Fluorescence detection electron excitation

Though we and others (27-29) have demonstrated the utility and the improved sensitivity of the peroxyoxalate chemiluminescence method for analyte detection in RP-HPLC separations for appropriate substrates, a substantial area for Improvement and refinement of the technique remains. We have shown that the reactions of hydrogen peroxide and oxalate esters yield a very complex array of reactive intermediates, some of which activate the fluorophor to its fluorescent state. The mechanism for the ester reaction as well as the process for conversion of the chemical potential energy into electronic (excited state) energy remain to be detailed. Finally, the refinement of the technique for routine application of this sensitive method, including the optimization of the effi-ciencies for each of the contributing factors, is currently a major effort in the Center for Bioanalytical Research. 

Fluorescent chemical sensors occupy nowadays a prominent place among the optical devices due to its superb sensitivity (just a single photon sometimes suffices for quantifying luminescence compared to detecting the intensity difference between two beams of light in absorption techniques), combined with the required selectivity that photo- or chemi-luminescence impart to the electronic excitation. This is due to the fact that the excitation and emission wavelengths can be selected from those of the absorption and luminescence bands of the luminophore molecule in addition, the emission kinetics and anisotropy features of the latter add specificity to luminescent measurements8 10. 

Time-resolved laser flash ESR spectroscopy generates radicals with nonequilibrium spin populations and causes spectra with unusual signal directions and intensities. The signals may show absorption, emission, or both and be enhanced as much as 100-fold. Deviations from Boltzmann intensities, first noted in 1963, are known as chemically induced dynamic electron polarization (CIDEP). Because the splitting pattern of the intermediate remains unaffected, the CIDEP enhancement facilitates the detection of short-lived radicals. A related technique, fluorescence detected magnetic resonance (FDMR) offers improved time resolution and its sensitivity exceeds that of ESR. The FDMR experiment probes short-lived radical ion pairs, which form reaction products in electronically excited states that decay radiatively. ... 

Beyer and Welge43 have photolyzed NO by use of very far ultraviolet radiation. Photodissociation was detected by fluorescence between 1100 and 1500 A. The primary processes depended on the exciting wavelength and formed electronically excited oxygen and... 

Dornhofer, Hack, and Langel (180), in a detailed study of the fluorescence that is induced by an ArF laser, have been able to show that an intense ArF laser can distort the observed vibrational distribution by photodissociating CS radicals with v" > 5. The ArF laser absorption by CS will also produce electronically excited CS which, when it emits, will redistribute the vibrational populations. Probing the CS quantum state population under these conditions could distort the CS ground state populations. The LIF measurements will underestimate the amount of CS radicals that are produced, while the direct detection methods will overestimate the amount of S(3p) atoms because of the secondary photolysis of CS. The vibrational distribution of Lu et al. (178) will be less prone to this secondary photolysis because very low laser powers (< 1 mj) were used. Dornhofer, et al. concluded from their results that the S(3p)/S(J-D) ratio was 3, which is in reasonable agreement with the LIF measurements of Lu et al. 

When deciding to study the dynamics of electronic excitation energy transfer in molecular systems by conventional spectroscopic techniques (in contrast to those based on non-linear properties such as photon echo spectroscopy) one has the choice between time-resolved fluorescence and transient absorption. This choice is not inconsequential because the two techniques do not necessarily monitor the same populations. Fluorescence is a very sensitive technique, in the sense that single photons can be detected. In contrast to transient absorption, it monitors solely excited state populations this is the reason for our choice. But, when dealing with DNA components whose quantum yield is as low as 10-4, [3,30] such experiments are far from trivial. 

---