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I have a dynamic assay whose absorbance changes with time. I recorded it’s spectrum initially after preparing it. Then I added my analyte and mixed the contents. Then I waited for 2 min and recorded the second absorbance soectrum. I generated a normalized standard calibration curve by dividing the absorbance maximas (captured 2 min post analyte addition) at different analyte concentrations (1M, 2M, 3M...) by an average value of control (initial absorbance maxima before analyte addition). Is this approach correct? My ordinate is normalized absorbance and abscissa is concentration of analyte.

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    $\begingroup$ I'm not sure that I understand your method entirely. If your measuring absorbance at a single wavelength, then typically I'd say that the absorbance would be measured on a blank (ie solution without analyte) and subtracted from the various solutions with analyte. If you taking a whole spectrum then you might be able to get background from each spectra, but typically UV-Vis peaks are so broad that this might not be possible. Also not sure if you have overlapping peaks. So all in all a lot more detail is needed to understand what you are doing. $\endgroup$ – MaxW Jul 14 '18 at 16:46
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I'm not sure whether calibration for a dynamic assay is any different from one that is stable. But let's think of the situation like this. What you have done is no different from what an operator would do to obtain a calibration curve with an internal standard (IS) correction (by plotting concentration against the ratio of the intensities of analyte/IS) except you have used the absolute intensity of your background signal (no analyte) instead of an internal standard for correction. This is not correct by any standard since the background is a bad choice for correction (the background is NOT stable, and has a terrible RSD due to low counts). What you should do is to plot your calibration curve without normalising it to the background.

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