Infrared spectroscopy in aqueous solution: Difficulties and accuracy of water subtraction

The long-term reproducibility of a set of water spectra in the infrared region with cell thicknesses of less than 10 mu m was tested. With the application of a computer routine for subtraction in the range from 2600 to 1100 cm(-1) and the use of additional phase and nonlinearity corrections, an accuracy better than one milliabsorbance unit (mAU) can he achieved, if the temperature is kept constant within a tenth of a degree or if a temperature correction factor is implemented in the subtraction routine. The subtraction of water from an aqueous protein solution reduces the spectral range for a correction to 2300-1800 cm(-1). In this region the protein absorption, as derived from dry protein films, is constant, and because of the association band at 2125 cm(-1) water exhibits good subtraction properties. However, the reduction of the spectral range for the water subtraction leads to an increase in the residual error to 12 mAU at 1650 cm(-1). This standard deviation can be reduced to 6-7 mAU by applying a second subtraction range from 4000 to 3650 cm(-1). A further reduction of this error to 3-5 mAU is obtained by implementing a constant ratio of the integrated intensities of the amide I and amide II bands in the procedure of subtracting water from aqueous protein solutions. The remaining inaccuracy is mainly caused by uncompensated phase error.