Another descriptor associated with absorption spectroscopy is the wavelength range of the radiation being used in the incident beam. Thus you will find references to infrared spectroscopy, near infrared spectroscopy, microwave spectroscopy; all of which are examples of absorption spectroscopy. On the other hand you will also find references to other wavelength ranges, such as x-ray spectroscopy, that usually denote an emission spectroscopy. This article deals primarily with UV-visible spectroscopy.
UV-visible spectroscopy refers to techniques where one measures how much light of a particular wavelength (color) is absorbed by a sample. Since color can often be correlated with the presence and or structure of a particular chemical, and since absorbance is often an easy and cheap measurement to make, absorbance spectroscopy is widely used for both qualitative (is a chemical present?) and quantitative (how much?) and structural (is it degraded?) work in a wide range of fields. For instance, DNA absorbs light in the UV range (which is partly why sunlight is dangerous) so the amount of DNA in a sample can be determined by measuring the absorbance of UV light.
The relation between the visible color and the absorbance color is complicated; a sample that appears red does not absorb in the red, but absorbs at OTHER wavelengths (colors) so that the light which passes through the sample is enriched in red.
The word "color" is placed in quotes to indicate that absorbance spectroscopy deals not only with light in the visible range - photons with a wavelength of roughly 400 to 700 nanometers, but also with wavelengths that lie outside of the range of human vision (IR, UV, X-rays). However, the principles are quite similar for both visible and nonvisible light.
More technically, absorption spectroscopy is based on the absorption of photons by one or more substances present in a sample, which can be a solid, liquid, or gas, and subsequent promotion of electron(s) from one energy level to another in that substance. Note that the sample can be a pure, homogeneous substance or a complex mixture. The wavelength at which the incident photon is absorbed is determined by the difference in the available energy levels of the different substances present in the sample; it is the selectivity of absorbance spectroscopy - the ability to generate photon (light) sources that are absorbed by only some of the components in a sample - that gives absorbance spectroscopy much of its utility. Typically, X-rays are used to reveal chemical composition, and near ultraviolet to near infrared wavelengths are used to distinguish the configurations of various isomers in detail. In absorption spectroscopy the absorbed photons are not re-emitted (as in fluorescence) rather, the energy that is transferred to the chemical compound upon absorbance of a photon is lost by non-radiative means, such as transfer of energy as heat to other molecules.
While the relative intensity of the absorption lines do not vary with concentration, at any given wavelength the measured absorbance () has been shown to be proportional to the molar concentration of the absorbing species and the thickness of the sample the light passes through. This is known as the Beer-Lambert law. The plot of amount of radiation absorbed versus wavelength for a particular compound is referred to as the absorption spectrum. The normalized absorption spectrum is characteristic for a particular compound, does not change with varying concentration and is like the chemical "fingerprint" of the compound. At wavelengths corresponding to the resonant energy levels of the sample, some of the incident photons are absorbed, resulting in a drop in the measured transmission intensity and a corresponding dip in the spectrum. The absorption spectrum can be measured using a spectrometer and by knowing the shape of the spectrum ,the optical path length and the amount of radiation absorbed, one can determine the structure and concentration of the compound.
Visible light absorption spectra can be taken in anything that is visibly clear. Polystyrene, quartz, and borosilicate (Pyrex) cells, often called cuvettes, are the most commonly used. UV light is absorbed by most glasses and plastics, so quartz cells are used. The Si-O moieties in glasses and quartz, and the C-C moieties in plastics absorb infrared light. Therefore, infrared absorption spectra are typically carried out with a thin film of the sample held in place between sodium chloride sample plates. Other methods involve suspending the compound in a substance does not absorb in the region of study. Mineral oil (Nujol) emulsions and potassium bromide glasses are perhaps the most common. NaCl and KBr, being ionic, do not have significant IR absorptions, and Nujol has a relatively uncomplicated IR spectrum.
A simple example: a cyanide standard at 200 parts per million gives an absorbance with an arbitrary value of 1540. An unknown sample gives a value of 834. The math could be stated as: "if 200 gives you 1540, what gives you 834?" Since this is a linear relation and goes through the origin, the unknown is easily calculated to be 108 parts per million. Note the beauty of the ratio method in that it is not necessary to know the values of the governing coefficients, or chromophores, or the experimental cell length - it all divides out.
In practice, use of a calibration curve rather than a single point of comparison reduces uncertainty in the final measurement by excluding random interference (noise) in the preparation of the standards.
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