Hello Paromik, the concentration established from the calibration curve is valid for the solution being analysed, hence if you quantitated an aliquot from the 2 ml solution, the volume you should use is 2 ml. If you diluted the aliquot prior to analyses, you should multiply your volume accordingly. For example, if you made a 1:5 dilution prior to spectrophotometer/HPLC analysis, you must consider a 10 ml volume ( 2 ml x 5 ).

Best regards

2 ml.... you put all flavonoids from 100 mg of dried sample in 2 ml of solvent. The intermedia step (the first extraction with 100 ml) is not important since you used the 2 ml solution to test the flavonoid content.

Good luck.

Thanks all, still one small doubt. Suppose My net crude extract which I got after concentrating the extract in a vacuum concentrator is 3ml. Now suppose for testing FC test for phenols I took 0.5ml from the extract. Now in that case what should be my Value of V in the equation C=c1XV/N [c=Total content of the phenol/flavonoid in mg/g; C1=the conc. of gallic/tannic acid established frm calibration curve in mg/ml; V=the volume of the extract in ml and N=the wt of the plant extract in gm]. will it be 0.5ml as I am testing with 0.5ml of extract or total concentrated extract volume of 2ml from where I am pipetting out 0.5 ml?Please explain

Dear Paromik,

Let me try to clear your confusion.

Let us suppose that you have taken 100 mg of sample and you used 300 ml of an apt solvent to extract phenol. That means whatever phenol that is present in 100 mg sample is now present in 300 ml solvent. After filtering, you have concentrated the phenol (and may be even other things) that were extracted out in the solvent through rotary evaporator and reduced volume to 3 ml. That means, now whatever phenol that was initially present in 300 ml is now present in 3 ml (if you further reduce volume to 2 ml - that means the phenol that was present in 300 ml is now present in 2 ml).

In the next step, you are taking 0.5 ml for assaying (must be as per standard protocol and according to which you must have prepared standard curve or using an extinction coefficient). That means you determined phenol present in 0.5 ml. Obviously, in the next step you will find out the amount of phenol present in 3 ml or 2 ml depending on the extent to which you concentrated. Finally, whatever value you will now be getting is present in 100 mg (or it can be any other quantity that one takes) of sample you had taken.