Fluorescence Excitation and Emission Spectra

The steady-state emission spectrum of the crystals obtained with unpolarized excitation light (Fig. 8.52A) does not overlap the emission spectrum of ai- acid glycoprotein - progesterone complex obtained in solution (Fig. 8.52B), but overlaps the spectrum of hydrophobic Trp residues of the protein (Fig. 8. 53). Thus, the fluorescence observed for the crystal is characteristic of Trp residues embedded in the protein matrix. Therefore, the two Trp residues surrounded by a hydrophobic environment have the same microenvironments in crystal and in solution. 

Since the fluorescence emission spectrum of the crystals is characteristic of Trp residues present within a hydrophobic environment, this means that the fluorescence of the surface Trp residue is completely quenched. This result may be explained by the fact that the protein-protein interaction in the crystal occurs via the surface of the protein. The Trp residue at the surface will be in contact with two proteins, facilitating high energy transfer to the neighboring amino acids. This result is in good agreement with that found by x-ray diffraction studies, i.e., the proteins are linked together at their surface by the carbohydrates. 

When excitation was performed with a vertical polarized light, whether on solution of ai- acid glycoprotein or on the crystal, the band widths of the spectra are 10 nm smaller than those observed when the spectra were recorded with unpolarized light. In the crystal, the bandwidth is equal to 35 1 nm instead of 43 1 nm, and in solution, it is equal to 41 1 nm instead of 54 1 nm. We do not know whether this phenomenon is due to the polarizer used or not. 

Excitation at 300 nm gives an emission maximum in the same range as that obtained with excitation at 295 nm. Therefore, there is no evidence of tyrosine fluorescence contribution at excitation wavelengths equal to or higher than 295 nm. This contribution may account for a blue-shifted spectrum. 

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Fig. 10.1.3 Fluorescence excitation and emission spectra (solid lines) and H2O2-triggered luminescence spectrum (dashed line) of Ophiopsila photoprotein (Shimomura, 1986b, revised). The dotted line indicates the in vivo bioluminescence spectrum of Ophiopsila californica plotted from the data reported by Brehm and Morin (1977).

Also bound to the UV-Vis spectral area is fluorescence spectrometry. It is most important with respect to those fluorescent food colorants that have been incorporated into food. In detail it helps to (1) identify a colorant by the spectral pattern of fluorescence excitation and emission spectra, (2) quantify its concentration by the fluorescence emission intensity, (3) qualify the enviromnent into which the colorant molecule is embedded, and (4) perform structural research on the food matter into which the colorant is incorporated. 

Photophysical Processes in Dimethyl 4,4 -Biphenyldicarboxy-late (4,4I-BPDC). The ultraviolet absorption spectrum of dimethyl 4,4 -biphenyldicarboxyl ate was examined in both HFIP and 95% ethanol. In each case two distinct absorption maxima were recorded, an intense absorption near 200 nm and a slightly less intense absorption near 280 nm. The corrected fluorescence excitation and emission spectra of 4,4 -BPDC in HFIP at 298°K shows a single broad excitation band centered at 280 nm with a corresponding broad structureless emission band centered at 340 nm. At 77°K, the uncorrected phosphorescence spectra shows a single broad structureless excitation band centered at 298 nm, and a structured emission band having maxima at 472 and 505 nm with a lifetime, t, equal to 1.2 seconds. 

Figure 23 Fluorescence excitation and emission spectra, (a) virgin EVA sample (excitation = 280 nm emission = 360 nm) (b) EVA sample degraded for lh at 180°C (excitation = 239 nm emission = 390 nm) (c) EVA sample degraded for 1 h at 180°C (excitaton = 301 nm emission = 361 nm) (d) EVA sample degraded for 2 h at 180°C (excitation = 238 nm emission = 388 nm). Reprinted from Allen [67]. Copyright 2000, with permission from Elsevier. (This figure has been reproduced from the original in reference [67], however it would appear that the labels excitation and emission have been incorrectly inserted and should be switched for parts (b), (c) and (d).)...

Griseofulvin exhibits both fluorescence and luminescence. A report by Neely et al., (7) gives corrected fluorescence excitation (max. 295 nm) and emission (max. 420 nm) spectra, values for quantum efficiency of fluorescence (0.108) calculated fluorescence lifetime (0.663 nsec) and phosphorescence decay time (0.11 sec.). The fluorescence excitation and emission spectra are given in Figure 7. 

Konev(10) and Vladimirov(n) were carrying out similar work in the Soviet Union. In 1957, Teale and Weber0 2) reported the first careful, thorough investigation of the fluorescence excitation and emission spectra of the aromatic amino acids. 

