SynchroPak

SynChropak GPC supports were introduced in 1978 as the first commercial columns for high-performance liquid chromatography of proteins. SynChropak GPC columns were based on research developed by Fred Regnier and coworkers in 1976 (1,2). The first columns were only available in 10-yu,m particles with a 100-A pore diameter, but as silica technology advanced, the range of available pore diameters increased and 5-yu,m particle diameters became available. SynChropak GPC and CATSEC occasionally were prepared on larger particles on a custom basis, but generally these products have been intended for analytical applications. 

SynChropak size exclusion supports are composed of spherical uniformly porous silica that has been derivatized with a suitable layer. SynChropak GPC supports are available in six pore diameters ranging from 50 to 4000 A and particle diameters from 5 to 10 /zm. SynChropak CATSEC supports are available in four pore diameters. Table 10.1 details the physical characteristics of the product lines. 

SynChropak GPC supports are bonded with y-glycidoxypropylsilane by a proprietary process that results in a thin, neutral hydrophilic layer that totally covers the silanol sites of the silica. The silica backbone prevents the supports from swelling. 

The neutral hydrophilic surface and the wide range of pore diameters available for SynChropak GPC allow many compounds from small peptides to nucleic acids and other polymers to be analyzed. Table 10.2 lists the approximate exclusion limits for both linear and globular solutes. Although this information... 

TABLE 10.2 Molecular Weight Range for SynChropak SEC Supports°... 

For proteins, the most useful columns are those with pores of 100-500 A, as seen in Fig. 10.2, because most proteins elute on the linear portions of the calibration curves. Figure 10.5 illustrates an analysis of a protein mixture on SynChropak GPC100. Small peptides can be analyzed on the 50-A SynChro-pak GPC Peptide column with appropriate mobile-phase modifications. Many peptides have poor solubility in mobile phases standardly used for protein analysis, as discussed later in this chapter. 

When cationic polymers are run on SynChropak CATSEC columns, the calibration curves, as shown in Fig. 10.4, are not identical to those produced... 

FIGURE 10.2 Calibration curves for proteins on SynChropak GPC columns. Mobile phase 0.1 M potassium phosphate, pH 7. (From MICRA Scientific, Inc., with permission.)... 

Quality assurance for size exclusion supports is based primarily on the reproducibility of molecular weight calibrations. Although the reproducibility of the exclusion and inclusion limits is important, the distribution coefficients (Ko) of included standards are a better indication of duplication. Table 10.3 (page 314) shows such data for the SynChropak GPC and CATSEC supports. 

SynChropak GPC and CATSEC columns are packed in either stainless steel or PEEK columns with standard inverted fittings so that they readily connect to most instruments. Stainless-steel columns are available in 2.1, 4.6, 7.8, 10,... 

SynChropak GPC supports can be packed by slurrying in methanol and packing upward with methanol to a pressure of 4000 psi. SynChropak CATSEC supports can be packed by slurrying in a mixture of isopropanol and methanol and packing upward with methanol to a pressure of 4000 psi. The high pore... 

Each SynChropak column is tested chromatographically to assure that it has been packed according to specifications. For SynChropak GPC columns, a mixture of a high molecular weight DNA and glycyltyrosine, a dipeptide, is used to evaluate internal volume and efficiency. The mobile phase used for the test is 0.1 M potassium phosphate, pH 7, and the flow rate is 0.5 ml/min for 4.6-mm i.d. columns. Minimum plate count values and operational flow rates are listed in Table 10.4 for 4.6-mm i.d. columns of all supports and the various diameters of the SynChropak GPC 100 columns. 

SynChropak CATSEC columns are evaluated similarly using a polyvinyl-pyridine standard of molecular weight 600,000 and cytidine. The mobile phase is 0.1 % trifluoroacetic (TEA) acid containing 0.2 M sodium chloride. Minimum plate counts are listed in Table 10.4. 

For many proteins, a simple buffer such as 0.1M phosphate, pH 7, produces excellent separations on SynChropak GPC columns. Generally, minimal interaction is achieved when the ionic strength is 0.05-0.2 M. To prevent denatur-ation or deactivation of proteins, the pH is generally kept near neutrality. For denatured proteins, 0.1% sodium dodecyl sulfate (SDS) in 0.1 M sodium phosphate, pH 7, is recommended. 

Although SynChropak GPC supports have excellent efficiencies for small molecules at various flow rates, macromolecules, because of their low diffusion constants, exhibit band spreading when linear velocities are increased. This effect increases with molecular weight, as seen in Fig. 10.11 (4). It should be noted that proteins are usually homogeneous in size and thus yield better efficiencies than polymers, which are usually heterogeneous. For preliminary analy-... 

FIGURE 10.14 Analysis of polyvinylpyridines on SynChropak CATSEC 100,300, and 1000 columns in series (250 X 4.6 mm i.d.). Flow rate 0.37 ml/min. Mobile phase 0.1 % trifluoroacetic acid in 0.2 N sodium nitrate. Detection by differential viscometry. (Reprinted from Ref. 9 with permission.)... 

The other restriction of SynChropak size exclusion columns is a general one for silica-based supports, that of pH. The most harmful pH is that above 7.5 due to silica dissolution. The bonded phase of SynChropak GPC has some polymeric properties therefore, it is not removed rapidly from the silica at pH 2-3. The bonded phase of SynChropak CATSEC is polymeric and stable at pH 2-7.5. 

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