The Complete Guide to Colorimetry

Introduction: What is Colorimetry and How Does it Work?

In clinical practice it is important to know the concentration of certain compounds is biological samples. Colorimetry is the most windly used technique for quantitative analysis of such compounds.

In colorimetric technique the concentration of compounds is determined by measuring the intensity of colour the intensity of colour is propotioned to the concentration of the compound being measured. However many compound of clinical important present in body fluid such as sugar proteins area creatinine etc are not coloured these compounds conform colour complexes by specific chemical reaction and the colour intensity is directly proportional to the concentration of the compound undergoing chemical reaction.

The relationship between concentration and colour intensity give by Beer's low and Lambert's Low.

Beer's low :- 

When monochromatic light passed through a light absorbing medium the intensity of the transmitted light decrease exponentially as the concentration of the light absorbing increase.

Lambert's Low:- 

When  monochromatic light passed through a light absorbing medium the intensity of the light transmitted decrease Exponentially as the length of the light path through the light absorbing material increases.

Thus if the length of the light path is kept constant then the intensity of the transmitted absorbing material increases.

Relationship between Absorbence and transmittenced

When light passed through coloured solution. Some amount of light is absorbed by the solution depending on the concentration of the light is absorbed by the solution depending on the concentration of the light absorbed compound while remaing ligh is transmitted the amount of light absorbed is termed as absorbance ( A ) or optical density (O.D ) where as the amount of light transmitted in termed as transmittance Transmittance ( T ) 

 Transmittance is defined as the ratio of the intensity of light energing ( I ) and the intensity of light incident ( Ic ) I.e.

The transmittance of a solution containing absobing 

Substance depends upon:-

The nature of light absorbing substance 

Water length of light 

Amount of light absorbing substance in the light path 

Which in turn depends concentration of light absorbing substance and depth of the solution through which light passed the lelation us expressed as 

 -log T = O.D = KCL

Where  K. = Constant characteristic of a substance

               C  = Concentration of light absorbing substance

                L  = length of depth of the solution through which light passed .

Conside two solution of the same substance but of different concentration. One acting as standard and the order of unknown concentration acting as unknown the 

Technique :- 

1. Colorimetry techniques in value three step:- 

Preparation of a colour solution by performing appropids chemical reaction if the compound to be estimated is not Colored.

Similar treatment to the standard solution of the same substance ( with unknow Concentration ) to abtain subtable density of standard and unknown solution. Colour comparison by measuring tranmett once of or optical dimeter of standard and unknown solution 

Part of Colorimetry :- 

The basic element of a colorimeter are shown in the diagram shown in appendix.

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