Comparison of actual vs. synthesized ternary phase diagrams for solutes of cryobiological interest

Abstract

Phase diagrams are of great utility in cryobiology, especially, those consisting of a cryoprotective agent (CPA) dissolved in a physiological salt solution. These ternary phase diagrams consist of plots of the freezing points of increasing concentrations of solutions of cryoprotective agents (CPA) plus NaCl. Because they are time-consuming to generate, ternary diagrams are only available for a small number of CPAs. We wanted to determine whether accurate ternary phase diagrams could be synthesized by adding together the freezing point depressions of binary solutions of CPA/water and NaCl/water which match the corresponding solute molality concentrations in the ternary solution. We begin with a low concentration of a solution of CPA+salt of given R (CPA/salt) weight ratio. Ice formation in that solution is mimicked by withdrawing water from it which increases the concentrations of both the CPA and the NaCl. We compute the individual solute concentrations, determine their freezing points from published binary phase diagrams, and sum the freezing points. These yield the synthesized ternary phase diagram for a solution of given R. They were compared with published experimental ternary phase diagrams for glycerol, dimethyl sulfoxide (DMSO), sucrose, and ethylene glycol (EG) plus NaCl in water. For the first three, the synthesized and experimental phase diagrams agreed closely, with some divergence occurring as wt% concentrations exceeded 30% for DMSO and 55% for glycerol, and sucrose. However, in the case of EG there were substantial differences over nearly the entire range of concentrations which we attribute to systematic errors in the experimental EG data. New experimental EG work will be required to resolve this issue.

Relationship between intracellular ice formation in oocytes of the mouse and Xenopus and the physical state of the external medium—A revisit

“…Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. The bottom three lines of Table 1 and the bottom half of Table 2 show the computed values for the right-hand five columns based on the synthesized ternary phase diagrams [3]. The chief conclusion is that now the values for the unfrozen fractions at the flash temperature for the oocytes in the EG solutions are in rather close agreement with the values obtained for oocytes in glycerol and in PBS alone.…”

“…In the ternary phase diagrams for the other CPAs, the downward curvature of the plots (isopleths) with increasing solute concentration becomes greater as the weight ratio of CPA to salt (R) decreases. That is so with the synthesized ternary EG plots in [3]; however, it is not the case, or is much less the case, with the Woods et al data in [8]. For these two reasons, we concluded that the synthesized phase diagrams are closer to reality than are the Woods et al data.…”

“…However, in the case of EG, the agreement is not good. In addition, as pointed out in [3] there is a substantive internal discrepency in the Woods et al experimental data for EG [8]. In the ternary phase diagrams for the other CPAs, the downward curvature of the plots (isopleths) with increasing solute concentration becomes greater as the weight ratio of CPA to salt (R) decreases.…”

“…Subsequently, Kleinhans and Mazur [3] have compared published experimental ternary phase diagrams for the cryoprotective agents (CPA) glycerol, sucrose, DMSO, and EG in NaCl with those that they synthesized by adding the melting points of the binary system CPA/water and the binary system NaCl/water. In the case of glycerol, sucrose, and DMSO, the agreement between the experimental and synthesized ternary phase diagrams is very good.…”

Non-ideal Solution Thermodynamics of Cytoplasm

“…3 Other research groups have applied our equation to v) a quaternary system of water-CPA-sugarsalt, 43 and vi) aqueous solutions of two micelle-forming non-ionic surfactants. 44 Although there have been other multi-solute solution theories able to make predictions directly from single solute information that apply accurately to specific examples, 49 our osmotic virial approach is the most broadly applied. 3,26,27,[42][43][44] It is important to point out that some nonlinear relationships have been presented for osmolality of cytoplasm in bacteria (i.e., Escherichia coli); 50,51 however, in those studies the nonlinearity arose from different phenomena than those being studied here.…”