Corrected emission spectrum

The development of methods to obtmn excitation and emission spectra corrected for wavelength-dependent effects has been the subject of nummous investigations. Overall, none of these methods is completely satisfactory, especially if the corrected spectra are needed on a regular basis. Prior to correcting spectra, the researcher should determine if such corrections are necessary. Frequently, one only needs to compare emission spectra with other spectra collected on the san instrument. Such comparisons are usually made between the technical (or uncor-... 

Absorption Spectrum 0 Emission Spectrum Emission Spectrum corrected for the sensitivity of the humer) eye... 

Figure 24.7 Intensity normalised Ols emission spectrum, corrected with respect to the snhstrate component, for increasing Ca film thicknesses. In addition to the snhstrate component, Ca-hydroxide and Ca-oxide components are identified in the adsorbate.

Since the measurement of die intrinsic quantum yield is very hard to perform, it is usually replaced by the calculation of the radiative lifetime. However, this calculation is also very complicated, except for the special case of europium where approximations yield a simple convenient formula. It comes from the very rare property of europium to exhibit a purely MD transition, the Dq, the intensity of which is practically independent on the chemical environment. The calculation then only needs the emission spectrum (corrected to take into account the sensitivity function of the detector) and uses die ratio of the total intensity of the lanthanide emission over the intensity of the MD transition, as well as the refractive index of the medium n, and a parameter Amd.o that defines the strength of the MD transition. For Eu +, Amd.o = 14.65 5 . ... 

Fig. 4.2.2 Left panel-. Uncorrected Ca2+-triggered bioluminescence spectrum of W92F obelin derived from O. longissima. Right panel Corrected bioluminescence spectrum of the same obelin (dotted line), and the fluorescence emission spectrum of the spent solution after luminescence (solid line). From Deng et al., 2001, with permission of the Federation of the European Biochemical Societies.

Luminescence of Pyrosoma. All species of the genus Pyrosoma (about 10 species) are bioluminescent. Pyrosoma is one of the few organisms reported to luminesce in response to light (Bowlby et al., 1990). The luminescence emission spectrum of Pyrosoma atlantica is bimodal according to Kampa and Boden (1957), with the primary peak near 482 nm, and the secondary near 525 nm. Swift et al. (1977) reported the emission maxima of two Pyrosoma species at 485 and 493 nm, respectively, and Bowlby et al. (1990) found an emission peak at 475 nm with P. atlantica. A corrected bioluminescence spectrum of P. atlantica (A.max 485 nm) reported by Herring (1983) is shown in Fig. 10.5.2. 

Figure 1. Average corrected emission spectrum (- -) and excitation spectrum (- -) for quinine sulfate In 0.1 mol/L HC10 obtained during round-robin test with ten laboratories coefficient of variation at each wavelength (-t).

Consider a sample with two fluorophores, A and B, whose lifetimes (lA and XB) are each independent of emission wavelength and are different fram one another. By setting 4>D = 4>A 90°, the contribution from A is nulled out and the scanned emission spectrum represents only the contribution from B. Similarly, the spectrum for A is obtained by setting 4>D = 4>B 90°. In practice, finding the correct value of for this... 

In Fig. 3 the total corrected emission spectrum of MBC in ethylcellulose filmsisshown(curve A)... 

Photophysical Processes in Pol,y(ethy1eneterephthalate-co-4,4 -biphenyldicarboxyl ate) (PET-co-4,4 -BPDC). The absorption and luminescence properties of PET are summarized above. At room temperature the absorption spectrum of PET-co-4,4 -BPDC copolymers, with concentrations of 4,4 -BPDC ranging from 0.5 -5.0 mole percent, showed UV absorption spectra similar to that of PET in HFIP. The corrected fluorescence spectra of the copolymers in HFIP exhibited excitation maxima at 255 and 290 nm. The emission spectrum displayed emission from the terephthalate portion of the polymer, when excited by 255 nm radiation, and emission from the 4,4 -biphenyldicarboxylate portion of the polymer when excited with 290 nm radiation. 

