of standard absorptivity value, calibration graph and single or double
point standardization. In standard absorptive value method, the use
of standard A (1%, 1 cm) or E values are used in order to determine
its absorptivity. It is advantageous in situations where it is difficult or
expensive to obtain a sample of the reference substance. In calibration
graph method, the absorbances of a number of standard solutions of
the reference substance at concentrations encompassing the sample
concentrations are measured and a calibration graph is constructed.
The concentration of the analyte in the sample solution is read from
the graph as the concentration corresponding to the absorbance of
the solution. The single point standardization procedure involves the
measurement of the absorbance of a sample solution and of a standard
solution of the reference substance. The concentration of the substances
in the sample is calculated from the proportional relationship that exists
between absorbance and concentration.
Ctest= (Atest×Cstd)/Astd
Where Ctest and Cstd are the concentrations in the sample and
standard solutions respectively and Atest and Astd are the absorbances of
the sample and standard solutions respectively. For assay of substance/s
in multi component samples by spectrophotometer; the following
methods are being used routinely, which includes [4],
• Simultaneous equation method
• Derivative spectrophotometric method
• Absorbance ratio method (Q-Absorbance method)
• Difference spectrophotometry
• Solvent extraction method
of standard absorptivity value, calibration graph and single or double
point standardization. In standard absorptive value method, the use
of standard A (1%, 1 cm) or E values are used in order to determine
its absorptivity. It is advantageous in situations where it is difficult or
expensive to obtain a sample of the reference substance. In calibration
graph method, the absorbances of a number of standard solutions of
the reference substance at concentrations encompassing the sample
concentrations are measured and a calibration graph is constructed.
The concentration of the analyte in the sample solution is read from
the graph as the concentration corresponding to the absorbance of
the solution. The single point standardization procedure involves the
measurement of the absorbance of a sample solution and of a standard
solution of the reference substance. The concentration of the substances
in the sample is calculated from the proportional relationship that exists
between absorbance and concentration.
Ctest= (Atest×Cstd)/Astd
Where Ctest and Cstd are the concentrations in the sample and
standard solutions respectively and Atest and Astd are the absorbances of
the sample and standard solutions respectively. For assay of substance/s
in multi component samples by spectrophotometer; the following
methods are being used routinely, which includes [4],
• Simultaneous equation method
• Derivative spectrophotometric method
• Absorbance ratio method (Q-Absorbance method)
• Difference spectrophotometry
• Solvent extraction method
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