Chromatography Forum

Hi,

Im using an HPLC system with an ELSD detector looking at lipids such as wax esters, cholesteryl esters etc and my retention times for all my target compounds have suddenly shifted, resulting in earlier retention times and also my peak shapes have degraded for the late eluting lipids (what looks like both shouldering and peak broadening). I have tried to isolate the cause of the shift by checking the method parameters on the system to the parameters set out in the in-house SOP, using a fresh batch of mobile phase, cleaning the column with IPA (Si 60 column), checking the lamp on the detector, but have not yet tried a new column as we only have one at the moment. Does this sound like the column is the likely cause? I don’t have much experience in HPLC but from my experience in GC-MS my first thought would be column issues. The change in RT’s is alarming as the method has been used by the company for years with little change, yet after two weeks of not being used this issue has arisen. Is the lack of use likely to be a factor in the deterioration of HPLC columns?

Any help or advice would be greatly appreciated.

Thanks

Contact - in your shoes I would (1) install a brand-new column of the same part number, to see if the situation has returned to "normal" or stays the same. I know you state you don't have one, but one should always have a new one in the drawer for situations like this.

(2) if a new column doesn't return the chromatography to normal, second thing I would do would be to prepare/re-fill reservoirs all mobile phases from scratch and re-try (you say you don't have much HPLC experience, maybe you filled wrong reservoir, or a buffer was made incorrectly

(3) to check out the HPLC, most install a C-18 column and run a known test mixture, and see how that looks. This should help identify if there are pump issues, solvent mixing issues, etc.

This is the fun stuff, and separates the men from the boys! You will learn a lot here.

A stop gap method to test the column is to back flush it with a strong compatible solvent. Do this only if the entrance and exit frits are the same size!!
Leave the column exit open and start with a low flow rate.
If the column is dead you can't make it any worse.

Welcome my friend to the all new world of liquid. It is like coming to water planet in Dune from the desert planet (in your case GC)...
Back to the problem now. Normally in LC, chromatography does not crash suddenly. Late eluting normally are not affected as much as first peaks. Sudden change due to column is a possibility if it was killed by previous injection of something wrong, eg high pH.
Please check connections they are more critical in LC than GC due to lower resolution of LC. Ferrules and fittings should be with correct specification, absence of leak does not mean that there is no problem in the connection.
Check flow and gradient composition as well that it is what is set in software.