HEPES: A Unique Case of Conformational Multi-ZwitterionicPolymorphism

Abstract HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane-1-sulfonic acid) is a widely used buffering agent in biological and biomedical research. Here, we reveal an exceptional solid-form landscape for HEPES. In addition to the two previously known polymorphs, we discover and characterize three new polymorphs and three new hydrates. These solid forms differ not only in crystal packing but also in conformation and protonation states, giving rise to a unique case of rich conformational multi-zwitterionic polymorphism. All forms are characterized using a comprehensive suite of experimental techniques, their phase transformations are examined, and their structures are further investigated through computational modeling. Energetic analyses uncover an unusual interplay between conformation, ionization state, and intermolecular interactions in determining the thermodynamically stable form. Together, the experimental and computational insights reveal the complex solid-state behavior of zwitterionic molecules and the resulting challenges for modeling, crystallization control, and overall early solid form and process development in the pharmaceutical and other industries.