Keikaku
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Registered: 15-1-2024
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Question regarding UV-VIS calibration curves
During one of my labs we had to prepare a calibration curve for the UV-VIS quantitative analysis of polyphenols present in grape skins using the
Folin-Ciocalteu method. Our stock solution was a 100ug/mL galic acid solution.
The lab paper instructed us to proceed as following:
1. In multiple test tubes, insert 100; 200 ;300 ... 600; uL (microliters) of stock solution.
2. Add distilled water so that all test tubes have 1700 uL total volume.
3. Calculate the concentrations of the solutions. These concentrations are to be used in the A=f(concentration) plot. (This part is what I
don't understand)
4. Add the reagents to the test tubes. ( 500 uL Folin's reagent and 1500 uL of 20% Na2CO3 solution. )
5. Proceed to transfer to cuvettes and measure absorbance.
Here's where my problem lays: After step 3, we are further diluting our concentrations in the test tubes by adding volume (more than doubling the
total volume.). I believe that when making calibration curves, the concentrations should be calculated as to include the total volume, including that
of the reagents. My teacher argues that this way, I would be correlating Absorbance with the concentration of my sample inside the reagent-sample
complex. Am I wrong here?
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Bedlasky
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Posts: 1219
Registered: 15-4-2019
Location: Period 5, group 6
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Always is used concentration before you add reagents. Dilution is always the same. So yeah, you have technically half of the original concentration
after adding reagents, but that doesn't matter. You always add the same amount of sample and the same amount of reagents, this doesn't change content
of gallic acid.
[Edited on 21-1-2024 by Bedlasky]
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