Luminescence fluorescence spectra

Europium, and to a lesser extent terbium, complexes of /3-diketones have been studied in solution and in the solid state by means of their fluorescence (luminescence) spectra. As explained further in Section 39.2.10, it is possible to relate the splitting of the Do—> F transitions of Eu " to the symmetry of the emitting complex, and studies of circularly polarized luminescence (CPL) spectra can give related information. Thus a study of EuCls and complexes of Eu with hexafluoroacetylacetone and four other /8-diketones in methanol or DMF showed that while EUCI3 itself had axial symmetry in solution, the complexes had orthorhombic symmetry. The emission spectra of solutions of adducts of Eu(dpm)3 with PhsPO or borneol have been studied at low temperatures where conformal lability is reduced the Ph3PO adduct has uniaxial symmetry but the bulky, less symmetrical bomeol molecule confers lower symmetry on its adducts. ... 

Fig. 1.5 Fluorescence emission spectrum of the luciferase-oxyluciferin complex in the same solution as in Fig. 1.4 (solid line), compared with the luminescence spectrum of firefly luciferin measured in glycylglycine buffer, pH 7.6 (dotted line). The former curve from Gates and DeLuca, 1975 the latter from Selinger and McElroy, 1960, both with permission from Elsevier.

According to Charbonneau et al. (1985), aequorin is a single chain peptide consisting of 189 amino acid residues, with an unblocked amino terminal. The molecule contains three cysteine residues and three EF-hand Ca2+-binding domains. The absorption spectra of aequorin and BFP are shown in Fig. 4.1.3, together with the luminescence spectrum of aequorin and the fluorescence spectrum of BFP. 

Fig. 4.1.3 Absorption spectra of aequorin (A), spent solution of aequorin after Ca2+-triggered luminescence (B), and the chromophore of aequorin (C). Fluorescence emission spectrum of the spent solution of aequorin after Ca2+-triggered bioluminescence, excited at 340 nm (D). Luminescence spectrum of aequorin triggered with Ca2+ (E). Curve C is a differential spectrum between aequorin and the protein residue (Shimomura et al., 1974b) protein concentration 0.5 mg/ml for A and B, 1.0 mg/ml for C. From Shimomura and Johnson, 1976.

Fig. 4.1.16 Luminescence spectrum of aequorin triggered by Ca2+ (solid line /.max 465 nm), and the fluorescence spectra of Aequorea GFP excitation (dashed line A.max 400 nm and 477 nm) and emission (dash-dot line 7max 509 nm). The dotted line is the fluorescence excitation spectrum of GFP in the light organs, showing that 480 nm excitation peak is almost missing — an evidence showing that GFP in light organs exists in an aggregated form having a very low E value at 480 nm.

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.

Fig. 6.1.5 Fluorescence spectra of the purple protein (1-4) and the luminescence spectrum measured with Latia luciferin, luciferase and the purple protein (5 Xmax 536 nm). Excitation spectra (1) and (2) were measured with emission at 630 nm and 565 nm, respectively. Emission spectra (3) and (4) were measured with excitation at 285 nm and 380 nm, respectively. From Shimomura and Johnson, 1968c, with permission from the American Chemical Society.

Fig. 7.1.5 Fluorescence spectra of purified Chaetopterus photoprotein (CPA) in 10 mM ammonium acetate, pH 6.7 (solid lines), and the bioluminescence spectrum of the luminous slime of Chaetopterus in 10 mM Tris-HCl, pH 7.2 (dashed line). Note that the luminescence spectrum of Chaetopterus photoprotein in 2 ml of 10 mM Tris-HCl, pH 7.2, containing 0.5 M NaCl, 5 pi of old dioxane and 2 pi of 10 mM FeSC>4 (Amax 453-455 nm) matched exactly with the fluorescence emission spectrum of the photoprotein. No significant change was observed in the fluorescence spectrum after the luminescence reaction.

Harvey (1952) demonstrated the luciferin-luciferase reaction with O. phosphorea collected at Nanaimo, British Columbia, Canada, and with O. enopla from Bermuda. McElroy (1960) partially purified the luciferin, and found that the luminescence spectrum of the luciferin-luciferase reaction of O. enopla is identical to the fluorescence spectrum of the luciferin (A.max 510 nm), and also that the luciferin is auto-oxidized by molecular oxygen without light emission. Further investigation on the bioluminescence of Odontosyllis has been made by Shimomura etal. (1963d, 1964) and Trainor (1979). Although the phenomenon is well known, the chemical structure of the luciferin and the mechanism of the luminescence reaction have not been elucidated. 

The absorption spectrum of the photoprotein showed a small peak (Xmax 423 nm, with a shoulder at about 450 nm) in addition to the protein peak at 280nm (Fig. 10.1.2). The peak at 423nm decreased slightly upon the FI202-triggered luminescence reaction. The photoprotein is fluorescent in greenish-blue (emission A.max 482 nm), which coincides exactly with the luminescence spectrum of the photoprotein... 

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).

In the presence of an activator, naturally, the spectrum, yield, and lifetime are characteristics of the activator molecule. Indeed, the luminescence spectrum of the oxidized ethylbenzene was found to be identical to that of activator fluorescence [221]. 9,10-Dibromanthracene, 9,7-dipropylanthracene, and derivatives of oxazole were used as activators [221,223]. 

The value of the phosphorescence quantum yield can be determined by measuring the total luminescence spectrum under steady irradiation. If the fluorescence quantum yield is known then the phosphorescence quantum yield may be found by comparing the relative areas under the two corrected spectra. 

