Precise Light-Driven Polarity of Stationary Phase for Regulating Gradient Separation of Liquid Chromatography

Generally, the traditional stationary phase for liquid chromatography is the key part, but with an in situ immutable property, leading to many separation limitations. Based on the former exploration of photosensitive gas chromatography, we successfully prepared a photosensitive monolithic capillary silica column with high light transmission, taking advantage of the reversible cis-trans isomerism of azobenzene. And the cis-trans isomerism has launched an effective, reversible, and precise control on the liquid chromatographic retention behavior just by photoinduction according to the theoretical basis of a good correlation between photoinduction time, <i>trans</i>-azobenzene ratio, and chromatographic retention factor (<i>k</i>) (<i>R</i>² > 0.9586). In this system, the plausible control mechanism of stationary phase's polarity is the synergistic effect of the self-molecular polarity difference and the occupation (or release) of polar groups on the stationary phase surface by cis-trans isomerism. Furthermore, a "light control-time ratio cycle" strategy was first reported to maintain an arbitrary ratio of <i>trans</i>-azobenzene corresponding to the polarity gradient of the stationary phase during the whole chromatographic separation process, which is verified by a close correlation of the retention time of the targets and the ratio of light-controlled time (<i>R</i>² > 0.9977). The "gradient elution" of the targets was also surprisingly realized due to the gradient regulation of stationary phase's polarity by the programmatic control of alternate light induction and time ratio. This work further advances the practical significance of photosensitive chromatography in "one column for multiple columns", even "one column for multidimensional chromatography", and truly provides research foundation for its development in the separation and analysis of complex biological samples.