Spectrophotometry involves the use of a spectrophotometer. "A spectrophotometer is a photometer (a device for measuring light intensity) that can measure intensity as a function of the color, or more specifically, the wavelength of light". There are many kinds of spectrophotometers. Among the most important distinctions used to classify them are the wavelengths they work with, the measurement techniques they use, how they acquire a spectrum, and the sources of intensity variation they are designed to measure. Other important features of spectrophotometers include the spectral bandwidth and linear range.
Perhaps the most common application of spectrophotometers is the measurement of light absorption, but they can be designed to measure diffuse or specular reflectance. Strictly, even the emission half of a luminescence instrument is a kind of spectrophotometer.
the sequence of events in a spectrophotometer is as follows:
1. The light source shines through the sample.
2. The sample absorbs light.
3. The detector detects how much light the sample has absorbed.
4. The detector then converts how much light the sample absorbed into a number.
5. The numbers are either plotted straight away, or are transmitted to a computer to be further manipulated (e.g. curve smoothing, baseline correction)
Many spectrophotometers must be calibrated by a procedure known as "zeroing." The absorbency of some standard substance is set as a baseline value, so the absorbencies of all other substances are recorded relative to the initial "zeroed" substance. The spectrophotometer then displays % absorbency (the amount of light absorbed relative to the initial substance
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