Colorimetry is the techniques that is frequently used in biochemical investigations. This involves the quantitative estimation of colors. This means that if you want to measure the quantity of a substance in a mixture, you could use the technique of colorimetry, by allowing the substance to bind with color forming chromogens. The difference in color results in the difference in the absorption of light, which is made use of here in this technique called colorimetry.
The instrument use for colorimetry is colorimeter. This appartus will comprise of the following parts:
- light source
- filter (the device that selects the desired wavelenght)
- cuvette chamber (the transmitted light passes through compartment wherein the solution containing the colored solution are kept in cuvette, made of glass or disposable plastic)
- detector (this is a photosensitive element that converts light into electrical signals)
- Galvanometer (measures electrical signal quantitatively)
The spectrophotometer also works on a similar principle.
- Beer’s Law
According to Beer’s law when monochromatic light passes through the colored solution, the amount of light transmitted decreases exponentially with increase in concentration of the colored substance.
It = Ioe-KC
- Lambert’s Law
According to Lambert’s law the amount of light transmitted decreases exponentially with increase in thickness of the colored solution.
It = Ioe-kt
Therefore, together Beer-Lambert’s law is:
IE/Io = e-KCT
IE = intensity of emerging light
Io = intensity of incident light
e = base of neutral logarithm
K = a constant
C = concentration
T = thickness of the solution
Steps for operating the photoelectric colorimeter:
- Choose the glass filter recommended (see table below) in the procedure and insert in the filter.
- Fill two of the cuvette with blank solution to about three-fourth and place it in the cuvette slot.
- Switch on the instrument and allow it to warm up for 4 – 5 minutes.
- Adjust to zero optical density.
- Take the test solution i another cuvette and read the optical density.
- Take the standard solution in varying concentration and note down the optical density as S1, S2, S3, S4, S5 and so on.
- A graph is plotted taking concentration of standard solution versus the optical density.
- From the graph the concentration of the test solution or the unknown solution can be calculated.
Table for choosing the wavelength of absorption: