Photoelectric Colorimeter , Principle, Uses.

Colorimetry, in simple terms, is the measurement of colors and is probably the most widely used method for determining the concentration of biochemical compounds. This important laboratory procedure is based on the principle that when white light passes through a colored solution, some wavelengths are absorbed more than others. 

Colorimeter (Photoelectric meter)

• Many compounds, though not coloured themselves, can be made to absorb light in the visible spectrum by reaction with suitable reagents. The coloured compounds absorb light at given wavelength at visible spectrum. The extent to which a solution absorbs light depends on the intensity of its colour.

• Light travels in the form of waves. Wavelength is the distance between two wave peaks in nanometers (nm). Amplitude is the height of each wave. The number of waves passing a fixed pointx per unit time is the frequency (Fig. 1.1). The wavelength of light is expressed as:

velocity of light

• The wavelength of light determines the color of the light seen by the naked eye. White light is a combination or a mixture of light energy of different wavelengths. Any color that is seen emits light of a particular wavelength, and is called monochromatic light.

• Wavelengths between 400 nm and 700 nm form the visible spectrum of light which is the visible band of light of electromagnetic spectrum. Table 1.1 shows the color spectrum of white light.

• Wavelengths of about 700 nm are visible to the eye as red colors while those of shorter wavelength give rise in turn to colors orange, yellow, green, blue and finally, violet, which has a wavelength of about 400 nm. 

• Wavelengths greater than 700 nm have vibrations known as infrared and are not visible to the eye; wavelengths shorter than 400 nm have vibrations known as ultraviolet and are also not visible.

Relationship between the wavelength and color

Absorption of Light

Light falling on a coloured solution is either absorbed, reflected or transmitted. A coloured solution absorbs all the coloured components of white light and selectively transmits only one colour. This is its own transmitted colour. 

working principle of Colorimeter (Photoelectric meter)

For example, if a solution absorbs light between 400-480 nm (blue), it will appear yellow to the eye. The colour yellow is therefore, the complementary colour of blue. This phenomenon can be summarised as

Absorption of Light

absorption and transmission of light through colored solution

The above equation obeys the Beer's law and Lambert's law.

Beer's law

• The proportion of the incident light absorbed by the molecules in a solution is directly proportional to the number of absorbing molecules in the light path. The ability of a substance to selectively absorb certain wavelengths of light while transmitting others is determined by its molecular and atomic structure. 

• Thus, it follows that the concentration of a substance, i.e., absorbing molecules,is directly proportional to the intensity of the colour of the solution. In Beer's law, the light path is constant while the concentration varies. As the concentration of the coloured substance in the solution increases, absorbance increases and the amount of light passing through (transmittance) decreases. 

Lambert's law

When monochromatic light passes through a coloured solution, the amount of light absorbed increases with the increase in the thickness of the layer of the solution through which the light passes. Thus the concentration of the solution is constant while the light path varies. 

Beer-Lambert law

• The basic principles of colorimetry, absorptiometry and spectrophotometry are established, by combining Beer's law and Lambert's law

• This joint law shows that under suitable conditions, the amount of light absorbed by a coloured solution, when illuminated with a light of suitable wavelength, is directly proportional to the concentration of the coloured solution and the length of the light path through the solution. 

• Therefore the amount of light decreases exponentially with the increase in the concentration of the solution and with increase in thickness of the layer of solution through which light passes.
• At this stage, we should define some of the terms used in colorimetry.

Light path

The intensity of light transmitted through a solution decreases as the distance of the path through the solution increases. The distance travelled by the light (light path) can be kept constant by using tubes or cuvettes identical in size and optical density. 

Transmittance (T) 

This is the ratio of the intensity of the transmitted light over the intensity of the incident light. This is usually multiplied by 100 to give percentage transmission.

Percentage transmission

where I, is the intensity of transmitted light and I is the intensity of the incident light. 

Absorbance

Absorbance is related to transmittance in the following way: A = 2-log %T

Absorbance increases linearly with concentration. This linear relationship between absorption and concentration is the basis of Beer-Lambert law. It is expressed as A = abc 

where 

• A is absorbance,a is absorption coefficienta constant)
• b is the length of the lightpath (centimetres)
• c is the concentration of the solution(mol/L)

When Beer's law is obeyed and solution concentrations are plotted against absorbance, a straight line graph is obtained ). If percentage transmission is plotted against solution concentration a curve is obtained 

Relationship between the wavelength and color

percentage transmission and concentration

 Application of Beer-Lambert Law

 The principle of Beer-Lambert law is applied in Clinical Chemistry quantitative tests. It shows that a linear relationship exists between the concentration of the solution being measured and its absorbance. Many instruments and methods are first of all calibrated before a test on clinical samples is carried out.

For manual methods, absorbance is normally used since if Beer's law is obeyed, a plot of concentration against absorbance is a straight line and therefore much easier to handle than theexponential curve given by plotting %T against concentration.

It should be remembered that a is usually constant since the same conditions apply for both the test and standard solutions. As the test and standand solutions are compared in the same or identical cuvettes or tubes, the same lightpath or thickness of solution is employed and so b is also a constant value. 

Thus in the equation A = abc,

This formula is the basis of calculation in photoelectric absorptiometry and spectrophotometry when Beer's law is obeyed. Absorbance scales or the special scales for absorptiometers and spectrophotometers are used for faster calculations.

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