, poverty rears its ugly head. Unfortunately, experience gained is often in direct proportion to equipment ruined.

Thanks so much everyone for your kind suggestions.