Silica stationary phase functionalized by 4-carboxy-benzoboroxole with enhanced boronate affinity nature for selective capture and separation of cis-diol compounds

4-Carboxy-benzoboroxole was designed and synthesized. It was then combined with the modification effect of polyethyleneimine (PEI) for the preparation of boronate affinity silica stationary phase. The stationary phase showed improved binding strength with dissociation constant (K_d) towards xanthosine as low as 2.48 × 10^-4 M. The column showed excellent selectivity, high binding capacities (88.3 μmol adenosine g^-1, pH 7.0) and the lowest binding pH (4.0 for cytidine and as low as 2.24 for xanthosine). These binding properties were superior to the existing boronate affinity materials, facilitating the selective extraction of trace cis-diol compounds in complex samples and greatly expanding the application scope of boronate affinity chromatography. In addition, the column showed secondary separation capability under acidic conditions and this secondary separation capability was investigated thoroughly. It was found that the separation was pH-dependent and mainly determined by binding strength with the possibility of involvement of other interaction, providing alternative strategy for the separation of cis-diol compounds. The feasibility and practicability were demonstrated through the selective enrichment of nucleosides in urine samples and the results indicated the excellent performance and great potential for the extraction of trace cis-diol compounds in complex samples.