Physical aging of glassy state: DSC study of vitrified glycerol systems.

After annealing during the glass transition temperature (Tg) range or at sub-Tg temperatures, a "Tg overshoot" has been observed by differential scanning calorimetry in vitrified 55 and 80% (W/W) glycerol solutions. The temperature dependence of the overshoot is most pronounced immediately below the Tg and always increases in amplitude with lengthened annealing periods, whether annealed during Tg range or below. Of particular note is the anomalous shift in devitrification temperature (Td) to lower temperatures following increased time of annealing at sub-Tg temperatures. The Tg obtained on warming also occurs at higher temperatures with increased annealing times. The results allow us to suggest the existence of the time and temperature dependency of the glassy state. Accordingly, it may be desirable to maintain vitrified biological systems at temperatures sufficiently below Tg so that the extent of relaxation in the glass system is avoided or minimized during cryopreservation. The relaxation effects must be taken into account prior to warming a biological system after a long-term cryopreservation. These effects should also be considered in studies of vitrification solutions.