Solute crystallization in frozen systems-use of synchrotron radiation to improve sensitivity

Purpose. To demonstrate the sensitivity of low temperature synchrotron X-ray diffractometry (SXRD) for detecting solute crystallization in frozen sodium phosphate buffer solutions. To determine the effect of annealing on solute crystallization in frozen solutions. Materials and Methods. Sodium phosphate buffer solutions, at initial buffer concentrations ranging from 1 to 100 mM (pH 7.4) were cooled to -50°C. The crystallization of disodium hydrogen phosphate dodecahydrate (Na₂HPO₄ •12H ₂O) was monitored using a laboratory as well as a synchrotron source. At selected concentrations, the effect of annealing (at -20°C) was investigated. Results. With the laboratory source, solute crystallization, based on the appearance of one diagnostic peak with a d-spacing of 5.4 Å, was evident only when the initial buffer concentration was at least 50 mM. In contrast, using SXRD, crystallization was detected at initial buffer concentrations down to 1 mM. In addition, the use of a high-resolution 2D detector enabled the visualization of numerous diffraction rings of the crystalline solute. At both 10 and 100 mM buffer concentration, there was no increase in solute crystallization due to annealing. Conclusion. By using synchrotron radiation, solute crystallization was detected with substantially increased sensitivity, making the technique useful for freeze-drying cycles of practical and commercial importance. Since numerous peaks of the crystalline solute appeared, the technique has potential utility in complex, multi-component systems.