Chromatography Forum

Sorry I think I posted this in the wrong forum earlier.


I'm running Benzocaine Injections on an Agilent 1100 series LC. My peaks typically come out at about 2.5 to 3 minutes. They are now appearing at 5 minutes. I moved the column, samples, and mobile phase to a separate sytem, and peaks are normal.

Any help is greatly appreciated!

Tank you! Josh

you might have problem with proportional valve or flow rate. Check flow rate and delivery from all channels. Pre-mix mobile phase and put all lines into this bottle and see what happens.

I moved the column, samples, and mobile phase to a separate system, and peaks are normal.

Do you mean that the retention times are normal on the second system? If so, follow Vlad's advice.

If you mean that the shape is normal, but the retention times are still longer than you expect, then you have a chemistry problem. Make up a fresh batch of mobile phase first and if that doesn't fix the problem put in a new column.

I've tried two different columns, and have the same result. It is an isocratic run. Mobile phase is fresh, and my samples are all correct. I ran this calibration method on an identical system, and the retention times are correct on the second system.

I performed a PM on the pumps, and I'm still getting the same results.

Okay, you've done most of the "heavy lifting", by exonerating the column and tying the problem to one system. So, next questions:

1. The first thing to do is to verify that the flow is correct. I know you did PM on the pump, but just because I'm paranoid doesn't mean the world isn't out to get me. Try and find the "dead time" marker (t0) on your chromatograms. If that's also longer than your retention times (and especially if the percentage error is the same), then you have a flow problem.

2. If you can't find t0, then time the collection of about 10 mL of mobile phase and confirm that it's within 1% of the nominal flow.

3. Are you running premixed ("off-line") mobile phase or are you using the pumps proportioning system ("in-line")? If it's the latter (i.e., you are mixing A & B solvents in the pump) go back and re-run the "stairstep" test (http://www.lcresources.com/resources/TSWiz/hs450.htm) to verify accurate proportioning.

If you have tried all the suggested remedies and still have no luck i recommend changing the AIV cartridge, when i have had similar issues it is nearly always my first port of call esp. if running isocratic and getting RT problems. It is also possible that you may have an issue with the vespel rotor seal, the little grooves may be worn and it may need replaced.

Good luck