Interplay of vitrification and ice formation in a cryoprotectant aqueous solution at low temperature

TL;DR

This study investigates how vitrification and ice formation occur in glycerol-water solutions at low temperatures, revealing that water crystallizes upon annealing even below the glass transition, impacting cryopreservation strategies.

Contribution

It demonstrates that water crystallizes in marginal glycerol solutions during annealing below the glass transition, challenging previous assumptions and providing detailed parameters for cryoprotection.

Findings

Water crystallizes upon annealing below the glass transition.

Crystallization parameters depend on annealing temperature.

No evidence of an iso-compositional liquid-liquid transition.

Abstract

The proneness of water to crystallize is a major obstacle to understanding its putative exotic behavior in the supercooled state. It also represents a strong practical limitation to cryopreservation of biological systems. Adding some concentration of glycerol, which has a cryoprotective effect preventing to some degree water crystallization, has been proposed as a possible way out, provided the concentration is small enough for water to retain some of its bulk character and/or for limiting the damage caused by glycerol on living organisms. Contrary to previous expectations, we show that in the ``marginal'' glycerol molar concentration $\approx18\%$, at which vitrification is possible with no crystallization on rapid cooling, water crystallizes upon isothermal annealing even below the calorimetric glass transition of the solution. Through a time-resolved polarized neutron scattering investigation, we extract key parameters, size and shape of the ice crystallites, fraction of water that crystallizes, crystallization time, which are important for cryoprotection, as a function of the annealing temperature. We also characterize the nature of the out-of-equilibrium liquid phases that are present at low temperature, providing more arguments against the presence of an iso-compositional liquid-liquid transition. Finally, we propose a rule-of-thumb to estimate the lower temperature limit below which water crystallization does not occur in aqueous solutions.