Basic Concepts in Clinical Biochemistry-A Practical Guide.7z

Absorbance¼ 2 log 10 T

¼ 2 log%T

5.1 Colorimeter and Spectrophotometer

Colorimeter and spectrophotometer work on principle of light absorption which is
integral part of a large number of instruments including semi-autoanalyzers and
autoanalyzer. Colorimeter is an instrument used to measure the concentration of
colored substances. It works in visible range of 400–800 nm of electromagnetic
spectrum of light. The working of colorimeter is based on Beer-Lambert law and is
based on the fact that any metabolite or chemical substance that undergoes a reaction
to form a colored product in the cuvette will absorb some amount of light. The
amount of the light absorbance will be proportional to the concentration of the
metabolite in the sample. The functioning of colorimeter is simple. The light source
which is generally afilament lamp emits light. Thefilter blocks all wavelengths of
light source except the desired wavelength at which sample is read. This light with
selective wavelength then passes through a sample holder which holds a special
glass cuvette with afixed thickness. Some portion of incident light is absorbed by the
sample in cuvette, and the light that is not absorbed by the sample (also called
transmitted light) falls on photoelectric detectors. These photoelectric detector cells
measure the intensity of incident light falling on it and calculate the amount of
absorbed light or absorbance (Fig.5.1).

Spectrophotometer It also measures absorbance of light intensity by a chemical
substance when incident light passes through sample solution. The basic principle of
colorimeter and spectrophotometer is the same except that spectrophotometer also
covers ultraviolet (UV) region range (200–400 nm) in addition to visible range of
electromagnetic spectrum. Unlike recording the absorbance readings at a set wave-
length as used in colorimeter, the absorbance can be recorded over a range of

Light Prism

Monochromator

Monochromatic incident light

I 0 I

Sample Photodetector

Recorder

Transmitted

light

Fig. 5.1Basic components of colorimeter

18 5 Photometry: Colorimeter and Spectrophotometer