3.3.1
Explanation 3-10 Auto Zero Method (without coloring reagent)
Elements causing absorbance without using coloring reagent (not containing interfering substance)
A measured absorbance value is given as a total of absorptions due to various elements (cell,
solvent, coloring reagent, analyte of interest, interfering substance). In other words, this value
does not simply stand for an absorption due to the analyte alone.
For generating a calibration curve according to concentration levels 0, 1 and 2, solutions of
concentration levels 0, 1 and 2 are prepared for STD1, STD2 and STD3, respectively (see <1> to
<3> in the above figure). These STD’s have absorptions due to cell, solvent and analyte.
However, the absorbance required for actual quantitation is the one deriving from the analyte.
Therefore, absorptions due to cell and solvent need to be subtracted (by auto zero operation).
In this case, auto zero is carried out with STD 1 and the absorbance values of STD1 to STD3 are
measured, thereby generating a calibration curve.
And, for sample quantitation, the concentration of the sample is determined using the absorbance
after exclusion of cell and solvent. When the absorption other than those due to cell and solvent
derives from the analyte alone, the determined concentration equals the concentration of the
analyte as illustrated at <4> in the above figure.
Elements causing absorbance without using coloring reagent (containing interfering substance)
In the case where a sample contains an interfering substance such as turbid or disturbing
component as illustrated at <6> in the above figure, the absorbance due to the interfering substance
will be added to the quantitative value of the analyte.
In this case, it is necessary to carry out a blank test (the same preparatory operation as for samples
by use of pure water or the like) and subtract the measured absorbance of the blank test solution by
auto-zero operation at the time of sample measurement or subtract the quantitative value of the
blank test solution from that of the sample in order to subtract the absorbance of the interfering
substance from that of the sample.
Cell
Absorbance
Solvent
Analyte of interest
<1>
STD1
<2>
STD2
<3>
STD3
<4>
Sample
Sample solution
Standard solution
Absorbance-zero line by auto
zero with <1>
Absorbance of
analyte
Cell
Absorbance
Solvent
Analyte of interest
Interfering
substance
<1>
STD1
<2>
STD2
<3>
STD3
<5>
Blank test
solution
<6>
Sample (interfering
substance contained)
Sample solution
Standard solution
Absorbance of
analyte
Absorbance-zero line by auto
zero with <5>
Absorbance-zero line by auto
zero with <1>
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