Please help me in solving wavelength issue

[pakhurimehta]

Hiii all of you,
I am facing a problem that in a company, where i am doing my project work, they gave me the method for analyzing the drug content by UV (assay) at 332 nm and My related substances method analysis is on 254 nm. But when i analyzed it by taking a scan of it by UV, my Lamba max is coming to be 272 nm. Now can you tell me.....why they choose different wavelength for assay and related substances method when diluent is same.

[giacomo56]

Sometimes companies buy equipment that may NOT be suited for the best results.....it could be that the original essay was done on a filter type of UV detector and the best wavelength on that type of detector was 254 also the strongest.
Just a thought.

[sepscientologist]

The best wavelength is not always lambda max. It could be that what you want to analyze has a good absorbance at 332 and the impurities
have no absorbance at 332. If the impurities are monitored at 254 there is a good chance that they also have a strong absorbance at 272
since that is relatively close.

[tom jupille]

When I see 254 nm, I am always suspicious of what giacomo56 suggested: that part of method was originally developed years ago on a fixed-wavelength detector. 254 nm was the standard because a low-pressure mercury arc provides a dirt-cheap source of monochromatic light at that wavelength.

All too often, methods are developed by adapting older methods, which are based on even older methods, which started out as still older methods.

You would have to go back to the original documentation to see *why* they chose the wavelengths they did.

[martsims]

The organic impurities may not all have the same UV profile as the main component so the stated wavelength could be a compromise across what is likely to be seen. If you have RRFs for all your impurities at this wavelength it's not a problem at all, if you're working with an RRF=1 for everything and reporting as area% then in most cases I've seen you can ask what results they'd like to see and change your wavelength to give this. For the assay you'd typically choose the UV maxima to give maximum sensitivity. Occasions where you might not are where this maximum is very sharp or if you can reduce the effect of any co-elution.

[WillyOne]

The reason to choose a so high wavelength can be:
The absorbance of the active ingredient is so high that affects the linearity of response form the UV detector.
Choosing a high wavelength is a nice way to protect yourself about coelution intereferences. As you go to a higher wavelength, there are less absorbing compounds.