Wavelength spectrophotometers

Block diagram for a single-beam fixed-wavelength spectrophotometer with photo of a typical instrument. 

On-line size exclusion chromatographic (SEC) analyses were performed with a Waters Model 401 differential refractometer (DR), a Waters Model 480 ultraviolet (UV) variable wavelength spectrophotometer and a Foxboro Miran lA infrared (IR) photometer, equipped with a zinc selenide ultramicro flowcell of 1.5 mm nominal pathlength and 4.5 /xl volume, purchased from the same supplier. A set of ten Mycrostyra-gel (Waters Associates) columns, regenerated by Analytical Sciences Inc. (ASI) and of nominal porosities 100, 500 (two) 10 (two), 10 (three), 10 and lO X, in the order given and a mobile phase flow rate of 1 ml/min was used. The column set had a specific resolution of 19.7 in 1,4-dioxane, as determined by the method of Yau(2). 

Several different experimental conditions are used, depending on the laboratory, without any consensus agreement on which of these should be used for screening. This in part renders difficult comparison of results between laboratories. However, whatever the recipes used, one has to fully understand the complex regulatory properties of each of these activities before proceeding. These are presented below for each MRC complex routinely measured in the context of screening procedures. It is preferable to use a (pseudo-)double-wavelength spectrophotometer, since such a machine can now be obtained at a reasonable price and results in better, more... 

The development by Chance of a dual wavelength spectrophotometer permitted easy observation of the state of oxidation or reduction of a given carrier within mitochondria.60 This technique, together with the study of specific inhibitors (some of which are indicated in Fig. 18-5 and Table 18-4), allowed some electron transport sequences to be assigned. For example, blockage with rotenone and amytal prevented reduction of the cytochrome system by NADH but allowed reduction by succinate and by other substrates having their own flavoprotein components in the chain. Artificial electron acceptors, some of which are shown in Table 18-5,... 

Chance and associates employed spectrophotometry on intact mitochondria or submitochondrial particles to investigate both the sequence of carriers and the sites of phosphorylation. Using the dual wavelength spectrophotometer, the light absorption at the absorption maximum (Aniax) of a particular component was followed relative to the absorption at some other reference wavelength (Aref). The principal wavelengths used are given in Table 18-6. From these measurements the state of oxidation or reduction of each one of the carriers could be observed in the various states and in the presence of inhibitors. The... 

Apparatus Use a suitable variable-wavelength spectrophotometer capable of measuring percent transmittance throughout the visible spectrum and designed to permit the use of sample and reference cells with pathlengths of 2 to 4 cm. The transmittance of all paired cells should agree within 0.5%. 

This procedure requires the use of a dual wavelength spectrophotometer, or a multi-wavelength computerized spectrophotometer, the... 

For the detection of peptide fractions a variable-wavelength spectrophotometer is used. For the purpose of analysis we favor a wavelength of 215 nm that detects peptide bonds. The less-sensitive wavelength of 238 nm can be used for preparative purposes. 

Two types of photometers are used in HPLC— the fixed-wavelength filter photometer and the variable-wavelength spectrophotometer. These use a low-pressure mercury arc lamp as a light source, a transmission or interference filter to isolate a narrow band of wavelengths of light with which to excite the sample, and a photodiode to detect the light transmitted through the sample. 

Fluorometer or dual-wavelength spectrophotometer with magnetic stirrer and thermostatically controlled chamber (37°C)... 

It is worth noting that, because of the enrichment of reaction-center components in the ether-extracted PS-I particles as prepared by Ikegami and Ke , it has been possible to measure the difference spectrum of a component of low concentration such as Aq directly in a conventional commercial instrument (Hitachi model 557 dual-wavelength spectrophotometer) as illustrated by the results in Fig. 2 (B) above. Ordinarily, such difference spectra are obtained from flash-induced transients measured with specially-built, high-sensitivity instruments. 

B. Chance, D. Mayer, N. Graham, V. Legallais, Compensation of light source noise in a sensitive dual wavelength spectrophotometer, Rev. Sci. Instrum. 41 (1970) 111. 

In a detailed study on the compensation of Schlieren effects using a diode-array spectrometer, Zagatto et al.[49 recommended the use of a dual-wavelength spectrophotometer as a best solution. Almost complete compensation of Schlieren signals as high as 1 absorbance has been achieved using this approach. The method has been used successfully by Ferreira et al.[28] in the spectrophotometric determination of zinc in plants, following an on-line column separation. This, however, calls for special instrumentation which is not always available in the laboratory. 

Spectrophotometric assay of P700 and light-induced absorption changes were carried out with a Hitachi 556 dual wavelength spectrophotometer(1,2). EPR measurements of Fe-S proteins were performed as described previously(2). 

The advent of low priced powerful microcomputers has enabled easy processing of spectrophotometric data. In addition, fast scanning detectors have allowed elimination of errors resulting from poor time stability when a dual wavelength method is used with a single wavelength spectrophotometer. Modem multiwavelength analysis utilizes the reversed matrix representation of Beer-Lambert s law. This can be combined with chemometric techniques such as principal component analysis (PCA) and partial least squares (PLS). 

Figure 3 Absolute and difference spectra of cytochrome c. A is the absolute spectrum of the oxidized (Fe ") form of purified horse heart cytochrome c, recorded with reference to a sample of buffer, with 500 nm as the reference wavelength in a dual wavelength scanning spectrophotometer. B is the absolute spectrum of the reduced (Fe ) fomi of the same sample after treatment with a few grains of sodium dithionrte. Isosbestic points occur wherever the spectra of the oxidized and reference forms cross. C (shifted up) is the difference spectrum obtained by subtraction of A from B. Spectra were recorded in a Johnson Foundation/Current Designs Inc. SDB4 Dual-Wavelength Spectrophotometer and manipulated using Soft SDB software. Conditions were room temperature (about 20 C) 10 mm pathlength scan speed, 4.25 nm s spectral bandwidth, 2 nm. The positions of the p (520.5 nm) and a (550.5 nm) bands are marked.

The most distinctive feature of the dual-wavelength spectrophotometer is the presence of two monochromators, the outputs from which are directed to a single sample, again traditionally via a chopper mirror. Consider first the operation of a dual-wavelength non-scanning spectrophotometer. Rather than comparing the absorbance of a sample and reference at the same wavelength (as... 

The experiments illustrated in Figures 1 and 2 employed a microprocessor-controlled, scanning, dual-wavelength spectrophotometer which was capable of storing and manipulating spectra. 

Figure 2 Difference spectrum of freeze-trapped cerebral cortex. Gerbil brains were freeze-trapped (43) 1 min after the anaesthetized animal began breathing either 5% O2, 90% N2. 5% CO2 or 95% N2, 5% CO2. Reflectance spectra were recorded with a bifurcated light guide attached to a scanning, dual-wavelength spectrophotometer (1, 24) from the right cerebral cortex at 77 K, and the difference spectrum was computed from the spectra from each sample. Figure redrawn from data originally published in (29).

The method of dual-wavelength spectrometry was already proposed by Chance in 1951 [37] Giese and French [6] constructed a double-beam dual-wavelength spectrophotometer for this purpose. But the merits of developing it were not pointed out until 1969 and later by Shibata et al. [38-41]. 

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