Calibrating ITC instruments: Problems with weak base neutralization

• Published in: Analytical biochemistry 2024-11, Vol.694, p.115602, Article 115602
• Main Authors: Medoš, Žiga, Bešter-Rogač, Marija, Leontidis, Epameinondas, Tellinghuisen, Joel
• Format: Article
• Language: English
• Subjects: Acid-base reaction; Calibration; Calorimetry; Calorimetry - methods; Diffusion; Hydrochloric Acid - chemistry; Isothermal titration calorimetry
• ISSN: 0003-2697; 1096-0309; 1096-0309
• DOI: 10.1016/j.ab.2024.115602

Modern isothermal titration calorimetry instruments give great precision, but for comparable accuracy they require chemical calibration. For the heat factor, one recommended process is HCl into the weak base TRIS. In studying this reaction with a VP-ITC and two Nano-ITCs, we have encountered some problems, most importantly a titrant volume shortfall Δv ≈ 0.3 μL, which we attribute to diffusive loss of HCl in the syringe tip. This interpretation is supported by a mathematical treatment of the diffusion problem. The effect was discovered through a variable-v protocol, which thus should be used to properly allow for it in any reaction that similarly approaches completion. We also find that the effects from carbonate contamination and from OH− from weak base hydrolysis can be more significant that previously thought. To facilitate proper weighting in the least-squares fitting of data, we have estimated data variance functions from replicate data. All three instruments have low-signal precision of σ ≈ 1 μJ; titrant volume uncertainty is a factor of ∼2 larger for the Nano-ITCs than for the VP-ITC. The final heat factors remain uncertain by more than the ∼1 % precision of the instruments and are unduly sensitive to the HCl concentration. [Display omitted] •ITC calibration with HCl + TRIS examined in VP-ITC and two Nano ITCs.•Problem: Heat calibration factor fc depends on titrant volume v.•Interpreted as diffusion volume loss Δv in syringe; supported mathematically.•Problem: Rx heat affected by OH− (hydrolysis) and carbonate (CO2 contamination).•Precisions (variance functions) of instruments estimated from replicate data.