Chromatography Forum

WE ARE USING WATERS 996 PDA WITH EMPOWER SOFTWARE. I JUST WISH TO KNOW IF WE INJECTS A SINGLE COMPOUND AT FOR E.G 254 WAVELENGTH. WE SET THE WAVELENGTH RANGE FROM 200-400 . NOW HOW COME I KNOW THE EXACT CONCENTRATION SUPPOSE IF ANY OTHER COMPOUND ELUTES AT SAME RETENTION TIME ON SAME MOBILE PHASE THEN HOW CAN I KNOW OR EXTRACT THE UNKNOWN COMPOUND.
LET ME PLACE AN EXAMPLE. IF THERE IS ONLY SINGLE COMPOUND FINISHED PRODUCT SOMEBODY DO THE MISTAKE IN PRODUCTION AND ADD SOMETHING IN IT. THE UNKNOWN COMPUND NEVER APPEARED APART FROM MY COMPOUND OF INTEREST ON UV VIS DETECTOR, SO IF WE USE PDA IT WILL SEE THE ADDITIONAL COMPOUND OR IMPURITY OR NOT.
BCOZ WHAT I SEE IS THE SINGLE PEAK STRENGTH BCOZ I SET THE HPLC CONDITION FOR MY COMPOUND OF INTEREST AND I WISH TO KNOW IS THERE ANY IMPURITY IS PRESENT OR ANY RELATIVE COMPOUND.
PLZ INFORM ME WITH ANY E.G
I HAVE WATERS EMPOWER PROJECT DATA IF REQUIRED.

Just look at the spectrum. If it is other than your standard compound it will likely
have a different UV spectrum unless it is an enantiomer. Better yet, subtract the
spectrum at the peak apex of your standard and subtract it from the spectrum of
your measured sample. If they are the same you should see random noise.

Thanks a lot for replying.
I have tried this. but no success.
Let me set the example for you.
Suppose i have a actual compound on wavelength is 230 with Mobile phase Meoh/h20 50:50.
Now we run the sample . The peak of interest appears with good resolution. The question is that if somebody adds any impurity or anything other than my compound in production process how come i know that something is added in my sample .
Since i do not get any other response except my compound of interest. Naturally the unknown impurity is not necessaraly will elute with my mobile phase combination. and still i wish to know any other response within the spectrul range of 200-400nm
I am using Waters 996 with Empower software which is a good software.

Please guide me from this point

Thanks

Fareed Tayyeb

Check the Empower on-line help for "maxplot". I believe that will plot the maximum absorbance that it sees at any wavelength in the range.

I'm not sure about what you're trying to achieve. Your method certainly won't detect ANY additional substance in your sample, especially since it's isocratic.
During validation of a method you usually check for separation of your analytes of interest from additional substances that are or might be included in the sample, such as matrix components, synthesis byproducts or degradation products.
If you're paranoid about coelution of an unknown substance with your analyte peak, you might want to check out the "peak purity" function of the PDA (although for small amounts of coeluters this is more or less an "alibi test" that's not really significant). However, you should be aware that generally you might only prove the presence of a coeluting substance, but never the absence.