Absorption Spectra and Extinction Coefficients

A Beckman DU spectrophotometer was used for all measurements. The slit width was chosen so that a half-intensity band width of 1.6 nm was obtained in the region X = 650-590 nm, 0.6 nm in the region X = 590-A60 nm, and 0.15 nm in the region of the Soret bands. The wavelength of the instrument had been checked with the aid of the mercury emission spectrum. Measurements at X 590 nm were made in a layer thickness of 0.0993 cm, at X 590 nm in a layer thickness of 0.0128 cm. The latter was obtained by inserting a plane parallel glass plate into the 0.0993-cm cuvette (Fig. 13). The layer thickness of the cuvette had been determined spectrophotometrically using HiCN solutions and a 1.000-cm cuvette as a reference. 

Assuming Lambert-Beer s law to be valid for all solutions over the entire spectral region, can be calculated from for any hemoglobin derivative, when the total hemoglobin concentration of the sample has been determined  

The hemolysate (Section 3.2) was further diluted with 0.7% Sterox SE solution until an optical density of about 0.500, measured in a layer thickness of 0.0128 cm at X = 577 nm, was reached. The total hemoglobin concentration (Chs tot.) of the diluted hemolysate was measured (as described in Section 3.1). To the sample, Na SjOt (4 mg/ml) was added for complete conversion to Hb, and the spectrophotometric measurements 

e/X curve of hemoglobin (Hb). Quarter millimolar extinction coefficients based on = 11.0. The solid part of the graph represents the mean value resulting from 

5 samples, the dashed part results from measurements of a single sample. Measurements made with a Beckman DU spectrophotometer, layer thickness 0.0128 cm 10-fold dilution of samples for measuring below X = 460 nm. 

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Bowen, W.J, 1949. The absorption spectra and extinction coefficients of myoglobin. J. Biol. Chem. 179 235-245. 

Radicals of the cyclohexadienyl type show moderately intense peaks at 300-350 nm (e 3000-4000 M -1 cm-1). Radicals of this type are produced by addition of H or OH to benzene and its derivatives, and their spectral parameters are affected by the substituent. In general, it is found that both the OH and H adducts have very similar absorption spectra and extinction coefficients. The electron adducts usually absorb at somewhat lower wavelengths. The effects of substituents on the absorption maxima, as expressed by the bathochromic shifts [( ArH — Arx)IvAth where v is the wave... 

Radical cations of methoxybenzenes efficiently oxidize phenols and other reductants. For example, the radical cations of anisole, 1,3-dimethoxybenzene (DMB), and 1,3,5-trimethoxybenzene (TMB), produced in >90% yield by reaction of OH with the methoxybenzenes at pH 1, can oxidize phenols and other reductants. The product radicals were identified in most cases by their known absorption spectra and extinction coefficients. The rate constants, determined by monitoring the buildup of the product radical and/or the decay of the radical cation as a function of the concentration of reductant, are summarized in Table 2. The rate constants are high for phenols bearing electron-donating substituents and much lower for phenols bearing strong electron-withdrawing substituents. 

Fluorescence spectra and quantum yields are generally more dependent on the environment than absorption spectra and extinction coefficients. For example, coupling a single fluorescein label to a protein reduces fluorescein s quantum yields 60% but only decreases its molar extinction coefficient by 10%. Interactions either between two adjacent fluorophores or between a fluorophore and other species in the surrounding environment can produce environment-sensitive fluorescence. 

Triplet-triplet absorption spectra and extinction coefficients (Ca, = 330, 750 nm, 40,000,... 

Absorption and extinction coefficients are generally less pH dependent than fluorescence spectra and quantum yields because the radiative rates often compete with intra- and intermolecular relaxation precesses. 

A number of investigators have studied the effect of ozone on the ultraviolet absorption spectra of proteins and amino acids. A decrease in the absorption of 280-nm light in a number of proteins was originally reported ly Giese et aV to be a consequence of ozone exposure they suggested that this was due to an interaction of ozone with the ring structures of tyrosine and tryptophan. Exposure of a solution of tryptophan to ozone resulted in a decrease in 280-nm absorption, whereas the extinction coefficient of tyrosine increased. Similar results with tyrosine were reported by Scheel et who also noted alterations in the ultraviolet spectra of egg albumen, perhaps representing denaturation by ozone. 

The synthetic GFP chromophore analogue (2-(4-nitrophenyl)-5-(4-cyanophenyl methylidene) imidazol-4-one ), was synthetized according to ref [6]. It was recrystallized from ethanol and characterized by 1H-NMR through their typical proton signal at 7.1 2 ppm. High concentrated solutions of approximately 3.10 3M were prepared by dissolution in dioxan. The photophysical characteristics of this analogue were determined from the UV absorption spectra and from steady-state fluorescence. An extinction coefficient of 20700 M cm 1 was determined at the maximum absorption wavelength at 406 nm. The fluorescence emission peaks at 508 nm. 

