Emission spectra, true

Programming a CAM for fluorometry is far more complex than for spectrophotometry. Spectrophotometry is simple because it is based on the ratio of light in to light out. But fluorometry creates many of the problems associated with true radiometry—measuring the emission spectrum of an unknown source. The logic may become circular. Radiometry to determine an emission spectrum requires the relative spectral sensitivity of the photometer to be known, but how can this be determined without a source with a known emission spectrum Fortunately, physicists in our national standardization organizations provide us with calibrated sources and photometers. 

We have shown that the radiant flux spectrum, as recorded by the spectrometer, is given by the convolution of the true radiant flux spectrum (as it would be recorded by a perfect instrument) with the spectrometer response function. In absorption spectroscopy, absorption lines typically appear superimposed upon a spectral background that is determined by the emission spectrum of the source, the spectral response of the detector, and other effects. Because we are interested in the properties of the absorbing molecules, it is necessary to correct for this background, or baseline as it is sometimes called. Furthermore, we shall see that the valuable physical-realizability constraints presented in Chapter 4 are easiest to apply when the data have this form. 

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. 

The luminescence emission spectrum of a specimen is a plot of luminescence intensity, measured in relative numbers of quanta per unit frequency interval, against frequency. When the luminescence monochromator is scanned at constant slit width and constant amplifier sensitivity, the curve obtained is the apparent emission spectrum. To determine the true spectrum the apparent curve has to be corrected for changes of the sensitivity of the photomultiplier, the bandwidth of the monochromator, and the transmission of the monochromator with fre-... 

For recording of the emission spectrum, the emitted radiation is focussed on the slit of a monochromator and intensities measured attach wavelength. Since sensitivities of photocells or photomultipliers are wavelength dependent, a standardization of the detector-monochromator combination is necessary for obtaining true emission spectrum This can be done by using a standard lamp of known colour temperature whose emission characteristics is obtained from Planck s radiation law. The correction term is applied to the instrumental readings at each wavelength. Very often substances whose emission spectra have been accurately determined in the units of relative quanta per unit wavenumber intervals are... 

In emission spectroscopy, we measure emitted irradiance rather than the fraction of incident irradiance striking the detector. Detector response varies with wavelength, so the recorded emission spectrum is not a true profile of emitted irradiance versus emission wavelength. For analytical measurements employing a single emission wavelength, this effect is inconsequential. If a true profile is required, it is necessary to calibrate the detector for the wavelength dependence of its response. 

Figure 21-3 A portion of the emission spectrum of a steel hollow-cathode lamp, showing lines from gaseous Fe, Ni.and Cr atoms and weak lines from Cr and Fe+ ions. The monochromator resolution is 0.001 nm, which is comparable to the true linewidths.

Activated chemiluminescence is observed from these secondary peroxy-esters as well. When the thermolysis of peroxyacetate [281 in benzene solution is carried out in the presence of a small amount of an easily oxidized substance the course of the reaction is changed. For example, addition of N,N-dimethyldihydrodibenzol[ac]phenazine (DMAC) to peroxyester [28] in benzene accelerates the rate of reaction and causes the generation of a modest yield of singlet excited DMAC. This is evidenced by the chemiluminescence emission spectrum which is identical to the fluorescence spectrum of DMAC obtained under similar conditions. Spectroscopic measurements indicate that the DMAC is not consumed in its reaction with peroxyester 28 even when the peroxyester is present in thirty-fold excess. The products of the reaction in the presence of DMAC remain acetophenone and acetic acid. These observations indicate that DMAC is a true catalyst for the reaction of peroxyacetate 28. The results of these experiments with DMAC, plotted according to (27) give k2 = 9.73 x 10-2 M-1 s-1. 

