Spectrophotometers choice

Hence, this assay is an extremely useful and selective assay to measure O2 secretion. Because of this selectivity and because it measures the initial product of O2 reduction, it is often used as the method of choice to detect NADPH oxidase activity. It is suitable for semi-automation because assays can be performed in 96-well microtitre plates (using ELISA plate readers with a suitable filter), or cytochrome c reduction can be detected using simple spectrophotometers. The assay, however, is not suitable for measuring O2 that may be generated intracellularly within activated neutrophils. 

Fiber-optic-coupled spectrophotometers (single beam and double beam) are the best choice for on-line analyses. The advent of nonsolarizing optical fiber has made possible on-line analyses in which the spectrophotometer may be located remotely from the process and light is carried to/from the process by the optical fiber. A rugged probe or flow cell provides the sample interface. 

Water-Soluble Sulfates, Chlorides, and Nitrates. The procedures are applicable to pigments and extenders. The choice of hot or cold extraction depends on the properties of the pigment and should be agreed. The anions are determined by the usual analytical methods. For standards, see Table 1 ( Sulfates , Chlorides , and Nitrates ). Apparatus Nessler tubes or spectrophotometer, equipment for pH measurement. 

Although the HRP/TMB system is usually a good, reliable, and sensitive combination, HRP has a number of alternative substrates, which can be used such as o-phenylene diamine. There are also number of options for the enzyme used other than HRP, such as alkaline phosphatase, which can be used in combination with the substrate p-nitrophenyl phosphate. It is important to note that if alkaline phosphatase is used, the wash buffer must not contain phosphate. Usually in this case a Tris-buffered rather than phosphate-buffered wash buffer is used. The choice of enzyme-substrate system depends on a number of factors, including price, sensitivity and whether a spectrophotometer filter is available for the substrate specific wavelength to be measured. 

The ratio /spectrophotometric measurement, and the value of a is then calculated from eq. (3.5) to yield the desired absorption constant. The numerous absorption constants found in the literature arise from the choice of quantities incorporated in the constant b. Some of the terms most commonly used to express absorption in minerals are summarized in table 3.2. Note that optical densities (O.D.), representing the direct output from many spectrophotometers, lack specificity about sample thickness and element concentrations. Absorption coefficients (a) indicate that sample thicknesses have been measured or estimated. Molar extinction coefficients (e) require chemical analytical data as well as knowledge of sample thicknesses. 

For compounds that nitrate rapidly at room temperature, two methods may be used. The substrate may be dissolved in sulfuric acid of appropriate strength and introduced into one limb of a two-limbed flask. A solution of nitric acid in sulfuric acid is introduced into the other limb, the flask is thermostatted, and the reaction started by vigorously shaking the flask. Aliquots are then withdrawn and quenched as before. However, by far the most convenient method is to carry out the kinetics directly in the thermostatted spectrophotometric cell and this would now be the method of choice in view of the automatic monitoring facilities currently available on spectrophotometers. For very fast reactions, stopped-flow techniques have been used. 

Consult your instructor on the proper use of the fluorescence spectrophotometer and the choice of appropriate collection parameters. You should understand how to optimize the following instrumental parameters in order to maximize the signal-to-noise ratio (S/N) of a luminescence spectrum ... 

The reaction can be observed in either the kinetic or the equilibrium mode. The Bacillus fastidiosus enzyme has the highest Michaelis constant (1.0 x 10" mol/L) and the hog fiver has the lowest (1,7 x 10 mol/L), the choice of enzyme influencing the incubation period required to reach equilibrium and the conditions for a pseudo first-order Idnetic approach. The decrease of absorbance as urate is converted may be monitored by a spectrophotometer at 293 nm and this forms the basis of a proposed reference procedure. However, at this wavelength, most of the absorbance is due to plasma proteins. Therefore there is a high signal-noise ratio, which can compromise the precision of the method. A high quality spectrophotometer with narrow bandpass is required and this is rarely satisfied with automated analyzers. 

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