How to choose the best wavelength choice for procedures?

<p> I have a question regarding the best approach to determine the proper wavelength to use in a procedure.  Do I blindly follow the the literature sources for the selection of a wavelength or should I review the PDA wavelength and work with the absorbance maxima of the compound of interest?  Sometimes the literature provides a wavelength selection not even close to the wavelength maxima of the compound.  Why would someone choose a wavelength other than the maxima?</p><p></p><p>Thanks</p>

Answers

  • I would certainly look at the PDA spectrum to choose a wavelength.  And I usually monitor a few wavelengths when I'm running samples.  It's not like it takes any more time to do so and if they aren't of any use you can just ignore them.

  • The  lambda max usually provides the best sensitivity but may not provide the best selectivity.

    If there is an interference that elutes at the same retention time as the compound of interest, using a wavelength that is selective for the compound of interest but still provides adequate sensitivity would be useful.  One does not typically consider UV absorbance to be terribly selective (versus say, fluorescence or mass spectrometry) but cases do exist.

    Wavelengths reported in the literature may also be a compromise so that multiple analytes in a sample can be detected with sufficient sensivity.  Compromising is not necessary if using a PDA since the lambda max of all analytes can be monitored.  Also, for any modern fixed wavelength UV detector, compromise can be avoided by programming wavelength changes during the run. This however will require very reproducible retention times and good resolution between peaks.

    Lastly, a wavelength that provides sufficient sensitivity and is transparent to the baseline drift caused by solvent gradients may be selected to make integration easier.

    Regards,

    Rich

  • Thanks for the replies.  To be more specific, I am working with salicylic acid in a product.  Using the PDA, salicylic acid shows two lambda max values, 229 and 296 nm.  The USP monograph has two procedures with salicylic acid present - one looking at related substances and the other doing an assay in a topical foam.  The wavelengths used are 270 and 280 nm, respectively.

    Why would the wavelengths chosen be towards (not at) a lamda min and not at a lambda max point?  My expectation is a lambda max would be used to maximize the sensitivity for the analysis.

    Using the PDA with my sample, there are no other components which "appear" at other wavelengths at or near the retention time of salicylic acid.  Technically, I could use any wavelength, so why not a wavelength that has the best potential to "see" the component?

  • In addition to Rich's comments, I will add that different assays for the same compound can easily have different detector parameters, determined by the purpose of the assay, the sample matrix, the mobile phase, etc. The absorbance maxima for a particular compound are not fixed in stone because they will be affected by the compound diluent. See the attached absorbane spectra of Salicylic Acid. Your PDA may generate different lambda maxes when you use different mobile phases. Without speaking with the method authors, it can only be speculated why they used 270 and 280 nm - which appear to be well away from any maxima for Salicylic Acid. Sometimes off maxima wavelengths are selected to reduce sensitivity, extend concentration linearity and avoid the necessity of sample dilution. Perhaps they wanted the simiplicity of specifying one wavelength and these wavelengths were more tailored to the other compounds in the method. Salicylic acid may have enough sensitivity and selectivity for their purposes.

  • Yes,i am sure this is a right way to choose the best wavelenth.i also follow this procedure.

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