Colorimetry: Principle and Instruments

In this article we will discuss about the principle and instruments of colorimetry.

Principle of Colorimetry:

Colorimetry is a widely used technique applied in biological system. It involves the measurement of a compound or a group of compounds present in a complex mixture. The property of colorimetric analyses is to determine the intensity or concentration of compounds in coloured solution.

This is done by passing light of specific wavelength of visible spectrum through the solution in a photoelectric colorimeter instrument and observe the galvanometric reading of reflection sensitizing the quantity of light absorbed.

Based on the nature of colour compounds, specific light filters are used. Three types of filters are available — blue, green and red — with corresponding light wavelength transmission rays from 470-490 nm, 500-530 nm and 620-680 nm, respectively.

There are two fundamental laws of absorption which are highly important in colorimetric estima­tion. These are Lambert’s law and Beer’s law. Lambert’s law states that when monochromatic light passes through a solution of constant concentration, the absorption by the solution is directly proportional to the length of the solution.

In contrary, Beer’s law states that when monochromatic light passes through a solution of constant length, the absorption by the solution is directly proportional to the concentration of the solution.

Thus both the laws can be expressed as:

Lambert’s law: log₁₀ I₀/I = K₁I

Beer’s law: log₁₀ I₀/I = K₂I

[where I₀ = Intensity of incident light (light entering a solution);

I = Intensity of transmitted light (light leaving a solution);

l = Length of absorbing solution;

c = Concentration of coloured substance in solution;

K₁ and K₂ = Constants.]

Both Beer-Lambert law are combined together for getting the expression transmittance (T).

T = I/I₀

(where I₀ is the intensity of incident radiation and I is the intensity of transmitted radiation).

A 100% value of ‘T’ represent a totally transparent substance, with no radiation being aborted, whereas a zero value of ‘T’ represents a totally opaque substance that, in effect, represents complete absorption. For intermediate value we can define the absorbance (A) or extinction (E) that is given by the logarithm (to base 10) of the reciprocal of the transmittance:

A = E = log₁₀ (I/T) = log₁₀ (I₀/I)

Absorbance used to be called optical density (OD) but continued use of this term should be discour­aged. Also, as absorbance is a logarithm it is, by definition, unit-less and has a range of values from 0 (= 100% T) to cc (= 0% T).

Thus the variation of colour of the reaction mixture (or system) with change of substrate concentra­tion forms the basis of colorimetric analysis.

The formation of colour is due to the reaction between substances and reagents in appropriate proportion. The intensity of colour observed is then compared with that of reaction mixture which contains a known amount of substrate. The optical spectrophotometry is based on identical principles of colorimetry.

Instruments of Colorimetry:

(A) Colorimeter:

The colorimeter instrument is very simple, consisting merely of a light source (lamp), filter, curette and photosensitive detector to collect the transmitted light. Another detector is required to measure the incident light; or a single detector may be used to measure incident and transmitted light, alternately.

The latter design is both cheaper and analytically better, because it eliminates variations between detectors. The filter is used here to obtain an appropriate range of wavelengths within the bands which it is capable of selecting.

(B) Spectrophotometer:

It is a more sophisticated instrument. A photometer is a device for measuring ‘light’, and ‘spectro’ implies the whole range of continuous wavelengths that the light source is capable of producing. The detector in the photometer is generally a photo cell in which a sensitive surface receives photons; and a current is generated that is proportional to the intensity of the light beam, reaching the surface.

In instru­ments for measuring ultraviolet/visible light, two lamps are usually required: one, a tungsten filament lamp which produces wavelengths in the visible region; the second, a hydrogen or deuterium lamp, is suitable for the ultraviolet.

There are two kinds of optical arrangements: a single-beam or a double beam type. Here, first the blank and then the sample must be moved into the beam, adjustments made and readings taken.

The details of optical arrangement in Spectrophotometer is given:

The major advantage of the spectrophotometer, however, is the facility to scan the wavelength range over both ultraviolet and visible light and obtain absorption spectra.

A large number of inorganic and organic compounds were quantitatively estimated by the use of colorimetric or optical spectrophotometric techniques: