Multiple-Use Microplate Assay for Submicromolar Ultra High-Throughput Separation of Amines Based on their Degree of Substitution.

Small-molecule amines, typically studied in their more stable and water-soluble protonated forms, are of central importance in drug discovery. Their structural diversification often relies on N-alkylation, yielding mixtures of analogs with varying degrees of substitution-posing a key challenge for purification. While advanced chromatographic techniques exist, no high-throughput, broadly applicable alternative has emerged that aligns with the capabilities of automated synthesis. Here, a reusable microplate-based assay enabling ultra-high-throughput, parallel separation of protonated amines-including alkyl-, aryl-, and aralkylamines-at submicromolar levels is reported. The method exploits a covalently immobilized tris(pyridino)-crown ether selector, which forms reversible host-guest complexes by H-bonds, which differ with the degree of N-substitution. This supramolecular recognition strategy eliminates the need for compound-specific method development, derivatization, or preparative-scale quantities. In addition, the present article introduces a generally applicable surface-functionalization protocol for customizing standard commercial microplates into molecular recognition platforms. The present approach resolves key limitations of current separation technologies-such as high energy use, low integration with liquid-handling systems, inevitable sample dilution, and time-intensive workflows-offering a transformative tool for rapid and efficient purification directly compatible with modern synthesis pipelines.