Chromatography Forum

Hello. I'm working with an HPLC system (Jasco) that is manifesting a smaller-than-normal peak. We were previously having problems with the pump having a suspected internal leak, and it was recently serviced. After being serviced, the system had a few issues with a noisy baseline, which I fixed by cleaning the cell of the UV detector. The detector now has precipitate forming in the cell at an abnormal rate (after, say, two or three injections of sample). Whereas before, we could usually do around 200-300 injections, at 1 microliter, before any cloudiness issues manifest in the cell. My questions are: Is the formation of precipitate at a quicker rate and the smaller-than-normal peak an indication of a pump that's still faulty? Or should I be looking elsewhere for answers? FYI: I looked at all junctions and saw no leaks.

Usually a precipitate is an indication that the sample and in particular the mobile phase has not been ultrafiltered. Many pH buffers (like phosphate) are very soluble in water but very less soluble in a common aprotic solvent like Acetonitrile. Thus, a gradient can rapidly change the buffer's solubility.

Hello

First of all...

1.If you are not sure about mobile phase miscibility just make simple test in test tube - mix them and see if there is any precipitate.
2.If your mobile phase A and B are not miscible you will have problems (precipitation) in pump at first. So check your pump for leaks, and any blockages.
3.Check if your sample solvent is miscible with mobile phase
4.Check if you have any washing solvents in LC system (needle wash etc.)
5.If you're using phosphate buffers never let the detector cell to dry out - do not stop your run (sequence) completly without flushing LC system with buffer-free solvent (especially detector cell)

And read LC manual to see how to keep instrument in good condition and what solvents should be avoided.

Regards

Tomasz Kubowicz