Absorption spectra Ultraviolet/visible spectroscopy

A solvent for ultraviolet/visible spectroscopy must be transparent in the region of the spectrum where the solute absorbs and should dissolve a sufficient quantity of the sample to give a well-defined analyte spectrum. In addition, we must consider possible interactions of the solvent with the absorbing species. For example, polar solvents, such as water, alcohols, esters, and ketones, tend to obliterate vibration spectra and should thus be avoided to preserve spectral detail. Nonpolar solvents, such as cyclohexane, often provide spectra that more closely approach that of a gas (compare, for example, the three spectra in Figure 24-14). In addition, the polarity of the solvent often influences the position of absorption maxima. For qualitative analysis, it is therefore important to compare analyte spectra with spectra of known compounds measured in the same solvent. 

The preceding empirical measures have taken chemical reactions as model processes. Now we consider a different class of model process, namely, a transition from one energy level to another within a molecule. The various forms of spectroscopy allow us to observe these transitions thus, electronic transitions give rise to ultraviolet—visible absorption spectra and fluorescence spectra. Because of solute-solvent interactions, the electronic energy levels of a solute are influenced by the solvent in which it is dissolved therefore, the absorption and fluorescence spectra contain information about the solute-solvent interactions. A change in electronic absorption spectrum caused by a change in the solvent is called solvatochromism. 

Molecular absorption spectroscopy deals with measurement of the ultraviolet-visible spectrum of electromagnetic radiation transmitted or reflected by a sample as a function of the wavelength. Ordinarily, the intensity of the energy transmitted is compared to that transmitted by some other system that serves as a standard. 

Most absorption spectroscopy is done in the ultraviolet, visible, and infrared regions of the electromagnetic spectrum. 

Spectroscopy and photochemistry of CF3NO in the visible region have been extensively studied by Roellig and Houston (113,114), Bower et al. (115), Jones et al. (116), and Spears and Hoffland (117). Trifluoronitrosomethane has a weak structured absorption spectrum in the visible near 700 nm. Absorption of light in this region induced fluorescence as well as dissociation, which is similar to the H2CO photochemistry in the ultraviolet described earlier. 

The FT direct absorption spectra [28] of OCIO provide an example of the capabilities of FT spectroscopy in the visible and ultraviolet regions for the study of short-lived species. In Figure 14, part of the near-UV absorption spectrum is... 

Solvents used in ultraviolet, visible, infrared, microwave, and radiowave spectroscopy must meet the following requirements transparency and stability toward the radiation used, solubility and chemical stability of the substance to be examined, and a high and reproducible purity ( optical constancy ). Normally, intermolecular interaction with the solute should be minimal. On the other hand, important information about the solute can be obtained from the changes in the absorption spectrum arising from such interactions. 

These devices are based on the anisotropic absorption of light. Usually molecular crystals exhibit this property and tourmaline is the classical example for this. For practical purposes, however, micro crystals are oriented in polymer sheets. Polymers containing chromophors become after stretching dichroic polarizers. The devices produced in this manner are called polawids. They have found a broad application in many technologies. Their application in spectroscopy is limited to the near ultraviolet and to the visible and near infrared range of the spectrum. In vibrational spectroscopy polaroids are employed as analyzers only for Raman spectroscopy. 

Using ultraviolet/visible (UV/Vis) absorption spectroscopy, it is possible to measure the protein concentration using Beer s Law A = e c, where A is the measured absorbance of a solution, e is the absorptivity of the protein, is the pathlength of the cell used to determine the absorbance, and c is the protein concentration. Proteins typically exhibit two strong, broad absorption bands in the UV/Vis part of the spectrum. The first and most intense band is centered at 214 nm and arises from absorption of light by the peptide backbone. The second absorption band is typically found at 280nm. This band arises from absorbance from the aromatic side chains of Trp, Tyr, and Phe. Disulfide bonds may exhibit weak absorption in this range as well. 

Analysis is an integral part of research, clinical, and industrial laboratory methodology. The determination of the components of a substance or the sample in question can be qualitative, quantitative, or both. Techniques that are available to the analyst for such determinations are abundant. In absorption spectroscopy, the molecular absorption properties of the analyte are measured with laboratory instruments that function as detectors. Those that provide absorbance readings over the ultraviolet-visible (UV-vis) light spectrum are commonly used in high-performance liquid chromatography (HPLC). The above method is sufficiently sensitive for quantitative analysis and it has a broader application than other modes of detection. 

The ultraviolet-visible spectra of most compounds are of limited value for qualitative analysis and have been largely superseded by the more definitive infrared and mass spectroscopies. Qualitative analytical use of ultraviolet-visible spectra has largely involved describing compounds in terms of the positions and molar absorptivities of their absorption maxima, occasionally including their absorption minima. Indeed, some organic compounds are still characterized in terms of the number of peaks in the UV-visible spectrum and their absorbance ratios. This is usually the case in phytochemistry and photodiode array chromatography and when the analyst has a limited range of compounds to work with whose spectra are known to differ. In the pharmacopeias, however, absorbance ratios have found use in identity tests, and are referred to as Q-values in the U.S. Pharmacopia (USP). 

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