Thank you for your suggestions Markus.

We have used the methods for more than ten years, and have never experienced the problem before, so I do not think that unadequate column equilibration or change of mobile phase are the reasons.

The degassing unit is switched on and seems to be working properly.

The pressure is stable, and the pumps have recently been replaced.

The column and guard column are changed regularly. The retention times change for each sample, but then revert back to normal when we start a new batch (24 samples per batch). I would not expect it to go back to normal, if the column was going bad, or what do you think?

I think perhaps there might be a problem with the Gradient Proportioning Valve. Do you have any experience with this?

I agree that the problem is column equilibration as the HPLC is prone to the current environment.  For Hexane in 'normal phase' I used a water saturated solution where I added 1.0 mL of HPLC water to 1 L of Hexane and stirred this mobile phase overnight.  The retention time was 'rock solid'.

Clearly, something is wrong. You did not provide us with any chromatography details which would help us provide suggestions. Is this a gradient analysis or isocratic? Flow rate, Temperature, Column Dimensions, Exact composition and Program % changes used, inj volume, detector settings and so on. Could be something simple such as a sticking valve in the proportioning valve, check valve, partially obstructed solvent inlet frit, temp variation, flow variation, poor equilibration and so on.

There are many different areas which need to be checked to find the cause. Please review the linked article for areas to check and correct and I think you will solve the problem.

"HPLC Retention Time Drift, Change, Variability or Poor Reproducibility. Common Reasons for it".

http://hplctips.blogspot.com/2015/11/hplc-retention-time-drift-change.html

Thanks for the linked article.

It is a gradient analysis: 0 - 18 min: 90% A (n-hexane) and 10% B (n-hexane:isopropanol:methanol 40:15:45), 18-22 min: linear change to 50%B, 22-35 min: linear change back to 10%B, 35-40 min: 10%B.

Flow rate: 1.5 ml/min, temperature: 25 C, column: spherisorb silica 250x4,6mm (5 uM), inj. volume: 300 uL, detector: PDA (280 nm).

The method has been used for more than 10 years without any drift in retention times until august this year. A service technician replaced one of the pumps in September - did not help. I've checked the proportioning valve, and it works as it should. I've replaced the column and guard column - did not help.

May I ask what types of samples are run using this very unusual method?

BTW: I think the solution to your observed issue is to incorporate a proper wash step, after analysis, which uses a high percentage of MeOH (98% with a pinch of water or 100% MeOH) after each analysis (or after 2 to 5 runs, as needed) to regenerate the silica column. IPA does not seem to work as well as Methanol and you must use a high percentage of methanol in the wash for it to be effective. Most methods which are centered around very high percentages of Hexane require that the column be periodically flushed with a much more polar solution such as methanol to restore the silica surface layer back to normal. I have analyzed hundreds of different compounds (esp fats, lipids, oils) and found that this step is needed to obtain reproducible results when silica columns are used with hexanes. Otherwise, after many runs, the Rt's start to change and the RSD gets worse. This simple flush fixes the problem (I published this technique as part of another method a few decades ago) This added flush step should cure the problem you have observed.

Other comments:

The method appears to be essentially an isocratic method (0-18 min) with a quick increase of methanol at the end (18-22 min), then a reverse back to initial conditions over the remaining 18 min. At 18 minutes, it would take about 2 minutes for the change in mobile phase to reach the head of the column (~ 20 min) so the quick methanol boost only has about 2 minutes (20-22) to effect elution before the method ends at 22 minutes. Does anything elute between 20-22 minutes? Why not use a linear gradient over time which gradually increases the MeOH % (in the Hex/IPA/MeOH soln), then holds it for a much longer time. The actual time points and compositional changes used would depend on the chromatogram obtained (which we do not have at this time).

*If possible, consider removing all of the steps to restore (re-equil) the column back to initial conditions and make them a separate wash method (run after the analysis). This is better overall and removes all of those baseline changes and extra peaks seen during the wash and re-equil portion of the run, saving lots of time).

Your 300 ul inj volume is ~ 10% of the column's volume. A general guideline is to use an injection volume which is less than 3% of the column volume (~ 87 ul in your case). Now there are some exceptions to this guideline, but column fouling, peak splitting, poor RSD and poor quality chromatographty often result from excessive injection volumes.

Thank you for your very thorough reply, Bill.

We are using the method to separate/fractionate environmental pollutants (e.g. PCBs, pesticides, and perfluorinated compounds) from endogenous hormones in human serum (extracted by SPE prior to HPLC). The method is not used for quantification of the environmental pollutants - we analyze the endocrine disrupting (estrogenic and androgenic) effects of the environmental pollutants in the serum extracts using cell culture assays. I have linked to our first article on this issue, if you are interested.

To my knowledge, nothing is eluted between 20-22 minutes, but the perfluorinated compounds are eluted between 22-26 minutes.

I was not involved in the establishment of the method, so I am not entirely sure why the gradient is as it is. However, upon HPLC we are evaporating the HPLC fractions, and the evaporation must be gentle to avoid losing the volatile environmental pollutants, so we do not want too much methanol in our fractions as it is harder to evaporate compared to hexane.

I will definitely consider introducing a wash step with methanol - but I do not think that it will solve my current problem, since replacing the column did not help.

The method that you describe and being used above does not appear to follow the basic fundamentals of chromatography and should be re-developed for your specific samples. It needs modification.

Example: You Wrote: "the perfluorinated compounds are eluted between 22-26 minutes." -

Your method ends at 22.0 minutes and you start to re-equilibrate your column at this point (you change back to your initial conditions at 22 minutes to re-equilibrate the column) so NO Sample PEAKS should be eluted during this time. In your case, peaks are being eluting during the column regeneration phase so the method is not finished and the data obtained is not valid. You need to develop a proper HPLC method for the proper retention and separation before you move on.

You Wrote: "I will definitely consider introducing a wash step with methanol - but I do not think that it will solve my current problem, since replacing the column did not help". The use of methanol as a wash step is not the same thing as replacing the column. It is used to re-condition the column after use of strong hexanes. It would be needed even if the column was new/replaced. Besides, we now know from your above statements that the sample peaks are still eluting off the column during your wash steps so that means your column may be overloaded over time (fouled) with samples from each run because the method is stopped before it is actually finished retaining, then eluting all of the samples. All of your data and observations point to the fact that the method should not be used as-is, and needs to be re-developed. Good HPLC methods first retain ALL of the samples, hold them, then elute them ALL off the column during the analysis. Your column wash and re-equilibration steps should be separate and run after the analysis method (a second, separate method). I have attached a link to a few basic articles on method development to assist you, but please find someone at your school with some expertise in liquid chromatography to help you with this project.

http://hplctips.blogspot.com/2016/07/modern-hplc-method-development-tips.html

http://hplctips.blogspot.com/2016/08/modern-hplc-method-development-tips.html