Chromatography Forum

Hi ! I am new here
I am a student currently working on hplc separation
The problem was the separation was fine a couple of weeks ago


However, when I try running the separation last week, all the peaks began showing a double peak at the tailing end




I have tried preparing fresh standards and mobile phases, but it doesn't seem to help though

Any advice is appreciated. Thanks

Hi kermit,

looks like the inlet of your column is contaminated/partly clogged.
If you use a guard column: replace it (if not: consider its use).
Try backflush the column. Maybe your column allows for the change of the inlet frit. I also would advise you to re-think your sample preparation.

Good luck

I agree with Friederike's post. Try a new column, see if that reproduces the good separation on the standard or sample, just to make sure the instrument is fine. Most column suppliers have a process to clean out columns, might include backflushing, stronger solvents, check with supplier.

You may need to inject less volume/material, clean up your samples more prior to injection, stuff like that. Your initial chromatography looks nice.

Is the instrument used regularly, or does it sit unused for several days at a time? If you are using buffers and the instrument sits for several days or weeks without being used you can have precipitation of buffer in the system and that can cause problems.

I would like to ask you if u washed the column after the last use that was Ok? cuz if your mobile phase contains a buffer then most probabaly there might be some distortion to the stationary phase....also i think that even if the instrument is used regularly, flushing the whole system after finishing your runs prevents the precepitation of any salts in the whole system's tubing.

We have a mini-seminar on our web site that addresses your problem exactly:
http://www.lcresources.com/more_resourc ... hp?f=3&t=3

You will have to register (free) to view it.