Elution behavior of short dsDNA strands in silicon micropillar array columns in ion pair reversed-phase chromatography mode

In the present paper, dsDNA separation has been studied in a silicon micro-pillar array column using ion-pair reversed-phase HPLC (IP-RP-HPLC). The deep-etched (32.0μm) silicon micro-pillar array was fabricated by advanced deep-UV lithography and by a dedicated Bosch etch process and then sealed by anodic bonding to a Pyrex glass. The pillar surface was subsequently conditioned hydrophobic. Working in isocratic mode under non-retained conditions, van Deemter curves of dsDNA and coumarin were established to assess the performance of the micro-pillar array column, resulting in plate heights of only a few μm. Working in gradient mode, separations of dsDNA fragments were evaluated. The relevant gradient operation parameters were studied to understand their influence on dsDNA separations. The correlation between DNA length and retention was measured and theoretically described in a length range of 50-500bp, promising for the determination of DNA of an unknown length. Finally, a separation example demonstrated the excellent separation power of on-chip IP-RP chromatography by achieving a large operation range of DNA length (10-300bp) with a 5bp difference among 11 dsDNA fragments