In order to obtain true emission and excitation spectra it is uaially necessary to apply conections for variations in excitation intentity and the wavelength sensitivity of the detection system. The correction needed may be calculated by comparing the instmment response for a standard compound of known corrected ectral characteristics with that of the sample under study, although q)ectrofluorimeters have been described which fully electronically compensate for intensity and wavelength response of the system Comparison of the area under the corrected emission spectrum with that of various standard fluorescence compounds allows the quantum yield of the luminescence process to be calculated ... 

Phosphorescence is also characterized by an excitation and an emission spectrum. Because the lowest triplet state invariably lies lower in energy than the lowest excited singlet state of the same molecule, phosphorescence will occur at wavelengths longer than those of fluorescence and, therefore, at wavelengths much longer than those of the excitation spectrum. As in the case of fluorescence, the phosphorescence spectrum and the phosphorescence excitation spectrum are distorted by the instrumental components and therefore do not represent true spectra. 

On the other hand, the excimer emission because it is 80% non-correlated with monomer trap emission and because it is effectively quenched in the copolymers even at low temperatures, must largely arise from a mobile precursor. The activation energy for hopping of this precursor is implied to be <10 cm l. This is not unreasonably low(12,17), and indeed, the zero-point energy of the phenyl chromo-phore could in principle allow completely activationless hopping (tunneling) at reasonable rates. Determination of the true situation will require measurements at still lower temperatures, which are now in progress. We note that the polystyrene emission spectrum at 4.2K reported in (Id) indicates a monomer/excitner intensity ratio nearly the same as our 20K spectra. 

On the other hand, the a—Pu My photoabsorption is almost similar in form to a transition metal p photoabsorption. The jump denotes the presence of transitions to / states hybridized with states belonging to a continuum. This is probably also true for the Sj and 83 structures. One resonance line has been observed in the My emission spectrum in coincidence with the R2 peak and none in the M,y spectrum. It seems that in a—Pu, only a part of empty 5/states are locahzed there are states toward which a 3 5 2 electron can be excited. All the others are 5/—6cf hybridized states these are, in particular, states toward which a 3 d- i electron has a large probability of being transferred. Thus, the absorption curves present a jump just at the Fermi level and their inflexion point gives the position of this level. 

True spectrofluorometcrs allow production of a fluorescence excitation spcclruni or a fluorescence emission spectrum. Figure 1.5-9a shows an excitation spectrum for anthracene in which the fluorescence emission was measured at a fixed wavelength while the excitation wavelength was. scanned. With suitable corrections for variations in source output intensity and detector response as a function of wavelengtli, an absolute excitation spectrum is obtained that closely resembles an absorption spectrum. 

In contrast, the fluorescence of PTPAF lb closely resembles its solution spectrum, with maxima at 428 run (2.90 eV) and 452 nm (2.74 eV), and no peak in the low-energy region. The same is true for the solid state emission spectrum of PBPF (Ic), i.e., another PF polymer with bulky side groups, which shows a peak at 457 nm (2.71 eV, pure blue emission), with no emission in the low-energy region. 

The red-shifted spectrum in Fig. 5a is due to self-absorption of the chemiluminescence by the aerosol particles while the lower curve is the true emission spectrum in solution. 

The donor contribution in the acceptor channel (crosstalk) should be as low as possible the impact of this contribution on a bioassay is not obvious to anticipate starting from a lanthanide complex emission spectrum, since many instmmental factors, such as the filter settings (bandpass width), have to be considered. The intensity distribution between the emission lines is critical, particularly for europium complexes, with a strong impact of the ligand structure and symmetry (for terbium complexes, this impact is reduced). Care must be exercised in comparing published emission spectra, since many of the published spectra are not corrected for the photomultiplicator sensitivity (which falls off rapidly between 650 and 800 nm even using a red PMT ). The consequence is that the 690-nm ( Dq p4) band seems much smaller than its true value. Some articles do indeed show spectra corrected for the sensitivity of the detection system (which contains contributions from the PMT, but also from the monochromators and optics). Whenever such corrections have been applied, this is usually indicated in the experimental section of the article. 

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