[veronika]

I use Waters Spherisorb ODS-2 column (4.6 x 250 mm; 5 um). Column is 4 months old. Mobile phase is 10% methanol:90% water. Column shipping solvent was acetonitrile. At the beginning of my work, my supervisor told me I could wash column in methanol at the end of day (and leave it in methanol). 3 weeks ago I start washing and keeping my column in acetonitrile. I wash column for 30-45 minutes (flow = 1 ml/min) at the end of day, with acetonitrile. 2 weeks ago I noticed increase in retention time of my analyte - each day I had changes in retention time. Now my analyte has retention time 11 minutes, and 3 weeks before, retention time was 5 minutes. I have cca 30 injections per week. What can I do to decrease RT ?
Thanks for advice.
Veronika

[Dude]

Likely possibilities in your liquid chromatography analysis:

1.  There is a leak.  This decreases the amount of pressure that is available to force your material out of the column.  Check all of the tubing first and then the pump.  The characteristics are typically no loss in chromatogram resolution and an increase in the retention time.

2.  Your column is spent (ie channeling).  The characteristics are typically peak broadening (loss in resolution) and potentially an increase in retention time.  This is unlikely since it is 4 months old.

3.  There is a plug restricting fluid flow but not stopping it.  This is typically accompanied by the need for very high pressures to get the material out.  The characteristic to look for is the pressure change using a set flow rate, column, temp and solvent at a time when things were working and comparing to the current condition.  The test for this is to confirm the amount of material coming out of the column with your specified flow rate (ie use a small graduated cylinder to check that you are truly getting 1 mL out in 1 minute).  If there is a plug, you will likely see less material coming out.  THis can be minimized by filtering the material through a 0.45 micron filter prior to analysis.

4.  This is under the assumption that the storage material has been flushed out thoroughly and is not a factor (ie this isn't the first run of the day and) and that it isn't an expected change from switching elution solvents.

If you are at a steady state 11 minute retention time and you don't want to fix what has been proposed as a problem, increase the fluid flow to decrease the retention time.  

[veronika]

Dear Dude,
Thank you very much for your answer.
I need an expert like you to be present in my lab all the time

I had service and system validation 4 weeks ago (authorized person did it) and everything was OK. I'll check once more everything maybe I'll find something. I always filter all samples and standards prior to analysis. I already increase flow of the mobile phase from 1 ml/min to 1,2 ml/min and decrease retention time from 11,5 minutes to 9,4 minutes. What do you recommend, how much I can increase flow ?

According to your opinion, do you think that I should wash the column with methanol instead of acetonitrile ? Is it possible that washing with acetonitrile cause changes in retention time ?

Thanks once more.
Veronika