Minute quantities of native cellular fluorophores can be quantitatively assayed using ultraviolet fluorescence detection with microcolumn separations, but spectral diversity of biological chromophores imposes serious limitations on the use of this strategy to investigate biological components. We present an approach for rapid characterization of picoliter samples containing dissimilar cellular fluorophoresincluding amino acids, monoamine neurotransmitters, flavins, and pyridine nucleotidesusing multiphoton excited fluorescence detection coupled to capillary electrophoresis separations. In this highly versatile approach, biological fluorophores are excited through the nearly simultaneous absorption of different numbers of low-energy photons. Because spectrally distinct species all can be excited with a single, long-wavelength laser source, fluorescence throughout the ultraviolet and visible regions can be detected efficiently with extremely low background. Samples containing serotonin, melatonin, FAD, and NADH can be reproducibly analyzed in 5-μm and 2-μm i.d. channels. Detection limits in 5-μm capillaries range from 350 zmols (38 nM) for FAD to 27 amols (1.0 μM) for serotonin. Use of 2-μm channels is shown to improve the mass detection limit for serotonin approximately as the decrease in capillary cross-sectional area (LOD ≈ 4 amol), and further reductions in mass detection limits are projected for analysis with even smaller diameter channels that better match the submicron size of the diffraction-limited multiphoton focal spot.

Methyl and CF3CF2 radicals were combined to form chemically activated CF3CF2CH3 with 104 kcal/mol of internal energy, and the experimental rate constant for unimolecular 1,2-dehydrofluorination was 4.5 × 105 s-1. Fitting the calculated rate constant for HF elimination from RRKM theory to the experimental value provided a threshold energy, E o, of 68.5 kcal/mol. Comparing this threshold energy to those for CF2HCH3, CH3CF2CH3, CF2ClCH3, and CF3CH3 shows that replacing the α-H of CF2HCH3 with CH3 lowered the E o by 7 kcal/mol and replacing with CF3, Cl, or F raises the E o about 8 kcal/mol. The CF3 substituent, an electron acceptor, increased the E o by an amount nearly equal to that with F and Cl substituents, suggesting that halogen substituents exert a similar inductive effect at the α-carbon that loses electron density as the transition state forms. These proposals will be compared to recent calculations of the carbon's atomic charges in the reactant and transition state.

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