I am doing analytical method development for to assaying the content of Vit-B2 in its ophthalmic formulation by following the BP procedure using specific absorbency. In that they used specific absorbency(328) for its calculation while reviewing literature I found the formula as follows.
% coloring mater present= AbsorbanceX5000X1.327 / 328X weight taken
in this why the value 5000 was used and factor 1.327 why they used. and how to calculate the content form this. Please any one knows this please help me to know it.
If you want to measure B2 concentrations spectrophotometrically, you want to use the Lambert Beer law:
E = e*c*d
E = extinction measured (your reading)
e = molar extinction coefficient, M-1cm-1
c = concentration, usually mol/L
d = width of the pathway (cuvette), commonly used cuvettes are 1cm
You need to know your e for your compound of interested at the wavelength you are measuring, then you can solve the equation for concentration.
Alternatively, you can also prepare a standard curve and measure the extinction based on those concentrations using the regression y = mx + b.
In both cases you need to be sure that there are no interference present in your sample that influences the the absorption at your target wavelength.
Good luck!
Dear MANI GANESH,
It is easy to calculate--
The specific absorbency (328) is usually mentioned in pharmacopoeia for 1 % w/v of solution.
Now you prepare the solution of your sample/drug to the working concentration as mentioned in p'copoeial procedure or suitable procedure. Measure its absorbance at same wavelength.
Next step is make calculations to expressed concentration in same unit ( microgarm/ml) and directly perform calculations by cross multiplication.
Thanks a lot Dr.Daniela Hampel and Mr Vishnu Prabhakar Choudhari for your suggestions.
I evaluated many applications for regulatory approval. It is a requirement to validate the method using a serial dilution of standard (your test subject of certified material). Depends your purpose of study, you could go simple way of calculating from published reading or validating with a reference standard.
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