The fluorescent components are denoted by I (intensity) followed by a capitalized subscript (D, A or s, for respectively Donors, Acceptors, or Donor/ Acceptor FRET pairs) to indicate the particular population of molecules responsible for emission of/and a lower-case superscript (d or, s) that indicates the detection channel (or filter cube). For example, / denotes the intensity of the donors as detected in the donor channel and reads as Intensity of donors in the donor channel, etc. Similarly, properties of molecules (number of molecules, N quantum yield, Q) are specified with capitalized subscript and properties of channels (laser intensity, gain, g) are specified with lowercase superscript. Factors that depend on both molecular species and on detection channel (excitation efficiency, s fraction of the emission spectrum detected in a channel, F) are indexed with both. Note that for all factorized symbols it is assumed that we work in the linear (excitation-fluorescence) regime with negligible donor or acceptor saturation or triplet states. In case such conditions are not met, the FRET estimation will not be correct. See Chap. 12 (FRET calculator) for more details. 

Figure 12.7 Electronic transitions giving rise to the emission spectrum of sodium in the visible, as listed in Table 12.1. The principal series consists of transitions from the 3s level to 3p or a higher p orbital the sharp series from 3p to 4s or a higher s orbital diffuse from 3p to 3d or above and the fundamental from 3d to 4/or higher. The terms below the lines [(R/(3-1.37)2, etc.] are the quantum defect corrections referred to in Section 10.4.

Alternatively, a standard fluorescent compound can be used whose corrected emission spectrum has been reported3 . Comparison of this spectrum with the technical spectrum recorded with the detection system provides the correction factors. The wavelength range must obviously cover the fluorescence spectrum to be corrected. Unfortunately, there is a limited number of reliable standards. 

Proper correction of the emission spectrum is a prerequisite for the measurement of the fluorescence quantum yield of a compound. Quantum yields are usually determined by comparison with a fluorescence standard4, i.e. a compound of known quantum yield that would ideally satisfy the following criteria (Demas, 1982) ... 

In order to minimize the effects of possible inaccuracy of the correction factors for the emission spectrum, the standard is preferably chosen to be excitable at the same wavelength as the compound, and with a fluorescence spectrum covering a similar wavelength range. 

The emitted spectral radiant power (or exitance) or the emitted spectral photon trradiance (or exitance) plotted as a function of the frequency, wavenumber, or wavelength. The corrected emission spectrum has been cor-... 

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... 

Figure 23-13 (A) Corrected emission and excitation spectra of riboflavin tetrabutyrate in w-heptane. Concentration, about 0.4 mg I-1. Curve 1 excitation spectrum emission at 525 nm. Curve 2 emission spectrum excitation at 345 nm. FromKotaki and Yagi.128 (B) Indole in cyclohexane, T = 196 K. 1, Fluorescence excitation spectrum 2, fluorescence spectrum and 3, phosphorescence spectrum. From Konev.125...

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 8 Corrected emission spectrum of pyrazolyl-bridged iridium(I) dimer, Ir2(jx-pz)2(CO)4, where pz = pyrazole. Fluorescence maximum is at 540 nm and phosphorescence maximum is at 750 nm. (Reprinted with permission from Ref. 109.)...

Figure 14.1 Normalized corrected emission spectrum of apoHb-ANS complex upon excitation at 350 nm. 2 Normalized absorption spectrum of apo FIA. Reprinted with permission from Sassaroli, M., Bucci, E., Liesegang, J., Fronticelli, C. and Steiner, R. F. (1984). Biochemistry, 2487-2491. Copyright 1984 American Chemical Society.

Silicate, nickel, and cobalt tend to interfere in the air-acetylene flame, although nickel and cobalt are rarely present in sufficient excess to cause a problem. Silicate interference may be eliminated at modest excesses by the use of lanthanum as a releasing agent or by using a nitrous oxide-acetylene flame. Very careful optimization is sometimes necessary, for example in the analysis of freshwaters, when concentrations are very low. It is important to use a narrow spectral bandpass and to make sure that the correct line is being used, because the hollow cathode lamp emission spectrum of iron is extremely complex. If you have any doubts about monochromator calibration, check the sensitivity at adjacent lines ... 

Figure 3. Representative emission of a dual-emitting heterocyclic-substituted platinum 1,2-enedithiolate. The emission spectra of [(dppelPtjSiCitCHiCHi-iV -pyridiniumlJllBPfr,], in dimethyl sulfoxide (DMSO) at 298 K (no instrument correction applied) Solid line, emission spectrum under N2 (emission from both the ILCT and 3ILCT ) Dashed line, emission spectrum under air (emissions from the iLCT ).

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