Packed-bed characterization of microfluidic liquid chromatography columns using breakthrough curve analysis

Microfluidic technologies enable the miniaturization of liquid chromatography, reducing reagent consumption and analysis time. In this work, a packed-bed microfluidic column was developed and evaluated using breakthrough curve analysis as an alternative to conventional pulse-based methods. The device incorporates a 1 cm microcolumn fabricated through a cleanroom-free process based on micromilling and soft lithography. Packed beds of silica particles with diameters of 45-75 µm and 10 µm were evaluated using ethanol as the mobile phase and rhodamine B as a model analyte. Breakthrough experiments performed over a range of flow rates enabled the determination of retention behaviour, peak variance, and plate height after correction for extra-column dispersion. Van Deemter analysis demonstrated that the apparent plate-height trends were strongly influenced by particle size and confinement effects imposed by the microchannel geometry. Larger particles exhibited lower reduced plate heights, suggesting that confinement-induced packing effects may alter conventional chromatographic trends. Independent packing experiments showed good column-to-column reproducibility, confirming the reliability of the proposed methodology. To validate the microfluidic approach, equivalent breakthrough experiments were conducted in a macroscale chromatography system packed with the same 45-75 µm silica stationary phase. Both platforms exhibited comparable efficiency trends, supporting the use of the microfluidic device for comparative packed-bed characterization. Preliminary measurements using integrated hydrogenated amorphous silicon photodiodes demonstrated the feasibility of compact on-chip optical detection. These results establish our tool as a proof-of-concept microfluidic platform for rapid chromatographic column screening and support its future development toward portable and fully integrated liquid chromatography systems.