Quantitative surface studies of protein adsorption by infrared spectroscopy. I. Correction for bulk concentrations

A method for correcting attenuated total reflectance (ATR) infrared spectra of surface-adsorbed proteins for the contribution of proteins in the bulk solution has been demonstrated. This procedure estimates the soluble protein contribution to the ir spectra from parameters determined from transmission experiments, and uses the absorbance of the water band in ATR as an internal reference. Based on this procedure, the soluble protein contribution to the total spectrum is estimated to range from approximately 3% for IgG or albumin in solution at 1 mg/ml, to approximately 35% when whole blood (from sheep) is adsorbed onto a 45 degrees germanium ATR crystal.