Digitally programmable microfluidic valving for autonomous, high-resolution continuous chromatographic purification

Continuous microscale purification requires analytical methods that provide deterministic fluid handling, precise temporal control, and contamination-free fraction discrimination. Existing microfluidic and benchtop chromatography systems only partially address these needs, leaving a gap for methods that support tightly coordinated, programmable purification cycles. This work presents a microfluidic continuous protein purification method that uses digitally programmable inlet (ICV) and collection (CCV) valves to establish a logic-driven chromatography operation. Sub-second buffer switching and deterministic routing across parallel affinity columns enable a reproducible and algorithm-defined purification sequence. Temporal gating through the CCV provides real-time, profile-guided fraction selection that isolates high-concentration eluates while effectively removing tailing segments. Using GFP-His₆ as a model substrate, the system maintains 70-89% purity over ten uninterrupted cycles, demonstrating strong cycle-to-cycle stability. Purification of His₆-tagged TRAIL further confirms compatibility with structurally sensitive biologics and preservation of functional activity. The compact, modular, and single-use architecture minimizes dead volume, prevents cross-contamination, and accommodates diverse chromatographic modes. By combining programmable valve logic with time-resolved elution control, this work advances microfluidic platforms from diagnostic tools toward autonomous and precision-controlled process operations. The method provides a broadly applicable analytical framework for microscale purification and supports the development of next-generation bioseparation and continuous biomanufacturing technologies.