Ph Shifts In Tris Buffer During Freeze Thawing

Abstract

Purpose: To study the phase behavior of tris buffer during freeze and thawing and monitor the resulting pH changes.Methods: Tris buffer, at concentrations of 10 mM and 100 mM, and room temperature pH values (pH0) of 7.3, 8.0 and 8.8 are studied. The phase behavior of tris buffers under subambient conditions was characterized by thermal (differential scanning calorimetry, DSC), spectroscopic (UV-Vis spectrophotometer) and X-ray diffractometric (synchrotron X-ray diffractometry, SXRD) techniques. Cresol red (CR) was added as the pH indicator to measure the pH of frozen tris solutions. Results: According to the DSC results, (i) both tris base and tris HCl crystallized during freeze thawing. (ii) Compared with tris base, tris HCl exhibited a higher crystallization propensity. (iii) The crystallization of tris HCl was more pronounced at higher solute concentrations. (iv) As the concentration of tris base increased (i.e., higher pH), there was more pronounced inhibition of tris HCl crystallization. In other words, tris base exhibited a concentration dependent inhibition of tris HCl crystallization. SXRD results confirmed that at subambient conditions, tris HCl has a substantially higher crystallization propensity than tris base. Its crystallization was often observed during heating. In the current study of buffers with pH0 7.3, 7.5, and 7.9, the increase in Hammett Acidity function (Hx) is ~ 0.3 to 0.4 unit per 10 °C decrease, possibly indicating that the positive pH shift of tris buffer at subambient conditions is attributable to increasing pKa rather than tris HCl crystallization. Conclusion: Tris base exhibited a concentration dependent inhibition of tris HCl crystallization. The positive pH shift of tris buffer at subambient conditions is attributable to increasing pKa rather than tris HCl crystallization.