Capillary fractionation of HPLC substrates by a microfluidic droplet generator for high throughput analysis

Biochemical samples are complex mixtures containing 1000´s of components which often must be fractionated prior to analysis. Conventional fraction collectors, which can only accommodate 10´s of fractions, are not well suited for high throughput analysis. This paper describes microfractionation in droplets (μFD), a scalable microfluidic technique for generating thousands of fractions. A drop generator, placed downstream from a high performance liquid chromatography (HPLC) column, encapsulates the separated components into a serial array of monodisperse droplets. The droplets can be stored in a capillary or immediately used in subsequent assays. Using μFD, a mixture of 3 dyes separated in a C18 column was fractionated into 2,160 droplets in <; 6 min. The volume and frequency of the droplet fractions are governed by the capillary number (Ca), which depends on the viscosity of the carrier fluid, flow rate, and interfacial tension. With HPLC-compatible flow rates of 0.38-0.7 mL/min, in a 1.5 mm Teflon capillary, fractions contain volumes of 1-6 μL and are generated at 2-10 drops/s. Droplet fractions can be mixed with a subsequent reagent using a downstream tee junction. In theory, μFD can be coupled to a wide variety of separation processes, enabling high throughput fractionation and screening of complex mixtures in μL to sub-nL volumes.