Absorption Spectroscopy of Biopolymers

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Absorption Spectroscopy of Biopolymers

• Experimental aspects
• Spectrum Analysis

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Solvents

• UV-visible spectra are normally recorded on
  dilute solutions (10-6 to 10-3 M) of a sample and
  referenced to the pure solvent that the solute is
  dissolved in
• Solvents are normally chosen so that their
  absorption does not interfere with the solute
  peaks, as the UV-visible spectrometer has
  difficulty subtracting the signal of the pure
  solvent from the solvent in the sample solution
• The solvent end absorption or solvent cut-off
  may extend up to 250 nm for some solvents, so
  that peaks below below this wavelength must be
  regarded with some caution
• In addition, the material that the cuvettes
  (cells) are made of also absorb light in the UV
  region.
• Glass cells are opaque to the UV light up to 300
  nm
• Quartz cells are transparent down to 170 nm
• Visible spectra may be obtained in cells of
  either material

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Solvent and Cuvette transparencies
material opaque material
transparent
Harwood and Claridge, p. 14
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Spectral Bandwidth
Benzene
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Chromophore absorptions
Chromophore Example Excitation lmax, nm e Solvent
CC Ethene p  __gt   p 171 15,000 hexane
C?C 1-Hexyne p  __gt   p 180 10,000 hexane
CO Ethanal n  __gt  p p  __gt  p 290 180 15 10,000 hexane hexane
NO Nitromethane n  __gt  p p  __gt  p 275 200 17 5,000 ethanol ethanol
C-X   XBr XI Methyl bromide Methyl Iodide n  __gt  s n  __gt  s 205 255 200 360 hexane hexane
http//www.cem.msu.edu/reusch/VirtualText/Spectrp
y/UV-Vis/spectrum.htm
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Organic Chromophores
Chromophore Transition Lmax(nm) Lmax(nm) log(e)
Nitrile (-CN) h to p h to p 160 lt1.0
Alkyne (-CC-) p to p p to p 170 3.0
Alkene (-CC-) p to p p to p 175 3.0
Alcohol (ROH) h to s h to s 180 2.5
Ether (ROR) h to s h to s 180 3.5
Ketone (-C(R)O) p to p p to p 180 3.0
Ketone (-C(R)O) h to p h to p 280 1.5
Aldehyde (C(H)O) p to p p to p 190 2.0
Aldehyde (C(H)O) h to p h to p 290 1.0
Amine (-NR2) h to s h to s 190 3.5
Acid (-COOH) h to p h to p 205 1.5
Ester (-COOR) h to p h to p 205 1.5
Amide (-C(O)NH2) h to p h to p 210 1.5
Thiol (-SH) h to s h to s 210 3.0
Nitro (-NO2) h to p h to p 271 lt1.0
Azo (-NN-) h to p h to p 340 lt1.0
http//www.chemistry.ccsu.edu/glagovich/teaching/4
72/uvvis/table.html
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Band Classification
B (for benzoid) E (for ethylenic) R (for
radical-like) K (for conjugated from the German
konjugierte)
lmax, nm e, M-1 cm-1 log10e Assignment
244 12,600 4.1 p ? p K
280 1,600 3.2 p ? p B
317 60 1.8 n ? p R