Measurement of absorbance of colored sample using Colorimeter - Biochemistry Lab (BIT12060), Btech Biotechnology - Adamas University
ADAMAS UNIVERSITY,
B.Tech BIOTECHNOLOGY, Semester III
Experiment Number - 02
Measurement of absorbance of colored sample using Colorimeter
Objectives:
• To learn handling the colorimeter
• To learn how to measure the absorbance of colored sample
INTRODUCTION:
A colorimeter is a light-sensitive device used for measuring the transmittance and absorbance of light passing through a liquid sample. The device measures the intensity or concentration of the color that develops upon introducing a specific reagent into a solution.
PRINCIPLE:
The colorimeter is based on Beer-Lambert's law, according to which the absorption of light transmitted through the medium is directly proportional to the medium concentration. In a colorimeter, a beam of light with a specific wavelength is passed through a solution via a series of lenses, which navigate the colored light to the measuring device. This analyzes the color compared to an existing standard. A microprocessor then calculates the absorbance or percent transmittance. If the concentration of the solution is greater, more light will be absorbed, which can be identified by measuring the difference between the amount of light at its origin and that after passing the solution. To determine the concentration of an unknown sample, several sample solutions of a known concentration are first prepared and tested. The concentrations are then plotted on a graph against absorbance, thereby generating a calibration curve. The results of the unknown sample are compared to that of the known sample on the curve to measure the concentration.
MATERIALS REQUIRED:
Colorimeter, Copper Sulphate solution, micropipette, Tissue paper, Washer, Beakers, Cuvettes
PROCEDURE:
4 ml of water is transferred to a cuvette using 1 ml micropipette. The outer surface of the cuvette is wiped properly using tissue paper and the cuvette is inserted into the colorimeter. Using the absorbance dial, it is set to 0.0 mark and will be used as a reference. The wavelength of the light is set to 700 nm as we are using copper sulphate. Now, the cuvette is carefully removed and the containing water is discharged. Next, using micropipette we are transferring 4 ml of CuSO4 into the dried cuvette and the outer surface is wiped using tissue paper. Then the cuvette is inserted into the colorimeter and the results are noted.
Reference Link: The working principle of Colorimeters by Ben Arnold
OBSERVATION:
It is observed that the absorbance value of the copper sulphate solution is between the range 0.16 to 0.18.
INTERPRETATION:
From this experiment, we learnt the working principle of colorimeter and the absorbance value of copper sulphate solution viz., 0.16-0.18.
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