[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.

[Arkcon]

You can tell if its just injector noise, or something else, by injecting a matrix blank.  That is, try to inject, as exactly as possible, what the sample is dissolved in, without the sample dissolved in it.  You can also try repeated injections, to see if this is reproducible.  If you keep seeing this negative peak in a sample you're testing for impurities, and it comes out so late its likely not an injector artifact, then you may very well have to quantitate this negative peak -- or more likely, adjust your parameters to try to see its absorbance.  Since you're using a PDA @ 254 nm, you can have other wavelength data at that point, if you set up your system to store that data.

[mandytan]

Thanks Arkcon.
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.The reference wavelength function was turned off from the start.
Can you kindly advise?

[mandytan]

A HPLC analysis for Impurities using polymeric reversed phase column was conducted. The diluent (i.e. Blank) contains phosphate buffer in methanol.
In the chromatogram for the Blank, 2 peaks were observed in the first few minutes. In the sample chromatogram, the peak areas for these two peaks were much larger. For quantitation of impurities, are these 2 peaks considered to be due to sample interaction with the diluent and ignored?

[Arkcon]

Yes.

The blank must be a matrix blank.  If standards and samples are dissolved in "mobile phase containing EDTA" then that's fine, other wise, try to inject the blank to see if the column is separating out some components.

Try to run some mobile phase blanks as wee.  To see if something weird is just happening at the 5 minute mark.

The PDA is capable of storing in the chomatogram a complete spectrum at each point.  You can use this information to help identify the unknown positive peaks, and the negative peak may well have a positive absorbance at another wavelength.  It may just give a spectrum of noise, which also helps us understand.  Unfortunately, I don't have an Agilient manual and instrument in my living room, so you will have to poke around with Chemstation's help files and manual to find out how to turn this function on.

[Arkcon]

No.  For impurity testing, say for a pharmaceutical or foodstuff, you will have to identify these peaks more rigorously.

[mandytan]

The matrix blank is the mobile phase. In this case, should we then quantitate the 2 peaks (inclusive of negative peak)?
Thanks!

[mandytan]

Hence, would it be correct to say that we should subtract the areas of these two peaks in the blank from that in the sample and report these values as impurities in the sample?

[Arkcon]

Ye-Ye -- Yes--ish.  That could work, depending on the regulations you work under.  But trying to resolve it better, would be a better choice.  Trying to use the extracted spectra at each pint, to see if the sample peaks contain something else, would also be helpful.  But in the end, yes, you can get acceptable quantitation of the impurities by subtracting blank runs that flank your samples runs.

[Arkcon]

Yes, and I'm going to merge this thread with your similar thread, in case someone else wants to add info.

[voidSetup]

Wait, so the blank is the mobile phase, then are the samples and standards also dissolved in the mobile phase or something else?  Are the retention times of the peaks the same between the blank and sample?