Compounds 1,2,3,5,10,11,12,13,14 were dissolved in EPIP (diethyl ether, petroleum ether, isopropanol 5 5 2)whereas compounds 4,6,7,8,9,15 were dissolved in THF-DE (tetrahydrofurane, diethyl ether 1 1). These solvent mixtures can be frozen as glassy samples at 77 K. The absorption spectra were recorded on a standard spectrophotometer SF-10 or Beckman-5270. The measurements of fluorescence excitation and emission spectra were made with the aid of a spectrofluorometer SLM-4800 with automatic correction of spectral response. Fluorescence lifetimes were measured with the aid of a pulse fluorometer PRA-3000. Magnetic circular dichroism (MCD) measurements were carried out in a 8 kG magnetic field using a JASCO J-20 circular dichrometer. Triplet state formation was observed for investigated compounds at the experimental set up, whose detailed description can be found in our paper (27). The optical experiments were carried out with a porphyrin concentration of 4.10- - 4.10 mol.l". In NMR investigations (Bruker WM-360) we used higher concentrations ( 5.10" raol.l ) and dried solvents (CDCl, C 2 and toluene-d0). 

Electronic absorption and fluorescence excitation and emission spectra of phenazines were determined in several solvents of various polarities <1995SAA603>, and the effect of the solvent upon the spectral characteristics was studied. 

Tryptophan, tyrosine, and phenylalanine are the three natural amino acids that give rise to the intrinsic fluorescence of peptides in the ultraviolet region. Reliable, corrected fluorescence excitation and emission spectra of these aromatic amino acids were first published by Teale and Weber.M The fluorescence emission maxima of tryptophan, tyrosine, and phenylalanine in water are at 348, 303, and 282 nm, respectively. The photophysics and photochemistry of tryptophan and tyrosine have been comprehensively reviewed.1910 ... 

The laser atomic fluorescence excitation and emission spectra of sodium in an air-acetylene flame are shown below. In the excitation spectrum, the laser (bandwidth = 0.03 nm) was scanned through various wavelengths while the detector monochromator (bandwidth = 1.6 nm) was held fixed near 589 nm. In the emission spectrum, the laser was fixed at 589.0 nm, and the detector monochromator wavelength was varied. Explain why the emission spectrum gives one broad band, whereas the excitation spectrum gives two sharp lines. How can the excitation linewidths be much narrower than the detector monochromator bandwidth ... 

Fluorescence excitation and emission spectra of the two sodium D lines in an air-acetylene flame, (a) In the excitation spectrum, the laser was scanned, (to) In the emission spectrum, the monochromator was scanned. The monochromator slit width was the same for both spectra. [From s. J. Weeks, H. Haraguchl, and J. D. Wlnefordner, Improvement of Detection Limits in Laser-Excited Atomic Fluorescence Flame Spectrometry," Anal. Chem. 1976t 50,360.]... 

Fig. 2 shows typical fluorescence excitation and emission spectra obtained from alcoholic solutions of Florida Valencia SSOJ s. Fluorescence spectra obtained from Pineapple and Hamlin orange juices were similar to but of lower intensity than Valencia. Early in the Pineapple season the emission spectrum may appear as a flat apex (from about 310 to 333 nm). Early season Hamlin juice may produce an emission spectrum with 310 nm as the maximum dropping to an inflection at 333 nm. As the fruit matured 333 nm became the emission maximum. Maximum excitation and emission (for the three varieties) were also evident at 290-93 and 343 nm, respectively, producing excitation spectra similar to Fig. 2 (shoulder at 283 nm and slight inflection changes at 270 and 302 nm). Valencia variety exhibited greater fluorescence than Hamlin or Pineapple varieties. 

Figure 4. Comparison of fluorescence excitation and emission spectra of orange juice (----) and orange pulpwash ( — )...

Visible and ultraviolet absorption and room temperature fluorescence excitation and emission spectra obtained from alcoholic... 

The fluorescence excitation and emission spectra of the electrogenerated fused benzothiophene oligomers [poly(39) and poly(41)] show the existence of dramatic red shifts of the fluorescence maxima and important increases of the fluorescence intensity relative to the parent monomers. These results suggest the existence of extended electronic conjugation in the oligomer chains. Poly(39) and poly(41) showed a well-structured excitation band with maxima at about 335 nm and 395 nm, respectively. These excitation maxima are strongly red shifted by about 50 and 108 nm, respectively, against the 39 and 41 excitation spectra. The emission spectra are characterized by a relatively wide, poorly structured band, centered at 410 nm and 445 nm, respectively. These emission maxima also present dramatic red shifts relative to the emission spectra of the parent monomers. 

The electropolymerization of 3-methoxythiophene (MOT) was performed in an aqueous micellar medium containing sodium dodecylsulfate (SDS) as a surfactant. The electronic absorption spectra, the fluorescence excitation and emission spectra, and the quantum yields of 546, were measured in different solvents of various polarities and hydrogen bond abilities (89SM(28)C487 98SM(93)175 00JF107 00POLLDG4047 ... 

