Operation of a Gel Column

The procedure for gel column chromatography is very similar to the general description given earlier. The same precautions must be considered in packing, loading, and eluting the column. A brief outline of important considerations follows. 

For fractionation purposes, it is usually not necessary to use columns greater than 100 cm in length. The ratio of bed length to width should be between 25 and 100. For group separations, columns less than 50 cm long are sufficient, and appropriate ratios of bed length to width are between 5 and 10. 

The sample volume is a critical factor in planning a gel chromatography experiment. If too much sample is applied to a column, resolution is decreased if the sample size is too small, the solutes are greatly diluted. For group separations, a sample volume of 10 to 25% of the column total volume is suitable. The sample volume for fractionation procedures should be between 1 and 5% of the total volume. Column total volume is determined by measuring the volume of water in the glass column that is equivalent to the height of the packed bed. 

The flow rate of a gel column depends on many factors, including length of column and type and size of the gel. It is generally safe to elute a gel column at a rate slightly less than free flow. A high flow rate reduces sample diffusion or zone broadening but may not allow complete equilibration of solute molecules with the gel matrix. 

A specific flow rate cannot be recommended, since each type of gel requires a different range. The average flow rate given in literature references for small-pore-size gels is 8 to 12 mL/cm2 of cross-sectional bed area per hour (15 to 25 mL/hr). For large-pore-size gels, a value of 2 to 5 mL/cm2 of cross-sectional bed area per hour (5 to 10 mL/hr) is average. 

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The operation of a gel filtration column is illustrated in Figure 3.9. The stationary phase consists of inert particles that contain small pores of a controlled size. Microscopic examination of a particle reveals an interior resembling a sponge. A solution containing solutes of various molecular sizes is allowed to pass through the column under the influence of continuous solvent flow. Solute molecules larger than the pores cannot enter the interior... 

A few precautionary measures will ensure the efficient operation of TSK gel columns ... 

In the case where only the length of a gel chromatography column is doubled, how is the resolution of two solutes changed under the same operating conditions with the same packed beads Ihe linear correlation of h = Hs,l lrQ versus v shown in Figure 14.7 can be applied to this system. 

T he partitioning of a solute between the stationary and mobile phases of a gel permeation column is a function of the molecular size and shape of the solute and the size distribution of gel pores separating the two phases. For a gel permeation column operating under conditions in which an equilibrium distribution of solute between the phases is ob-... 

The refractive index (RI) of a mixture is a function of the composition of the mixture and the respective refractive indices of the constituents [8]. The mixture refractive index follows mixture laws such as the Lorentz-Lorenz law. Operational measuring instruments are usually differential refractometers or critical-angle re-fractometers [4]. A large disadvantage in the method is that it only provides meaningful results when a two-component system is considered. However, a differential refractometer is commonly used as a concentration detector in the effluent of a gel permeation chromatography (GPC) column for molecular weight determination. 

It is clear that the separation ratio is simply the ratio of the distribution coefficients of the two solutes, which only depend on the operating temperature and the nature of the two phases. More importantly, they are independent of the mobile phase flow rate and the phase ratio of the column. This means, for example, that the same separation ratios will be obtained for two solutes chromatographed on either a packed column or a capillary column, providing the temperature is the same and the same phase system is employed. This does, however, assume that there are no exclusion effects from the support or stationary phase. If the support or stationary phase is porous, as, for example, silica gel or silica gel based materials, and a pair of solutes differ in size, then the stationary phase available to one solute may not be available to the other. In which case, unless both stationary phases have exactly the same pore distribution, if separated on another column, the separation ratios may not be the same, even if the same phase system and temperature are employed. This will become more evident when the measurement of dead volume is discussed and the importance of pore distribution is considered. 

The authors repeated the experiment with two, more strongly retained, solutes m-dimethoxy benzene and benzyl acetate. These solutes were found to elute at (k ) values of 10.5 and 27.0 respectively on a silica column operated with the same mobile phase. The results obtained are shown as similar curves in Figure 13. The m dimethoxy benzene, which eluted at a (k ) of 10.5, also failed to displace any ethyl acetate from the silica gel even when more than 0.5 g of solute resided on the silica surface. Consequently, the m-dimethoxy benzene must have also interacted with the surface by a sorption process. 

