Hello everyone,
Please help me in calculation of total phenolic and flavonoid content, may be iam missing some steps in calculation.my experiment was like follows_
50 μl extract (mg/ml)+450μl dw+2.5 ml FC reagent+2 ml Na2Co3 (7.5%), in ratio of 1:5:4. Incubated for 15 min, OD taken at 765 nm.
standard was gallic acid in conc of 5 μl gallic acid+455 μl DW to 25μl +425μl dw+ 2.5 ml FC reagent+2 ml Na2Co3 (7.5%), for blank dw used instead of gallic acid and remaining same. one of my reading was 0.072 (absorbance)
Formula is TPC=C*V/M.
Please suggest me how to take values in my case.
TPC=?
For total flavonoid content experiment was as follows:
100μl extract (mg/ml) + 900μl dw + 3ml methanol + 0.200 ml aluminium trichloride + 0.200 ml potassium acetate+ 5.6 ml dw= final 10 ml
ratio was 1:3:0.2:0.2:5.6. mixture incubated for 30 min OD taken at 415 nm.
standard wad Quercetin 20μl Quercetin + 980 μl.dw to 100μl Q+ 900μl dw and remaining same as sample.
Blank dw insted of quercetin and remaining same as sample.
one of my reading was 0.013 (absorbance)
TFC=? in my case
Please suggest
Which value should consider by graph or by excel? or both?
Thank you
You need first to provide the equation of standard curve of both gallic acid and quercetin. Then you can use the absorbance reading of sample in this equation. After that you can apply your equation provided in your question.
If you write these equations (standard curve), I can help you.
Hi Bhagyashali Karle,
I suppose the unit of standard solutions was mg/mL:
1) TPC:
Sample volume = 50 uL
Total volume (reaction medium) = 5 mL
Sample absorbance value = 0.072
Gallic acid curve equation: Y= 0.0113*X+0.0439
So:
0.072=0.0113*X + 0.0439
X=2.487 mg/L (reaction medium)
Or:
= 2.487 mg /1000 mL (reaction medium)
Or:
= 0.0124 mg/5 mL (reaction medium)
Or:
= 0.0124 mg/50 uL extract
= 0.2487 mg/1000 uL extract
= 248.7 mg gallic acid/L extract
2) TFC:
Sample volume=100 uL
Total volume (reaction medium)= 10 mL
Sample absorbance = 0.013
Quercetin curve equation: y= 0.057*X - 0.079
So:
0.013 = 0.057*X - 0.079
X= 1.614 mg/L (reaction medium)
= 0.01614 mg/10 mL (reaction medium)
Or:
= 0.01614 mg/100 uL extract
= 161.4 mg quercetin/L extract
I hope these calculations are enough clear to explain the answer.
Thank you so much sir for your detail explanation its really helpfull.
Thanks,
Bhagyashali
Hi Bhagyashali Karle , if your stock concentration was 1 mg/mL, the concentration of your standard gallic acid solutions ( 5-25 μl gallic acid) will range from 1ug/mL (1 mg/L) to 5 ug/mL ( 5 mg/L). So, the unit of your gallic acid in the standard curve could be ug/mL or mg/L. So, this matter will not change the calculations explained previously.
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