A Complementary Type of Zero Dead Volume Connection for Capillary Column Liquid Chromatography

A notable trend in modern liquid chromatography is the growing use of narrow bore, high-efficiency columns to expedite high resolution separations of small-volume complex mixtures. In microscale separations, the extracolumn band broadening (ECBB) becomes especially pronounced and has detrimental effects on peak shape, efficiency, and achievable resolutions. In practice, however, dead volumes at column connections are often overlooked, despite their non-negligible contributions to ECBB. In this technical note, we report a complementary type of connection to eliminate dead volumes between microcolumns, based on elastic macroporous polyacrylamide hydrogel septum (MAPS). When integrated into the connector, the septum reformed under pressure, compensating for the microgaps between columns, resulting in minimized dead volumes and therefore diminished ECBB. In comparison with the commonly used through-type zero dead volume connections, the MAPS connection effectively improved peak shape (28% reduction in tailing factor) and column efficiency (27% increase in theoretical plate number). Furthermore, it exhibited a good performance stability in repeated use (RSD < 11% for peak efficiency, n = 15) and in a time span of one month. When applied for serially connecting multiple capillaries, the MAPS connection enabled an almost linear increase in column efficiency (25,000 plates on a 48 cm connected column vs 4200 plates on an 8 cm column), making it ideal for high resolution separations. Meanwhile, as demonstrated, the MAPS connection did not cause significant nonspecific adsorptions in analysis of biomolecules. The macroporous hydrogel-based microdevice presented in this work may provide an effective solution toward dead-volume free connections in microfluidics, securing high resolution in miniaturized liquid chromatography of small volume complex mixtures.