Mode selection size exclusion

Let us consider the separation of polymethylmethacrylate (PMMA) on a nonmodified silica column as an example. In THE (medium polar eluent) the PMMA eludes in size exclusion mode because the dipoles of the methylmethacrylate (MMA) are masked by the dipoles of the THE. Using the nonpolar toluene as the eluent on the same column, the separation is governed by adsorption because the dipoles of the carbonyl group in the PMMA will interact with the dipoles on the surface of the stationary phase. The separation of PMMA in the critical mode of adsorption can be achieved by selecting an appropriate THF/toluene mixture as the eluent. In this case all PMMA samples... 

A more complicated, but flexible, system has been reported by Blomberg et al. (46). Here, size exclusion chromatography (SEC), normal phase EC (NPLC) and GC were coupled for the characterization of restricted (according to size) and selected (according to polarity) fractions of long residues. The seemingly incompatible separation modes, i.e. SEC and NPLC, are coupled by using an on-line solvent-evaporation step. 

Non-silica-based RP-HPLC stationary phases have also been developed and their separation capacity has been compared with those of silica-based ones. The porous structure of crosslinked polymer gels may be responsible for the markedly different selectivity and retention characteristics. Up till now, the mode of separation on polymer stationary phases is not entirely understood at the molecular level. It has been established that the size-exclusion effect may influence the retention of analyses on polymer gels. 

Selecting a column for an HPLC separation is a matter of asking yourself a series of questions (Fig. 5.4). You first must determine how much material you wish to separate in a single injection (preparative vx. semipreparative vs. analytical). The next question involves the separation mode to be employed (size exclusion vx. ion exchange vx partition). Finally, there is the question of solubility controlling solvent and column selection in all modes. 

In the case of gel permeation or size-exclusion HPLC (HP-SEC), selectivity arises from differential migration of the biomolecules as they permeate by diffusion from the bulk mobile phase to within the pore chambers of the stationary phase. Ideally, the stationary phase in HP-SEC has been so prepared that the surface itself has no chemical interaction with the biosolutes, with the extent of retardation simply mediated by the physical nature of the pores, their connectivity, and their tortuosity. In this regard, HP-SEC contrasts with the other modes of HPLC, where the surfaces of the stationary phase have been deliberately modified by chemical procedures by (usually) low molecular weight compounds to enable selective retardation of the biosolutes by adsorptive processes. Ideally, the surface of an interactive HPLC sorbent enables separation to occur by only one retention process, i.e., the stationary phase functions as a monomodal sorbent. In practice with porous materials, this is rarely achieved with the consequence that most adsorption HPLC sorbents exhibit multimodal characteristics. The retention behavior and selectivity of the chromatographic system will thus depend on the nature and magnitude of the complex interplay of intermolecular forces... 

Specific and essentially stand-alone mode of liquid chromatography is associated with the absence or suppression of any analyte interactions with the stationary phase, which is called size-exclusion chromatography (SEC). In SEC the eluent is selected in such a manner that it will suppress any possible analyte interactions with the surface, and the separation of the analyte molecules in this mode is primarily based on their physical dimensions (size). The larger the analyte molecules, the lower the possibility for them to penetrate into the porous space of the column packing material, and consequently the faster they will move through the column. The schematic of this classification is shown in Figure 1-1. 

Size Exclusion Chromatography A mode of chromatography using porous particles having pores of a size comparable to that of the molecules to be separated. Large molecules do not access the pores and elute at the external porous volume. Small molecules access all pores and elute at the total pore volume. Intermediate size molecules access part of the pores. This method applies to polymers, and especially to proteins. The solvent and the porous soUd must be selected to avoid adsorption of the feed components. 

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