The Use Of Infrared Spectroscopy Biology Essay


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When exposed to infrared radiation, organic molecules may absorb infrared radiation and convert it into molecular vibrations. The molecular vibrations and rotations within a molecule must result in a change in the dipole moment of the molecule for a molecule to be IR active. The electromagnetic radiation consists of an oscillating electrical field and an oscillating magnetic field, perpendicular to each other. When IR radiation impinges on a molecule, the radiation will only be absorbed if the frequency of the radiation matches the vibrational frequency of the molecule. The absorption of the IR radiation results in an increase of the vibration amplitude for the molecule. If the wavelengths of light that are absorbed by a molecule can be determined and those wavelengths can be associated with a specific mode of motion, it is then possible to determine what modes of vibration and rotation a molecule is exhibiting.  This information then makes it possible to determine what types of bonds are present in the molecule. (Chamberlain 1979)

The IR spectrum of a sample is recorded by passing an infrared beam through the sample. Detection and analysis of the transmitted radiation can then determine the magnitude of the energy that was absorbed at each discrete wavelength. There are typically two ways to collect this data. In older spectrometer designs, the sample is exposed to a monochromatic beam of IR radiation whose wavelength can be shifted to obtain a transmission spectrum. Newer instruments use a Fourier transform to collect data at all wavelengths, emitted from a source, at once. Through the analysis of the radiation transmitted through the sample, an absorption or transmittance plot can be constructed. From these plots, the wavelengths at which the molecule absorbed radiation, and therefore information about the chemical bonds that the molecule contains, can be determined(Pavia, Lampman et al. 1996)

Transmittance or absorbance plots are constructed by graphing the wavenumber, the reciprocal of wavelength, on the X-axis in the order of increasing energy and either %transmittance or absorbance on the Y-axis. In a transmittance plot, an absorption is indicated by a trough or downward pointing peak whereas in an absorption plot the peaks are positive.

Michelson Interferometer

Most modern IR instruments use Fourier-transform techniques with a Michelson interferometer. Different from the dispersive infrared spectrometer based on the refraction and diffraction of light, FTIR is an interferometric infrared spectrometer design based on the principle of optical coherence. FTIR is known as a third-generation infrared spectrometer with the prism and grating infrared spectrometers being the first and second generation instruments. However, an interferometer can only get the interference pattern of the source, but not the spectrum that people are familiar with. To this end, the interferogram as a function of optical path difference is converted to spectra as a function of wavelength by Fourier transform (FT). And hence the interference infrared spectrometer is known as a FTIR spectrometer. (Stuart 2004)

The heart of most modern FTIR spectrometers is the two-beam Michelson interferometer, which consists of a beamsplitter, a fixed mirror, and a moving mirror, as illustrated in Figure 2. (Griffiths and deHaseth 1986; Jiang 2003) The moving mirror oscillates back and forth, whereas the fixed mirror is stationary. The beamsplitter is a laminate material that transmits 50% of the light to the moving mirror and reflects 50% to the fixed mirror. The two IR beams are then reflected back to the beamsplitter by the mirrors. The transmitted beam from the fixed mirror and reflected beam from the moving mirror are simultaneously recorded by the detector. The two combined beams interfere constructively or destructively depending on the wavelength of the light and the optical path difference introduced by the moving mirror. On reflection from these mirrors, the beam splitter recombines the light which is then guided on towards the sample. After the IR radiation passes through the sample, it enters a detector and is converted into electrical signals. The information collected from the detector allows the path length difference between the two interferometer beams to be determined. The path length difference is known as the retardation of the interferometer. By changing the retardation of the interferometer and collecting the detector signal at these different levels of retardation, an interferogram can be constructed. This background interferogram is then used to determine what changes are made to the detector signal due to the presence of a sample.

Sample Preparation

The sample preparation and handling is important in IR spectroscopy. If the sample is prepared inappropriately, even if the instrument is in good condition, an acceptable IR spectrum cannot be obtained, and even may lead to wrong conclusions. In general, the following points should be given attention to during sample preparation.

