As per Beer's law: A = abc
Where A = absorbance
a = proportionality constant defined as absorptivity
b = light path in cm
c = concentration of the absorbing compound
When b is 1 cm and c is moles/liter, the symbol a
is substituted by a symbol Ε (epsilon).
Ε is a constant for a given compound at a given wavelength under prescribed
conditions of solvent, temperature, pH and is called as molar absorptivity. Ε
is used to characterize compounds and establish their purity.
Example:
Bilirubin dissolved in chloroform at 25°C should have a molar absorptivity (Ε)
of 60,700.
Molecular weight of bilirubin is 584.
Hence, 5 mg/liter (0.005 g/l) read in 1 cm cuvette should have an absorbance of
A = (60,700)(1)(0.005/584) = 0.52 {A = abc}
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Conversely, a solution of this concentration showing absorbance of 0.49 should have a
purity of 94% (0.49 ÷ 0.52).
In most biochemical and toxicological work, it is customary to list constants based on
concentrations in g/dl rather than mol/liter. This is also common when molecular weight
of the substance is not precisely known.
Here for b = 1 cm; and c = 1 g/dl (1%), A
can be written as A1%1 cm
This constant is known as absorption coefficient.
The direct proportionality between absorbance and concentration must be established
experimentally for a given instrument under specified conditions. Frequently there is a
linear relationship up to a certain concentration. Within these limitations, a
calibration constant (K) may be derived as follows:
| Therefore, |
A = abc. |
| c = A/ab = A x 1/ab |
The absorptivity (a) and light path (b) remain constant
in a given method of analysis. Hence, 1/ab can be replaced by a constant (K).
Then,
c = A x K; where K = c/A. The value of the constant K
is obtained by measuring the absorbance (A) of a standard of known
concentration (c).
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