In the first attempts to overcome the background problem using decay time, the variation of the fluorescence decay time as a function of wavelength across the entire emission profile for a variety of materials have been used (Measures 1985). For a variety of rocks and minerals, it was proved that this information represents a new kind of signature, the so called fluorescence decay spectrum, that possesses considerable discrimination power, being able to characterize the irradiated material with far superior precision than the normal luminescence spectrum (Fig. 7.2). 

As the removal of a proton from thymine results in the establishment of a tautomeric equilibrium between its two monoanionic forms,3425 the emission spectrum of singly ionized thymine may consist of overlapping spectra of both monoanions. In fact, Gill344 has observed some inconsistencies between the absorption and the fluorescence excitation spectra of thymine in 0.01 N NaOH at room temperature. These inconsistencies were of the same kind as those found later by Berens and Wierzchowski,345 who suggested that at room temperature only the thymine monoanion tautomer (34) fluoresced, while at 77°K emissions of both monoanionic species contributed to the observed luminescence spectrum. 

Broad band laser oscillation from Coumarin 153 doped ORMOSIL gels was easily obtained in the free-running laser cavity. The laser emission and the luminescence spectrum both peak at nearly the same wavelength as shown in Figure 4. The laser emission peak was at 526 nm with an oscillation bandwidth of approximately 20 nm FWHM. (For comparison, the reported total oscillation bandwidth in ethanol pumped at 308 nm is 75 nm.) The fluorescence spectrum has a broad peak at about 530 nm with a bandwidth (FWHM) of about 80 nm. 

The laser emission peak from R6G doped ORMOSIL gels occurred at 571 nm with a bandwidth of 4 nm. The laser emisison band is narrower than the FWHM fluorescence band. The doped ORMOSIL sample exhibited a luminescence peak at 565 nm with a bandwidth of 55 nm (FWHM) In contrast to the C153 gel, the solid state rhodamine doped sample did not oscillate over the FWHM range of die fluorescence emission spectrum. The R6G samples exhibited detectable oscillation over a total range of about 38 nm (559 to 587 nm). 

Study of these and other transitions can thus yield valuable information. Crystals of the nine-coordinate complex [Eu(tmhd)3(terpy)] contain two slightly different molecules present in the crystal, its luminescence spectrum showing a broad but imresolved Do Fo transition. In solid [Eu(tmhd)3(Me2phen)] (Ln = La, Eu, Tb, Ho), there are two different square-antiprismatic isomers in the unit cell, and in this case emissions from both isomers can be distinguished in the fluorescence spectrum of the europium complex, which shows an unusually high splitting of the Dq Fq transition. ... 

Commercial spectrometers, such as the Perkin-Elmer MPF-43A fluorescence spectrometer, that allow interlocking of excitation and emission monochromators lately have become available for utilizing this underexploited analytical technique. The synchronous luminescence technique reduces the complexity of the luminescence spectrum of a compound compared with a conventionally obtained luminescence spectrum. One can, therefore, better tackle the analysis of fairly complex mixtures without resorting to techniques that are expensive or excessively time consuming. 

Emission of phosphorescence by 1,2-dioxetanes and a-peroxylactones has also been observed, but is quite rare. Thus, in degassed acetonitrile the 430 nm emission exhibited by the tetramethyl-l,2-dioxetane (7) has been assigned to acetone phosphorescence. Similarly, this acetone phosphorescence has been detected for the dimethyl-a-peroxylactone. ° For the acetyl derivative (19), both the n,TT fluorescence and phosphorescence of 2,3-butanedione have been reported. Thus, if the photoexcited luminescence spectrum of the carbonyl product is known or can be readily measured, the chemiluminescence spectrum can be used as corroborative structure confirmation of the 1,2-dioxetane or a-peroxylactone. 

Luminescent standards have been established for use in calibrating fluorescence spectrometers and have been suggested for Raman spectroscopy in the past (18). The standard is a luminescent material, usually a solid or liquid, that emits a broad reproducible luminescence spectrum when excited by a laser. Once the standard is calibrated for a particular laser wavelength, its emission spectrum is known, and it can provide the real standard output , d)i(AF) depicted in Figure 10.8. In practice, a spectrum of the standard is acquired with the same conditions as an unknown then the unknown spectrum is corrected for instrument response function using the known standard... 

Molecular fluorescence spectroscopy is a commonly employed analytical method that is sensitive to certain chemical properties of FA (9-13). Fulvic acid s molecular fluorescence is principally due to conjugated unsaturated segments and aromatic moieties present in the macromolecule (14). Several types of fluorescence spectra can be measured, including an excitation emission matrix or total luminescence spectrum, constant offset synchronous fluorescence, excitation spectra, and emission spectra, furnishing the researcher with useful data. The ability to resolve and select multiple fluorescent species makes these approaches extremely useful for studying FA relative to its chemical reactivity. 

Become familiar with the operation of the fluorescence spectrophotometer in your laboratory. In particular, you should understand how the following instrumental parameters affect the intensity and signal-to-noise ratio (S/N) of a luminescence spectrum ... 

In order to explain the data one needs at least a two-level scheme. These two states may reside on the same molecule or be representative of two distinct excited species in solution. Consider the case in which two distinct excited species exist in solution. In this context, the appearance of the 660 nm absorption change between 290 and 270 K would indicate a shift in the relative equilibrium concentration of the two species. If both species were fluorescent, the emission spectrum from each species would almost certainly be different. Thus one would expect that the luminescence spectrum would change with temperature. Moreover, unless the fluorescence lifetimes of the two species were identical the observed emission lifetime... 

---