Chromatography Forum

Peak of interest is decreasing by 10% in retention time over the course of 15 injections with 30 min run time.

10 mM PO4 to pH 3; 30% buffer 70% methanol

c18 column; no pressure fluctuation.

This phenomena has been reproducible, that is, the retention time at the begining of each NEW sequence begins around 4 min and decreases.

What could be causing this? We reviewed the mobile phase prep and it looks fine.

Question: if you start a sequence and keep injecting samples (beyond the 15 injections) does the RT ever stabilize?

Additional questions: What happens between sequences? Do you clean/regenerate the column at the end of each sequence and if so, how? Also, do you start with a freshly prepared eluent every time you set a new sequence or do you use one and the same eluent in more than one sequence?

Please note that column temperature could cause that kind of phenomena if you start a sequence with a cold (ambient temperature) column which is warmed up to a higher temperature over time - during the sequence.

Best Regards

The peak's retention time stabilizes after ~ 15 (30 minute) injections.

Methanol and water washes in between sequences were used and the column was equilibrated with mobile phase until a stable baseline was reached.

We are going to investigate temp effects by running at 25C and again at 20C because the method is at ambient.

We used the same MP for each sequence that gave shifting retention times.

Recently we tried a fresh MP and no shift.

I think it is something with the methanol. When new mobile phase was prepared using different methanol than before (same buffer) we acheived consistent RT. I don't know what kind of impurities in methanol could cause a slow decrease in retention time like that. Could it be a metal?

A temperature dependance investigation is by far the most rational action in mi mind.
Impurities in the methanol seams quite exotic to me

Best Regards

Does your lab get warmer thoughout the day?

I know ours does, especially if the AA is running. I find it best to ALWAYS control column and sample temp, mostly because my lab and the lab I transfer methods to can have a 10 degree (celsius) difference in the summer. We need A/C.