SynChropak columns

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. 

Scientific (Northbrook, IL) contain a silica support with a -y-glycidoxypropylsi-lane-bonded phase to minimize interaction with anionic and neutral polymers. The columns come in five different pore sizes ranging from 100 to 4000 A. The packing material has a diameter from 5 to 10 /cm and yields in excess of 10,000 plate counts. With a rigid silica packing material, the columns can withstand high pressure (maximum of 3000 psi) and can be used under a variety of salt and/or buffered conditions. A mobile phase above pH 8, however, will dissolve the silica support of the column (21). A summary of the experimental conditions used for Synchropak columns is described in Table 20.8. 

TABLE 20.8 Summary of Experimental Conditions Used with Synchropak Columns... 

FIGURE 20.7 DRI chromatograms of anionic polymers using Synchropak columns with an aqueous mobile phase of 0.10 N sodium nitrate. 

Synchropak columns are very useful for characterizing hydrophilic, anionic, and nonionic, water-soluble polymers, CATSEC columns work best for characterizing cationic polymers utilizing both light scattering and/or differential viscometry detection over a wide range of molecular weights. 

A 25 cm X 4.6 mm ID long 300A SynChropak column was used to evaluate temperature effects. Injections were made with 5% D2O and 1.3 mg/ml glucose solutions. D2O gave a negative refractive index response. 

TABLE II. ELUTION CHARACTERISTICS OF D2O AND GLUCOSE ON SYNCHROPAK COLUMNS ... 

S in/min lOOA Synchropak column, other conditions. See Table II for... 

The retention times of glucose and D2O as a function of column temperature using a 300A SynChropak column are in Table VII. 

TABLE VII. EFFECT OF COLUMN TEMPERATURE ON THE ELUTION TIME OF D2O AND GLUCOSE USING A 300A SYNCHROPAK COLUMN ... 

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. 

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.)... 

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 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. 

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