Page 34 - Instrumental Analysis - Pharm D Clinical- 07-PA403

UV-visible Spectrophotometry

1- Using standard absorptivity value

A=εbC

A = A(1%,1 cm) b C

• This method is used for official stable compounds where ε and
A(1%,1 cm) are given by the pharmacopoeia.

• Advantage:
No need to prepare a standard solution of the reference substance.
This is important especially if the reference substance is not
commercially available or expensive.

• Disadvantage:
The measured absorbance of a given solution will usually vary
somewhat from one instrument to another, so the instrument should
be calibrated.

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2- Using single point standardization

Procedure: measure the absorbance at λmax for the sample solution
and for a standard solution of the reference substance. The standard
and sample solutions are prepared in the same solvent. The sample
concentration can be calculated by the following equation:

Atest = a b ctest Astandard = a b cstandard

─A──tes─t = ───A──st─andard
ctest cstandard

ctest = ───A─t─es─t ─x─c─s─tandard
Astandard

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