Entrapment is a noncovalent immobilization method that enables a large biological binding agent, such as a protein, to be put within a support without modifying the structure of the binding agent. This chapter describes an on-column entrapment method that can be used with proteins and HPLC-grade silica to prepare columns for high-performance liquid chromatography. In this method, a protein is trapped within a dihydrazide-activated silica support by using oxidized glycogen as a capping agent. This method allows the protein to be placed within the support in a soluble form and with little or no loss of activity. The approach and reagents needed for this method are described in this chapter, along with some applications reported for columns that have been made using on-column protein entrapment.