WATER (H2O AND D2O) MOLAR ABSORPTIVITY IN THE 1000-4000-CM(-1) RANGE AND QUANTITATIVE INFRARED-SPECTROSCOPY OF AQUEOUS-SOLUTIONS

Water (H2O and D2O) molar absorptivity was measured by Fourier transfo rm infrared transmission spectroscopy in the 1000-4000 cm(-1) range at 25 degrees C. A series of assembled cells with path lengths from 1.2 to 120.5 mu m was used for these measurements. The optimal path length (the path length of aqueous solution at which the IR spectrum of solu te, corrected for water absorbance, has the highest signal-to-noise ra tio) was calculated for all water absorbance bands. The results presen ted here show that the optimal path length does not depend on solute p roperties and is inversely proportional to the solvent (water) molar a bsorptivity. The maximal signal-to-noise ratio for measurements of IR spectra of aqueous solution in the 1650 cm(-1) spectral region, of pri mary interest in biological applications, can be obtained at an optima l cell path lengths of 3-4 mu m (H2O) and 40-60 mu m (D2O). As an exam ple, the signal-to-noise ratio was calculated as a function of the cel l path length for the amide I (H2O) and amide I' (D2O) bands of an aqu eous lysozyme solution. The molar absorptivities of water bands are se veral orders of magnitude weaker than those of the strongest bands of biological macromolecules in the same spectral regions. High net water absorbance in aqueous solutions is due simply to the very high molar concentration of water. A method is proposed for the quantitative meas uring of the path length of the cell which exploits the molar absorpti vity of the strongest water bands (stretching vibrations) or of bands which do not overlap with solute absorbance. A path length in the rang e from similar to 0.01 mu m to similar to 1.0 mm can be determined wit h high precision using this technique for a samples of known concentra tion. Problems involved in the proper correction of strong water absor bance in IR spectra of aqueous solutions of biomolecules are discussed , including multiple reflections within the cell, the effects of pH, t emperature, and perturbation of water spectral properties by polar sol utes, as well as the selection of optimal spectral regions in which on e may obtain the most precise absorbance corrections. (C) 1997 Academi c Press.