There are a number of advantages of using colloidal, semiconductors in artificial photosynthesis. They are relatively inexpensive. They have broad absorption spectra and high extinction coefficients at appropriate band gap energies. Nevertheless, they can be made optically transparent enough to allow direct flash photolytic investigations of electron transfers. They can be modified by derivatization or sensitizer adsorption. Importantly, electrons produced by band gap excitation can be used directly without relays for catalytic water reduction (Figure IB). 

The reactivity of (20) is more typicai of anthraquinones in genera) whiie the formation of Dewar structures (21) and (22) is the first observation of this type of reaction for the anthraquinone system. The authors note that (18) and (19) show large perturbations in their U.V. absorption spectra compared with anthraquinone (20). These perturbations include bathochromic shifts and extinction coefficient enhancements of the longest wavelength, presumably n->7r , bands which may reflect steric interactions between one of the anthraquinone carbonyls and the adjacent ferr-butyl group. Anomalous reactivity has also been found for 9-ferf-butyl-lO-cyanoanthracene (23) which upon irradiation at -20 C gave the dibenzobicyclo(2.2. llheptane (24). This is in constrast to the photochemistry of 9-ferf-butylanthracene (25) which forms the Dewar isomer (26). The latter reaction and its reversal have been examined for their... 

Ultraviolet-Visible Spectroscopy Ultraviolet-visible (UV-VIS) molecular absorption spectrophotometry (often called light absorption spectrophotometry or just UV-visible spectrophotometry) is a technique based on measuring the absorption of near-UV or visible radiation (180-770 nm) by molecules in solution.35,36 Reference standard characterization by UV-VIS spectophotometry includes determining the absorption spectra and the molar extinction coefficient. These two spectral characterizations are used as identifiers of reference standards. 

With phloroglucinol, the reaction (also in basic solution) leads to di- and if done with excess diazonium chloride even to tri-benzotriazolization. It is obvious that these compounds are of interest because of their very broad ultraviolet absorption spectra and their extremely high extinction coefficients, while at the same time having a sharp cutoff at 370 nm. 

The optical absorption spectra of the high mobility solvent holes resemble those for the radical cations isolated in freon matrices [20,22-25]. All of these spectra are bell-shaped featureless curves with maxima in the visible and/or near IR regions. In pulse radiolysis studies, the absorption signal from the solvent hole always overlaps with the signals from the fragment (and/or secondary) radical cations ("satellite ions"), even at the earliest observation times [22-25,57]. Therefore, complex deconvolutions are needed to extract the spectra of the solvent holes. This leaves large uncertainty as for the exact shape of the absorption spectra and the extinction coefficients. 

Also, since the intensity and thus the quantum yield are dependent on the number of emitted photons, they will be lower when emission occurs from a polar environment. One should mention that fluorescence parameters are more sensitive to the environment than absorption spectra and / or extinction coefficients. 

Rieche (28) published some years ago the absorption spectra and molecular extinction coefficients of hydrogen and alkyl hydrogen peroxides that are reproduced here (Fig. 2). These data may be used for routine assays of peroxide solutions, for routine assays of catalase activity, and for studies of catalase activity in connection with the mechanism of catalase action. 

Broderick et al indicated the presence of high-spin Fe(III) in a rhombic environment with tyrosine coordination, based on the results obtained from electronic absorption, EPR, and resonance Raman spectroscopies [100]. No evidence has been obtained for the histidine coordination which is verified by the solution of the X-ray crystal structure of Pseudomonas aeruginosa 3,4-PCD [33]. The visible spectrum is similar in both Amax (430 nm) and extinction coefficient (e= 3095 M cm" ) to those reported for the 1,2-CTD and 3,4-PCD enzymes. (Bhat et al also observed a band at 425 nm, e = 4700 M cm with 3,5-dichlorocatechol 1,2-dioxygenase [104].) EPR spectra exhibit resonances at = 4.25 and 9.79 (X-band) and those at 4.3 (gx = 4.21, gy = 4.18, gz = 4.32) and g = 9.83, indicating nonheme Fe(III) in a rhombic environment. The resonance Raman spectra (Agx = 647.1 nm) exhibit tyrosine ring vibrations characteristic of Fe(III) tyrosinate sites at 1604 and 1507 cm (C-C stretch), 1266 cm" (broad, C-0 stretch of two different tyrosine ligands), and 1183 cm (C-H stretch). 

Ultraviolet Spectra. Only one absorption above 300 nm is clearly visible in the UV spectrum of amides in NH3 solution. A second band at 240 nm or shorter wavelengths is obscured by the onset of the absorption of the solvent [41, 42]. At a given temperature the absorption shifts to higher wavelengths as the size of the cation increases from Li to K and then remains constant. A similar solvation of the larger cations (K, Rb, Cs) indicates electrostatically associated ion pairs without appreciable perturbation of the solvent sphere around the anion. The smaller cations, however, have a stronger influence on the solvent sphere of the amide ion [43]. The positions of the maxima of the absorption and extinction coefficients... 

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