I have had my HPLC for approx. 6 mos. Everything has been going fine until now. When a working standard is injected signal and area responses are what they should be and are reproducible. But when samples are run the peak area responses are much smaller (by far) than what they should be. This problem started occuring after new types of Whatman vials were tried. Could this be some sort of indication of a column failure? I wouldn't think so because working standard results are fine. Also, new lots of solvents have been tried (methanol), but mobile phase (MeOH:H20) has not been changed. Any help would be greatly appreciated :)

If the problem started after changing the type of vial used, start there. Get some of the old style vial and try them. If they work properly, the new vial are not appropriate for the analysis for some reason.

The thing is we have gone back to the old vials once we saw they wouldn't work (we saw this problem with the Whatman vials and have seen it ever since!)

If you are sure all of your working standards are good, then it is probably not your system/column/mobile phase.

Have you changed any element in your sample prep? Do you filter your samples, is there any particulate? Are all samples affected or is the problem sporadic?

I would also check the data acquisition method and parameters and ensure the same values are being used for standards and samples. Are both stds and samples preped in mobile phase? Are there interspersed std injections during your run that are all fine? Look at the things that you do differently for samples than for standards.

Good Luck.

how viscous are your samples? once we had a problem with an diluter. it diluted aquaus samples right, but when diluting blood samples big air bubles came in the injector. this problem came from a leak seal. maybe this is happening to you in the autosampler?

If your HPLC is operated from its front panel rather than remotely computer controlled:

Have you altered any factor on your HPLC set-up to account for the new vials? i.e. vial size or some other such parameter.
Alternatively has someone else programmed in a dilution factor or different injection volume for sample vials and you are only checking the standard setup when you run?

I have seen on some instruments various parameters that may be changed deep in the programming innards that are not necessarily checked every time an analysis is run.

Good luck

is there your samles having different ph than

the your stds.

We have seen something similar before. Our STD was very fine particles but our samples were much bigger ones. During sample preparation (sonication to dissolve), the STD was well dissolved but the samples were not. Results: sample potency was shown to be lower than expected. After we increased the sonication time, everything was fine.

IS this related to your story?

Good luck!