  Vitrification of aqueous cryoprotectant mixtures is essential in
cryopreservation of proteins and other biological samples. We report systematic
measurements of critical cryoprotective agent (CPA) concentrations required for
vitrification during plunge cooling from T=295 K to T=77 K in liquid nitrogen.
Measurements on fourteen common CPAs including alcohols (glycerol, methanol,
isopropanol), sugars (sucrose, xylitol, dextrose, trehalose), PEGs (ethylene
glycol, PEG 200, PEG 2 000, PEG 20 000), glycols (DMSO, MPD), and salt (NaCl)
were performed for volumes ranging over four orders of magnitude from ~nL to 20
mkL, and covering the range of interest in protein crystallography. X-ray
diffraction measurements on aqueous glycerol mixtures confirm that the
polycrystalline-to-vitreous transition occurs within a span of less than 2% w/v
in CPA concentration, and that the form of polycrystalline ice (hexagonal or
cubic) depends on CPA concentration and cooling rate. For most of the studied
cryoprotectants, the critical concentration decreases strongly with volume in
the range from ~5 mkL to ~0.1 mkL, typically by a factor of two. By combining
measurements of the critical concentration versus volume with cooling time
versus volume, we obtain the function of greatest intrinsic physical interest:
the critical CPA concentration versus cooling rate during flash cooling. These
results provide a basis for more rational design of cryoprotective protocols,
and should yield insight into the physics of glass formation in aqueous
mixtures.
