Measurement of Ionization Difference Spectrum

Approximately 0.05 g of the LiAlH4-reduced lignin accurately weighed to 0.001 g is dissolved in methylcellosolve-ethanol (1 4, v/v) or in another suitable solvent (Table 5.3.2) in a 100-ml volumetric flask and diluted to the mark with the same solvent. 

Using a 1-ml pipet (0.01ml subdivisions), a suitably sized aliquot (e.g., 0.3 ml) of the above solution is transferred to a 1-cm path quartz (reference) cuvette ( 3-ml capacity) and diluted tenfold (i.e., to 3.0 ml) with the same solvent used to dissolve the sample. A second aliquot, identical in volume to the first, is similarly transferred to another 1-cm path quartz (sample) cuvette and diluted exactly tenfold with an aqueous sodium hydroxide solution of sufficient strength to raise the pH of the diluted solution to 12. The difference spectrum is measured immediately (Note 1) as the absorbance of the alkaline solution relative to that of the neutral solution in the 210-400 nm range. The difference in absorbance, A A, at approximately 378 nm should fall between 0.2 and 0.8 (Note 2). 

Note 1. The absorbance of alkaline solutions of certain stilbenes has been reported to decrease rapidly initially as a consequence of photochemical (Falkehag et al. 1966) and/or air oxidation (Lin et al. 1974) reactions. The latter authors have speculated on the underlying chemical changes responsible for the decreased absorbance. 

Note 2. If the absorbance reading fails to fall within the prescribed range, the sample concentration should be adjusted accordingly and the spectrum re-measured. 

---