Modern Fluorescence Spectroscopy

The principles of fluorescence spectroscopy are by now well established, and, after a rather lengthy gestation period, the technique is now routinely applied to a broad spectrum of problems, ranging from mechanistic photo­ chemistry to chemical analyses in biomedical and environmental systems to probes of structure and function in biological macromolecules. Phosphor­ escence spectrometry and chemiluminescence are also well-known tech­ niques; they are somewhat less well established than fluorescence (at least in analytical chemistry), but they too are receiving greatly increased appli­ cation to both laboratory and "real" problems. This is not to imply that luminescence spectroscopy, viewed in its broadest sense, is a static field. In fact, recent advances in instrumentation make it feasible to apply fluorescence to problem areas in which its use five years ago would have been unthinkable. Advances in hardware generate advances in application, and very significant progress is being recorded in the application of fluorescence (and its close relatives, phosphorescence and chemiluminescence) in the biochemical, biomedical, and environmental spheres.

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.