Dual wavelength spectrophotometer

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]. 

---

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... 

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

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). 

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).

Figure 8.19 Optical arrangement oja dual wavelength spectrophotometer...

Conditions as described in Fig.l. Carotenoid band shift was monitored using a dual wavelength spectrophotometer at 528-512 nm. The oscillations between the values of the carotenoid signals observed before and after the single flash are indicated by the shaded areas. 

Diffusion potentials were measured from the carotenoid absorption change with a Perkin-Elmer 356 chopped, dual-wavelength spectrophotometer and flash-induced absorption changes were measured with a home-built, crosse beam dual wavelength instrument. 

Chromatophores were prepared from cells of Rhodopseudomonas sphaeroides green mutant as described in S. Itoh (1982). Absorption changes of carotenoids and merocyanine dye (NK2274, see Fig. 1 for the structural formulae) were measured with Hitachi 557 dual wavelength spectrophotometer or with a split beam flash spectrophotometer constructed in the Institute. A merocyanine dye was purchased from Nippon Kanko Shikiso Laboratory, Okayama. 

Chloroplast suspensions were prepared from spinach leaves by the method of Arnon and Chain (1977). Cytochrome absorbance measurements were carried out in an AMINCO DW-2a dual wavelength spectrophotometer at 570 and 559 nm as reference and measurement wavelengths, respectively. Redox potentials were determined with a ME-TROHM-HERISAU E512 potentiometer equipped with a combined Pt-Ag/AgCl INGOLD electrode, previously calibrated against a saturated solution of quinhydrone (Eq pH 7 = +280 mV). The titrations were carried out in a 3-ml cell thermostatized at 20 . 

---