Dear All!

Perhaps somebody here can help me:
I get a strange HPLC trace from time to time, which appears to be independent of solvents, isokratic, gradient run, detection wavelength and anything else I could think of.
How it looks like: appr. 2 min after switching the injector from load to inject I get a cross section of the rocky montains followed by a hill. The detector goes to nearly the maximum.
You can see a picture of a trace here:
http://www.embl.de/~schleife/hplc/

I would be really greatful if somebody could help me !!!

Thanks a lot,
Andreas

Hello,

The first thing to do is to isolate the problem checking each module one by one.
- Check the pump pressure and verify the flow.
- Disassemble the flow cell and clean it as well as the tubing.
- Renew your solvents and wash tubing filter.
- Change injection syringe.
- Clean injection loop
- Try another column

If you have another detector on another HPLC try to switch them.

Laurent

Dear Anon,
Dose this rocky mountain appears even when there is no flow of mobile phase??

DK

Hi Andreas

I'm afraid that I cannot offer you a solution to your problem but having looked at your trace, your problem looks very similar to one I encountered a few months ago. This was on a ThermoSeparation Products (TSP) HPLC system (they subsequently changed their name to ThermoFinnegan and are currently ThermoElectron), consisting of a degasser, pump, autosampler and a PDA detector. The assay was isocratic. Like your situation, the only thing that was consistent was the time at which the first baseline spike appeared (although in my case it was nearer to 6 mins post-injection). To me, the style of the trace looked like the result of air in the system, perhaps from attempting to inject from an autosampler vial with not enough sample remaining, although I regularly encountered this spiky chromatogram when clearly there was more than sufficient sample volume. Also, noise from air contamination tends to be rather random, not appearing at a consistent time?!

The HPLC system (apart from the bizarre trace) appeared to be functioning properly throughout (normal range of back pressure, stable flow etc.)

After bugging me intermittently for a few days, the problem disappeared without me changing any assay reagents or cleaning the HPLC system, so I never did get to the source of the problem. I will watch this topic with interest! :o)

Best wishes, Ann

Andreas,

Where is the waste line from your injector going to? Is the waste lower than the injection on the Rheodyne injector? I have seen this type of thing happen when this is the case. I believe what happens is that when you switch from inject to load, there is a siphon effect from the injection port to the end of the waste tube. If your syringe is not in the port, air is drawn into the loop and is therefore injected. There are 2 ways to fix this if this is the case, raise the waste line to be even or slightly higher than the injection port, or make sure your syringe is in the injection port when you go from inject to load. Good luck.

Because of what last Anon described we removed all hoses (capillary) from Rheodyne outlets, and keep things horizontal. The "time to time" could be due to different amount of inj. vols. If you inject more than the loop volume you will ejet the air into waste. Air can also be a problem (normally to a lesser extent) if you inject less than the loop volume: One inadvertendly pushes in air when inserting the syringe needle (in every case that we tested for this; the air peak can disappear in the normal fronting peaks at tm).