Colorimetry

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  • It is the most common analytical technique used in clinical laboratories and is based on the quantitative estimation of color.
Sonal Shinde, CC BY-SA 4.0 https://creativecommons.org/licenses/by-sa/4.0, via Wikimedia Commons

  • Components-
    • Light source: Tungsten lamp
    • Slit: To adjust the light passing through
    • Condensing lens: To provide parallel beam of light
    • Monochromatic filter: Which absorbs all and transmits only complimentary wavelength color.(Blue-Red, Purple-green, yellow-violet, orange-bluish green)
    • Cuvettes: A special glass tube with uniform thickness, inner diameter, and refractive index and 1cm light path to measure the absorbance of colored solution
    • Photosensitive detectors: To convert incident light to electricity.
    • Measuring Device: Like galvanometer, to measure current generated which gives optical density.
  • Working Principle-
    • When a beam of monochromatic light (I) passes through a colored solution, a part of the incident light is absorbed by the colored solution and remaining light is transmitted (I0).
    • The intensity of the transmitted light depends on:-
      • The concentration of the substance in the solution.
      • Path length through which it traverses.
      • Wavelength of the incident light.
    • Transmittance(T) is the proportion of the total incident light which is transmitted by the solution.
      • T = I0/I
      • T% = I0/I(100)
    • Beer’s Law-
      • When a beam of monochromatic light passes through a colored solution, the absorbance (A) (optical density OD) of the solution is directly proportional to the concentration (C) of the substance in the solution.
      • A α C or A= kC where  k = absorption coefficient
    • Lambert’s Law:-
      • The absorbance (A) is directly proportional to path length (L) of the light through which it passes.
      • A α L or A = kL
    • When these two laws are combined, A = kCL.
  • Applications:-
    • Widely used in hospitals and laboratories for routine estimation of various compounds like glucose, urea, uric acid, lipids, etc. in various samples like blood plasma, serum, CSF, urine, etc.