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Absorption Spectroscopy

Absorption Spectroscopy. Topic 4 (abridged) Biophysics. Energy and Absorption. Absorption is proportional to Get resonance at hf = ħ w = E b – E a. www.andor.com/chemistry/?app=189. Absorption Broadening.

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Absorption Spectroscopy

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  1. Absorption Spectroscopy Topic 4 (abridged) Biophysics

  2. Energy and Absorption • Absorption is proportional to • Get resonance at hf = ħw = Eb – Ea www.andor.com/chemistry/?app=189

  3. Absorption Broadening • Every electronic state (eg S0 and S1) has several vibrational and rotational substates. • Each of the sharp peaks is further broadened by Doppler shifts andsolvent effects and quantum probabilities. pubs.rsc.org/ej/DT/2000/B002123G/

  4. I1 l I0 Reference Beam Splitter monochromator Detector I2 l Light Source I0 Sample with pathlength l Detector I l monochromator I0 Sample with pathlength l Detector Light Source Extinction Coefficient Or A(l) = log (I0/I) A(l) = Ce(l)l

  5. Biopolymer absorption Protein Secondary Structure Protein Aromatics Blood http://life.nthu.edu.tw/~lslpc/biophys/hyperchorism.gif

  6. C t Chemical KineticsZero Order • Rate independent of concentrations • -dC/dt = k • C(t) = C0 – kt Reaction of nitrite with deoxyhemoglobin

  7. ln(CA) t Chemical KineticsFirst Order • -dCA/dt = kCA , CA = CA0 e-kt • t1/2 = ln(2)/k; t = 1/k = lifetime NO binding to Hb

  8. Chemical KineticsSecond Order • -dCA/dt = -dCB/dt = kCACB • Make one species in excess so get pseudofirst order kinetics, kobs = kCB so CA = CA0 exp(-kobst)

  9. Hemoglobin • Cooperative Binding of Oxygen • Linked to quaternary structure • Explained by MWC Model

  10. On the Nature of Allosteric Transitions:A Plausible Model Jacques Monod, Jeffries Wyman, Jean-Pierre Changux J. Mol. Biol. 1965

  11. Time Resolved Optical DensityTROD Pump-probe Stopped-flow http://fig.cox.miami.edu/~cmallery/255/255enz/ecb3x28.jpg Perturb System, measure change in absorption over time.

  12. SVD and data analysis(eg. for Hofrichter paper) • Truncated X (or A) data matrix is fit to exponentials or other model • Get noise filtering Basis Specta Time Course of Basis Specta www.math.tau.ac.il/~nin/Courses/BioInfo04/svd.ppt

  13. Circular Dichroism and Optical Rotary Dispersion CD • CD is the differential absorption of circularly polarized light • ORD rotates linearly polarized light (and other) • Both result from chiral structures as can be used as structural probes (eg Protein 2ndary Structure) ORD http://upload.wikimedia.org/wikipedia/commons/thumb/d/d1/Circular_dichroism.png/622px-Circular_dichroism.png

  14. Polarized Absorption Arrangements can be made to measure elements directly, using PEM or using ellipsometric techniques.

  15. Ellispometric Technique • Key is crossed (or nearly crossed) polarizers as in this schematic for TRORD. • Used for protein folding (Goldbeck paper).

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