Spectrophotometer dual beam

Optical absorption (OA) absorption spectra for all the samples were collected with a CARY 5E UV-VIS-NIR or a Jasco V570 dual-beam spectrophotometers in the 200-800 nm wavelength range at the Department of Mechanical Engineering (Materials Division) in Padova (Italy). 

The first measurement we make when starting a fluorescence study is not usually a fluorescence measurement at all but the determination of the sample s absorption spectrum. Dual-beam differential spectrophotometers which can record up to 3 absorbance units with a spectral range of 200-1100 nm are now readily available at low cost in comparison to fluorimeters. The wide spectral response of silicon photodiode detectors has made them preeminent over photomultipliers in this area with scan speeds of a few tens of seconds over the whole spectral range being achieved, even without the use of diode array detection. 

DOX concentration was determined spectrophotometrically based on the molar extinction coefficient of 125000 OD M (38) in a dual-beam spectrophotometer (either Perkin-Elmer Lambda 3B or Kontron Uvikon 860). The DOX quantification was confirmed by HPLC (49-51). Purity of DOX and its degree of degradation during the processes of liposome preparation and liposome storage were determined by a combination of HPLC and TLC, as described by Barenholz leave et al. (38,49,50). 

Typical standard curves are shown in Figures B1.1.2, B1.1.4, B1.1.6, and B1.1.8 for each of the four assay methods. In each case, the tube protocols were performed in duplicate on diluted BSA or BGG standard. The color in each tube was measured at the appropriate wavelength in a dual-beam spectrophotometer. The net absorbance for each sample was plotted versus its protein concentration. 

Absorption spectra of 2-nitroanisole in supercritical C02, N20, Freon-13, ammonia and C02-methanol mixtures were obtained on a Cary model 1605 spectrophotometer operated in the dual beam mode. The gases used as supercritical solvents were of the highest purity available from the supplier (Matheson) and were further filtered prior to use. The mixed solvent system of C02-methanol was obtained from Scott Speciality Gases (15.4 wt% methanol), and other mixtures were made in the laboratory. Spectra of 2-nitroanisole in n-pentane, methanol, tetrahydrofuran and acetonitrile (Burdick A Jackson) were obtained using quartz cells with a 1-cm light path and with a pure solvent blank in the reference beam. Vapor phase and supercritical fluid spectra were obtained using an air reference. 

Figure 6 The structure of the potent quasi-irreversihle CYP3A4 inhibitor troleando-mycin (top panel), the metabolic steps required to convert troleandomycin into a nitroso metabolite that coordinates with the ferrous-heme of CYP3A4 (side panel), and the spectral changes associated with MI complex formation (bottom panel). Troleandomycin (50 pM) was incubated at 37°C with a human liver microsomal sample with high CYP3A4/5 activity (1 mg/mL) andNADPH (1 mM) for the times indicated. The reference cuvette contained the same components minus troleandomycin. Scans from 380 to 520 nm were recorded on a Varian Cary 100 BIO IJV/Vis dual beam spectrophotometer. Abbreviation MI, metabolic intermediate.

In spectrophotometric analyzers, interference filters are selected for desired wavelengths, as determined from the spectral relationship curves. Photodetectors are least sensitive in the blue end of the spectrum. This can be dealt with by using prefilters or narrow spectral ranges, which are calibrated for more sensitivity. Improvements in spectrophotometers include a flashed xenon light source with dual-beam measurement. Dual-beam machines measure the spectrum of both the light source and the reflected light for each measurement. 

Figure 5.2 Schematic of typical single- and dual-beamed spectrophotometers.

Prepare absorbance spectra of the pH 2.40, 3.80, and 5.20 samples. These spectra should be made in a dual beam spectrophotometer using a cuvette filled with glass-distilled water as a reference. The spectral measurements should be performed between 660 nm and 310 nm. From these spectra (Figure 2-20) it should be clear why wavelengths of 430 and 590 nm were selected in step 2-69. 

