Liquid chromatography with rapid equilibration

The popularity of reversed-phase liquid chromatography (RPC) is easily explained by its unmatched simplicity, versatility and scope [15,22,50,52,71,149,288-290]. Neutral and ionic solutes can be separated simultaneously and the rapid equilibration of the stationary phase with changes in mobile phase composition allows gradient elution techniques to be used routinely. Secondary chemical equilibria, such as ion suppression, ion-pair formation, metal complexatlon, and micelle formation are easily exploited in RPC to optimize separation selectivity and to augment changes availaple from varying the mobile phase solvent composition. Retention in RPC, at least in the accepted ideal sense, occurs by non-specific hydrophobic interactions of the solute with the... 

High performance liquid chromatography (HPLC). All HPLC analyses were performed with a model HP1090 analytical HPLC equipped with a diode array detector (Hewlett Packard, Mountain, View, CA). A Protein PAK 3000 SW column (Waters, Bedford, MA) was used for each size exclusion experiment. The column was equilibrated with ten column volumes (100 ml) of elution buffer (2.0 M GuHCl, 50 mM Tris sulfate, 5 mM EDTA, pH 7.5) prior to operation. A sample volume of 25 gl was applied to the column and eluted at a flow rate of 1.0 ml/min to facilitate rapid separation. For equilibrium experiments, each sample was equilibrated for three to eight hours before column separation. 

Many natural materials are porous but also proton-rich such as wood or other plant products. Relaxation of liquids in these materials has features in common with both inorganic matrices and the protein systems discussed above. The class of porous polysaccharide materials used for size exclusion chromatography provides an example one commercial product is Sephadex. The material swells on solvation to form a controlled pore gel. The main application involves excess liquid, generally water, which flows through the gel bed carrying solutes of various size. The large solutes are excluded from the pore interior and elute rapidly while the smaller ones equilibrate with the pore interior and elute later. The solvent generally samples the pore interior as well as the bulk phase. 

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