Probing Chemical Equilibrium in Frozen Sodium Phosphate Buffer Solution by 31P Solid-State NMR.

ABSTRACT

Phosphate buffers are crucial for cryopreservative stability in pharmaceuticals, food processing, biomedical sciences, and biology. However, their freeze concentrates lack quantitative characterization, especially regarding the physicochemical properties of phosphate salt species in equilibrium at subzero temperatures. This study employs 31P solid-state NMR (ssNMR) to analyze frozen sodium phosphate (NaP) solutions, providing insights into phase composition, ionic strength, and pH. For the first time, we have directly quantified phosphate species in frozen NaP buffer, including crystallized disodium phosphate dodecahydrate (Na2HPO4·12H2O) content and the concentrations of H2PO4- and HPO42- in the freeze concentrate. This enabled the calculation of the pH as well as the ionic strength in the freeze concentrate. Trehalose effectively mitigated pH shifts in buffer solutions by preventing the selective crystallization of salt, a spectroscopic phenomenon not previously observed experimentally.