In physics, spectrophotometry is the quantifiable study of electromagnetic spectra. It is more specific than the general term electromagnetic spectroscopy in that spectrophotometry deals with visible light, near-ultraviolet, and near-infrared. Also, the term does not cover time-resolved spectroscopic techniques.
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 use of spectrophotometers is not limited to studies in physics. They are also commonly used in other scientific fields such as chemistry, biochemistry, and molecular biology. They are widely used in many industries including printing and forensic examination.
There are two major classes of spectrophotometers; single beam and double beam. A double beam spectrophotometer measures the ratio of the light intensity on two different light paths, and a single beam spectrophotometer measures the absolute light intensity. Although ratio measurements are easier, and generally more stable, single beam instruments have advantages; for instance, they can have a larger dynamic range, and they can be more compact.
Historically, spectrophotometers use a monochromator to analyze the spectrum, but there are also spectrophotometers that use arrays of photosensors. Especially for infrared spectrophotometers, there are spectrophotometers that use a Fourier transform technique to acquire the spectral information quicker in a technique called Fourier Transform InfraRed...
The spectrophotometer quantitatively measures the fraction of light that passes through a given solution. In a spectrophotometer, a light from the lamp is guided through a monochromator, which picks light of one particular wavelength out of the continuous spectrum. This light passes through the sample that is being measured. After the sample, the intensity of the remaining light is measured with a photodiode or other light sensor, and the transmittance for this wavelength is then calculated.
In short, the sequence of events in a spectrophotometer is as follows:
- The light source shines through the sample.
- The sample absorbs light.
- The detector detects how much light the sample has absorbed.
- The detector then converts how much light the sample absorbed into a number.
- The numbers are either plotted straight away, or are transmitted to a computer to be further manipulated (e.g. curve smoothing, baseline correction)