Excitation and emission monochromator

The synchronous spectra (SF) were collected in the 260-460 nm excitation wavelength range using bandwidth of AA=20 nm between the excitation and emission monochromators. All SF and emission spectra were recorded with a 10 nm slit width on both monochromators. The scan speeds of spectra were 500 nm/min. 

Wakeham [14] has discussed the application of synchronous fluorescence spectroscopy to the characterization of indigenous and petroleum derived hydrocarbons in lacustrine sediments. The author reports a comparison, using standard oils, of conventional fluorescence emission spectra and spectra produced by synchronously scanning both excitation and emission monochromators. 

Figure 5.25 — Flow-through ion-selective optrode based on a multilayer lipidic membrane prepared by the Langmuir-Blodgett method. (A) Cross-sectional view of the composite six-layer membrane (four layers of arachidic acid/ valinomycin covered by an arachidic acid and rhodamine dye bilayer). (B) Optical arrangement integrated with the sensor, which is connected to a flow system. LS light source Ml and M2 excitation and emission monochromator, respectively FI and F2 primary filters M mirror LB lipid-sensitive membrane in a glass platelet FC flow-cell A amplifier D display P peristaltic pump. (Reproduced from [107] with permission of the Royal Society of Chemistry).

All steady-state measurements are performed on a SLM 48000 modified to accommodate the optical cells (72). A Xe-arc lamp is used for excitation, and both excitation and emission monochromators are used for wavelength selection. 

Fig. 7. Excitation, fluorescence, and synchronously-scanned spectra of oestrone in ethanol, and their second derivatives. A, excitation spectrum monitored at a fluorescence wavelength, Af B, fluorescence spectrum obtained at an excitation wavelength, Aex C, synchronously-scanned spectrum obtained with a constant interval, AA, between the excitation and emission monochromators. (From A. F. Fell, in Proc. 1st Symp. Anal. Steroids, Eger, Hungary, S. Gorog (Ed.), Amsterdam, Elsevier Press, 1982, pp. 495-510.)...

The calibration of excitation and emission monochromator wavelengths should be checked regularly by the use of sharp lines from the instrument s own radiation source (e.g. xenon lines at 450.1, 462.4,... 

Commercial spectrometers, such as the Perkin-Elmer MPF-43A fluorescence spectrometer, that allow interlocking of excitation and emission monochromators lately have become available for utilizing this underexploited analytical technique. The synchronous luminescence technique reduces the complexity of the luminescence spectrum of a compound compared with a conventionally obtained luminescence spectrum. One can, therefore, better tackle the analysis of fairly complex mixtures without resorting to techniques that are expensive or excessively time consuming. 

Fluorometers and fluorescence spectrophotometers are available that offer a variety of features. These features include ratio referencing, microprocessor-controlled excitation and emission monochromators, pulsed xenon light sources, photon counting, rhodamine cell for corrected spectra, polarizers, flow cells, front-surface viewing adapters, multiple cell holders, and microprocessor-based data reduction systems. 

It may be also useful to scan the excitation and emission monochromators simultaneously (synchronous fluorimetry). Often, this is carried out by scanning the... 

Spectroscopists have shown many powerful variations on the fluorescence experiment, which can generate additional selectivity and structural information. These include two-photon excitation (55) supersonic jet expansion (56) constant-energy synchronous fluorescence, where the excitation and emission monochromators are scanned synchronously to maintain a constant energy difference (57) and pulsed excitation for the real-time measurement of fluorescence lifetimes (58). However, it is far from certain that these techniques will ever be useful for the practicing analyst. 

Synchronous scanning techniques have also been applied to the quantitative analysis of fluorescent substances. Synchronous scanning involves scanning both the excitation and emission monochromators simultaneously, while maintaining a constant wavelength interval between them. The technique has been employed in the analysis of multicomponent preparations. The technique is reported to simplify the spectra of multicomponent samples and reduce the bandwidths of fluorescence spectra. The equation relating the measured fluorescence to concentration is given by... 

