Principle of colorimetric technique for measuring unknown analyte concentration

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Principle of colorimetric technique for measuring unknown analyte concentration

This method is performed using the photometric technique (i.e. colorimeter or a spectrophotometer whose wavelength range lie in the visible region (i.e. >400 nm)) to make a quantitative determination for the substance under investigation. Therefore, this technique depends on the absorption of light by a colored substance where the amount of the absorbed light is directly proportional to the concentration of the substance we want to detect according to Beer’s law.

A = ε b C

Where,

  • (A) is called absorbance and it measures the amount of light absorbed by the colored compound
  • (ε) is the molar absorptivity (i.e. measuring the amount of light absorbed per mole when the light pass through a path length of 1 cm)
  • (b) is the path length that the light passes through.
  • (C) is the concentration of the substance under investigation.

How to make the colored compound? In this method, the substance under investigation is reacted with another compound to form a colored compound or to form a compound that will react with a third one to form the colored compound. This colored compound is measured against the same colored compound resulted from the same reaction of a standard solution of known concentration from the analyte under testing. The concentration of this colored compound is directly proportional to the concentration of the analyte. Then, to calculate the concentration of the sample, we use the Beer’s law equations like this: –

As-Ast22

Where,

  • (AS) is the amount of light absorbed by the colored compound resulted from the reaction of the analyte of the unknown concentration in the sample.
  • (Ast) is the amount of light absorbed by the colored compound resulted from the reaction of the analyte of the known concentration in the standard solution.
  • (CS) = the concentration of the colored compound resulted from the reaction of the analyte of the unknown concentration of the sample.
  • (Cst) = the concentration of the colored compound resulted from the reaction the analyte of the known concentration of the sample.

Because the (ε) is the same for the colored compound in the unknown sample and in the standard solution and because the path length is constant during the test, then the equation will be as follow:-

As-Ast-ep-b

THUS

sample conc

Thus, if you know the concentration of the colored compound resulted from the reaction of the analyte of the standard solution and measure its absorbance, then by measuring the absorbance of the colored compound resulted from the reaction of the analyte of the unknown sample, you can know the concentration of the substance you want to measure in the unknown sample.

Some colorimetric methods make a calibration curve in which we measure the absorbance of serial known concentrations and make a graph; the absorbance on the vertical axis and the concentration in the horizontal axis and then the absorbance of the sample is measured and the concentration is found from the graph.



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