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Using this monolithic column, hydrophilic separations are achieved even at a lower level of 50% organic solvent. The separation efficiency up to 1.41 × 105 N m-1 and 1.19 × 105 N m-1 is achieved for the separation of theophylline and phenol, respectively. For a real sample, cytochrome C digestion, the monolithic column shows good separation performance, which offers the potential application of the monolithic column on proteomics study.In the present document, we report the development of an analytical method consisting of a sequentia