Systematic errors in isothermal titration calorimetry: The role of feedback power and effects of mixing and diffusion on concentrations

Using experiments based on the NaCl dilution calibration method, we examine several previously unstudied instrumental effects that can lead to systematic errors in isothermal titration calorimetry (ITC). We confirm earlier results that power feedback can affect estimated heats and find further that it can also affect cell temperature and introduce temperature "noise." To estimate effects from loss of titrant from the syringe through diffusion, and concentration changes in the cell from mixing with the overflow volume, we conduct experiments with injections separated by time intervals Δt from 8 to 60 minutes, giving run durations from 6 hrs to over 2 days. Because the effects are small, we have developed a global least-squares fitting algorithm to analyze 20 datasets simultaneously, from which three parameters governing diffusion and mixing ("leakage") are estimated, along with calibration parameters for heat and cell volume. Expressed as an effective volume loss from the syringe, Δvdif is found to be of order ∼0.03 μL per injection for NaCl into water. Its effect is predicted to be negligible in many ITC applications. The effects from leakage are more significant but their neglect is still predicted to affect both NaCl calibration and 1:1 binding results by ∼1%, which is marginally significant for calibration but seldom so for 1:1 binding. The calibration results for the cell volume V₀ are particularly sensitive to temperature and to the literature source for the relative molar enthalpy function for NaCl(aq).