I am standardizing a UV-VIS screening method that allows me to quantify provitamin A carotenoids. The results will be compared with samples quantified by HPLC, which is my Gold Standard. Nevertheless; the results that I've gotten so far using both methods aren't comparable between each other. I was expecting the concentration using both methods would be similar, sadly this is not happening. Interestingly, when I multiply the concentrations I get in my colorimetric method by a factor of 4.5 ish, they are very similar to the ones in HPLC. You might say that maybe the calculations are not correct, but I considered the dilution factor as well.
Has anybody encountered this issue, if so... Let's talk about it
In HPLC you will see the specific peak for that particular compound, but when you derivatize a compound with the colorimetric agent, that can react with other compounds as well. In spectrophotometry, these compounds can not be separated, and thus the absorbance will be higher; and can show higher concentrations than the real values.
Absolutely the result will not match because, first need to understand the basic principle of both techniques. Separation takes place in HPLC hence all components get separated and individual results shall be obtained whereas colorimetry is measurement of solution based on absorbance and hence reading is sum of all compounds. Hence both the results will not be matched.
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