Monochromator excitation

Figure 2. Schematic of the SLM 8000 fluorometer. Excitation occurs through the excitation monochromator, and light emitted from the sample is observed in as many as four different positions. Photomultiplier tubes (PMTs) A, B, and C can be used to monitor fluorescence or right-angle light scatter through the monochromator (PMT A) or through filters (PUT B and C), and position D measures transmittance. Three channels can be monitored simultaneously with measurements being acquired at intervals of 1 s or less. The data are stored by the computer for subsequent manipulation.

In the equation, the subscripts 1 and 2 refer to the reference compound and the compound of interest, respectively, is the intensity of the fluorescent signal of each compound measured as peak height in centimeters, 8 is the molar absorptivity, c is the concentration in moles per liter, and is the fluorescence quantum yield. In this application, i is set at 1.00. The concentrations of the solutions that were tested ranged from 10 to 10 M. The solutions run at the higher concentrations were all checked for self-quenching, but none was found. All measurements, except the fluorescence-versus-solvent study, were made in 0.1-N phosphate buffer, pH 7.4. Slit settings on the Perkin-Elmer MPF-2A were 10 mp (nm) for both emission and excitation monochromators. 

The basic instrumentation used for spectrometric measurements has already been described in the previous chapter (p. 277). Methods of excitation, monochromators and detectors used in atomic emission and absorption techniques are included in Table 8.1. Sources of radiation physically separated from the sample are required for atomic absorption, atomic fluorescence and X-ray fluorescence spectrometry (cf. molecular absorption spectrometry), whereas in flame photometry, arc/spark and plasma emission techniques, the sample is excited directly by thermal means. Diffraction gratings or prism monochromators are used for dispersion in all the techniques including X-ray fluorescence where a single crystal of appropriate lattice dimensions acts as a grating. Atomic fluorescence spectra are sufficiently simple to allow the use of an interference filter in many instances. Photomultiplier detectors are used in every technique except X-ray fluorescence where proportional counting or scintillation devices are employed. Photographic recording of a complete spectrum facilitates qualitative analysis by optical emission spectrometry, but is now rarely used. 

Fluorescence Instrumentation and Measurements. Fluorescence spectra of the FS samples were obtained on a steady state spectrofluorometer of modular construction with a 1000 W xenon arc lamp and tandem quarter meter excitation monochromator and quarter meter analysis monochromator. The diffraction gratings In the excitation monochromators have blaze angles that allow maximum light transmission at a wavelength of 240 nm. Uncorrected spectra were taken under front-face Illumination with exciting light at 260 nm. Monomer fluorescence was measured at 280 nm and exclmer fluorescence was measured at 330 nm, where there Is no overlap of exclmer and monomer bands. 

Equation (6.4) is still valid if the compound and the standard are not excited at the same wavelength, provided that the instrument is well corrected for the wavelength dependence of the lamp intensity and the excitation monochromator efficiency. Otherwise, the second term of Eq. (6.4) must be multiplied by the ratio JoC eV-IoC er)-... 

Let Ix, Iy and Iz be the intensity components of the fluorescence, respectively (Figure 6.3). If no polarizer is placed between the sample and the emission monochromator, the light intensity viewed by the monochromator is Iz + Iy, which is not proportional to the total fluorescence intensity (Ix + Iy + Iz). Moreover, the transmission efficiency of the monochromator depends on the polarization of the incident light and is thus not the same for Iz and Iy. To get a response proportional to the total fluorescence intensity, independently of the fluorescence polarization, polarizers must be used under magic angle conditions (see appendix, p. 196) a polarizer is introduced between the excitation monochromator and the sample and... 

The sample is excited with a lamp, which is followed by a monochromator (the excitation monochromator) or a laser beam. The emitted light is collected by a focusing lens and analyzed by means of a second monochromator (the emission... 

Figure 1.8 A schematic diagram showing the main elements for measuring photoluminescence spectra. The excitation can also be produced using a laser instead of both a lamp and an excitation monochromator.

The observed excitation spectrum is distorted because the light intensity of the excitation source is a function of the wavelength and the transmission efficiency of the excitation monochromator is a function of wavelength. The emission spectra are distorted by the wavelength-dependent efficiency of the emission monochromator and the photomultiplier (PMP) tubes. Thus both... 

A fluorescence emission spectrum is generally measured by setting the excitation monochromator, Mi, to the chosen wavelength and scanning the second monochromator, M2, with constant slit width. The fluorescent screen monitor, F-P2, now serves to correct for variations in the intensity of the exciting light caused by fluctuations in lamp output. The emission spectrum so recorded has to be corrected for the spectral sensitivity of the apparatus to give the true emission spectrum. 

Fig. 4. Arrangement for calibrating fluorescence spectrometer.17 L, xenon arc lamp Mi, excitation monochromator B, silica plate beam splitter. F, 0.5 mm. silica optical cell containing fluorescent screen solution Pi, monitoring multiplier phototube S, screen coated with MgO Mj, fluorescence monochromator Pa, fluorescence multiplier phototube.

