The colorimeter is a device used in physiology and clinical laboratories to measure the concentration of a substance in solution. this is accomplished by the application of Beer's Law which states that the concentration of a substance in solution is directly proportional to the amount of light absorbed (absorbance, A) by the solution and inversely proportional to the logarithm of the amount of light transmitted (percent transmittance, %T) by the solution.

These relationships can be expressed graphically.

Beer's Law will be followed only if the incident light (the light entering the solution) is monochromatic, that is, light composed of photons of a single wavelength. By means of a prism or diffraction grating, the colorimeter can separate white light into its component wavelengths. You select incident light of any wavelength by simply turning the appropriate dial to that wavelength. This light enters a special tube, the cuvette, which contains the test solution. A given fraction of the incident light is absorbed by the solution (the amount absorbed depends on the concentration of the solution), and the remainder of the light, the transmitted light, passes through the cuvette. The transmitted light generates an electric current by means of a photoelectric cell, and the amount of this current is registered on a galvanometer scale.

A needle on the meter indicates the percent transmittance (%T). Since the amount of light that goes into the solution and the amount of light that leaves the solution are known, a ratio of the two indicates the light absorbance (A) of the solution. An absorbance scale is provided in the colorimeter adjacent to the percent transmittance scale.

Standardizing the Colorimeter

The following procedure is intended specifically for the Spectronic 20 colorimeter. The general procedure is similar for all colorimeters, the specific details may vary among the different models.

1. Turn on the colorimeter by rotating knob b to the right.
2. Set the monochromator dial, d, so that the correct wavelength in nanometers is lined up with the indicator in the window adjacent to this dial. (Start here with 500 nm.)
3. When there is no cuvette in the cuvette holder, a, the light source is blocked. The pointer should thus read zero transmittance or infinite absorbance at the left end of the scale. Turn know b until the pointer is aligned with the left end of the scale.
4. Place in the cuvette holder the cuvette that contains all the reagents present in the other tubes, except the test substance (such as the glucose or starch solutions). this tube is called the blank because it has a concentration of test substance equal to zero. It should therefore have an absorbance of zero (or a transmittance of 100 %). this is at the right end of the scale. Set the pointer to the right end of the scale using knob c.
5. Repeat steps 3 and 4 to confirm settings.
6. One at a time, place the other cuvettes that contain the standard solutions and the unknown, in the cuvette chamber. Close the hatch, read, and record the absorbance value of each solution.
7. Fill four cuvettes with the colored solutions used for the pipette exercise. Place them in the colorometer one at a time. For each solution determine the frequencies of maximum and minimum absorbance. You may do this by rotating the freqency dial. Rotate the dial to determine at which frequency the movement of the needle is minimum. This is the maximum absorbance. Rotate the frequency dial to determine at which frequency the reading is miximal. If the needle goes above 100% transmission, adjust the right hand know to lower the reading onto the scale. Remember to zero the blank when done before proceeding to the next tube.

Note: Before placing each cuvette in the chamber, wipe it with a lint-free, soft paper towel (chemwipe). If the cuvette has a white indicator line, place the cuvette so that this line is even with the line in the front of the cuvette holder.