3D-printed serpentine columns for size-exclusion chromatography with mass spectrometric detection

LC-MS is an important tool for analyzing various biomolecules. In recent years, 3D printing has shown great potential in customizing and improving LC-MS experiments. However, the direct coupling of stereolithography-based (SLA) LC column with mass spectrometry (MS) for biomolecule analysis has not been demonstrated.

In this work, we report the development of compact 3D-printed serpentine size exclusion chromatography (SEC) columns, which are effectively coupled with MS detection for biomolecule separations under native conditions. We utilized a simple surface coating strategy that effectively prevents chemical leaching in our compact 3D-printed serpentine size exclusion chromatography (SEC) columns. The 3D-printed serpentine SEC column (1.60 mm diameter x 150 mm length) shows highly reproducible peak retention times with a relative standard deviation (RSD) of less than <1.80 %. Additionally, it maintains a peak asymmetry factor <1.50 (RSD <8 %), confirming the reliability and consistency of the column performance. This column also demonstrates compatibility with high pressures, reaching up to 1.1 × 10⁷ Pa (110 bar). Finally, we demonstrated the separation of a mixture of ubiquitin and angiotensin and a mixture of myoglobin and insulin with baseline resolutions and a tailing factor below 1.40.

This work presents the first 3D-printed serpentine SEC columns compatible with spray-based MS analysis. It also paves the way for creating LC columns with custom geometry and integrating additional functional units for various LC-MS applications.