Preparation and use of in situ chemically bonded small-particle silica as packings in high-pressure liquid chromatography

Abstract Previously we have demonstrated the feasibility of forming bonded phases on porous layer beads (Corasil II) by a completely in situ process. A logical extension of this in situ method has been its application to small-particle silica columns. Such columns have all the advantages of the bonded phases formed on porous layer beads with the added feature of much higher plate counts. In addition, when compared to commercially available small-particle bonded phase columns, the cost is reduced significantly. In this paper, both the initial column preparation procedure and the in situ technique applied to completely porous small-particle silica columns are presented. Columns were packed with a constant-pressure pump using a dibromoethane slurry procedure. After initial packing, all columns were conditioned with a solvent series of decreasing polarity and evaluated for performance using a test mixture of biphenyl ( k ′ ≈ 0.5–1.0) and nitrobenzene ( k ′ ≈ 5–10). After evaluation, suitable columns ( H = 0.008 ± 0.002 cm at a flow velocity of 0.55 cm/sec) were selected for in situ modification. By this procedure, differences due to variation in column packing were minimized. Subsequently, various modification procedures were investigated. As in the case of porous layer beads, control of pre-reaction surface hydration was found to be critical in determining the consistency of surfaces formed.