spectrophotometry

spectrophotometry

[spek-troh-foh-tom-i-ter]

Branch of spectroscopy dealing with measurement of radiant energy transmitted or reflected by a body as a function of wavelength. The measurement is usually compared to that transmitted or reflected by a system that serves as a standard. In chemistry and physics, different types of spectrophotometers cover wide ranges of the electromagnetic spectrum: ultraviolet (UV), visible light, infrared (IR), or microwave. UV spectrophotometry is particularly useful in detecting and quantifying colourless substances in solution. IR spectrophotometry is used mostly to study the molecular structures of complex organic compounds. In astronomy and astrophysics, spectrophotometric studies also include the X-ray and gamma ray ranges of the spectrum. Seealso colorimetry.

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In physics, spectrophotometry is the quantitative 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.

Design

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 measures quantitatively 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:

  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).

UV and IR spectrophotometers

The most common spectrophotometers are used in the UV and visible regions of the spectrum, and some of these instruments also operate into the near-infrared region as well.

Visible region 400-700nm spectrophotometry is used extensively in colorimetry science. Ink manufacturers, printing companies, textiles vendors, and many more, need the data provided through colorimetry. They usually take readings every 20 nanometers along the visible region, and produce a spectral reflectance curve. These curves can be used to test a new batch of colorant to check if it makes a match to specifications.

Traditional visual region spectrophotometers cannot detect if a colorant has fluorescence. This can make it impossible to manage color issues if one or more of the printing inks is fluorescent. Where a colorant contains fluorescence, a bi-spectral fluorescent spectrophotometer is used. There are two major setups for visual spectrum spectrophotometers, d/8 (spherical) and 0/45. The names are due to the geometry of the light source, observer and interior of the measurement chamber. Scientists use this machine to measure the amount of compounds in a sample. If the compound is more concentrated more light will be absorbed by the sample; within small ranges, the Beer-Lambert law holds and the absorbance between samples vary with concentration linearly.

Samples are usually prepared in cuvettes; depending on the region of interest, they may be constructed of glass, plastic, or quartz.

IR spectrophotometry

Spectrophotometers designed for the main infrared region are quite different because of the technical requirements of measurement in that region. One major factor is the type of photosensors that are available for different spectral regions, but infrared measurement is also challenging because virtually everything emits IR light as thermal radiation, especially at wavelengths beyond about 5 μm.

Another complication is that quite a few materials such as glass and plastic absorb infrared light, making it incompatible as an optical medium. Ideal optical materials are salts, which do not absorb strongly. Samples for IR spectrophotometry may be smeared between two discs of potassium bromide or ground with potassium bromide and pressed into a pellet. Where aqueous solutions are to be measured, insoluble silver chloride is used to construct the cell.

Spectroradiometers

Spectroradiometers, which operate almost like the visible region spectrophotometers, are designed to measure the spectral density of illuminants in order to evaluate and categorize lighting for sales by the manufacturer, or for the customers to confirm the lamp they decided to purchase is within their specifications. Components:

  1. The light source shines onto or through the sample.
  2. The sample transmits or reflects light.
  3. The detector detects how much light was reflected from or transmitted through the sample.
  4. The detector then converts how much light the sample transmitted or reflected into a number.

See also

References

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