10/8/11 1 Lecture 14 – Oct 10 Chem 4101 – Fall 2011 Fluorescence 1- Fluorescence and phosphorescence Theory 2- Excitation and emission spectra 3- Molecular features of fluorophores 4- Instrumentation -Sections 15A – 15B, 15C-6 Suggested exercises: 15-1, 15-2, 15-3, 15-4 Lecture 14 – Oct 10 Chem 4101 – Fall 2011 Energy diagram – Jablonski diagram Figure 15-2, Section 15A Lecture 14 – Oct 10 Chem 4101 – Fall 2011 Excitation and emission spectra for quinine Figure 15-3, Section 15A10/8/11 2 Lecture 14 – Oct 10 Chem 4101 – Fall 2011 Molecular rigidity – favors fluorescence transitions involving π π* Section 15A Φ = 1.0 Φ = 0.2 Lecture 14 – Oct 10 Chem 4101 – Fall 2011 pH Effect Section 15A High pH, more stable first excited state, more fluorescent Low pH low fluorescence Lecture 14 – Oct 10 Chem 4101 – Fall 2011 Fluorescent (Fluorophores) and non-fluorescent molecules Figure 7-4, Section 7B10/8/11 3 Lecture 14 – Oct 10 Chem 4101 – Fall 2011 Molecular features and fluorescence Section 15A Know the chemical structures Lecture 14 – Oct 10 Chem 4101 – Fall 2011 Fluorometer Figure 15-10, Section 15B What circle can be used to correct for source variations? Lecture 14 – Oct 10 Chem 4101 – Fall 2011 Spectrofluorometer Figure 15-11, Section 15B10/8/11 4 Lecture 14 – Oct 10 Chem 4101 – Fall 2011 Instrument Standarization Figure 15-14, Section 15B 10 µM Quinine sulfate, 0.2 M H2SO4 Standarization is need to compare spectra obtained with different instruments Quantum counter Digital reference spectrum Lecture 14 – Oct 10 Chem 4101 – Fall 2011 Epifluorescence Microscopy Wikipedia commons Lecture 14 – Oct 10 Chem 4101 – Fall 2011 Fluorescence microscopy Figure 15-16, Section 15C-6 Calcium transients in a cerebellar Purkinje cell Lasser-Ross, et al. J. Neurophysiol., 1992, 68, 1167 Read the article (Methods) for possible bonus
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