Optical Spectroscopy

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Optical Spectroscopy

• Physical Biochemistry, November 2004
• Dr Ardan Patwardhan, a.patwardhan_at_ic.ac.uk,Dept.
  of Biological Sciences, Imperial College
• http//www.cbem.ic.ac.uk/ardan/os-1/OpticalSpectro
  scopy2004-1.ppt

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Course Plan

• Absorbtion spectroscopy
• Circular dichroism
• Rotational and vibrational spectroscopy
• Fluorescence and phosphoresence

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Absorption and emission spectra (atoms vs
molecules)
Atoms!
Molecules!
Atoms!
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Complementary Colors

• The absorbed and perceived spectral band are
  diametrically opposite each other in the color
  wheel

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Electronic (UV) spectroscopy

• Light absorbed electron excited to higher
  molecular orbital

DEhn
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Definitions etc

• Chromophores- Part of molecule responsible for
  absorption
• Auxochromes- Groups that modify absorption of
  neighboring chromophores- Often have lone pairs,
  e.g. OH, -OR, -NR2, -halogen- Bathochromic
  shift towards longer wavelength- Hypsochromic
  shift towards shorter wavelength- Hyperchromic
  effect increase in peak absorbance- Hypochromic
  effect decrease in peak absorbance
• Typical for unconjugated organic molecules? ?
  ? below 150 nm (vacuum UV)n ? ?, ? ? ? below
  200 nm (quartz UV)n ? ? 200-400nm (quartz UV)

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Effect of conjugation

• Increased conjugation leads to longer absorption
  wavelengths
• Ex Butadiene absorbs at 217nm whereas lycopene
  absorbs at 470nm

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Polycyclic aromatic hydrocarbons

• Extensive conjugation leads to large bathochromic
  shift

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Auxochromes

• Auxochromes with lone pairs often lead to
  increased delocalization (and conjugation) and
  therefore a bathochromic shift

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Solvent pH effect on phenol spectrum

• Increasing pH shifts equilibrium to right
• More non-bonding electrons in phenoxide ion ?
  higher extinction coefficient ? greater
  delocalization ? bathochromic shift(l,e)(270,145
  0) (287,2600)

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Solvent pH effect on aniline spectrum

• Decreasing pH shifts equilibrium to right
• No non-bonding electrons in anilinium ion ?
  lower extinction coefficient ? less
  delocalization ? hypsochromic shift(l,e)(280,143
  0) (254,169)

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Amino acids with significant absorbtion
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Dependence of tyrosine spectrum on pH

• pH titration can be used to determine - whether
  Tyr is internal or external- environment

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Polarity effects of the solvent

• p more polar than p in polar molecules? p
  better stabilized than p in polar solvent? p ?p
  transition undergoes bathochromic shift with
  increasing polarity of solvent
• Hydrogen bonding stabilizes n more than p in
  polar solvents ? n ?p undegoes hypsochromic
  shift ?peak absorbance is reduced due to
  stabilization of non-bonding electrons

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Effect of solvent polarity on tyrosine spectrum

• Solid line solv water
• Dashed line 80 water and 20 ethylene glycol

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Difference spectrum

• Perturbing solvent 80 water 20 substance of
  reduced polarity (e.g. ethylene glycol)
• DAAps Awater
• DA0 ? sample unaffected by perturbant
• Ex DA1 for free tryptophan but in a protein
  with 5 Trp it is 0.6 when measured using eth.
  gly. (4.3Å) and 0.4 when measured using
  glycerol(5.2 Å)for eth gly (0.6/1) x 5 3 Trp
  accessiblefor glycerol (0.4/1) x 5 2 Trp
  accessible? 3 2 1 Trp lies in a crevice
  which is between 4.3 Å and 5.2 Å in diameter

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Linearly, circularly and elliptically polarised
light
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Linear and elliptical from circular

• Combination of L and R circularly polarised
  light- of equal amplitudes gives linearly
  polarised light- of unequal amplitudes gives
  elliptically polarised light

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Optical activity

• Enantiomers are optically active
• Optically active molecules have different
  refractive indices, and different extinction
  coefficients for L and R circularly polarised
  light

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Optical rotatory dispersion (ORD)

• Measurement of rotation as a function of
  wavelength
• Refractive index is wavelength dependent

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Circular dichroism (CD)

• Measurement of difference in extinction
  coefficient for L and R as a function of
  wavelength
• Sign yields handedness information for absorbing
  chiral centre
• Easier to interpret than ORD

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ORD versus CD
CD
ORD
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Study of proteins with CD

• Peptide bond absorption (200nm) mainly studied
  (asymmetry imposed by environment)
• CD good for determining alpha helical content
• Excellent at monitoring conformational changes-
  changes in enzyme structure upon binding-
  protein denaturation- protein folding

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Trends

• Shorter wavelengths
• Improved prediction software for proteins
• Better understanding of electronic transitions
  involved