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

2. 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

3. 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

4. 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

5. 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

6. Biophysical Tools '02 - UV/Vis spectroscopy
Absorption bands
Solvent effects – blue/red shifts
peptides
p  190 nm (e ~ 10,000)
n  nm (e ~ 100)
Sidechains
Trp, Phe, Tyr (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* 190 nm
(two p orbitals side-byside) : allowed, large charge displacement, , (e ~ 10,000)
n  p* transition 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

7. 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

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