Preparation and application of monolithic columns with narrow immobilized pH gradients

Monolith with immobilized pH gradient (M-IPG) is a novel separation matrix for amphoteric substances, such as peptides and proteins. To improve the properties of the newly designed column, efforts were made to optimize the preparation procedure, including the concentrations of the monomers, glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA), as well as the kinds of porogens, which led to a monolith with improved permeability, uniformity and continuity. In addition, different diamines, including ethylenediamine, 1,3-diaminopropane, 1,4-diaminobutane and 1,5-diaminopentane, were subjected to aminate the polymer, and the last two exhibited excellent reactivity with epoxy on the polymer surface, which could obviously make the immobilization of pH gradient facilitated. Under the optimal conditions, a simple method to prepare M-IPG column with narrow pH gradient was developed with commercial carrier ampholytes (CAs) solution. Such columns were applied into the analysis of proteins and peptides, and showed the improved resolution compared to the traditional one with a wide pH distribution.