Optical Spectroscopy: UV/Vis Biophysical Tools '02 - UV/Vis spectroscopy 11/11/2018

Principles of Spectroscopy Electromagnetic radiation plane and circularly polarized Interaction of electromagnetic wave with matter Scattering Absorption Emmision Principles of Spectroscopy Electromagnetic radiation alternating electric and magnetic vectors   polarization plane and circularly polarized Biophysical Tools '02 - UV/Vis spectroscopy 11/11/2018

Biophysical Tools '02 - UV/Vis spectroscopy Scattering oscillating E-field  electrons oscillate  radiation Elastic – same frequency Inelastic – lower frequency (Raman) Small and large molecules Small: Rayleigh  M/4 Large: interference P()  RG (Guinier plots) Dynamic Scattering ln A()  D  Interaction of electromagnetic wave with matter electromagnetic radiation can be : scattered, absorbed, or emitted. Scattering oscillation of E field induces oscillation of electrons in a molecule which emits radiation in all direction. The extent of induced oscillation proportional to polarizibility. elastic scattering frequency of emitted radiation same as the frequency of exciting radiation inelastic scattering: Raman energy loss resulting in lower frequency of emitted radiation change of vibrational state during scatter: shifted refraction Small Molecules: Rayleigh No interference between particles Scattering  M/4 Can determine molecular weight Large Molecules internal interference: interference between light scaterred from different points in the molecule angular dependence of scaterring (Guinier plots) - P() for any angle other than 0, scaterring from extended particel will be smaller than from “point” scaterrer due to internal interference P()  RG can determine radius of gyration (hydrodynamic size) of a biomolecule Dynamic Scattering fluctuations of local concentration as molecules diffuse in and out of the beam  diffusion coefficient   Autocorrelation function ln A()  D  can obtain diffusion coefficient Biophysical Tools '02 - UV/Vis spectroscopy 11/11/2018

Biophysical Tools '02 - UV/Vis spectroscopy Absorption Lineshape Position Intensity Width Absorption Resonance: absorption energy Oscillating electric field induces displacement of electric charge between the states: resonance Transition between energy levels when energy of electromagnetic wave = energy difference between states: Transition probability: absorption intensity Selection rules Probability of transition proportional to the extent of charge displacement: transition dipole (m2),. linear displacement: electric dipole me rotation of charge: magnetic dipole mm no charge displacement  no transition dipole  no absorption example: transition between spherical ground and excited states s  s’ transition is forbidden, s  p is allowed Population difference Oscillating electromagnetic field is just as likely to stimulate transition from the lower (ground) state upper (excited) energy state (absorption) as the emission from the excited  ground state (stimulated emission). If the two are equally populated be electrons there is no net absorption. Probability of absorption proportional to the population difference between the states: (Boltzmann) Relaxation the excited state relaxes to the ground state by: radiation (emission), interaction with lattice (heat), or by energy transfer to another electron. saturation If the rate of stimulation exceeds the rate of relaxation then the population difference is annihilated resulting in no absorption: saturation. Width of resonance The lifetime of the excited state ( t ) defines the uncertainty in the energy difference according to Heisenberg Principle. Spread of energies leads to spread of frequencies of exciting electromagnetic which evokes absorption  lifetime broadening Boltzmann Biophysical Tools '02 - UV/Vis spectroscopy 11/11/2018

Electronic absorption (UV/Vis) Spectrophotometers Single beam Double beam Diode array Beer-Lambert Law: dI ~ I*dl*sample Beer-Lambert Law each successsive slice (dz) of absorbing material absorbs a fraction of incoming light (dI/I): rule of thumb: absorbance should be below 1 OD (ie. 10% of light gets through) OD(190) more accurate than protein assays Spectrophotometers Single beam Double beam Diode array Biophysical Tools '02 - UV/Vis spectroscopy 11/11/2018

Biophysical Tools '02 - UV/Vis spectroscopy Absorption bands Solvent effects – blue/red shifts peptides p  p* @ 190 nm (e ~ 10,000) n  p* @ 210-220 nm (e ~ 100) Sidechains Trp, Phe, Tyr (p  p* @ 280nm ) Hypochromism and hyperchromism Electronic energy levels delocalized electrons in double bonds, bound (liganded) metal ions peptides dominant group is the amide p  p* transition @ 190 nm (two p orbitals side-byside) : allowed, large charge displacement, , (e ~ 10,000) n  p* transition 210-220 nm (s and p orbitals): forbidden, no linear charge displacement (rotation of charge will interact with the magnetic field), very low extinction coeff., (e ~ 100) sidechains overpowered by the amide except for Trp, Phe, Tyr with p  p* transition at 280 nm  conjugated systems porphyrins charge transfer transfer of electrons from and to metals due to oxidation/reduction e.g. Fe(III) in hemoglobin Solvent effects - Wavelength shifts for blue and red shifts – changes in absorbtion maximum due to interaction with solvents p  p* transition – blue shift in nonpolar sovents n  p* transition – red shift in nonpolar sovents  Ionization state Red shift and increase in extinction coefficient for charged states of titratable groups (-OH of Tyr, imidazole of His, or –SH of Cys)  Broadening solvent reorients slowly on the timescale of electronic absorption (10-15 sec) leading to non-uniform environment  broadening Hypochromism and hyperchromism For transitions involving multiple dipoles, the absorption intensity is either less (hypo-) or more (hyper-) than the sum of the individual absorptions. One dipole induces another dipole in the neighboring molecule. If the transition dipoles are stacked side-by-side the orientation of induced dipole is opposite to the exciting dipole  hypochromism If the dipoles are stacked head-to-tail, the resulting dipole is strengthened Ionization state Biophysical Tools '02 - UV/Vis spectroscopy 11/11/2018

Biophysical Tools '02 - UV/Vis spectroscopy Dichroism Polarized light: Different reffractive index for L &R = rotation (ORD) Different light absorption = ellipticity (CD) Circular dichroism/ORD – secondary structure Absorption Instruments Dichroism Direction of transition dipole of a molecule is defined by the direction of charge displacement which in turn is determined by the symmetry of the electron clouds in the two energy states The absorption of polarized electromagnetic will depend on the angle between oscillating E field and the transition dipole. Linear dichroism anisotropy in absorption of linearly polarized light Circular dichroism anisotropy in absorption of circularly polarized light Circular dichroism or Optical Rotary Dispersion CD curves are sensitive to the secondary structure of proteins for comparison - absorption not much Biophysical Tools '02 - UV/Vis spectroscopy 11/11/2018

Vibrational Spectroscopy Infrared Raman Spectroscopy Biophysical Tools '02 - UV/Vis spectroscopy 11/11/2018