Enthalpic relaxation in frozen aqueous trehalose solutions

Our object was to investigate the effect of annealing on the glass transition temperatures and enthalpic recovery of frozen aqueous solutions of trehalose. Trehalose solutions were subjected to differential scanning calorimetry wherein they were first cooled from room temperature to −60 °C, and heated to the annealing temperature, which ranged between −34 and −48 °C. Following isothermal annealing for the desired time period, the glass transition temperatures and the enthalpic recovery were determined in the final heating scan. Frozen unannealed trehalose solutions were characterized by two glass transition events. At a heating rate of 2 °C/min, the observed Tg₁′ and Tg₂′ were ∼−45 and −31 °C, respectively. Annealing resulted in an increase in the lower transition temperature, Tg₁′, while the higher transition temperature, Tg₂′, was unaffected. Enthalpic recovery due to annealing was associated only with Tg₂′. Annealing at −36 °C resulted in the highest value of Tg₁′ and the maximum enthalpic recovery. Irrespective of the heating rates, the magnitude of enthalpic recovery and Tg₁′ showed a similar trend (first an increase, followed by a decrease) as a function of annealing temperature. This suggests that annealing led to crystallization of ice and subsequently the system became maximally freeze-concentrated. Annealing, at temperatures higher than −36 °C, led to a reduction in enthalpic recovery associated with Tg₂′ and a lowering of Tg₁′. These observations are consistent with the hypothesis that the higher transition temperature coincides with the onset of ice melting. We have attempted to bridge two conflicting schools of thought regarding the origin of multiple glass transitions in frozen aqueous sugar solutions. The two glass transitions are attributed to the formation of two “populations” in the freeze-concentrated phase during “non-equilibrium” or rapid cooling—one, that is maximally freeze-concentrated and the other that contains a higher amount of water. The higher transition temperature also overlaps with the onset of ice melting.