Abstract
Obtaining high-quality small-molecule crystals suitable for high-resolution structural analysis remains a significant challenge across many areas of crystallography, including pharmaceuticals, natural products, organic compounds, and other molecular materials. We present a general crystallisation workflow based on two complementary strategies implemented using a microbatch-under-oil approach on the Douglas Instruments Oryx8 crystallisation platform: (i) phase diagram-guided optimisation to identify metastable growth conditions and (ii) controlled seeding to enhance crystal growth and quality.
Paracetamol was selected as a model small molecule to evaluate the workflow. Nanolitre-scale crystallisation experiments enabled systematic exploration of metastable and supersaturation regions within the phase diagram. Phase diagram studies successfully identified conditions that supported crystal growth without the need for seed crystals, providing a rational approach for compounds where initial crystals are unavailable. Once seed crystals were obtained, finely prepared microseed stocks were introduced into metastable conditions to promote controlled crystal growth.
Comparative experiments demonstrated clear differences between seeded and unseeded systems. Unseeded drops initially remained clear and formed crystals only after gradual solvent evaporation increased supersaturation over approximately 72 hours. In contrast, seeded drops initiated crystal growth within minutes, producing larger, well-defined crystals with improved morphology and reproducibility.
Selected crystals grown under seeded conditions were characterised by single-crystal X-ray diffraction. The resulting datasets achieved exceptional resolutions of approximately 0.38–0.37 Å with excellent refinement statistics, demonstrating the effectiveness of the crystallisation strategy.
Together, phase diagram-guided optimisation and controlled seeding provide complementary approaches for producing high-quality small-molecule crystals. This workflow is broadly applicable to pharmaceuticals, natural products, organic compounds, coordination complexes, and other small molecules requiring high-quality crystals for structural characterisation.