HPLC-negative peaks or noise

[mandytan]

In a reversed phase HPLC analysis for impurities, the sample chromatogram showed a peak followed by a negative peak during the first 5 minutes. This was not observed for the standard.
Should these two peaks be quantified?
How can we tell if peaks are due to injector noise?

[mandytan]

Sorry, I forgot to add that Agilent HPLC Diode Array Detector is used and the analysis is carried out at wavelength 254nm.

[tom jupille]

Check to see if the reference wavelength function is enabled. If it is, turn it off.

It may be that you sample contains an impurity which has a higher absorbance at the reference wavelength than it does at 254.

[mandytan]

Thanks Tom.
The reference wavelength function was turned off from the start. This situation was observed in the first 5 minutes in the sample chromatogram.No such situation occurred in the the blank (mobile phase containing EDTA) and the standards.
Can you kindly advise?

[tom jupille]

Possibly some early-eluter is fluorescing? Since you have a PDA, the next thing to do would be to look at a 3-D plot of response vs both time and wavelength (or simply look at the spectra when those negative peaks are eluting. That may give some insight as to what's going on.

[HPLCaddict]

In a reversed phase HPLC analysis for impurities, the sample chromatogram showed a peak followed by a negative peak during the first 5 minutes. This was not observed for the standard.
Should these two peaks be quantified?
How can we tell if peaks are due to injector noise?

What are the retention factors of these peaks? (i.e. are we talking about t0-noise?)

[Klaus I.]

Is it possible to show us a typical chromatgrams? Also informations about your mobile phase, column and chromatographic conditions would be very helpful.

Another Question: Do you need really EDTA in your mobile phase?