https://www.reddit.com/r/askscience/comments/w7rt65/why_is_bioassay_absorbance_measured_at_2/
created by Remarkable_Quarter_6 on 25/07/2022 at 15:27 UTC
0 upvotes, 3 top-level comments (showing 3)
I am reading a lab procedure and the absorbance for the bioassay is measured at 2 wavelengths. Why are bioassays measured at two different wavelengths? Is this standard? When reporting results, should both figures be reported or just at the maximum wavelength?
Comment by sometimesgoodadvice at 25/07/2022 at 16:56 UTC
7 upvotes, 0 direct replies
This really depends entirely on the assay. Assuming that only one absorbance is needed to measure whatever quantity you are trying to measure, typically a second reading is performed on the part of the spectrum where you have minimal absorbance (typically far red) and helps control for various apparatus/pathlength effects. Again, it depends on the assay how you would report, if the second wavelength is to function as that control, then you would use it to normalize your absorbance value.
However, there are many assays where you would want multiple readings and how to report will depend on what you are doing. It could be that you are trying to get a purity calculation out of the spectrum (with a pure compound, the ratio of absorbance between two wavelength should be fixed, so deviation from the ratio could be a quantitative way to measure impurities). It could be that you are measuring two compounds that are somewhat distinct spectrally and the measurements are independent. And it could be any number of other reasons.
Comment by Pink_Axolotl151 at 26/07/2022 at 02:35 UTC
1 upvotes, 0 direct replies
It’s done that way in a lot of plate-based assays because impurities such as dust particles, scratches on the bottom of the assay plate, fingerprints, etc., will absorb light equally at both wavelengths, whereas the thing you are measuring (ie, the color of the sample) will only absorb light at one wavelength. So the reference wavelength is subtracted from the measurement at the other wavelength to control for that optical interference.
Comment by swat_08 at 27/07/2022 at 16:11 UTC
1 upvotes, 0 direct replies
Recently, I did an experiment where we used spectrophotometric analysis results, that was to determine the Lambda max of a particular protein *we used Bovine serum albumin.* At first, we made several different concentrations of that protein and we measured the absorbance of the protein at OD660nm and noted down the results that would help us in plotting the standard curve for determining the Absorbance via calculations. Then we selected a certain concentration of diluted protein and measured its absorbance from 200nm through 400nm and interpreted at what wavelength the absorbance was the highest and hence that was the absorption maxima/lambda max.