Apparatus. Fluorimetric measurements were recorded on SPEX Fluorolog-2 spectrofluorimeter equipped with a xenon lamp of 450W. The fluorescence intensities of solutions were obtained using 1 cm quartz cells. The excitation and emission monochromators were fixed with 0.25 mm slits. Fluorescence was collected and detected by photomultiplier tube (Hamamatsu Model R 928) powered at 950V. All spectral data were obtained by SPEX DM 3000F... 

Apparatus. Spectrofluorometer SPEX Fluorolog-2 (Edison, NJ, USA). Xenon lamp 450-W (OSRAM, Germany) and photomultiplier tube (R 928 Hamamatsu Co.) powered at 950 V as the detector. Excitation and emission monochromator slits, wavelength increment, and integration time were set at 1 mm, 1 nm and 1 second respectively. A pH meter (Model Orion 520A, USA) was used for pH adjustment. Basic procedure. Benserazide solution 2 mL (1.0 x 10 6 1.0 x 10 4mol/L) was added to 2 ml of Triton X-100 solution (4.0 10 4 mol/L) and 2 mL pH 4.0... 

Figure 5 A block diagram of a typical fluorescence spectrophotometer. L represents the light source ExM and EmM, the excitation and emission monochromators, respectively BS is a quartz glass beam splitter F/P is a filter or polarizer Sa is the sample holder Sle and Six are the entrance and exit slits of the monochromators RB is the rhodamine B sample used in the reference channel F is the red filter in that channel PMT, represents the photomultiplier tubes which detect the fluorescence and reference fluorescence signals, S and ff, respectively the shaded arrows represent the excitation light beam the solid arrow pointing towards RB represents c. 10% of the light reflected from BS and the arrows from Sa represent a portion of the fluorescence light intensity that is emitted in a spihere surrounding the sample.

Hgure 8 A demonstration of the overlap of excitation and emission monochromator bandpass when and Xem are close to one another thus explaining the observation of Rayleigh scattering of Xex-... 

In an independent titration, titrate the buffer with the DnaB protein solution and determine the contribution of the protein fluorescence to the observed fluorescence of the eADP sample for each titration point. At high protein concentrations such a contribution will become signiflcant, in spite of the fact that the excitation is set at 325 nm (far from the protein absorption spectrum), due to the impurities in the protein sample and the discrete band pass of the excitation and emission monochromator. This is your background, 6. 

Synchronous fluorescence. Synchronous fluorescence stands for a type of fluorescence measurement technique in which the excitation and emission monochromator are scanned simultaneously and the constant wavelength difference between excitation and emission is maintained throughout the process. Synchronous spectra are a characteristic property of a molecule or compound and so the analyte can be detected selectively from a mixture or solution. 

All fluorescence studies were performed on a Fluorolog-2 spectrofluorometer, Spex Industries (Edison, NJ), equipped with a 450-Watt Xenon arc lamp. The excitation and emission monochromator slit widths were both set at 2 mm. The excitation and emission wavelengths of the various fluorophores used are indicated in Figure 2. All data were acquired at room temperature using quartz cuvettes with sample volumes of 1.5 mL. 

Spectrofluorimeter. Preferably capable of synchronous scanning, with wavelength reproducibility of excitation and emission monochromators +2nm or better typical monochromator gratings of 6001ines/nm blazed at 300 nm or better, spectral resolution with bandpass of 2.5 nm or less, maximum bandpass of 10 nm and a response of S-20 or S-5 on the photomultiplier tube. 

Generally, filter fluorimeters are more sensitive than those with monochromators. However, they lack scaiming options and hence cannot be used for investigations related to molecular structure. Nevertheless, for a number of routine analyses filter instruments are quite satisfactory. If a filter fluorimeter is used, the excitation filter should be a narrow band filter (+5nm) centred at 325 nm. On the emission side, a wide band pass filter ( 10-20 nm) centred at 420 nm should be used. Scanning fluorimeters should have excitation and emission monochromators with slit widths of 5 nm or smaller. 

Place IRBP (0.5-2 iM m buffer A) in a fluorescence cuvet in the sample compartment of a fluorometer. Measure the fluorescence of the sample with excitation and emission monochromators set at 330 and 480 nm, respectively (see Note 8)... 

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