Spectral subtraction and baseline correction via interactive graphics programs have been extensively used for the analysis of weak IR absorption signals which are under strong bands, such as occur in aqueous protein solutions. However, personal bias and uncertainty can result in significant differences in the spectra of the same protein substracted and baseline corrected by different persons. This is demonstrated in the case of albumin adsorbed on the surface of a germanium ATR crystal in contact within pure water. A simple computer algorithm to automatically subtract the water spectrum and remove any baseline tilt or curvature, and thus remove this individual bias and uncertainty, is presented, with a comparison of the results between different persons and the algorithm.

An infrared/attenuated total reflection (ATR) technique has been utilized to study the structural changes in proteins induced by nonaqueous solvents, without the need of dissolving the protein in the nonaqueous solvent. For the two proteins studied, methanol and ethylene glycol caused similar changes in albumin, i.e., an increase in helix secondary structure. However, the two solvents had dissimilar effects on immunoglobulin G (IgG). Changes in the pH of aqueous solutions of IgG produced a third effect. By dissolving some IgG in ethylene glycol and then adsorbing IgG from this solution onto an ATR crystal, the time behavior of the adsorption process could be studied and a mechanism for the structural changes proposed.

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A vibrational assignment has been made for most of the observed bands of aqueous solutions of albumin. This assignment is based not only on past assignments, but on experimental justifications and the use of both the infrared and Raman spectra. With the use of these assignments, changes in the secondary structure of both myoglobin and albumin with adsorption can be followed. Changes in the adsorbed protein films indicate that both reversible and irreversible protein adsorption takes place under certain conditions. The presence of reversibly adsorbed proteins can affect the extent of unfolding of the irreversibly adsorbed protein film.

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