Modern Fluorescence Spectroscopy

of Volume 2.- 1 Applications of Centrifugal Fast Analyzers to Fluorescence and Chemiluminescence Analyses.- A. The Centrifugal Fast Analyzer: A New Concept in Automated Analytical Instrumentation.- B. Basic Components and Principles of Operation of Centrifugal Fast Analyzers.- 1. Basic CFA Components.- a. Analytical Module.- b. Sample-Reagent Loading Module.- c. Computer Module.- 2. Basic Operating Principles.- C. Optimization of the Centrifugal Fast Analyzer Optical System for Fluorescence Measurements.- 1. Inner-Filter Effect.- 2. Instrumental Design of the CFA and MCFA Fluorometric Analyzers.- D. Dynamic Fluorescence Referencing for Direct Calculation of Concentration and Enzyme Activity Units from Intensity Data.- 1. Referencing Fluorescence Measurements.- a. Calculation of Enzyme Activity.- b. Calculation of Substrate Concentration.- c. Determination of the Effectiveness of Dynamic Referencing.- E. Applications of the Centrifugal Fast Analyzer to Fluorescence, Light-Scattering, and Chemiluminescence Measurements.- 1. Fluorescence Measurements.- a Enzyme Activity Assays.- b. Enzymic Substrate Analysis.- c. Kinetic Enzyme Parameter Evaluation.- d. General Fluorometric Analyses.- e. Physical Fluorescence Measurements.- 2. Light-Scattering Measurements.- 3. Bioluminescence and Chemiluminescence Measurements.- F. Summary and Conclusions.- References.- 2 Applications of Luminescence Spectroscopy to Quantitative Analyses in Clinical and Biological Samples.- A. Introduction.- B. General Requirements of Clinical and Biological Analyses.- 1. Time Required for Analysis.- 2. Sensitivity of the Analysis.- 3. Selectivity of the Analysis.- 4. Size and Stability of the Sample.- 5. Normal Levels and Clinical Analysis.- C. Methods of Clinical Fluorometry.- 1. Clean-up of the Sample.- 2. Formation of Fluorescent Derivatives.- 3. Kinetic Methods of Analysis.- D. Modern Methods of Fluorescence Detection.- 1. Approaches toward the Stabilization of Reagents.- 2. Fluorescence Detection with Chromatographic Methods of Separation.- E. Automated Analysis.- 1. The Autoanalyzer.- 2. The Centrifugal Fast Analyzer.- 3. Automation of Kinetic Methods of Analysis.- F. Chemiluminescence and Bioluminescence.- 1. Chemiluminescence.- 2. Bioluminescence.- G. The Future of Fluorometry in Clinical and Biological Analysis.- References.- 3 Fluorescent Probing of Dynamic and Molecular Organization of Biological Membranes.- A. Introduction.- B. Theory and Practice.- 1. Origins of Fluorescence Phenomena.- a. Electronic and Vibrational Transitions.- b. Medium Effects on Fluorescence.- c. Resonance Energy Transfer.- d. Polarized Intensities.- e. Polarization of Fluorescence.- f. Fluorescence Quenching.- 2. The Measurement of Fluorescence in Membrane Systems.- a. General Aspects.- b. Apparatus and Methods.- 3. Summary of the Information Available from Fluorescence Measurements on Membrane Systems.- a. Environmental Information.- b. Distances.- c. Probe Orientations.- d. Rotational Motion.- e. Lateral Diffusion.- f. Complexes and Binding.- g. Site Accessibility.- h. Structural Transitions.- i. Electric Fields.- j. Empirical Correlations.- C. Applications to Model Membrane Systems.- 1. Lipids.- a. The Response to Lipid as a Solvent.- b. The Location in the Bilayer.- c. Orientation and Dynamic Distribution.- d. Response to Different Lipid Components.- e. Other Approaches to Probe Behavior.- f. General Conclusions.- 2. Proteins.- 3. Carbohydrates.- 4. Lipid-Protein Interactions.- D. Applications to Biological Membranes.- E. Concluding Discussion.- 1. Pitfalls and Problems.- a. Impurity Nature of Probes.- b. Interpretation of Data.- 2. Location of Probes.- 3. “State of the Art”.- 4. Future Trends.- F. Addendum.- References.- 4 The Application of Fluorescence Techniques to the Study of Micellar Systems.- A. Introduction.- B. Experimental Techniques Used in Fluorescent Probing of Micelles.- C. Measurement of the Time Dependence of Fluorescence.- D. Theory of Fluorescence Quenching Processes in Micellar Systems.- E. Dynamics of Pyrene Fluorescence in Solutions of Simple Detergent Micelles.- F. Influence of Additives upon the Permeability of Sodium Lauryl Sulfate Micelles.- G. Pyrene Fluorescence in Aqueous CTAB Micelles. Dynamics of Motion of Solubilized Pyrene Molecules.- H. Fluorescent Probing of Specific Regions of Micelles.- 1. Fluorescence Studies of Pyrenesulfonic Acid Solutions.- 2. Fluorescence Studies in Pyrenebutyric Acid Solutions.