The use of mixed gel technology has become widespread since the late 1980s. If the column is well designed it should not he necessary to supplement resolution in certain areas of the operating range (particularly the extremes) hy the addition of individual pore size columns. This practice is not necessary or recommended for PLgel mixed gel columns, although it is a common practice for other commercial products. 

Figures 13.25-13.28 show the ultrahigh resolution separations in chloroform of polystyrene standards, polytetramethylene glycol, urethanes and isocyanates, and epoxy resins, respectively. Multiple column sets of anywhere from two to six columns in series have been used for well over a year with no apparent loss of efficiency. The 500- and 10 -A gels can easily tolerate 15,000 psi or more. In fact, the limiting factor in the number of columns that can be used in series is generally the pump or injector in the FIPLC system. A pump capable of 10,000 psi operation should allow the use of a column bank of 10-12 50-cm columns with a total plate count of 500,000 or more.

As the porosities of PDVB gels increase above 10 A, the pressure limits drop, with 2500 psi being the maximum usable pressure for 10 A, 10 A, and mixed-bed columns. Because the normal operating pressures in most solvents for these columns tend to be in the range of 1000 psi or less for a 10 X 500-mm column, there is seldom an operational problem. Figure 13.8 shows the resolution of a typical mixed-bed column run in chloroform at 1.5 ml min yielding a back pressure of 700 psi and running polystyrene standards. 

It is evident from these results that the interactive properties of the investigated SEC PS/DVB or DVB gels are very different. Because polar electroneutral macromolecules of PMMA were more retained from a nonpolar solvent (toluene) than from polar ones (THF, chloroform), we conclude that the dipol-dipol interactions were operative. Columns No. 1 and No. 2 were very interactive and can be applied successfully to LC techniques that combine exclusion and interaction (adsorption) mechanisms. These emerging techniques are LC at the critical adsorption point (18), the already mentioned LC under limiting conditions of adsorption (15,18), and LC under limiting conditions of desorption (16). In these cases, the adsorptivity of the SEC columns may even be advantageous. In most conventional SEC applications, however, the interactive properties of columns may cause important problems. In any case, interactive properties of SEC columns should be considered when applying the universal calibration, especially for medium polar and polar polymers. It is therefore advisable to check the elution properties of SEC columns before use with the... 

An example of the efficacy of the resin phases used as an alternative to a conventional silica based reverse phase is shown in figure 12 where the separation of the three tocopherols are shown separated on the Cl 8 Polymer Column and The ODA-A 120A silica gel based columns. The columns were 15 cm long, 4.6 mm i.d., operated at a flow rate of 0.5 ml/min at 30°C with a mobile phase of 98% methanol/2% water. 

An example of a separation primarily based on polar interactions using silica gel as the stationary phase is shown in figure 10. The macro-cyclic tricothecane derivatives are secondary metabolites of the soil fungi Myrothecium Verrucaia. They exhibit antibiotic, antifungal and cytostatic activity and, consequently, their analysis is of interest to the pharmaceutical industry. The column used was 25 cm long, 4.6 mm in diameter and packed with silica gel particles 5 p in diameter which should give approximately 25,000 theoretical plates if operated at the optimum velocity. The flow rate was 1.5 ml/min, and as the retention time of the last peak was about 40 minutes, the retention volume of the last peak would be about 60 ml. 

It is fruitless to attempt detailed study of a phenomenon whose products are not well identified. It is unfortunately frequently noted in the literature, especially in cases of column chromatography, that fractions are only identified as to the chemical operations which brought them to light. Fractions are identified, for example, only by the solvent used. Speculations as to the composition of the radioactive solutes in such solutions can seldom be really reliable, and the presence of an unexpected radioactive species is in such cases undetectable. It is also important in reading the literature to watch out for cases in which the chemical yields of the carriers have not been measured. Extensive decomposition can often occur on silica gel and alumina columns, especially when photosensitive or moisture sensitive compounds are used. For these reasons much of the information now existing in the literature must be regarded as only exploratory, awaiting the development of better analytical methods for separation, purification, identification and determination of the products —known or expected. 

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