Silica-based SEC column

The attributes of a SEC column packing material are listed in Table 2. As indicated, the support must be optimized with respect to specific resolution, efficiency, column pressure, and mechanical, chemical, and thermal stability. Recovery of mass and activity is particularly important in the analysis and purification of biopolymers. It also plays a role in the analysis of nonbiochemical synthetic polymers on silica-based SEC columns. In addition to recovery losses by adsorption, the recovery for both groups of polymers can also be reduced by polymer degradation as a result of, for instance, mechanical shear. 

Aqueous buffers around pH 6-8 are a good environment for many proteins and are suitable for silica-based SEC columns. The most common nondenaturing... 

Historically, silica-based SEC columns have been used most widely. This is because many practical grafting procedures for silica are already known and some of them can be readily utilized in preparating SEC columns based on silica [ref. 8]. Among SEC... 

Silica-based SEC columns commercially available at present are summarized in Table 2, together with their basic properties. LiChrospher 100 Diol series have also been used for many years and cited in published papers [ref. 29-33]. The other three are newly introduced. In Table 2, column efficiency of the columns is not shown because this factor, which is usually obtained by use of ethylene glycol as a sample, does not always reflect the separation efficiency of the columns for biologically complex samples. 

A useful point about silica-based SEC columns is that such columns can be used with high polarity organic solvents, such as methanol. Therefore, they can be filled with the solvent and thus avoid fatal damage to the columns caused by bacteria. On the other hand, however, there is a strict limitation for silica-based columns in the practical pH range (pH 3-7.5). 

The silica-based SEC packings which are currently being employed were Introduced on the market in 1980 and there has since been very little improvement In their properties in terms of pore size distribution, particle size etc. Now, initiated by theoretical Investigations of the resolution of SEC columns, a new family of SEC packings Is beginning to be produced and marketed. The new packings are characterized by a particle size smaller than 5 / m, adequate porosity, and superior bonding chemistry. The Bio-Series 6F 250 column from DuPont de Nemours Is a typical example. However, the performance - with reference to resolution, analysis time and peak capacity — of this novel material has not yet been evaluated. 

In a previous paper [ref. 34], a comparison of the separation behaviors between silica- and acrylate-based SEC columns was reported. Elution profiles of polyethylene glycols with molecular weights of 200 and 400 on TSKgel G2000PW and G2000SW are depicted in Fig.l and 2, respectively. This comparison shows a difference in the separation mechanism between these columns, which have the same pore size. It should be noted, therefore, that base materials should be considered in choosing columns as well as pore size of columns. 

Because of the limitations of silica, several manufacturers offer SEC columns based on hydrophilic organic polymer. These include polymethacrylate supports, proprietary hydrophilic polymers, and semirigid cross-linked agaroses and dextrans. These materials are more stable under high-pH operation. 

SEC-HPLC on silica-based columns is a fast and versatile technique for the characterization of complexes between molecular chaperones and substrate proteins. The technique provides an invaluable tool for a rapid determination of their hydrodynamic properties and. in combination with SDS-PAGE, can be used to determine the stoichiometry of such complexes. One limitation is the fact that SEC-HPLC can be applied only to the study of stable complexes, i.e. complexes that will not dissociate significantly during the chromatographic run. 

These non-SEC modes use high efficiency silica-based columns and binary, ternary and quaternary miscible mixtures of organic solvents and water to achieve fast, selective separations (Figure 9.6). In general, a reverse phase mode of operation is most suitable for lipophilic samples, whilst normal partition or adsorption is used for samples which are lipophobic. 

A mobile phase is primarily chosen for its effectiveness in solubilizing and stabilizing the sample. Because of the short contact time related to the isocratic conditions, proteins remain stable if the appropriate mobile phase and column are used. As discussed earlier, nonideal SEC behavior may be observed on silica-based columns. Mobile phase considerations therefore play an important role in SEC. Elimination of protein adsorption is crucial, but the effect of the eluant on protein structure must also be considered. Additionally, polyelectrolytes expand and condense with changes in macroion concentration within the buffer (118). 

Secondary amine-functionalized polystyrene was prepared by normal addition of N-benzylidenemethylamine to PSLi (A n = 2100gmor ) in benzene (eqn [21]). No unfunc-tionalized polystyrene was observed by TLC on silica gel plates using benzene as eluent. The SEC chromatogram of the functionalized polymer showed no indication of dimer formation, in contrast to the analogous functionalization reaction with N-benzylidenetrimethylsilylamine that formed 19% of dimeric products discussed in the previous section. The Mn of the functionalized polymers determined by SEC was appreciably smaller compared to that of base polymer due to interactions of the functionalized polymer with the SEC columns, which could be deduced from the larger retention volume of the functionalized polymers compared to that of base polymers and tailing of the functionalized polymers. The functionality determined by end-group titration was 96%. 

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