By bluejay168 on Friday, April 16, 2004 - 08:04 pm:
Our impurities test method uses a diluted active as the external reference standard and found an unknown peak in the chromatogram. From the UV spectra of the active and the unknown we believe the quantity calculated is inflated since the wavelengh in the method is at the maxima for the unknown. The unknown is not stable enough to be separated. How can we determine the RRF of the unknown compound against the active? Or how to deal with the unknown quantitation without RRF data? Thanks for any input.
By tom jupille on Sunday, April 18, 2004 - 10:34 pm:
If the unknown is present at high enough concentration, you might try using an evaporative light-scattering detector. Response is more or less independent of structure. The catch is that you may have to re-work the separation if you have anything non-volatile (like a buffer) in the mobile phase.
By DR on Monday, April 19, 2004 - 05:38 am:
If you're not separating the unknown from other analytes, you're in a tough spot. Either change run conditions to get the separation or try MSD detection or (last resort) scan unknown peak w/ PDA and subtract out scan of coeluting known at same concentration. Even if you do this, you will be left with the spectrum of an unknown, so you can't assign a RRF until you have some idea of what it's extinction coefficient is. MSD can help with determining what the unknown is. Other tests can also help, but you will probably have to run in a semi-prep mode to collect more of the unknown (which gets you back to fixing the separation).
By A.Mouse on Monday, April 19, 2004 - 05:15 pm:
I am not sure if we understood you correctly. Therefore let me try to rephrase:
You got a new unknown peak in the chromatogram. It is separated well from other compounds. You seem to think that the collection of the impurity (with prep or whatever) won't work, because the new unknown is too unstable. From the UV response and the spectrum that you have, you believe that the UV response factor of the unknown is higher than that of the parent peak. Now you are asking, how you can quantitate this unknown compound in a way that hopefully makes the problem go away.
Please correct me if I misunderstood something!
By DR on Tuesday, April 20, 2004 - 06:46 am:
I don't think that you can base a response factor on a UV spectrum without an associated weight or structural informationprovided by an orthagonal technique.
What makes you think it's unstable?