SynChropak, GPC

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... 

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. 

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-... 

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. 

Elevated temperatures may be used with SynChropak GPC or CATSEC supports if necessary for solubilization, speed, or reduction of band spreading. Such conditions are not recommended for routine analyses, however, because column degradation is enhanced as temperatures are raised. 

Many proteins and polymers have been analyzed on SynChropak GPC and CATSEC columns. Table 10.6 lists some of the published applications. The use of a surfactant to analyze the caseins in milk is illustrated in Eig. 10.12. Viruses have also been analyzed on SynChropak GPC columns, as seen in the chromatogram from Dr. Jerson Silva of the University of Illinois (Pig. 10.13). Dr. Nagy and Mr. Terwilliger analyzed cationic polymers on a series of CATSEC columns using differential viscometry as detection (Pig. 10.14) (9). 

Comparison of surface areas as determined by the BET and t-curve methods (21) Is another measure of mlcroporoslty since the latter technique will estimate the surface area of pores under ISA In diameter. A SynChropak GPC-lOO sample gave 201 m /g by the BET method and 216 m /g by the t-curve method. 

Porous silica Zorbax PSM Li Chrospher Li Chrospher Diol Synchropak GPC Chromegapore Sphere sil p-Bondagel E Protein Column TSK Type SW... 

Solute and ionic strength TSKgel G3000SW LiChrosorb Diol SynChropak GPC 100 TSKgel G2000SW Waters I-... 

SynChropak, also sold as Aquapore and Blo-Sil GFC, Is applied in the SEC separation of water-soluble polymers, proteIns and pectins . The SynChropak GPC 100 Is compared with other commercial products in ref. 44. 

The most extensive comparison of commercial SEC columns for water soluble polymers was carried out by Pfannkoch et al. The packings examined were TSK gel SW 3000 and 2000, SynChropak GPC 300 and 100, Waters 1-125, -Bondage I, Shodex OH-Pak B 804 and LiChrosorb DIOL. All columns exhibited pronounced ion exclusion characteristics for citric acid, which were, however, reduced by increasing the ionic strength. Hydrophobic effects were smailest... 

However, decreasing column efficiencies may be observed as the sample load approaches Its maximum level. For exasiple, Gooding et al. (4) reported that Increasing the sample load on a SynChropak GPC-lOO column from 0.1 mg to 1 mg of chymotrypslnogen per 0.1 ml sample had negligible effects on the observed plate height (and thus resolution efficiency) of the column. 

Figure 2 Typical profile obtained from high-performance size exclusion chromatography. Column SynChropak GPC 300, 300 x 7.8 mm ID Mobile phase 0.1 M KH2PO4, pH 7. Flow rate 1.0 mL/min.

Figure 4 Displacement of protein caiibration curve in the presence of 0.1% SDS in the mobiie phase. Column SynChropak GPC 100, 250 x 4.6 mm D. Flow rate 0.5mL/min. Standard proteins.

Figure 5 Effect of solute shape on calibration curves. Coiumn SynChropak GPC 500,250 X 4.6 mm ID. Flow rate 0.5 mL/min. (A) Sulfonated polystyrenes in 0.1 M sodium sulfate mobile phase. (O) Standard proteins in 0.1 M potassium phosphate, pH 7.

Figure 7 Dependences of the size exclusion distribution coefficient, K, for the small globular protein hen egg white lysozyme versus the ionic strength, /, of the mobile phase for several notional size exclusion sorbents of different average pore diameter, particle size, and surface chemistry characteristics. The sorbents employed in these investigations were 1, Synchropak GPC 100 2, Waters I-125 3, Shodex OH Pak B-804 4, Lichrosorb Diol 5, Tosoh TSK SW 3000 and 6, Tosoh TSK SW 2000. The Interplay of hydrophobic interaction and electrostatic phenomena, superimposed upon the size exclusion effect due to the differences in the pore sizes of the support materials, is particularly evident with these sorbents at high- and low-ionic-strength conditions. (Data ad ed from Ref. 98.)...

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