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Spectroscopy techniques date back to Isaac Newton’s first studies of light and today provide researchers with a better understanding of what happens at the atomic and molecular level when matter interacts with light. Advances in electro-optics, high-speed array detectors, inexpensive optical fibers and powerful computers have spurred the growth of miniature spectroscopy. This miniaturization has increasingly made optical spectroscopy the sensing technique of choice for many real-world applications.

Fundamental Physics of Spectroscopy

Figure 1. UV/VIS Absorbance Spectra for Heme Proteins.
A critical component of any spectroscopic measurement is the breaking up of light into a spectrum showing the interaction of light with the sample at each wavelength. Two of the most common interactions of light and matter are light that is absorbed by the atoms and molecules in the sample and light that is emitted after interacting with the atoms and molecules in the sample.

Absorbance

Absorption spectroscopy studies light absorbed by molecules. For absorbance measurements, white light is passed through a sample and then through a device (such as a prism) that breaks the light up into its component parts, a.k.a. a spectrum. When white light is passed through a sample, under the right conditions, the electrons of the sample absorb some wavelengths of light. This light is absorbed by the electrons so the light coming out of the sample will be missing those wavelengths corresponding to the energy levels of the electrons in the sample. The result is a spectrum with black lines at the wavelengths where the absorbed light would have been if it had not been removed by the sample.

Emission

Emission spectroscopy is the opposite of absorption spectroscopy. The electrons of the sample are promoted to very high energy levels by any one of a variety of methods (e.g., electric discharge, heat, laser light, etc.). As these electrons return to lower levels they emit light. By collecting this light and passing it through a prism, it is separated into a spectrum. In this case, the spectrum is a dark field with colored lines that correspond to the electron transitions resulting in light emission.

Using Spectroscopy

While spectroscopy is conducted in nearly all regions of the electromagnetic spectrum, practical considerations make the infrared, visible and ultraviolet regions the most useful in chemical laboratories. Infrared spectroscopy is particularly useful for studying the vibration of bonds between carbon, hydrogen, oxygen and nitrogen atoms that predominate in organic compounds. Infrared spectra can indicate the presence of particular functional groups in unknown organic compounds by the presence of characteristic features. They can also be used to confirm the identity of compounds by comparison with reference libraries of known spectra.

Visible light spectroscopy is useful for studying some of the organic compounds and elements that have electrons in d-orbitals, such as transition metals. Ultraviolet spectroscopy is useful for studying some organic compounds and most biological samples. All proteins have useful ultraviolet spectra as do nucleic acids. Furthermore, UV spectroscopy can be used to follow biochemical reactions and this tool is commonly found in biochemical laboratories. In clinical laboratories, ultraviolet spectroscopy is often the means for making quantitative determinations on plasma and urine samples.

In these applications, both absorbance and emission spectroscopy techniques can be used either qualitatively or quantitatively.

Qualitative Spectroscopy

One of the most useful aspects of spectroscopy derives from the fact that the spectrum of a chemical species is unique to that species. Identical atoms and molecules will always have the same spectra. Different species will have different spectra. For this reason, the spectrum of a species can be thought of as a fingerprint for that species. Qualitative spectroscopy is used to identify chemical species by measuring a spectrum and comparing it with spectra for known chemical species to find a match.

Quantitative Spectroscopy

Quantitative spectroscopy is one of the quickest and easiest ways to determine how many atoms or molecules are present in a sample. This is because the interaction of light with matter is a stoichiometric interaction. At any given temperature, the same number of photons will always be absorbed or emitted by the same number of atoms or molecules in a given period of time. This makes spectroscopy one of the few techniques that can provide a direct measure of the number of atoms or molecules present in a sample.

Quantitative emission spectroscopy requires heating samples to very high temperatures to enable electrons to emit light. Most often, this is done by feeding the sample into a burner flame. As a result, it is not practical for use with most molecular compounds but is frequently employed for elemental analysis.

Using Absorption Spectroscopy

Absorption spectroscopy is performed by passing light through a sample and measuring how much of the light at each wavelength is absorbed. Complex interactions of atoms and molecules in solution make the absorbance of light in solutions a very complex phenomenon. Nevertheless, the observed spectra are repeatable and predictable, thus making them useful. By making absorbance measurements at various wavelengths and then plotting the result, one can create what is known as an absorbance spectrum.

In Figure 1 the absorbance spectra for different heme containing proteins is shown. Even though the proteins are closely related, the absorbance spectra are distinct enough to enable discrimination of the different proteins.

Absorbance spectra are like fingerprints. Each compound has its own unique spectrum. In some cases the spectrum can be used to identify the presence of certain compounds in a sample. More often, it is used to determine the amount of a compound present.

Due to the nature of the electronic changes that give rise to absorption spectra, the peaks are generally broad. Therefore, absorption spectroscopy is less useful than other molecular techniques for the purpose of identifying compounds. For example, absorption spectroscopy in the infrared, or Raman spectroscopy, is much better for identifying the component species compared to UV-Visible absorption spectroscopy.

The type of data required–qualitative or quantitative–will dictate the type of spectroscopic instrument needed for analysis.

Spectroscopy Instrumentation

Figure 2. Typical cross Czerny-Turner monochromator.
A range of instruments are available for spectroscopy measurements. The optimal spectrometer depends upon the application; the choice of optical bench and components can greatly affect the measurement results.

Spectroscopy necessarily involves a series of tradeoffs among different optical parameters. In order to understand these tradeoffs, an understanding of the components of the optical bench and the role of each in segregating and measuring the spectrum is needed. A typical cross Czerny-Turner monochromator (a combination of parts typically known as an “optical bench,” which forms the core of any spectrometer) is shown in Figure 2. The optical bench comprises an entrance aperture, or slit (1); a collimating mirror to make parallel the light entering the bench (2); the grating, which disperses the light (3); a focusing mirror to direct the light onto the detector (4); and the detector (5).

More specifically, the wavelength range and the resolution are influenced by (a) the size of the entrance slit, (b) the groove density of the grating and (c) the number of active elements (pixels) in the array detector.

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