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All spectrophotometers rely on the 150-year-old observation that a material, like an invisible gas or any liquid, will absorb some amount of specific wavelengths of light while passing some amount of other wavelengths. One might think that this observation is, at best, an interesting phenomenon, but developing this technology has given the research and clinical laboratories one of the most important and versatile laboratory instruments ever devised. Between the 1950s and the 1970s, spectrophotometers were a fundamental instrument found in every hospital-based clinical laboratory in the country.
Today, they are found primarily in research facilities and reference laboratories, This technology did not disappear from clinical ratories, however, since nearly every hospital laboratory contains automated analyzers employing the same basic principles embodied in these legacy manual instruments, At the heart of many of these modern instruments lies a spectrophotometer, albeit enveloped by some complex automated sample preparation hardware, sample flow plumbing, and, often, computer-based flow and output controls. Likewise, dry chemistry analyzers utilizing light reflectance technology, a cousin of spectrophotometry, utilize these same optical principles.
Before describing the spectrophotometer, some knowledge of its use in performing basic laboratory tests must be attained to understand how this instrument is used and what it does. As the spectrophotometer evolved, aside from merely being able to measure absorbance (the amount of light absorbed by the sample expressed as a percentage) or transmittance (how much light is passed, the mathematic reciprocal of absorbance), two important observations were made about the sample. First, the amount of light absorbed or transmitted at a particular wavelength is proportional to the concentration of a material. That is, the more of a particular material that exists in the sample, the greater the light absorbance. If the material does not absorb light itself, one or more reagents can be mixed into the solution and its absorbance or transmittance will be proportional to the concentration of the material.
Second, through prior testing, using titrated aliquotes of a material, a correlation can be drawn between the absorbance or transmittance and the concentration of the material. What is "the material"? It can be a plethora of things from the amount of iron in the blood, alkaline phosphatase in the serum, or the amount of copper in tissue. The "secret,"...