Page 15 - Instrumental Analysis - Pharm D Clinical- 07-PA403

UV-visible Spectrophotometry

Fundamental Laws

Beer’s Law

• Beer’s law is defined as follows: when a monochromatic light passes through
an analyte solution, absorbance of light by this solution is directly proportional
to concentration of the analyte.

• Plotting the transmittance (T) or absorbance (A) of the solution against
concentration, we get the following curves.

Exponential Linear
relationship relationship

Concentration Concentration

Relationship between Relationship between
% transmittance and concentration absorbance and concentration

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Beer’s Law

• Mathematically, Beer’s law can be Concentration
represented by the equation:
A = k’ c This relationship is called:
Calibration curve
• Absorbance of a solution is directly or Standard curve
proportional to concentration provided or Beer’s plot
that the pathlength is constant.

• At certain λ, if we plot absorbance (A)
against concentration, a straight line is
obtained passing through the origin
and possessing a slope equal to k’. The
latter is a constant for the absorbing
substance at a certain wavelength.

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