HPLC concentrations

[jane]

Hello,
i would be very gratefull if you can help can anybody tell what is the cause of obtaining different concentration of the same sample, with large variation .For example, i have made a 1000 ppm solution and the concentrations after 3 injection were: 895, 763,740 ppm.

[Consumer Products Guy]

If the peaks have the same retention time and the same peak shape (and assuming that you're really measuring the peak of interest and not a solvent blip), then I'd say your issue is with sample introduction. If you're using an autosampler, maybe the rotor seal or the metering seal needs replacing (Agilent units have both of these). If you're doing manual injection, then it could be your technique, partial filling of the sample loop....

Check your equipment by running a KNOWN assay under KNOWN conditions, eliminate equipment issue first. It might help to also post your chromatograms, your conditions, and your analyte.

[thohry]

Hello,
i would be very grateful if you can help can anybody tell what is the cause of obtaining different concentration of the same sample, with large variation .For example, i have made a 1000 ppm solution and the concentrations after 3 injection were: 895, 763,740 ppm.

1000 ppm is too concentrated so it may get overloaded and the results are not reproducible

[danko]

1000 ppm is too concentrated so it may get overloaded and the results are not reproducible

Depends on the injection volume, column diameter, nature of the substance etc.

Best Regards

[carlos.teixeira]

Dear Jane,

1) Did you verify if your sample analyte is degrading during your analysis? It may be photosensitive and you are not using none amber vial and flask. Or it is suffering some oxidation.

2) Are you sure about your injection system efficiency?

3) If your areas are so large it is possible that your detector is saturated and the HPLC system lose the method linearity. So, you should dilute your sample or reduce the injection volume.

4) Are you using a validated method?

Have you nice elutions!!

Carlos Teixeira

[jane]

First i would like to thank you for all the answers.
I work on the separation of phenolic acids from different vegetal resources and the system i use is a Dionex UltiMate 3000, the column is an Acclaim 120 RP, 5um, 2,1 mm.
Probably you wonder why i use such high concentrations.I'm trying to establish a purification method using ion exchangers so i need to know what is the adsorption capacity of the resins. Now I use standards of phenolic acids for tests and I chosed HPLC over spectrophotometric assays for the precision.
The substance i was talking about is gallic acid, i figured that was a problem of liniarity since the concentration was too high so today i was trying to establish the liniarity between 40 and 1000 mg/L. I made several dilutions and i chosed some points for the calibration curve corresponding to the correlation coefficient of 0.999. The problem is that for the other known concentrations that i injected i have still obtained
different values of the calculated concentrations. I mention that I weighted the substance exactly (4 decimals) and the dilutions were also prepared exactly.
Another problem that i have, is a peak that appers on the debut of chromatogram, it has a high intensity (200-2500 mAU) and it's not the same all the time, furthermore ,sometimes doesn't appers at all.

Carlos, i'm not using a validated method but i'm workin on it.
Danko, the injection volume was 10 ul and the colum was mentioned before.
Thank you all and I hope I made myself understandable.