I want to determine the concentration of a flavonoid molecule. The error due to weighing is quite high as the amount of sample is less. So, I thought of trying biochemical tests like Aluminium chloride test for quantifying. My query is, the binding sites and affinity of the reagent (in this case, Aluminium ions) to the standard might be different from that of the unknown molecule leading to variations in colorimetric readings. We can to an extend predict the sites but not know for sure. Will that cause error in quantification? And also, the standards need to weighed, again causing additional error due to weighing.
How reliable are such biochemical tests?
This reference seems to indicate that the aluminum chloride test is specific to certain compounds, so it may not be accurate for yours.
Article Evaluation of Aluminium Complexation Reaction for Flavonoid ...
If you have at least a few mg, you can weigh the compound accurately on a balance that goes out to 0.01 mg, if you can locate one.
Another option is do the quantitation by 1H-NMR, if the equipment is available. You dissolve the compound in deuterated DMSO along with an internal standard of known concentration. You compare the integrated peak of any distinct resonances to that of the internal standard to get the concentration.
If you know the extinction coefficient of the compound at a UV or visible wavelength, you can measure the concentration of a solution by its absorbance using Beer's Law.
According to my experience in this field
The color method using aluminum ions is the approved method and there is no other way to measure the total amount of flavonoids including chromatography HPLC or GC-MS
Because the latest equipment needs to diagnose all unknown COMPOUNDS before estimating their quantities compared to the standard compound.
All chromatic methods such as alkaloids (BCG) or folin-sc. reagent for phenols will developed and estimated by some scientists of with errors and deviations from the real weights
But there is no alternative
High weights overlap colors with the color of the detector so you should dilute the solutions to be examined
Thank you for the suggestions.
Dear Mr. Shapiro,
We have a balance of 0.01 mg accuracy, but the values fluctuates a lot at less weight measurements.
And even for estimating extinction coefficient for the first time, we need to know the concentration of the compound. Quantification by NMR seems to be a good option. Thank you very much.
Dear Mr. Kadhum,
My sample has one compound, a flavonoid molecule and the structure is known. Although the interaction with the reagents can be predicted but how far they are same as that with the standard is not known.
Thank you.
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