Self-digitization of sample volumes.

This paper describes a very simple and robust microfluidic device for digitizing samples into an array of discrete volumes. The device is based on an inherent fluidic phenomenon, where an incoming aqueous sample divides itself into an array of chambers that have been primed with an immiscible phase. Self-digitization of sample volumes results from the interplay between fluidic forces, interfacial tension, channel geometry, and the final stability of the digitized samples in the chambers. Here, we describe experiments and simulations that were used to characterize these parameters and the conditions under which the self-digitization process occurred. Unlike existing methods used to partition samples into an array, our method is able to digitize 100% of a sample into a localized array without any loss of sample volume. The final volume of the discretized sample at each location is defined by the geometry and size of each chamber. Thus, we can form an array of samples with varying but predefined volumes. We exploited this feature to separate the crystal growth of otherwise concomitant polymorphs from a single solution. Additionally, we demonstrated the removal of the digitized samples from the chambers for downstream analysis, as well as the addition of reagents to the digitized samples. We believe this simple method will be useful in a broad range of applications where a large array of discretized samples is required, including digital PCR, single-cell analysis, and cell-based drug screening.

What is syncrystallization? States of the pH indicator methyl red in crystals of phthalic acid.

The concept of syncrystallization was reinvestigated by focusing on phthalic acid (PA) grown with methyl red (MR). Crystals are alternately red and yellow in adjacent growth sectors. X-ray structures of MR and its cocrystals, revealing MR in the neutral, zwitterionic, and protonated states, as well as measurements of linear birefringence and linear dichroism of mixed crystals, were used to investigate mechanisms of PA coloring. These experiments were complemented by force field calculations of the lowest energy stable surfaces of expressed facets and energies of MR on and in crystals, as well as molecular orbital calculations of MR. Two MR species were detected in PA having distinct energies, polarizations, and face selectivities. Assignments of structures to these MRs, previously thought to be neutral and protonated, required a nuanced analysis of hydrogen bonds. The essential difference between yellow and red species is whether the MR carboxylic acid proton is inter- or intramolecularly hydrogen bound. Inferences about mixed crystal structure drawn from an examination of cocrystals of PA and MR are inconsistent with polarization spectroscopy signaling caution when using stoichiometric compounds as models of dilute solid solutions. Upon heating mixed crystals, linear dichroism diminishes and oriented, elongated pools of MR separate and pass through the bulk in directions perpendicular to the direction of elongation. These bâtonnets subsequently crystallize leaving macroscopic oriented crystals of a MR-rich phase within PA. No evidence was found for the simultaneous crystallization of MR and PA; however, the MR reorientation on heating as well as the separation and recrystallization of a MR-rich phase are distinct processes that could be embraced by the literal meaning of syncrystallization.

Structural analysis of polymorphism and solvation in tranilast.

Five polymorphic forms of tranilast were characterized by thermal, diffractometric, and spectroscopic techniques. The crystal structures of the most stable anhydrous form (Form I), a chloroform solvate, and a dichloromethane solvate were determined from single-crystal X-ray analysis. Two additional anhydrous forms of tranilast (Forms II and III) were also studied, but were not amenable to SCXRD. All five forms were also analyzed using solid-state nuclear magnetic resonance, Fourier transform infrared, and Fourier transform-Raman spectroscopy, and thermal methods. From the trends observed in the crystal structures and the spectral data, some conclusions can be made about hydrogen bonding, molecular conformation, and crystal packing differences in the polymorphs and solvates. Form II was found to be a spectroscopically distinctive polymorph that is probably missing an important intramolecular hydrogen bond coupled with a conformational change. In contrast, Form III was found to be more similar to the crystallographically characterized forms, and is more likely a packing and hydrogen-bonding polymorph with a weakened intermolecular hydrogen-bonding interaction relative to the other forms. From a pharmaceutical development perspective, it is shown that although the anhydrous forms of tranilast have similar thermal properties, they can be reliably distinguished by spectroscopic methods.