Wavelength and absorbance calibrations

The most common type of absorption cell or cuvette is the square cell, open at the top, with a pathlength of 10 mm and working volume of 2-3 ml. Only fluorescence cuvettes are polished on all faces. Manufacturers catalogues illustrate designs for special purposes, with pathlengths ranging fh)m 1 to 100 mm. 

The choice of glass, silica (quartz) or plastic cuvettes will be primarily dictated by whether they will be used in the UV or visible spectral regions. Only silica is suitable below about 360 nm. Plastic disposable cuvettes are cheap, popular and can give perfectly acceptable results. Their less-than-perfect optical faces may be of little consequence when working with highly light-scattering samples. 

Examples of cuvettes for special purposes and which must usually be constructed by the user are those for retaining standard e.p.r. tubes in a single beam, for low-temperature work and photodissodation spectra, and for 

The importance of clean cuvettes is self-evident. Routinely, all non-disposable cuvettes should be emptied immediately after use, rinsed repeatedly in the solvent (e.g. water), then with clean ethanol or acetone and dried with low pressure air or nitrogen from a cylinder. It is prudent to install a filter (such as those with pore sizes of 0.45 pm used in filter sterilization) in the gas line. Cuvette washers (e.g. Aldrich) wash, rinse, and dry cuvettes. Cotton wool buds can also be usefiil for dislodging interior, stubborn marks and for drying. The outside optical surfoces should be polished with clean lens tissue. Note that plastic squeety bottles generally used for solvents contain plasticizers such as butyl phthalate, which can interfere with critical UV spectra. 

The Perspex windows of low-temperature cuvettes easily become scratched and Avill eventually crack. However, provided they are still liquid-tight (at least for as long as it takes to pltmge them and their contents into liquid nitrogen) they are quite serviceable in this condition. The opacity of the Perspex is negligible compared with that of the frozen sample. 

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Wavelength and absorbance calibration can be checked using standard filters. However, even with a well-calibrated instrument and properly prepared samples, things can go wrong. Look at the graph in Figure 2.11, which shows the actual measured absorbance at 280 nm for a series of standard protein solutions of different concentrations. 

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