1. Absorption Spectroscopy of Biopolymers Experimental aspects & Spectrum Analysis

2. 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

3. Solvent and Cuvette transparencies material opaque material transparent Harwood and Claridge, p. 14

4. Spectral Bandwidth Benzene

5. Chromophore absorptions Chromophor e Example Excitatio n max, nm  Solvent C=CEthene     17115,000hexane CCCC 1-Hexyne     18010,000hexane C=OEthanal n         290 180 15 10,000 hexane N=ONitromethane n        275 200 17 5,000 ethanol C-X X=Br X=I Methyl bromide Methyl Iodide n     205 255 200 360 hexane http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/spectrum.htm

6. Organic Chromophores Chromophore Transition  max (nm) log(  ) Nitrile (-C ≡N)  to   160<1.0 Alkyne (-C ≡C-)  to   1703.0 Alkene (-C=C-)  to   1753.0 Alcohol (ROH)  to   1802.5 Ether (ROR)  to   1803.5 Ketone (-C(R)=O)  to   1803.0  to   2801.5 Aldehyde ( – C(H)=O)  to   1902.0  to   2901.0 Amine (-NR 2 )  to   1903.5 Acid (-COOH)  to   2051.5 Ester (-COOR)  to   2051.5 Amide (-C(=O)NH 2 )  to   2101.5 Thiol (-SH)  to   2103.0 Nitro (-NO 2 )  to   271<1.0 Azo (-N=N-)  to   340<1.0 http://www.chemistry.ccsu.edu/glagovich/teaching/472/uvvis/table.html

7. Band Classification max, nm , M -1 cm -1 log 10  Assignment 24412,6004.1    * K 2801,6003.2    * B 317601.8 n   * R B(for benzoid) E (for ethylenic) R (for radical-like) K(for conjugated – from the German “konjugierte”)