Aim:

          The low concentration of many substances in body fluids and the difficulty of isolating them from such complex mixtures make colorimetric methods based on specific chemical reactions particularly suitable and they have been extensively used.

- They are easy to perform.

- Require small amounts of blood and give results of high order accuracy.

Principle:

          It depends on the intensity of colour produced during a chemical reaction.

The laws of light absorption:

          When light passes through a solution some may be absorbed. When monochromatic light passes through a solution, the intensity of light transmitted decreases exponentially with increasing path length (Lambert's law) and with increasing concentration of the absorbing substance (Beer's law). The combination is often termed the Beer-Lambert law.

          From Lambert's law, the proportion of light absorbed or transmitted is independent of the intensity of light entering the solution. A constant proportion of absorbed light entering each successive layer of the solution is absorbed although the absolute amount in each layer diminishes progressively.

Scales:

          Some photoelectric instruments are provided with two scales, one from 0 to 100 showing %T, the other from infinity to 0 showing the absorbance. In practice the instrument is set to zero absorbance with the appropriate blank in position. The test solution is then introduced and the absorbance read again. In most cases the path length is constant so that provided Beer's law is obeyed, absorbance is linearly related to concentration. On the other hand, if %T is read from the scale and is plotted against concentration, a curve results.

0...10...20...30...40...50...60...70...80...90...100     <-----T

2...1.5........0.4......0.3........0.2.......0.1...........0     <-----A

Scales used in photoelectric colorimeters.

The upper is for %T, the lower for absorbance.

Some recent instruments are provided with digital scales or are even computerized.

If a suitable standard is prepared and the absorbance of this and the unknown solution are read, then provided Beer's law is obeyed, then

Concentration of unknown    =    Absorbance of unknown

Concentration of standard        Absorbance of standard

and therefore,

Concentration of unknown = Absorbance of unknown X Conc. of st.

                                                    Absorbance of standard

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