Chromatography Forum

I am developing HPLC method for a drug and its metabolites. I have to use gradient since the great difference between the parent compound and its metabolites.

The mobile phase is A: 30% acetonitrile (0.2% acetic acid, pH 3.6), and B: 90% acetonitril. The gradient is:
keep 90% A for 5 min, gradually increased to 90% B within 25 min, then stay at 90% B for another 10 min before return to the 90% A.

The parent compound and its four major metabolites (M1-4) were perfectly separated. Retention time was about 6, 7, 9, 11 and 42 min for M1-4 and parent compound, respectively.

The problem is, as you see, the parent compound come out too late. And there are several unknow peaks come before the parent compound. There are also unknown peaks when I run the mobile phase alone. I also wash the HPLC system but they are still there. So, what can I do to get rid of these unknown peaks?

Another problem is the baseline droped steeply during the gradient running, from 0 to -1.5 AU. It's so ugly chromatogram.

Very appreciate any kind of help.


Ru Yan
UTMB, Texas

re baseline: what wavelength are you working at? If it is in the low UV region, it is possible that you see the acetic acid. If this is the case, you could either try to go to a higher wavelength, or you could add acetic acid to the mobile phase B.

Interferences could come from the quality of your mobile phase. Run your composition at the beginning for different times: 5 minutes, 10 minute 15 minutes, then inject your sample and run the notmal gradient. If the junk peaks increase, they come from your solvents. Most often, the water is dirty.

Why does it bother you that the parent compound comes out late? Is there a problem with the peak? Is the run time too long?