Making Good's buffers good for freezing: The acidity changes and their elimination via mixing with
sodium phosphate

                       Lukáš Veselý^1, Behera Susrisweta^1, Dominik Heger^*1

^1Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech
Republic

Solutions of three Good’s buffers (HEPES, MOPS, and MES), both pure and mixed with sodium phosphate
buffers (Na-P), are investigated in terms of the freezing-induced acidity changes in their
operational pH ranges. The Good’s buffers have the tendency to basify upon freezing and, more
intensively, at lower pHs. The acidity varies most prominently in MES, where the change may reach
the value of two. Importantly, the Good’s buffers are shown to mitigate the strong acidification in
the Na-P buffer. Diverse concentrations of the Good’s buffers are added to cancel out the strong,
freezing-induced acidity drop in 50 mM Na-P that markedly contributes to the solution’s acidity^[1,
2]; the relevant values are 3 mM HEPES, 10 mM MOPS, and 80 mM MES. These buffer blends are
therefore proposed to be applied in maintaining approximately the acidity of solutions even after
the freezing process and, as such, should limit the stresses for frozen chemicals and biochemicals.

                         Shape, square Description automatically generated

Graphical representation of the useful ranges (below one unit of acidity change) relating to the 50
mM Good’s buffers suitable for the freezing processes^ ^[3].

1.            Thorat, A.A. and R. Suryanarayanan, Characterization of Phosphate Buffered Saline
(PBS) in Frozen State and after Freeze-Drying. Pharm Res, 2019. 36(7): p. 98.

2.            Gomez, G., M.J. Pikal, and N. Rodriguez-Hornedo, Effect of initial buffer composition
on pH changes during far-from-equilibrium freezing of sodium phosphate buffer solutions.
Pharmaceutical Research, 2001. 18(1): p. 90-97.

3.            Veselý, L., B. Susrisweta, and D. Heger, Making good's buffers good for freezing: The
acidity changes and their elimination via mixing with sodium phosphate. Int J Pharm, 2021. 593: p.
120128.