Biochemical Techniques and Instrumentation

Procedure

1. Dissolve 200 mg of colored compound (eg. Potassium chromate) in 100 ml of distilled water to get a solution with a concentration of 2mg/ml OR Dissolve 2 mg of dye (eg. Methyl red) in 10 ml ethanol first and then make up to 100 ml with distilled water to get a concentration of 20 µg/ml.
2. Take a series of test tubes, number them and pipette out the solution following the given schedule:

   	1	2	3	4	5	6
   Dye/colored solution	0	0.5	1.0	1.5	2.0	2.5
   Water	5.0	4.5	4.0	3.5	3.0	2.5

3. Mix the solution well.
4. Place the highest dilution of the each colored compound and measure the absorption maxima in the Spectrophotometer
5. Take note of the absorbance maxima for each colored solution.
6. Set the wavelength at absorption maxima for the solution at spectrophotometer.
7. Then, measure the absorbance (O.D) for each dilution (Nos. 2 to 6) by keeping tube (No.1) as blank.
8. Draw a standard graph taking the concentration of colored solution (mg/ml or µg/ml) in X-axis and absorbance or OD in the Y-axis to get the standard graph.
9. As the concentration of coloured compound increases the absorbance (O.D) also increases and thus Beers law is verified.