A simple, rapid, sensitive, and selective spectrofluorimetric method (2ex/ lem = 345/455nm) has been developed for the determination of zaleplon. Tang et al. have studied the influence of micellar medium on the absorption, fluorescent excitation, and emission spectra character of zaleplon The nonionic surfactant of Triton X-100 showed a strong sensitizing effect for the fluorescence of zaleplon in a pH 5.0 buffer. The possible enhancement mechanism was discussed. Based on the optimum conditions, the linear range was 1.32 x 10 8-1.00 x 10 mol/1. The detection limit was 4.0 x 10 mol/1 with a relative standard deviation (RSD) of 0.06%. The proposed method was successfully applied to the determination of zaleplon in tablets, serum, and urine. 

Figure 10.5 Fluorescence excitation and emission spectra of (a) donor and (b) acceptor molecules.

Figure 13.4 Structure of the IRE RNA with A7 and G8 indicated (Hall and Williams, 2004). Top right fluorescence excitation and emission spectra of IRE 2AP7 and IRE 2AP8. Bottom fluorescence emission intensity of each IRE as a function of temperature, 4 jiM RNAs, 30 mM NaCl, 10 mM potassium phosphate, pH 7.0, 20 °C. The buffer baseline was subtracted from each spectrum (the Raman line is at 350 nm).

Figure 1.2. Comparison of the fluorescence excitation and emission spectra of hypericin (a) and hypocrellin (b) in a 1 1 ethanol/methanol mixture at room temperature. The excitation spectra were measured at 295 K (solid line) and at 77 K (dashed line). The excitation spectra were monitored at 650 nm at 295 K and at 620 nm at 77 K.

Some fluorophores are used to detect the presence of sodium or potassium in cells. For example, the maxima of the fluorescence excitation and emission spectra of SBFI increase, and its emission maximum shifts toward short wavelengths in the presence of sodium (Figure 7.17). 

Corrections of the fluorescence excitation and emission spectra for the optical densities (inner filter effect) yield an increase in the intensities without inducing any shift in any of the maxima. This is true for all recorded spectra. At the optical densities we are suggesting, you may find that these corrections are not significant and even not necessary for all fluorophores but ethidium bromide. Its intensity increases without any modification in the peak position. 

The results obtained are identical to many data obtained by different authors. However, one should remember that the literature also shows a fluorescence spectrum of bound ethidium bromide located at 605 nm to the difference of the peak observed for free ethidium bromide (see, e.g. the fluorescence excitation and emission spectra published by Molecular Probe) (Figure 12.5). However, this is not always observed, as is the case here. 

Polymeric Systems.—Absorption, fluorescence excitation, and emission spectra of soluble poly(diacetylene) at room and liquid nitrogen temperature has been recorded for different solvents. The excimer-to-monomer emission ratio for polystyrene depends upon the polymer molecular weight but is independent of the solvent. Monolayer films of the 27-carbon-chain-length alkyldiace-... 

Fig. 11.5. Fluorescence excitation and emission spectra of ZnSe nanocrystals showing the band edge emission. Adapted from [17].

Figure 1 shows the fluorescence excitation and emission spectra of 1 in CH2CI2. The excitation spectrum was found to be independent of the monitoring wavelengths and was identical to the absorption spectrum. Three emission bands at Xp 645, 660 and 702 nm are observed in the emission spectrum and they are designated as the a-, the j5- and the y-band respectively. Controlled experiments showed that the multiple emission is from 1 rather than from any impurities or any aggregational states of 1. 

Figure 1. Corrected fluorescence excitation and emission spectra of 1 in methylene chloride ([1] 3x1O 7 M).

Fluorescence Spectra. frons-Stilbene and some derivatives show fluorescence emission in the range from 340 to 400 nm [187], the fluorescence maximum (lf) is sensitive to substituents (Table 10). m-Stilbenes, in contrast, exhibit no fluorescence in solution at ambient temperatures [95]. Only recently, the fluorescence excitation and emission spectra (A = 275 and 409 nm, respectively) of ris-stilbene in n-hexane at room temperature have been reported [240,241], A very short lifetime (<10ps) has been assumed for the excited cis singlet state in fluid solutions (Section A.5). At lower temperatures (below — 120°C) and in rigid media c/s-stilbene shows a broad emission around 450 nm [113, 176, 242 245]. It does not originate from the trans isomer or a cyclization product and is hence assigned to fluorescence [246], Increased viscosity of the solvent likewise results in an increase of the fluorescence quantum yield of the cis isomer ([Pg.39]

Figure 11. Absorption, fluorescence excitation, and emission spectra (dotted, dashed, and full lines, respectively) of frans-4,4 -NAS in (a) toluene, (b) n-pentane, and (c) MTHF at RT [152, reproduced with permission].

Hexane Extraction and Bromination. Some of the fluorescent enones are extractable from the amorphous regions of the polymer matrix by cold hexane (1,13). This prpcess enables us to perform a simple chemical test for the presence of unsaturation, i.e., bromination (18). Figure 4 shows the fluorescence excitation and emission spectra of the cold hexane extract of commercial polypropylene before and after treatment with bromine. After bromination there is a significant reduction in the fluorescence intensity, confirming the presence of unsaturation. A similar result was obtained on brominating a cold hexane extract of poly-4-... 

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