Hi everyone,
I am following a reaction by HPLC. My starting material and my product have different max absorbances (respectively 260 and 232 nm). I would like to do a calibration curve of both compounds to determine the conversion. My question is :
Should I pick the area from the max-plot chromatogram (both peaks are at their max absorbance) or should I set up one wavelength (let’s say 232 nm). Of course, I would use the same method (max-plot or one defined wavelength) for the calibration of both compounds but I was wondering if one or the other was more accurate.
Thanks for your help ^^
I suppose that you use a PDA detector. I think that it is too risky to use the max plot chromatogram, because you may loose wavelength selectivity. I strongly would recommend to use fixed wavelengths for each compound. Supposed the maximum absorbances are specific for your compounds, that would be 260 nm for your starting material and 232 nm for your product. (Anyway, please remember that the maximum absorbance may not be the best due to spectral interferences.) Deviation from maximum absorbance results in loss of sensitivity and error increase, but you may gain selectivity. It's always a compromise.
If you set up one wavelength for both compounds (say 232 nm) you would loose sensitivity for your starting material. It's not important here that you have maximum sensitivity, but that your sample data fall into your calibration curve, which should meet your QC requirements (you should have an idea about your LODs and CIs).
you can go with both of the options , with using one or individual wavelength you need to consider the interference at the retention time of your starting material and product also you need to consider the LOQ or Detection limit of your sample.
thank you so much for those answers. I will go with the same wavelength. I may try both methods to compare and see which one match the best with a known sample. Thank you again !
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