On-Chip Step-Mixing in a T-Nanomixer for Liquid Chromatography in Extended-Nanochannels

Miniaturization of liquid chromatography separation columns is a key trend in chemical and biochemical areas, particularly in genomics, proteomics, and single-cell analysis. The work at this level relies upon a novel analytical platform that can deal with sample volumes that are much smaller than a cell. An extended-nanospace is within a scale of 10¹-10³ nm and defines the space between a single molecule and normal liquid. Our group has realized high-performance liquid chromatography (HPLC) separation in extended-nanospace with sample injections of hundreds of attoliters and a separation efficiency of hundreds of thousands of plates/m that can overcome the limitations of a conventional packed column by a magnitude of several orders. However, gradient flow is needed to improve the separation performance, and in this work we present reversed-phase chromatography with step-mixing in extended-nanospace and describe its application. Six fluorescently labeled amino acids were separated in 16 s, followed by separation of 17 labeled amino acids in only 50 s with a plate height for most of the peaks of less than 1 μm.