Ultraviolet/visible spectroscopy cells used

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. 

Using matched cuvettes for solvent and analyte is seldom practical for infrared measurements because it is difficult to obtain cells with identical transmission characteristics. Part of this difficulty results from degradation of the transparency of infrared cell windows (typically polished sodium chloride) with use due to attack by traces of moisture in the atmosphere and in samples. In addition, pathlengths are hard to reproduce because infrared cells are often less than 1 mm thick. Such narrow cells are required to permit the transmission of measurable intensities of infrared radiation through pure samples or through very concentrated solutions of the analyte. Measurements on dilute analyte solutions, as is done in ultraviolet or visible spectroscopy, are usually difficult because there are few good solvents that transmit over appreciable regions of the infrared spectrum. 

The biochemist is quite familiar with ultraviolet and visible spectroscopy, in which a compound is frequently dissolved in an aqueous solution, a good spectrum is obtained, and quantitative analysis can be readily applied. In the case of infrared spectroscopy a common method of obtaining a spectrum is to dissolve the sample in an appropriate solvent, place the solution in a suitable cell, and record the spectrum. Certainly the solvent must have reasonable transparency to infrared radiation in the region to be used. This method is used widely in qualitative analysis, and is the most commonly used method in quantitative analysis. 

In ordinary infrared measurements, the requirements for the optical properties of cell windows and other materials (flatness, transparency, etc.) are not as severe as needed for ultraviolet and visible spectroscopy. Sometimes it is even necessary to make the window surface slightly coarse or to make its surfaces slightly deviated from parallel to avoid interference effects. However, such measures should never be taken in VCD measurements in order to ensure that circularly polarized radiation may be generated without fail. In VCD measurements, cell windows must be flat and parallel. A mirror cannot be used for any purpose. Samples should be transparent. VCD will not be observed in samples such as colloids because of scattering. 

UV-vis refers to absorption spectroscopy in the ultraviolet-visible spectral region. The absorption in the visible range directly affects the perceived color of the chemicals involved. The UV-vis spectra are measured using spin casted films, or dilute polymer solutions. Films are more representative for the active layer behavior in SCs, while solutions are more reliable when comparing the different polymers or blends. For example, the film deposited on ITO substrate may be dissolved in a suitable solvent (e.g., in chloroform at concentration of 25 pg/mL, in 10 mm quartz cell) and either directly used in a spectrometer, or spin casted on glass slides, vacuum dried and measured at the wavelength 280 to 900 nm at a rate of 300 nm/min.i ... 

Commercial cylindrical quartz cells can be adapted for gas-phase work as illustrated in Fig. 9.18. Such a cell finds use in the near infrared for the determination of overtone vibrational frequencies, and also in visible and ultraviolet spectroscopy. A much less expensive cell which is adequate for most gases may be constructed from Pyrex along the lines of the cell shown in Fig. 9.18. Quartz windows may then be attached by epoxy resin. A cell which is filled from a conventional vacuum line will generally contain mercury vapor which absorbs at 2537 A. Once the origin of this absorption is recognized, it causes little difficulty because of its narrow bandwidth. 

Over the past decade, there has been considerable development in imaging type detectors for the measurement of ultraviolet (UV) and visible light. These new detectors have attracted the interest of a number of analytical spectroscopists. For absorption spectroscopy, analytical chemists have traditionally used such instruments as the photometer, which uses a narrow-band light source (for example the 254 nm emission line from a low pressure Hg lamp or a continuous source with a filter), a sample cell and a photomultiplier tube (FMT) as the detector. While useful for many specific applications, the single-wavelength photometer cannot determine multiple sample components simultaneously or provide a general absorbance characterization of the system. When information at multiple wavelengths is desired,... 

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