for about 5 hours and the product is then filtered on a sintered glass filter, washed sequentially with toluene, tetrahydrofuran (THF), methanol, methanol water (50:50 v/v) and finally with methanol and dried under suction. The bonded phase now needs end-capping; that is, any unreacted silanol groups are treated with a small molecular weight silanizing reagent to react with those hydroxyl groups that were stearically unavailable to the larger reagent due to exclusion. To end-cap the product, thebonded phase is refluxed for two hours in a mixture of 100 ml of toluene and 25 ml of hexamethyldisilazane. The product is again filtered free of the reaction liquid mixture and washed sequentially with, toluene tetrahydrofuran, methanol, methanol water (50:50 v/v) and finally with methanol and then dried under vacuum. It should be pointed out that end-capping cannot eliminate the hydroxyl groups that are stearically hindered by the bonded moiety, or at best, only a small proportion of them will

Choosing a Bonded Phase Different organic moieties can be bonded to all types of silica gel particles including the very popular spherical variety. One of the most important features to consider when choosing a bonded phase is the reproducibility of the product. The packing must obviously achieve the separation that is required, but unfortunately any bonded phase has a limited lifetime that may range from a few hours, if operated at extremes of pH, to several months if operated under mild conditions. It follows, that all columns will eventually need replacing and if a specific analytical procedure has been established on a particular column, then the replacement must have as near identical

area. In addition, the amount of available stationary phase on a bonded phase can be modified by adjusting the molecular size (chain length) of the bonded material. The chain length of the bonded material can be critical when separating proteins as dispersive interactions between the alkane chains and the dispersive (hydrophobic) groups of the protein can be strong enough to cause structural deconformation; (i.e., the protein becomes denatured). Reducing the chain length of the bonded material, the dispersive forces can be reduced significantly and the deconformation diminished. In practice, carbon chains only two or four carbon atoms long are among those most commonly used for separating labile proteins. Stationary Phase Limitation by Chiral Selectivity. The extent to which an enantiomer can interact with the stationary phase depends on how close it can approach the molecules of the stationary phase. If the stationary phase is also chiral in nature

Types of Bonded Phase There are three basic types of bonded phases, the brush phases, the bulk phases and the oligomeric phases. The different phases are produced by the use of the mono, di- and tri- substituted silanes respectively in the bonding process, e.g. the monochloro, dichloro and tricoloro silanes. The monochlorosilanes, for instance octyldimethyl-chlorosilane, react with the hydroxyl groups on the silica surface to produce dimethyloctylsilyl chains attached to the silica. Brush Phases

nbsp; The Fluidized Bed Method for Bonded Phase Synthesis The concept of synthesizing bonded phases by means of a fluidized bed reactor was first suggested by Unger (23) and was later implemented by Simpson and Khong (23a). The term fluidization describes a contact process in which particulate matter is transformed into a fluid-like state by a stream of gas or liquid. This state is achieved by the upward passage of a fluid through the bed of particles to a velocity at which the drag force acting on the surface of the particles

matter on which the stationary phase is bonded or coated. The mobile phase (which may be a gas or a liquid) passes under pressure through the column to elute the sample. The column form may also be a long, small-diameter open tube that has the stationary phase coated or bonded to the internal surface. Alternatively, the chromatographic system may take the form of a plate (usually glass) the surface of which is loaded with particulate matter to which the stationary phase is coated or bonded. The mobile phase (a liquid) is arranged to percolate up the plate (usually by surface tension forces) to elute the sample. The sample is injected into the mobile phase stream just before the front of the columns. The column is designed to allow two processes to take place that will produce the separation. Firstly, as a result of different forces between each molecular type and the stationary phase, each solute is retained to a different extent and, thus, the more