Combined kinetic osmometry and pyrometric microcalorimetry on protein solutions: Setup and data evaluation

A novel principle to measure simultaneously the equilibrium solvent vapour pressure (solvent activity) and the solvent heat of evaporation of aqueous macromolecular (protein) solutions is presented. These measurements are based on the simultaneous recording and evaluation of both the course of the sample mass and sample temperature during the sample evaporation. A setup is described where the mass and the temperature of the sample are continuously measured by weighing and contactless pyrometry, respectively. The sample in the μl-range is stored in a container placed within a box where temperature and relative humidity is stabilized up to 10−2 K and 10−1%, respectively. The incoming data reflect the effect of the continuously increasing solute concentration (changing at a ratio of about 2:1 up to 3:1) provided that, in practice, the solvent only evaporates. The protein concentration-dependent part μA(s,P) and hA(s,P) of the solvent chemical potential and the solvent molecular enthalpy, respectively, can be simultaneously determined (the latter for the first time). For appropriate protein concentrations, the second-order and third-order contributions in solute concentrations can be determined by a regression analysis. The data evaluation utilizes reference measurements on the pure solvent and upstream measurements on the solution of the precipitant only. Supported by FE simulations, a prodecure to minimize statistical errors and to eliminate systematic errors is derived and applied. For conditions of an ordinary laboratory, the equipment described enables a measurement of hA(s,P)/kBT and μA(s,P)/kBT with an absolute accuracy ≤10−1 and ≤10−3, respectively (kB: Boltzmann constant; T: temperature). We present measurements on aqueous solutions of Insulin Glargine (Hoe901) with ammonium sulfate as main precipitant component to demonstrate basic steps of the data evaluation procedure and the use of our measuring method.