Chromatography Forum

I think that you're dealing with column overloading for those compounds (in some extent).

Why would the same column be overloaded when installed on an Agilent LC and not overloaded on a Varian LC?

Krystinakk, if you have explored all the possibilities of inappropriate fittings and plumbing in general, maybe you should focus on the injector valve – it could be malfunctioning.

Best Regards

I think you have a problem with "detector" overload, if only the large peaks are split. On most absorbance detectors, the peak will eventually flatten (become rounded on the top) as you go above 1000 mAU. It is never wise to try to do any important analytical work when your peak absorbance is this high.

On some detectors, when the absorbance gets very high (3000 mAU would be a good example), the detector is not seeing much light come through, and the calculations of absorbance become sensitive to noise and other factors. I have seen peak splitting happen in these cases. The fact that it doesn't happen every time is disturbing, but maybe your peak heights are not always the same, and maybe there are other noise and background factors that change slightly from run to run. If you dilute the sample, you should see the problem go away as you do more dilutions.

i was using directly pharmacopeia method, so if it is 'tested' why peaks split?
(they did not say that peaks will be splitted)

anyway sometimes, I have 2000-3000 mAU peaks with no splitting

the DAD cuvette lenght is 10 cm, is it good or not? Should I change it for another?

As I mentioned, peak splitting due to detector overload is not always predictable. Also, USP-type methods are often very old, and were developed using equipment that have very poor performance by current standards.

As other have mentioned, the design of the injector, tubing length and diameter, and flow cell design can all contribute to band broadening. That is, they will cause the peak to be spread out over a larger volume. This will reduce the concentration, which means the peak height is smaller. If you compare an old Varian LC with a new Agilent LC, I would expect better peak shape (and taller peaks) from the Agilent system.

You should also check your detector settings for the Agilent. Specifically, verify that the PeakWidth (ResponseTime) setting matches the actual peak width in your chromatogram.

If you still have problems, write to us again. But first I would find a sample that causes splitting. Injected it four times to see if the splitting for the same sample changes for each injection. Then dilute the sample with an equal volume of solvent, and inject again to compare peak shape.