Excitation wavelengths

The use of reversibly-bound photoaffinity labels presents unique problems that are not presented by ordinary affinity labels or indeed by those reagents in which the photoactivatable groups are attached covalently to the protein prior to photolysis. However, one problem common to both the latter type modification reagents as well as the reversibly-bound photoaffinity labels is the choice of the wavelength for photolysis. 

Clearly, the choice is restricted by the absorption spectrum of the label. However, diazoketone and diazoacetyl derivatives, for example, possess two absorption bands - one at 254 nm (s 7000) and one at 350 nm e 10). Since carbenes may be produced by irradiation at either wavelength the question which immediately arises is which absorption should be used. The potential gain from irradiating with the shorter wavelength is that the intermediate formed is more reactive than that generated at the longer wavelength. This would enhance the likelihood 

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Sun Y-P, Wang P and Hamilton N B 1993 Fluorescence spectra and quantum yields of Buckminsterfullerene (Cgg) in room-temperature solutions. No excitation wavelength dependence J. Am. Chem. Soc. 115 6378-81... 

Compound Solvent pH Excitation wavelength, nm Emission wavelength, nm... 

The fluorescent emission for quinine at 450 nm can be induced using an excitation frequency of either 250 nm or 350 nm. The fluorescent quantum efficiency is known to be the same for either excitation wavelength, and the UV absorption spectrum shows that 250 is greater than 350- Nevertheless, fluorescent emission intensity is greater when using 350 nm as the excitation wavelength. Speculate on why this is the case. 

Although intended for the biochemistry lab, this experiment provides analytical students with a practical characterization analysis. Of particular interest is the use of Job s method to determine the number of TNS (2-p-toludinylnaphthalene-6-sulfonate) binding sites on calmodulin, fluorescence is measured at 475 nm using an excitation wavelength of 330 nm. 

Samples of urine are analyzed for riboflavin before and after taking a vitamin tablet containing riboflavin. Concentrations are determined using external standards or by the method of standard additions, fluorescence is monitored at 525 nm using an excitation wavelength of 280 nm. 

Resonance Raman Spectroscopy. If the excitation wavelength is chosen to correspond to an absorption maximum of the species being studied, a 10 —10 enhancement of the Raman scatter of the chromophore is observed. This effect is called resonance enhancement or resonance Raman (RR) spectroscopy. There are several mechanisms to explain this phenomenon, the most common of which is Franck-Condon enhancement. In this case, a band intensity is enhanced if some component of the vibrational motion is along one of the directions in which the molecule expands in the electronic excited state. The intensity is roughly proportional to the distortion of the molecule along this axis. RR spectroscopy has been an important biochemical tool, and it may have industrial uses in some areas of pigment chemistry. Two biological appHcations include the deterrnination of helix transitions of deoxyribonucleic acid (DNA) (18), and the elucidation of several peptide stmctures (19). A review of topics in this area has been pubHshed (20). 

Raman spectrometry is another variant which has become important. To quote one expert (Purcell 1993), In 1928, the Indian physicist C.V. Raman (later the first Indian Nobel prizewinner) reported the discovery of frequency-shifted lines in the scattered light of transparent substances. The shifted lines, Raman announced, were independent of the exciting radiation and characteristic of the sample itself. It appears that Raman was motivated by a passion to understand the deep blue colour of the Mediterranean. The many uses of this technique include examination of polymers and of silicon for microcircuits (using an exciting wavelength to which silicon is transparent). 

Fig. 29 Fluorescence scans of polycyclic aromatic hydrocarbons at various excitation wavelengths in combination with various secondary filters.

Fig. 15. Stern-Volmer plots for (O) po-ly(A/St/Phen) (29) and ( ) APh-2 (8 with x = 2) with MV2+ in aqueous solution excitation wavelength, 297 nm [119]...

Incident wavelength 1.60 A. Photon energy. 1.24f 10 15) watt-seconds.8 Excited wavelength Cobalt Ka, 1.79 A. 

In general, the choice of a laser for use as a Raman excitation source is based on a number of considerations. The laser excitation wavelength, for experimental and theoretical reasons, must lie in the visible region, i.e. 400-700 nm. The laser should have many emission lines over a wide range of the visible region and the excitation frequency should not correspond... 

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