Monochromator emission

To determine the fluorescence excitation spectrum, one selects a wavelength on the emission monochromator (generally the fluorescence A 1 and... 

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. 

When the emission monochromator of the spectrofluorometer is set at a certain wavelength AF with a bandpass AAF, the reading is proportional to the number of photons emitted in the wavelength range from AF to AF + AAF, or in the corresponding wavenumber range from to 1/AF to 1/(AF + AAF). The number of detected photons satisfies the relationship ... 

Polarization effects The transmission efficiency of a monochromator depends on the polarization of light. This can easily be demonstrated by placing a polarizer between the sample and the emission monochromator it is observed that the position and shape of the fluorescence spectrum may significantly depend on the orientation of the polarizer. Consequently, the observed fluorescence intensity depends on the polarization of the emitted fluorescence, i.e. on the relative contribution of the vertically and horizontally polarized components. This problem can be circumvented in the following way. 

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... 

In some cases, filters are used instead of the emission monochromator. In principle, no G factor is then considered, but in practice, effects may be due to the sensitivity of the photomultipliers to polarization (in particular, photomultipliers with side-on photocathodes). 

It is sometimes difficult to totally remove (by the emission monochromator and appropriate filters) the light scattered by turbid solutions or solid samples. A subtraction algorithm can then be used in the data analysis to remove the light scattering contribution. 

Figure 1.9 The energy-level and transition schemes and possible luminescence spectra of a three-level ideal phosphor (a) the absorption spectrum (b, c) emission spectra under excitation with light of photon energies hvi and /iV2, respectively (d, e) Excitation spectra monitoring emission energies at /i( V2 — vi) and at /i vi, respectively. Arrows mark the absorption/emission transitions involved in each spectrum. Eixed indicates that the excitation or emission monochromator is fixed at the energy (wavelength) corresponding to this transition.

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).

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... 

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.

Light emitted at right angles to the incoming beam is analyzed by the emission monochromator. In most cases, the wavelength analysis of emitted light is carried out by measuring the intensity of fluorescence at a preselected... 

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... 

When a luminescence spectrum is obtained on an instrument such as that used to produce the spectra in Figure 7.23, it will depend on the characteristics of the emission monochromator and the detector. The transmission of the monochromator and the quantum efficiency of the detector are both wavelength dependent and these would yield only an instrumental spectrum. Correction is made by reference to some absolute spectra. Comparison of the absolute and instrumental spectra then yields the correction function which is stored in a computer memory and can be used to multiply automatically new instrumental spectra to obtain the corrected spectra. The calibration must of course be repeated if the monochromator or the detector is changed. 

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])...

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. 

If an emission wavelength was absent for a sample under investigation the procedure was to set the emission monochromator at the desired wavelength and scan the excitation. 

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. 

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. 

Figure 12. Relative spectral distribution of a low-pressure mercury vapor lamp with visible light cut-off filter as measured through the emission monochromator of a spectrofluorometer (2 nm slit width). (Typical of lamps used to view fluorescence/ fluorescence quenching on thin-layer chromatographic plates.)...

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