The Development And Uses Of Spectrophotometry
In the 1920's, G
In the 1920's, G. M. B. Dobson developed the first spectrophotometer used for research on the earth's atmosphere. It was based on the spectra, or color bands, of visible light, as can be seen with a rainbow or prism. This led to the development of other uses, including analysis in biochemistry. Light absorption studies began in earnest, and soon the first spectrophotometers used in a medical laboratory were born.The principle behind spectrophotometry can be explained simply. First a specific wavelength of light is passed through a material to be analyzed and the resulting refraction, reflection, or absorption is read by a photovoltaic sensor. This sensor is electrically attached to a computer system that can recognize a variety of elements or molecules by the way the light is changed by the material. In essence, there are color bands from the visible light that are absorbed by the material, leaving behind other aspects of the visible wavelength.
This is oversimplifying the explanation quite a bit, but the principle is just that: The absorption of light. This absorption is then quantified on a machine and the results are then shown on a screen or printed out. For example, when human blood gasses are analyzed using this principle, Oxygen, Carbon Dioxide, Nitrogen or other essential blood elements have each a different absorption wavelength; they will leave a dark band within each color of the prismatic bands, which leads to a final result.
The spectrophotometer has this ability, and the technological advances that have been made in this field are enormous. The first spectrophotometers were quite large, and had minimal computing power. It would take sometimes hours to get an end result, and there were many interfering substances. Now, however, spectrophotometers can sit on a laboratory bench and take only seconds to calculate the elements within a substance.
The mechanics involved in this process can be simplified for a single-beam spectrophotometer thusly: A light source is provided that has the visible light bands needed for the analysis. This is in turn sent through a monochromator, or prism. This divides the light up into the constituent bands of light, which is then sent through a cuvette, or tubule, containing the sample, which is normally a liquid. The light that results from passing through the specimen is then sent to a photoresistor, or photosensor, that reads the wavelength of light that remains from transmission through the cuvette.
This resulting reading is then amplified and sent to the analytical instruments computer, where the results are compared with a large database of results, thus when a match is made, the analysis is complete. With a single-beam spectrophotometer, one beam of light is sent through, and the beam before and after transmission is compared, giving a cleaner result.
In a double-beam spectrophotometer, two light beams are sent through, one for the specimen, the other for comparison. The light beam used in comparison is sent through a reference material within its own cuvette, making for a more accurate reading.
Spectrophotometers are ever-advancing in technology, and their uses are countless. From the atmosphere of the 1920's, to the biochemistry of human beings, spectrophotometry has a strong place in analytical chemistry.
by: Andrew Long