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give a mixed gel with definitive exclusion properties for specific separations. The exclusion properties of a silica gel cannot be obtained with sufficient accuracy for chromatographic use from nitrogen adsorption data or mercury porosity tests. It is necessary to determine the range of pore diameters and pore volume of a silica gel by a special experimental procedure that is designed to obtain accurate retention volume measurements for solutes eluted in relatively small elution volumes. In exclusion chromatography, all the peaks will be contained in a mobile phase volume equivalent to that of the total pore volume of the column. Consequently, the column volume itself must be large and a column 25 cm long and 4.6 mm I.D. is a practical size to obtain results having adequate accuracy. The sample volume used should be 0.5 to 1.0 ml in volume and the detector should have a low sensor volume (cf. 2-4 ml). A mobile phase that would be suitable for exclusion measurements with silica gel

by blending different silica gels. Northrop et al (17) examined the exclusion properties of a number of silica gels all having a particle size of 10m They choose two specific gels, one with a mean pore diameter of 80� and the other with a mean pore diameter of 500� and blended them to provide an exclusion packing that had a molecular weight range extending from 100 to about 1,000,000. The packing was intended for use in the separation of high molecular weight materials of biological origin. The exclusion curves obtained for the two silica gels and the blend having 38%� 80� silica and 62% 500� silica are shown in figure 31.     Figure 31. Graph of Exclusion Volume against Molecular Weight

the latter in exclusion chromatography. In practice, it is rare that either procedure is exclusive in any given separation as both retentive processes are usually present to some extent, particularly in liquid chromatography (LC). Retention in gas chromatography (GC), using coated open tubes, is, perhaps, the exception, as, in this distribution system, exclusion processes (aside from chiral phases) are normally absent and, consequently, retention control is purely interactive. Interaction and exclusion processes, when they do occur together, act independently. Although, together they determine overall the retention of a solute, the exclusion properties of a stationary phase do not effect the magnitude of any of its interactive properties. The mechanisms of retention have been discussed briefly in Book 1, but will now be considered in greater detail.� Although both retention processes (interaction and exclusion) are usually active, because they contribute to retention

Retention and Exclusion All stationary phases based on silica gel exhibit exclusion properties.� Polystyrene stationary phases are broadly similar in physical form but their exclusion properties tend to be less significant than those of silica gel. The exclusion properties of silica gel arise from its method of formation and so the processes involved will help reveal the physical nature of the gel. Silica gel is an amorphous, highly porous, partially hydrated form of silica� made from the two most

phase or supporting material contains pores of size commensurate with those of the solute molecules, exclusion will still partly control retention. This is because the larger molecules will interact with less stationary phase and be eluted relatively faster than if they had interacted with the same amount of stationary phase as the smaller molecules. The two most common exclusion media used in LC are silica gel and macroporous polystyrene divinylbenzene resins. Figure 19 shows an exclusion chromatogram of a series of molecular weight standards obtained on silica gel.   The column length was 50 cm and the mobile phase tetrahydrofuran (THF). The THF would be strongly adsorbed on the silica surface and thus the solutes would be distributed between pure THF in the mobile phase and THF on the surface of the silica. As a consequence, the interactions are virtually identical in the two phases and the retention was determined almost exclusively by

nbsp;  Courtesy of the Analyst (ref.11)   Figure 8. Graph of Retention Volume of n-Alkanes against Carbon Number    In figure 8, all the points lie on the same straight line, irrespective of the operating temperature and, thus, the enthalpy term (see The Thermodynamics of Chromatography ) is close to zero and the solutes are not retained by differential molecular forces. Consequently, the retention of the hydrocarbon solutes is solely due to exclusion.. It is clear that the exclusion effect is significant and, by extrapolating to a carbon number of zero, the total volume of mobile phase in the column is obtained. In this case,  the total interstitial volume plus the total pore volume is about 2.9 ml. This value agrees well with the data for the retention of water and methanol.   The problems associated with exclusion and dead volume measurement cannot be avoided but they can be minimized. Probably the best