- I. Individual and Specific Micelles.- 1. Bile Salt Micelles.- 2. N-Dodecylbetaine Micelles.- 3. Oleic Acid Micelles.- 4. Triton X-100 Micelles.- 5. Phenyl Decanoic Acid.- J. Fluorescence Studies of Phase Transistors in Micellar Systems.- K. Fluorescence Studies of Inverted Micelles.- References.- 5 Fluorescent Probe Studies of Binding Sites in Proteins and Enzymes.- A. Introduction.- B. Fluorescence of Pyridoxamine-5-P.- C. Binding of 4-Pyridoxic-5?-P to Ribonuclease A.- D. Fluorescence Indicators of Enzyme-Enzyme Interactions.- E. Energy Transfer in Pyridoxyl-5-P Protein Complexes.- References.- 6 Acid-Base Chemistry of Excited Singlet States: Fundamentals and Analytical Implications.- A. Introduction.- B. Chemical Structure and the Acid—Base Properties of the Ground and Lowest Excited Singlet States.- C. Dynamic Aspects of Proton Exchange in the Lowest Excited Singlet State.- 1. Excited-State Proton Exchange Is Much Slower than Fluorescence in Acid or Conjugate Base.- 2. Excited-State Proton Exchange Is Much Faster than Fluorescence in Acid or Conjugate Base.- 3. Excited-State Proton Exchange and Fluorescence Are of Comparable Rates.- 4. The Effect of Buffers.- 5. Photoautomerism.- D. Prediction of Excited-State Proton Exchange.- 1. Calculation of pKa* from Absorption Spectral Shifts.- 2. Calculation of pKa* from Fluorescence Spectral Shifts.- References.- 7 Use of Fluorescence to Study Structural Changes and Solvation Phenomena in Electronically Excited Molecules.- A. Introduction.- B. The Franck-Condon Principle.- C. Nuclear Conformation and Spectroscopic Behavior for Ring and Ring-Chain Systems.- 1. Relative Spectral Position, Width, and Structure.- 2. ?max and ?f/?f.- 3. Stokes Shift and Mirror Similarity.- 4. Concentration Sensitivity.- D. Examples of Spectroscopic Studies on Ring Systems.- 1. Biphenyl.- 2. Substituted Biphenyls.- 3. p-Oligophenylenes.- 4. 2-Phenylnaphthalene and Derivatives.- 5. Ring-Substituted Anthracenes.- 6. Vinyl-Substituted Anthracenes.- E. Examples of Spectroscopic Studies on Ring-Chain Systems.- 1. Stilbenes.- 2. Tetraphenylethylene (TPE).- 3. Trans-1,1,4,4-tetraphenyl-2-methylbutadiene (TPMB).- F. Polar Substituent Rotations in the Excited State.- 1. 9-Anthroic Acid and Its Esters.- 2. 1-Naphthoic Acid and Its Esters.- 3. 2-N-Arylamino-6-naphthalene Sulfonates.- G. Studies on Solvation of the Excited State.- 1. Excitation Wavelength-Dependent Fluorescence Spectra.- 2. Nanosecond Time-Resolved Fluorescence Spectroscopy.- 3. Subnanosecond Solvent Relaxation Studies by Oxygen Quenching of Fluorescence.- H. Conclusion.- References.- 8 The Study of Excited State Complexes (“Exciplexes”) by Fluorescence Spectroscopy.- A. Introduction.- B. On the “Charge-Transfer” Nature of Singlet Exciplexes.- 1. Dipole Moments.- 2. Correlations of Fluorescence Frequencies with Electron-Transfer Energies.- 3. Correlation of Heats of Exciplex Formation with Electron-Transfer Energies.- 4. Absorption Spectra of Exciplexes.- 5. Formation of Ions as Decay Products.- 6. Correlations of Fluorescence Quenching Rates with Electron-Transfer Energetics.- 7. Some Exceptions and Some Precautions.- a. Hydrocarbon Quenchers and Quenchees.- b. Exciplexes Involving Hydrogen Bonding or Hydrogen-Atom Transfer.- c. Fluorescence Quenching by Inorganic Anions.- C. The Formation of Exciplexes.- 1. Direct Measurement of Exciplex Formation Rates.- 2. Geometric Requirements for Exciplex Formation.- 3. Intramolecular Exciplexes.- 4. Is an “Excited Complex” Different from an “Exciplex”?.- 5. Does It Matter Which Exciplex Partner Is Initially Excited?.- 6. Thermodynamics of Exciplex Formation.- D. The Decay of Exciplexes.- 1. Relaxation.- 2. Dissociation to M* and Q (“Feedback”).- 3. Ionization and Pair Formation.- a. Competition of Ionization and Exciplex Formation “Charge Transfer” versus “Electron Transfer”.- b. Quantum Yields for Ion Formation.- c. Energies of Electron Transfer.- d. Exciplex Formation by Ion Annihilation. Nonphotochemical Exciplex Production.- 4. Formation of Triplet States.- 5. Quenching of Exciplexes by Other Solutes.- 6. Chemical Reactions. Product Formation.- E. Triplet Exciplexes.- 1. Phosphorescence Spectroscopy.- 2. Flash Photolysis.- 3. Chemical Decay of Triplet Exciplexes.- a. Addition of Triplet Carbonyls to Olefins.- b. Photoreduction of Triplet Ketones by Amines.- F. The Significance of Exciplexes.- 1. Biological Systems.- 2. Exciplex Dye Lasers.- 3. Synthesis.- 4. Analytical Chemistry.- 5. Prospectus.- References.- Author Index.