Chromatography Forum

Hi all,

I run a HP 1100 water HPLC for environmental analysis (UV-Vis). Lately I have been having numerous problems with the retention times shifting leftwards. I wouldn't mind so much if it didn't cause my continuing calibration to fail on my Fl detector.

I am using 60/40 ACN/H2O mobile phase with a flow of 1ml/min and temperature seems to be stable. No problems in pressure either.

Outside of temperature change, what causes shifting retention times.

If you are doing the mixing with the pump (and I assume that you run isocratically), I would suggest you to premix your mobile phase and try again with 100%A and 100%B. This will give you an indication if the problem comes from the pump or if it is downstream...

What about the column? Is it a new one?

I am doing mixing with the pump, but I am running a gradient.

The column is fairly new, I just replaced it about 2 to 3 weeks ago.

Since I do not use a buffer I am ruling out the piston seals as being the cause of the problem, I may consider changing the active inlet valves, but am still looking for ideas.

If you are concerned with the pumps on your 1100, you can always run a pressure or leak test from the control module. That can point you in the right direction.

You are running a gradient - what is your gradient profile?
Is your equillibration step long enough at the end of your gradient? If it's too short - the mobile phase composition will have too much organic at the beginning of your next injection, and that can cause a loss in retention.

Are your analytes neutral? If they are neutral, then ACN/H20 should be fine - but if they are ionized, then you should consider using a buffer to control retention.

Well, you got to be more specific...

What is your column, what is the mobile phase, especially pH etc...?

Thanks for the input. I ran the pressure test and found the problem to be my active inlet valve. Once I replaced that and the inlet filter I was good to go.

One quick question though, does in matter if I used expired IPA for the pressure and leak test would that affect my data analysis (or column) ?

I would say a general rule is to always use reagents within expiration date on your HPLC instrument.

You're ok if you used expired IPA, just rinse your instrument out (no column) for 30 min or so with 50:50 ACN:H20 if you are going to be using the instrument for RP.

If you are going to be using normal phase - find some fresh IPA and rinse out the instrument.