Split beam spectrophotometer

H6. Hansen, R. E., van Gelder, B. F., and Beinert, H., Attachment to a split-beam spectrophotometer for recording extinctions in round tubes of small diameter. Anal. Biochem. 35, 287-292 (1970). 

UV-visible spectra were determined using a Varian Cary 100 Bio-UV-Visible split-beam spectrophotometer running with Cary WinUV scan application with a capacity of measuring six samples at a time. Samples were scanned at 500 nm. A high-intensity Xe flash lamp was used as the source for UV light, which permits taking 80 data per second. 

Selected entries from Methods in Enzymology [vol, page(s)] Absorption Spectrophotometer Application, 24, 15-25 baseline compensation, 24, 8-10 computerized, 24, 19-25 light scattering, 24, 13-15 monochromator, 24, 4 photometer, 24, 5-8 recorder, 24, 8 sample compartment, 24, 5 single-beam, 24, 3-4 spectral characteristics, 24, 10-12 split-beam, 24, 3 stray light, 24, 12-13. 

Absorption spectra were obtained with a Shimadzu model UV-3000 spectrophotometer in the split beam mode. Fluorescence spectra were scanned with the same instrument supplemented with a fluorescence attachment and a Hamamatsu R473 multiplier. A solution of 3 gL 1 rhodamine B in polyethyleneglycol was used as a photon counter. The digitized spectra were transferred to a Hewlett Packard microcomputer (model 226) for storage and processing. 

In this study, dynamic experiments were performed using a differential method of dichroism. The sample was strained in the common beam of a Perkin-Elmer model 180 double-beam spectrophotometer, with the transmitted radiation split into two beams polarized parallel to and perpendicular to the direction of stretch. The recorded output was the difference between the two absorptions. The orientation function was calculated using Equation 3, where A0 is the unpolarized absorption of... 

Figure 4. Difference spectrum of P-450 CO measured with the Cary model 14 split-beam recording spectrophotometer. A suspension of P-3 particles containing 0.95 mg. of protein per ml. of 10 mM phosphate buffer (pH 74) was equally divided between two anaerobic optical cuvettes having Na2S20 in the sidearms. Both cuvettes were gassed for 5 min. with Ng, and a base line of equal optical density was established. Experimental cuvette was then gassed with CO for 2 min., dithionite was added to both cuvettes from the sidearms, and the change in optical density was recorded...

Alternatively, the sample and reference may be compared many times a second, as in double-beam instruments. The light from the source, after passing through the monochromator, is split into two separate beams—one for the sample and the other for the reference. Figure 7.14 shows two types of double-beam spectrophotometers. The measurement of sample and reference absorption may be separated in space, as in Figure 7.14A this, however, requires two detectors which must be perfectly matched. Alternatively, the sample and reference measurement may be separated in time as in Figure 7.14B this technique makes use of a rapidly rotating mirror or... 

The double-beam system is used extensively for spectroscopic absorption studies. The individual components of the system have the same function as in the single-beam system, with one very important difference. The radiation from the source is split into two beams of approximately equal intensity using a beam splitter, shown in Fig. 2.28. One beam is termed the reference beam, the second beam, which passes through the sample, is called the sample beam. The two beams are then recombined and pass through the monochromator and slit systems to the detector. This is illustrated schematically in Fig. 2.28. In this schematic, there is a cell in the reference beam that would be identical to the cell used to hold the sample. The reference cell may be empty or it may contain the solvent used to dilute the sample, for example. This particular arrangement showing the monochromator after the sample is typical of a dispersive IR double-beam spectrophotometer. There are many commercial variations in the optical layout of double-beam systems. 

DPC supported DCIP photoreduction was measured continuously at 600 nm using an Aminco DW2a spectrophotometer in the split beam mode. The amount of high-affinity Mn-binding site in the different PS II membrane preparations was determined using the assay of Hsu gt aL [6], by adding /xM concentrations of MnC to the DPC -> DCIP assay buffer. The details of these determinations are described elsewhere [5]. 

FIguie S Components and their arrangement in a split beam ( double beam ) spectrophotometer. For details, see text. 

Split-beam scanning spectrophotometer (or almost any other kindl)... 

Split-beam or dual-wavelength scanning spectrophotometer... 

Some lamps can be used for several elements. A low pressure mercury lamp can be used for the determination of mercury. However, because of some instability of die lamp output, a double beam spectrophotometer gives more reliable results than single beam instruments. In the former, a rotating sector mirror chopper splits die beam from the lamp into a reference beam and a sample beam, which passes through die burner or atomiser. A mirror combines the two beams which pass through the monochromator. Then the ratio of the intensity of the two pulses is electronically measured, thus elminating any fluctuation of the lamp output. Instruments display absorbance and/or u.v. transmission. 

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. 

Some spectrophotometers are single-beam instruments, and some are double-beam instruments. In a double-beam instrument the light beam emerging from the monochromator is split into two beams at some point between the monochromator and the detector. The double-beam design provides certain advantages that we will discuss shortly. 

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