Light emitted from the source converges on the excitation monochromator, which allows a narrow band of wavelengths to be selected (15 nm) to induce fluorescence of the sample solution in the measurement cell. The emitted light, observed perpendicular to the direction of the incident beam, passes through the emission monochromator, allowing the selection of a narrow band of wavelengths for measurement (Fig. 12.9). The simplest instruments have a double compartment for measurement. This allows the sample solution and a standard fluorescent reference solution to be put into the optical path. 

Figure 18-20 Essentials of a luminescence experiment. The sample is irradiated at one wavelength and emission is observed over a range of wavelengths. The excitation monochromator selects the excitation wavelength (X ) and the emission monochromator selects one wavelength at a time (Xem) to observe.

Figure 7.22 (a) Outline of a spectrofluorimeter. L, light source Mn(ex), excitation monochromator S, sample Mn (em), emission monochromator PM, photomultiplier tube A, amplifier X-Y, recorder, (b) Right-angle (left) and front-face (right) excitation. E, excitation beam L, luminescence R, reflection... 

Similarly, the instrumental excitation spectrum depends on the wavelength characteristics of the excitation monochromator and of the light source. Mercury arcs which emit line spectra are not suitable and xenon arcs are normally used. Correction is made against a reference such as a solution of Rhodamine which has a wavelength-independent fluorescence quantum yield. 

Figure 10. Excitation (left) and emission (right) spectra optimized for aleurone tissue showing intensity differences between aleurone, endosperm, and pericarp tissues. The emission monochromator was set at 445 nm for excitation spectral scans and the excitation monochromator was set at 350 nm for emission spectral scans. RFI = relative fluorescence intensity. (From [29])...

Figure 16.35. Layout of a typical fluorescence spectrometer, (i) Source, (ii) excitation monochromator, (iii) optical system, (iv) sample, (v) filter, (vi) emission monochromator, (vii) detector, and (viii) data acquisition system.

Emission Spectrum. Excitation monochromator is maintained in a specific wavelength, and the data acquisition system scans the emission monochromator measuring all wavelengths that the sample emits. 

Synchronous Scan. Emission and excitation monochromator scan at the same speed with a determined wavelength difference. This kind of scan is usually applied to study complex materials with several fluorophors or in mixtures of several fluorescent substances. The result of a synchronous scan is a product of excitation and emission spectra. This tool produces more defined peaks for interpreting the behavior of chemical structures during a dynamical process. 

Time-Resolved Fluorescence. Emission and excitation monochromators are maintained in a specific wavelength, but the excitation is chopped off and fluorescence decay is measured as a function of time. This kind of spectroscopy is interesting for studying structural changes or different complexation sites. 

A Perkin-Elmer MPF-2A Fluorescence Spectrophotometer was used to determine the excitation and emission wavelengths required for achieving maximum fluorescence intensity for the pesticides studied. The MPF-2A contained a 150 watt xenon arc and an excitation monochromator with a grating blazed at 300 nm as the excitation unit a Hamamatsu R 777 photomultiplier tube (sensitivity range 185 - 850 nm) and an emission monochromator grating blazed at 300 nm as the emission detection unit. A DuPont Model 848 Liquid Chromatograph was used for HPLC (Figure 2). The accessory injection device included a Rheodyne Model 70-10 six-port sample injection valve fitted with a 20 y liter sample loop. A Whatman HPLC column 4.6 mm x 25 cm that contained Partisil PXS 1025 PAC (a bonded cyano-amino polar phase unspecified by the manufacturer) was used with various mobile phases at ambient temperature and a flowrate of 1.25 ml/minute. 

The standard solutions were diluted with hexane or methanol to prepare solutions that contained 2 yg/ml pesticide for the initial fluorescence measurements. Excitation and emission band widths on the spectrofluorometer were adjusted to 4 nm. A solution of quinine sulfate, 1 yg/ml in 0.1 N sulfuric acid, was used as a reference in determining the relative fluorescence intensity of the pesticides. The wavelengths for excitation and emission that would give the maximum fluorescence intensity in both hexane and methanol were obtained next by using 1 cm quartz fluorometer cells. Finally the excitation monochromator was set at 254 nm, and the fluorescence intensity was again measured at wavelength of maximum emission in both hexane and methanol. 

Since we have to excite the sample and record the emitted intensity at different wavelengths, the layout of a fluorometer consists of an excitation source (a lamp or a laser), an excitation monochromator or a filter (if a lamp is used as the source of excitation), a cuvette holder into which we can put the sample, an emission monochromator or a filter... 

When we want to record the fluorescence emission spectrum, the excitation wavelength is kept fixed, and the emission monochromator is run (Figures 7.3c and d). The excitation spectrum is obtained by running the excitation monochromator at a fixed emission wavelength (Figures 7.3a and b). 

The fluorescence excitation spectrum characterizes the electron distribution of the molecule in the ground state. Excitation is, in principle and for a pure molecule, equivalent to absorption. The fluorescence excitation spectrum is obtained by fixing the emission wavelength and by running the excitation monochromator (Figure 7.3). 

The basic instrumentation used for spectrometric measurements has already been described in the previous chapter (p. 274). Methods of excitation, monochromators and detectors used in atomic emission and absorption techniques are included in table 8.1. Sources of radiation physically separated... 

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