Transcript Slide 1
States and transitions Spectroscopy—transitions between energy states of a molecule excited by absorption or emission of a photon hn = DE = Ei - Ef Energy levels due to interactions between parts of molecule (atoms, electrons and nucleii) as described by quantum mechanics, and are characteristic of components involved, i.e. electron distributions (orbitals), bond strengths and types plus molecular geometries and atomic masses involved Spectroscopic Regions Typical wavelength (cm) -11 10 -8 10 -5 10 -5 3 x 10 -5 6 x 10 -3 10 -2 10 -1 10 0 10 10 Approximate energy (kcal mole-1) 8 3 x 10 5 3 x 10 2 3 x 10 2 10 3 5 x 10 0 3 x 10 -1 3 x 10 -2 3 x 10 -3 3 x 10 -4 3 x 10 Spectroscopic region Techniques and Applications -ray X-ray Vacuum UV Near UV Visible IR Far IR Microwave Microwave Radio frequency MÖssbauer x-ray diffraction, scattering Electronic Spectra Electronic Spectra Electronic Spectra Vibrational Spectra Vibrational Spectra Rotational Spectra Electron paramagnetic resonance Nuclear magnetic resonance Adapted from Table 7-1; Biophysical Chemistry, Part II by Cantor and Schimmel Optical Spectroscopy - Processes Monitored UV/ Fluorescence/ IR/ Raman/ Circ. Dichroism Excited State (distorted geometry) Ground State (equil. geom.) Diatomic Model Analytical Methods Absorption UV-vis absorp. hn = E - E & Fluorescence. grd n0 nS ex Fluorescence hn = Eex - Egrd Raman: DE = hn0-hns = hnvib Infrared: DE = hnvib Q molec. coord. move e- (change electronic state) high freq., intense CD – circ. polarized absorption, UV or IR Raman –nuclei, inelastic scatter very low intensity IR – move nuclei low freq. & inten. Optical Spectroscopy – Electronic, Example Absorption and Fluorescence Essentially a probe technique sensing changes in the local environment of fluorophores eg. Trp, Tyr Change with tertiary structure, compactness (M-1 cm-1) What do you see? [protein example] Intrinsic fluorophores Amide absorption broad, Intense, featureless, far UV ~200 nm and below Optical Spectroscopy - IR Spectroscopy Protein and polypeptide secondary structural obtained from vibrational modes of amide (peptide bond) groups Aside: Raman is similar, but different amide I, little amide II, intense amide III What do you see? Model peptide IR Amide I (1700-1600 cm-1) a b Amide II (1580-1480 cm-1) rc Amide III (1300-1230 cm-1) I II