Purpose. (1) To determine the effect of solution pH before lyophilization, over the range of 1.5 to 10, on the salt and polymorphic forms of glycine crystallizing in frozen solutions and in lyophiles. (2) To quantify glycine crystallization during freezing and annealing as a function of solution pH before lyophilization. (3) To study the effect of phosphate buffer concentration on the extent of glycine crystallization before and after annealing. Materials and Methods. Glycine solutions (10% w/v), with initial pH ranging from 1.5 to 10, were cooled to -50°C, and the crystallized glycine phases were identified using a laboratory X-ray source. Over the same pH range, glycine phases in lyophiles obtained from annealed solutions (0.25, 2 and 10% w/v glycine), were characterized by synchrotron X-ray diffractometry (SXRD). In the pH range of 3.0 to 5.9, the extent of glycine crystallization during annealing was monitored by SXRD. Additionally, the effect of phosphate buffer concentration (50 to 200 mM) on the extent of glycine crystallization during freezing, followed by annealing, was determined. Results. In frozen solutions, β-glycine was detected when the initial solution pH was ≥4. In the lyophiles, in addition to β- and γ-glycine, glycine HCl, diglycine HCl, and sodium glycinate were also identified. In the pH range of 3.0 to 5.9, decreasing the pH reduced the extent of glycine crystallization in the frozen solution. When the initial pH was fixed at 7.4, and the buffer concentration was increased from 50 to 200 mM, the extent of glycine crystallization in frozen solutions decreased with an increase in buffer concentration. Conclusion. Both solution pH and solute concentration before lyophilization influenced the salt and polymorphic forms of glycine crystallizing in frozen solutions and in lyophiles. The extent of glycine crystallization in frozen solutions was affected by the initial pH and buffer concentration of solutions. The high sensitivity of SXRD allowed simultaneous detection and quantification of multiple crystalline phases.
Bibliographical noteFunding Information:
The authors thank Dr. Douglas Robinson of Midwestern Collaborative Access Team for the beam-line management and valuable support during the experiments. This work was supported, in part, by a Research Challenge award from the Ohio Board of Regents and from the National Science Foundation grant DMR-0312792. Use of the Advanced Photon Source (APS) was supported by the US Department of Energy, Basic Energy Sciences, Office of Science, under contract no. W-31-109-Eng-38. The Midwest Universities Collaborative Access Team (MUCAT) sector at the APS is supported by the US Department of Energy, Basic Energy Sciences, Office of Science, through the Ames Laboratory under contract no. W-7405-Eng-82. We thank Linda Sauer for her assistance in setting up the instrumentation. Any opinions, findings, and conclusions or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.
- Phosphate buffer
- Polymorphs and salts
- Synchrotron XRD