I performed an aluminum chloride colorimetric assay for Total flavonoid content calculation. Quercetin was used as standard (Stock 1 mg/ml; dilutions:10-100 ug/ml conc.). The stepwise procedure was followed as given below :
Quercetin dissolved in methanol
Standard Quercetin dilutions
0.3 ml of 10% AlCl3
0.3 ml of 1 M potassium acetate
2 ml of 1% NaOH
3 ml methanol
3.4 ml distilled water to make the volume up to 10 ml
Abs. was taken @ 417 nm
I performed the assay in triplicate, but the readings are not in proper increasing order. Even the readings of three replicates are not in close proximity.
I performed this assay many times but every time I am facing the same problem. Please suggest that what would be the probable absorbance range for quercetin, because every time the range of abs. varies.
I also tried the protocol of having sodium nitrite in place of potassium acetate but didn't get satisfactory results. Kindly share your valuable suggestions.
Thanks & Regards
Ana Reis Dear Ma'am I used this protocol also as well note down the readings at 510 nm. By this also, I didn't get a standard graph.
I used Quercetin instead of epicatechin/ rutin. Does it make any difference to use quercetin for this protocol?
Thanks
Dear Preety,
Shouldn't make any difference .Both Quercetin and epicatechin should give a linear response. with increasing conc values. The only difference would be to have the results expressed in Epicatechin equivalents (ECE).
Since you do not see that, at this point you should look for other sources of error, for instance is the laser in your spectrophotometer aligned? are the cuvettes dirty or scratched? one other possible source may be the difference in addition times you take to prepare replicates in each conc point. Possibly by using a multi-channel pipette you may decrease the difference in times.
Hope this helps,
Ana
https://doi.org/10.1134/S106193481204003X
https://doi.org/10.3390/plants8040096
https://doi.org/10.1155/2014/253875
https://doi.org/10.1063/5.0002657
Good luck.
Measurement of flavonoids using AlCl3 with spectrophotometric methods can use various methods, including HAC-AlCl3; AlCl3-KAC, NaNO2-Al(NO3)3-Al(NO3)3-NaOH, and the NaNO2-AlCl3-NaOH method. Comparing all these methods, it is worth to try the use of AlCl3 acetate buffer because it will produce a more stable absorbance; better standard deviation, standard error, variance, kurtosis, and skewness. It is recommended to measure the absorbance of the mixture reaction (Quercetin + Methanol + AlCl3 + Ac. buffer) first. The maximum absorbance is used to determine at which the absorbance of the samples will be measured. Hope that can help..
There are many assays, but the most popular is aluminum chloride colorimetric assay.
https://ejchem.journals.ekb.eg/article_66023.html
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