Chromatography Forum

hi,
I am facing a problem of negative peak after he positive peak of my degradation product

Anamika,

You need to be more specific. What is your MP and diluent? Are you using UV or CAD? Try to give as much info as you can.

Murali

my MP is 70% acetonitrile, 30% ammonium acetate (pH 5) and 0.1% triethylamine.
I am using UV detector and my lambda max is 232.
The diluent for my sample is mobile phase plus 0.1 N HCl (0.1 ml).

Hi Anamika
I fill you try to avoid the 0.1 HCl in final dilution and see is there any effect. Also try to monitor at dual wavelength.
I think this will work.


ALL THE BEST

i cant avoid using 0.1 N HCl because i have to perform the degradation studies of my drug and i have tried the dual detectopr or pda system but my problem continues.

I think you can neutralise the solution befor injecting with NaOH then you inject and see
Hopes it will work.

Anamika,

A negative peak looks just plain wrong but is likely just as reliable for quantitation. Does doubling the standard concentration produce twice the peak area and height?

If you balance the detector then the baseline will be arbitrartily set to '0' absorbance. A negative peak means that there is less absorbance while the peak is passing through the detector than when the mobile phase is passing through. Two likely reasons for this are:

1) The mobile phase has more absorbance than the analyte at the monitored wavelength. Inject a sample of pure water. If you don't get a large negative peak then this is not your problem.

2) Your absorbance at the refence wavelength is larger than at the measured wavelength. Since you have a PDA, measure the spectrum of the peak of interest. If the analyte absorbs more at the reference wavelength than at the measured wavelength then you should be getting negative peaks.

So... try injecting water. Try a shorter wavelength. Try changing your reference wavelength.