Principles of Spectrophotometry

A spectrophotometer consists of two instruments, namely a spectrometer for producing light of any selected color (wavelength), and a photometer for measuring the intensity of light. The instruments are arranged so that liquid in a cuvette can be placed between the spectrometer beam and the photometer. The amount of light passing through the tube is measured by the photometer. The photometer delivers a voltage signal to a display device, normally a galvanometer. The signal changes as the amount of light absorbed by the liquid changes.

If development of color is linked to the concentration of a substance in solution then that concentration can be measured by determining the extent of absorption of light at the appropriate wavelength. For example hemoglobin appears red because the hemoglobin absorbs blue and green light rays much more effectively than red. The degree of absorbance of blue or green light is proportional to the concentration of hemoglobin.

When monochromatic light (light of a specific wavelength) passes through a solution there is usually a quantitative relationship (Beer's law) between the solute concentration and the intensity of the transmitted light, that is,

where I sub 0 is the intensity of transmitted light using the pure solvent, I is the intensity of the transmitted light when the colored compound is added, c is concentration of the colored compound, l is the distance the light passes through the solution, and k is a constant. If the light path l is a constant, as is the case with a spectrophotometer, Beer's law may be written,

where k is a new constant and T is the transmittance of the solution. There is a logarithmic relationship between transmittance and the concentration of the colored compound. Thus,

The O.D. is directly proportional to the concentration of the colored compound. Most spectrophotometers have a scale that reads both in O.D. (absorbance) units, which is a logarithmic scale, and in % transmittance, which is an arithmetic scale. As suggested by the above relationships, the absorbance scale is the most useful for colorimetric assays.

Using a Spectronic 20 spectrophotometer

The Spectronic 20 spectrometer is widely used in teaching laboratories. The specific instructions will differ with other models, but the principles remain.

1. The instrument must have been warm for at least 15 min. prior to use. The power switch doubles as the zeroing control.
2. Use the wavelength knob to set the desired wavelength. Extreme wavelengths, in the ultraviolet or infrared ranges, require special filters, light sources, and/or sample holders (cuvettes).
3. With the sample cover closed, use the zero control to adjust the meter needle to "0" on the % transmittance scale (with no sample in the instrument the light path is blocked, so the photometer reads no light at all).
4. Wipe the tube containing the reference solution with a lab wipe and place it into the sample holder. Close the cover and use the light control knob to set the meter needle to "0" on the absorbance scale.
5. Remove the reference tube, wipe off the first sample or standard tube, insert it and close the cover. Read and record the absorbance, not the transmittance.
6. Remove the sample tube, readjust the zero, and recalibrate if necessary before checking the next sample.

Why use a reference solution? Can't you just use a water blank? A proper reference solution contains color reagent plus sample buffer. The difference between the reference and a sample is that the concentration of the assayable substance in the reference solution is zero. The reference tube transmits as much light as is possible with the assay solution you are using. A sample tube with any concentration of the assayable substance absorbs more light than the reference, transmitting less light to the photometer. In order to obtain the best readability and accuracy, the scale is set to read zero absorbance (100% transmission) with the reference in place. Now you can use the full scale of the spectrophotometer. If you use a water blank as a reference, you might find that the assay solution alone absorbs so much light relative to distilled water that the usable scale is compressed, and the accuracy is very poor.