A method is described for the quantitative subtraction of water from the transmission infrared spectra of aqueous solutions of proteins. The 2125-cm<sup>−1</sup> association band of water is used as an internal intensity standard, and the scaling factor is determined with the use of a second-order least-squares fit. This method eliminates the user bias encountered with interactive methods and takes into account small baseline variations due to instrument drift. Statistical analysis of the results obtained demonstrates that at 10% w/w protein concentration, the error is of the order of 2% in the region of the amide I and II bands.

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