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, the solute molecules are continually transferring from the mobile phase into the stationary phase and back from the stationary phase into the mobile phase. This transfer process is not instantaneous, because a finite time is required for the molecules to traverse (by diffusion) through the mobile phase in order to reach, and enter the stationary phase. Thus, those molecules close to the stationary phase will enter it almost immediately, whereas those molecules some distance away from the stationary phase will find their way  to it a significant interval of time later. However, as the mobile phase is moving, during this time interval while they are diffusing towards the stationary phase boundary, they will be swept along the column and thus dispersed away from those molecules that were close and entered it rapidly. The dispersion resulting from the resistance to mass transfer in the mobile phase is depicted in figure 7.The diagram shows 6 solute molecules in the mobile phase

from the mobile phase to the stationary phase. This transfer is not instantaneous; time is required for the molecules to pass (by diffusion) through the mobile phase to reach the interface and enter the stationary phase. Those molecules close to the stationary phase enter it immediately, whereas those molecules some distance away will find their way to it some time later. Since the mobile phase is continually moving, during this time interval, those molecules that remain in the mobile phase will be swept along the column and dispersed away from those molecules that were close and entered the stationary phase immediately. This process is depicted in figure 22. The diagram shows 6 solute molecules in the mobile phase and the pair closest to the surface, (1 and 2), enter the stationary phase immediately. While molecules 3 and 4 diffuse through the mobile phase to the interface, the mobile phase moves on. As a consequence, when molecules 3 and 4 reach the interface, they

the Performance of a Chromatographic System The stationary phase content of a column can affect a separation in two ways. The more stationary phase there is in a column, the more the solutes will be retained, the further they will be apart and the greater the separation. Any change in stationary phase, however, will change the retention of all solutes proportionally and thus the separation will only increase, if the peak widths remain unchanged. Increasing the amount of stationary phase will usually increase the thickness of the stationary phase film, which, as is shown in Dispersion in Chromatography Columns will increase peak dispersion. It follows that there will be a specific stationary phase loading that provides the best compromise between separation and band dispersion (6) and thus provides the maximum resolution. The loading can be quite critical for open tubular columns in GC. Thus, the stationary phase loading cannot be increased indefinitely to

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, it is likely that one enantiomer in the sample will fit closely to the stationary phase surface whereas the other will be stearically excluded and thus have less stationary phase with which to interact. The first chiral separations in GC were reported by Gil-Av et al. as in 1966 (7), but, surprisingly, the use of GC for the separation of enantiomers has only recently been investigated and developed into a practical system. The use of chiral stationary phases in GC has been dogged by entantiomeric instability arising from the racemization of both the chiral stationary phase and the chiral solutes at elevated temperatures. In addition, at the

(a reversed phase) to promote dispersive interaction with the solutes and provide adequate retention and selectivity. If the solutes are strongly polar then a polarizable stationary phase (one containing aromatic rings or cyano groups) would be appropriate to separate the solutes by polar and induced polar interactions. If the solutes are weakly polar then a strong polar stationary phase would be required (such as silica gel) to separate the solute by polar interactions. The mobile phase must be chosen to complement the stationary phase so that the selected interactions are concentrated in the stationary phase. Thus, a reversed phase having strong dispersive interactions would be used with a strongly polar mobile phase (e.g., mixtures of methanol and water acetonitrile and water or tetrahydrofuran and water). In contrast, if the strongly polar silica gel is selected for the stationary phase then a strongly dispersive mobile phase would be appropriate (e.g., n-heptane, n-

The Control of Chromatographically Available Stationary Phase (Vs) The volume of stationary phase that is made available to the solutes can be controlled in a number of ways. Firstly, the stationary phase loading on the column can be varied to adjust the retention as required. A specific stationary phase loading may be selected, to either improve the resolution, or to reduce the analysis time, or in some instances, to increase the sample load. Sometimes, the stationary phase loading is reduced so the column is more amenable to specific compounds (e.g. to prevent proteins from being denatured). Secondly, the stationary phase can contain molecules of a special shape that can only make close contact with molecules having a complementary shape. Other molecules can not interact so closely with the stationary phase and consequently, the stationary phase available to them will be restricted. This approach is exploited in chiral chromatography