Turn on a dual beam recording spectrophotometer and allow it to warm up as directed by the manufacturer. Both the tungsten and deuterium bulbs should be turned on. 

The coupling ratio is determined most easily with a dual beam spectrophotometer with solutions containing equal concentrations of unmodified and ribonucleoside conjugated keyhole limpet hemocya-nin in the reference and sample cuvettes, respectively. Protein concentrations can be determined by standard colorimetric assay (e.g., 18), and the concentration of coupled ribonucleoside can be calculated using published extinction coefficients (e.g., 19). An alternate method, based on the extinction coefficients of the carrier protein and of the coupled ribonucleoside, is described in ref. 20. 

Measurement is performed under constant stirring at 492 nm using 530 nm as a reference with the Aminco dual beam spectrophotometer. 

HGA-70 Procedure. In the development of this procedure, a HGA-70 graphite furnace atomizer was used with a Perkin-Elmer 403 dual-beam spectrophotometer equipped with a deuterium background corrector. With the HGA-70 it is necessary to use grooved-type furnaces for petroleum samples to prevent the sample from running out the end of the furnace. The grooved furnaces attain a maximum temperature of 1950°C. At this temperature beryllium forms a stable carbide which prevents quantitative atomization and drastically reduces the beryllium... 

Ferric-ion concentrations were determined by measuring optical density at 3020 A. with a Cary 15 dual-beam spectrophotometer at 25°C. 

The probe molecule used for the studies on the solvatochromic behavior of supercritical fluids was 2-nitroanisole, which has an S value of -2.428 0.195 (15) and a reference absorbance maximum of 32,560 cm-1 in cyclohexane ( )o) Absorption spectra of 2-nitroanisole in the supercritical fluids were obtained with a Varian Model 2200 spectrophotometer operated in the dual beam mode with an air reference. The gases used as supercritical solvents were SFC grade C02 high purity grade N2O and NH3, and research grade for the other gases. 

FIGURE 26.2 Dual-beam spectrophotometer. The diffraction grating is rotated to select the desired wavelength. The beam splitter consists of a half-silvered mirror which passes half the light while reflecting the other half. A rotating mirror with cut-out sections (chopper) alternately directs one beam and then the other to the detector. 

Pigment mutant C-2A of Scenedesmus obliquus (11) was grown heterotrophically in the dark as described (12). Isolation of pigments The pigments were extracted with hot methanol. PChlides were isolated and separated by a TLC-system with the method of (10). Chlide b was isolated with the method of (13). The PChl isolation and the determination of PChl/Chl and PChlide/Chlide photoconversion were carried out as described (14). Preparation of membrane particles The preparation of membrane fraction from C-2A mutant cells of Scenedesmus obliquus followed the modified method of (15,16). Spectroscopic methods Absorption spectra were recorded with a Kontron dual beam spectrophotometer (Uvikon 820). Fluorescence data were obtained with a Shimadzu spectrofluorophotometer RF-540. 

Analysis of the samples after photooxidation was carried out using a UVIKON 930 UV-VIS dual-beam spectrophotometer. The degradation of model pollutant (salicylic acid) was monitored by UV-VIS measurement. 

Fig. 14.9 Schematic of a dual-beam UVAf is absorption spectrophotometer...

In a spectrophotometer the absorption of a molecular sample (gas, liquid or solid) can be measured as a function of the wavelength. The absorption normally occurs in not too narrow bands and therefore a continuum light source can be used in connection with a spectral resolving apparatus, which is normally a grating monochromator but can also be a Fourier transform spectrometer [6.111,112]. Spectrophotometers based on grating monochromators are frequently of the dual-beam type, with one beam passing the sample cell and the other one a reference (empty) cell of the same type before the transmitted intensities are compared at the detector. The lay-out for such an instrument is shown in Fig. 6.66. 

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