Hello. We have a sample with an unknown concentration of polyphenols. We're diluting the sample in order to perform the Folin Ciocalteau method.
0.2 grams sample / 4 mL 70% methanol and 30% water = initial
200 microliters initial /800 microliters solvent = next dilution
100 microliters initial /900 microliters solvent = final dilution
We performed this several times. However, the final concentration calculated from our standard curve for each dilution was different. Additionally, all the replicates had a general (but not mathematic) trend of increasing concentrations with increasing dilutions. I was wondering where we could be going wrong in our approach and what could be causing this (assuming it's an error in our calculations).
Thanks.
I see you are performing Folin-Ciocalteu in very small volumes, this can be an issue as it's rather a complex process that also involves CO2 and darkness. My guess that it's maybe not a calculation error but a lack of reproducibility. Have you tried doing triplicates of each dilution? That will tell you if the trend you observed is really true or if you are observing random variations around the same average value.
However, if you want to post your calculations I can check. Either way, doing triplicates of the analyses is a must.
I cannot do much with a bad quality capture of a non-explained table. What I can see is that you have high RSD values (values above 5% are considered bad reproducibility), and considering that, the signals obtained for each dilution seem consistent to me. The dilutions and the decrese in signal are more or less proportional. Appart from the Non Diluted sample, which doesn't follow the trend. This may indicate that there is a systematical error in the dilution process.
Nevertheless, why are you using different dilutions for predicting a sample? You should simply choose the dilution whose signal lays in the middle of the linear range of your calibration. When you fix the lack of reproducibility issue, of course.
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