Infrared spectroscopy (IR). Figure 2

Schematic diagram of a classic Michelson interferometer

Firstly, the concentration or thickness of the sample should be carefully selected. If the sample concentration or thickness is too small, weak absorption peak will result and the more subtle peaks in the spectrum may not be displayed. If the sample concentration or thickness is too large, the strong absorption peaks will be beyond the ruler scale and may not determine the peaks' true location. Secondly, samples should not contain free water. Thirdly, the sample should be a pure substance with a single component.

There are a number of ways in which solid samples can be prepared. One commonly used method involves the crushing of the sample into an oily mulling agent (usually Nujol). A thin film of this mixture is then smeared onto salt plates and measured. One method is to grind a quantity of the sample with transfer agents like KBr or KCl, the common feature of which is they are completely transparent and hence do not absorb in mid-IR region. This powder mixture is then pressed into a translucent pellet in a mechanical press. "Drop cast film" technique is mainly used for polymeric materials. The third method is to cut a thin (20~100 µm) film from a solid sample using a microtome. Liquid samples can be sandwiched between two plates of a salt forming a liquid film (film method). Gaseous samples require a gas absorption cell. The cell must first be evacuated the air and then the sample is introduced into the cell.

Key Applications

IR spectral analysis is used to determine the attribution of absorption bands and to confirm moieties or bonds contained in molecules based on the location, intensity and shape of absorption bands, and the relationship between moiety vibration frequency and molecular structure. And further presume molecular structure according to characteristic vibrational frequencies of the displacement and changes of the intensity and shape of absorption bands.

The vibrational frequency of the same type of chemical bond is always present in a certain range. For example, the CH3 moiety in CH3CH2Cl has specific absorption bands, and many compounds containing CH3 moieties have absorption bands in the vicinity of the frequency range of 3000~2800 cm-1. The frequency of the absorption peak can be taken as the characteristic frequency of CH3 moieties. The vibration frequency associated with a structural unit is called the moiety frequency. The location and intensity of the absorption bands usually reflect the environment of constituents in a material, which can reflect the structural features. For example, the stretching vibration frequency range of C=O is 1850 ~ 1600 cm-1. When it is connected with atoms C, O or N, the C = O band appears at 1715 cm-1, 1735 cm-1 or 1680 cm-1, according to which, ketones, esters and amides can be distinguished.

Determination of Moiety or Bond

Each molecule has its characteristic IR spectrum, and each absorption band is on behalf of each of the vibrations a bond or a moiety can perform in a molecule. The type of moiety or bond can be determined by the location, intensity, and shape of the characteristic absorption bands. For example, methyl groups have four characteristic absorption bands near 2960 cm-1, 2870 cm-1, 1450 cm-1, 1380 cm-1, which are attributed to the asymmetric and symmetric stretching vibration and the deformation vibration absorption of C-H. The four characteristic absorption bands can be used as the "fingerprint" of a methyl group to confirm the presence or absence of methyl groups in a sample. The compounds containing the methyl groups can be further deduced due to the position of the characteristic absorption bands which shift according to the molecular structure and measurement environment. The characteristic frequency of moieties or bonds of organic compounds has been measured and pooled into the characteristic frequency of the table. So now the type of organic moiety or bond can be checked by means of the "dictionary" method.

In the actual analysis, spectra are usually analyzed starting from the moiety area (4000~1350 cm-1) in the order of strong peaks to weak peaks, combined with the fingerprint area (1350 ~ 850 cm-1) absorption to make sure. The fingerprint area reflects the characteristics of the molecular structure, and is especially sensitive to the molecular skeleton vibration absorption. The hydroxyl moiety in alcohol, for example, may be confirmed by the stretching vibration absorption near 3400cm-1. However, what can not be definitely determined is whether the alcohol is a primary, secondary, or tertiary alcohol. This can be confirmed however, by the position of the absorption bands 1040~1160 cm-1 in the fingerprint area: primary alcohols absorb at 1050 cm-1, secondary alcohols absorb at 1100 cm-1, and tertiary alcohols absorb at 1150cm-1.

Presumption of Molecular Structure

Comparing the IR spectra of unknowns with known compounds requires that the IR spectra of the sample and that of standard materials be recorded under the same conditions. It is necessary to use an instrument with similar performance and the same format of spectra representation, and the same sample preparation methods.

The negation and affirmation method: Based on the relationship between the absorption bands and molecular structure, can be used to confirm or deny the existence of certain moieties.

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