Chromatography Forum

Hi,

sorry for another newbie post. We recently acquired a Waters 2695/D separation system. We have 3 systems hooked up to a PC in the lab running empower. All now communicate fine and can be run. Our original systems Water 2695 have passed their change control for the hook up but, this new system will not.

It seems that the system is taking up more injection volume than required.
My method I have used on the other systems requires 50uL inj vol.
When looking at the vials used for SST on the new LC it appears that only 25% of an ~1mL HPLC vial is left!
I have taken the syringe out and cleaned it - could it be a seal that has gone?
Other than going through the manual and step-by-step faults I am a little lost.

Any advice would be greatful, I have never take the seals out before!

Thanks

Just because solvent is missing from the vial, it doesn't mean it has been injected. A pulled loop type injection system will draw 2-3x the injection volume from the vial to fill the loop properly.

You should try doing an OQ, at least this is a test for our OQ's, on the unit. Use a vial with water and weigh it to at least 6 places. Have the unit draw your sample volume out and then rewiegh the vial. Usually you can have it do 6 from the same vial with a runtime of .1 minutes and then get an average injection volume.

Hi,

What tests if any have been done on the new system when it was installed? Usually the issue is not enough sample is injected. Prime the system with Methanol, have a back pressure of around 600 to 800Psi, allow the delta to stable. Then do an adjust seals. Also do a compression check. Then the next step would be to do a lift test, fill a vial to the sholuder with Water, weigh it and then do 6 injects from it, use the front screen. run time 0.01, inject volume 50.0, 6 inject. post weigh it and subtract and then divide by 6 and you should get close to 50.0 +/- 2.
If it is still failing then the syringe and inject valves would need to be looked at.

IIRC, Waters 2695 systems come with a ~100 µL loop as standard, Check to see if someone has replaced it with something else.

One thing you did not say was if the peaks produced in your SST were different on this machine as opposed to the two others.

The 2695 only withdraws what is asked when it is working correctly.

Make sure that the actual syringe volume and the configured volume are the same.

Do the things mentioned by Bull76

If you are removing too much, you may need a new syringe or a new valve 3.

Thanks guys - I will try these today.

The sample is been injected as I get a peak the first time round, but not the second etc as there is no sample left to inject.....

The peak area is huge! So this leads me to believe the syringe is injecting far too much.

does your HPLC have an injection type setting (i.e. push, pull, full)? As if it is a bigger loop and it is set to "push" or "full" it will draw up far more of the sample than is needed and leave you with far less in the vial than expected.
lynz x

Someone had changed my syringe settings D'OH! I changed it as soon as it arrived to the 250uL (correct) on its arrival....so not sure what happened there.

It thought it had a 25uL syringe rather than a 250uL!

All sorted!