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.