Phase transitions in frozen systems and during freeze-drying: Quantification using synchrotron X-ray diffractometry

Dushyant B. Varshney, Prakash Sundaramurthi, Satyendra Kumar, Evgenyi Shalaev, Shin Woong Kang, Larry A. Gatlin, Raj Suryanarayanan

Research output: Contribution to journalArticlepeer-review

21 Scopus citations


Purpose: (1) To develop a synchrotron X-ray diffraction (SXRD) method to monitor phase transitions during the entire freeze-drying cycle. Aqueous sodium phosphate buffered glycine solutions with initial glycine to buffer molar ratios of 1:3 (17:50 mM), 1:1 (50 mM) and 3:1 were utilized as model systems. (2) To investigate the effect of initial solute concentration on the crystallization of glycine and phosphate buffer salt during lyophilization. Methods: Phosphate buffered glycine solutions were placed in a custom-designed sample cell for freeze-drying. The sample cell, covered with a stainless steel dome with a beryllium window, was placed on a stage capable of controlled cooling and vacuum drying. The samples were cooled to -50°C and annealed at -20°C. They underwent primary drying at -25°C under vacuum until ice sublimation was complete and secondary drying from 0 to 25°C. At different stages of the freeze-drying cycle, the samples were periodically exposed to synchrotron X-ray radiation. An image plate detector was used to obtain time-resolved two-dimensional SXRD patterns. The ice, β-glycine and DHPD phases were identified based on their unique X-ray peaks. Results: When the solutions were cooled and annealed, ice formation was followed by crystallization of disodium hydrogen phosphate dodecahydrate (DHPD). In the primary drying stage, a significant increase in DHPD crystallization followed by incomplete dehydration to amorphous disodium hydrogen phosphate was evident. Complete dehydration of DHPD occurred during secondary drying. Glycine crystallization was inhibited throughout freeze-drying when the initial buffer concentration (1:3 glycine to buffer) was higher than that of glycine. Conclusion: A high-intensity X-ray diffraction method was developed to monitor the phase transitions during the entire freeze-drying cycle. The high sensitivity of SXRD allowed us to monitor all the crystalline phases simultaneously. While DHPD crystallizes in frozen solution, it dehydrates incompletely during primary drying and completely during secondary drying. The impact of initial solute concentration on the phase composition during the entire freeze-drying cycle was quantified.

Original languageEnglish (US)
Pages (from-to)1596-1606
Number of pages11
JournalPharmaceutical research
Issue number7
StatePublished - Jul 2009

Bibliographical note

Funding Information:
The authors thank Dr. Douglas Robinson for the beam-line management and support during the experiments. This work was supported, in part, by a Research Challenge award from the Ohio Board of Regents. Use of the Advanced Photon Source at Argonne National Laboratory was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W-31-109-Eng-38. The Midwest Universities Collaborative Access Team (MUCAT) sector at the APS is supported by the U.S. 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.


  • Disodium hydrogen phosphate dodecahydrate
  • Glycine
  • In situ freeze-drying
  • Phase transitions
  • Phosphate buffer
  • Synchrotron X-ray diffraction


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