Topics in Biological Spectroscopy: A Comprehensive Course Outline for BIOC 6740 (2008), Study notes of Biology

Download Topics in Biological Spectroscopy: A Comprehensive Course Outline for BIOC 6740 (2008) and more Study notes Biology in PDF only on Docsity! Course Outline: BIOC 6740 (2008) Topics in Biological Spectroscopy I. Review on Interaction of Radiation and Matter Properties of Electromagnetic Waves: • Frequency, phase, polarization. • Superposition of EM waves • Plane, circular and elliptically polarized radiation. • Coherence Interaction of Electromagnetic Radiation with Matter • The dipole oscilator • Scattering, Reflection and Refraction • Polarizability and refractive index • Dispersion curves and birefringence Absorption of Light • Energy of Radiation and Transitions • The Transition Dipole Moment and the Probability of Absorption • The Direction of the Transition Dipole Moment • Polarized Absorption Spectra • Einstein Relationships for Absorption and Emission of Radiation • Boltzmann's Distibution • Lambert-Beer Law • Lasers II. UV-Visible Absorption Spectroscopy Theory • Rate of Electronic Transitions • Frank-Condon Principle • Spectrum Shape • Linewidth: Natural and the uncertainty principle • Effect of Intermolecular Interactions on Linewidth • Effect of Intermolecular Interactions on the Energy of the Transition: Solvent-induced spectral shifts • Transitions in the carbonyl and amide chromophores • Interactions between chromophores: Band splitting and Changes in Intensity Instrumental Aspects of UV-Visible Absorption spectroscopy • Single Beam Spectrophotometers • Split-Beam Spectrophotometers • Dual wavelength Spectrophotometer • Diode Array Spectrophotometer • Resolution, accuracy and sensitivity of a spectrophotometric measurement • Effect of stray light and Noise • Comparison Different Spectrophotometers: Resolution, accuracy, precision, speed and sensitivity Analytical Methods used in UV-Visible Absorption Spectroscopy • Sample: preparation and stability • Selection of cuvette • Temperature control: effect on pH • Single Wavelength determination of concentrations • Multi-component samples and the use of Multiple wavelengths • Isosbestic Points • UV absorption Spectra of Proteins and chromophores of biological importance. • Difference Spectra • Derivative Spectroscopy. III. Fluorescence Spectroscopy 1. Principles: • Jablonsky Diagram • Nonradiative Relaxation of Electronically Excited Molecules: Vibrational Relaxation and Internal Conversion. • Deactivation of the thermally-equilibrated lowest excited singlet state: radiative and nonradiative processes • Fluorescence • Singlet-triplet Intersystem Crossing • Phosphorescence • Delayed Fluorescence. • Rates of Different Processes of Deactivation • Stokes Shift • Fluorescence Lifetime • Quantum Yields 2. Steady-State Fluorescence Spectroscopy: • The steady-state spectrofluorometer. • Excitation and emission spectra: Acquisition and Corrections • Steady State Fluorescence Intensity: Effects of: quantum yield, absorbance, intensity of exciting radiation, and slit widths • Relationship between fluorophore concentration and fluorescence intensity Ideal Relationship, Inner Filter Effect • Experimental Considerations: Concentration of Fluorophores and Chromophores Scattering: Rayleigh and Raman Selecting the Excitation Wavelength Sensitivity of fluorescence: comparison with UV absorption techniques. 3. Fluorescence in Proteins Most common Fluorophores: Phe, Tyr and Trp Applications to study environment of fluorophores Position of Maximum and Quantum Yields Effect of pH, denaturants and Environment