Zorbax

Chromatographic conditions elution with 50 50 methanol/water solvent at the rate of 1.5 ml,/min through a DuPont Zorbax ODS column using a Waters R-401 Refractive Index Detector. 

Table 2 Summary of the Physical and Chemical Properties of the Zorbax Column...

A summary of the data for the Zorbax column obtained by Alhedai et al. [11] is shown in Table 2. It is seen that the distribution of the various chromatographically important volumes within a column is neither simple nor obvious. It would seem that about 70% of the column volume is occupied by mobile phase but only about 50% of that mobile phase is actually moving. 

ZORBAX POROUS SILICA MICROSPHERE COLUMNS FOR HIGH-PERFORMANCE SIZE EXCLUSION CHROMATOGRAPHY... 

Zorbax PSM particles are made from small (80-2000 A), extremely uniform colloidal silica sol beads. In a patented polymerization process, these beads are agglutinated to form spherical particles. The size of the Zorbax PSM particles is controlled by the polymerization process, and the pore size is determined by the size of the silica sol beads. After polymerization, the silica is heated to remove the organic polymer and sinter the particles. The result is a spherical, porous, mechanically stable, pure silica particle that provides excellent chromatographic performance (Pig. 3.1). 

FIGURE 3.1 Formation of Zorbax porous silica microspheres. 

Zorbax PSM packings are produced in three forms unmodified, trimethyl-silane modified, and diol modified. Modified Zorbax PSM packings are produced by chemically bonding a layer on the silica surface through siloxane bonds (Table 3.1). Silanized Zorbax PSM packings suppress adsorption effects and are the preferred choice when the mobile phase contains organic solvents. Unsilanized and diol modified Zorbax PSM packings should be used when the mobile phase consists of aqueous solvents. 

The mechanism of separation is the same for Zorbax PSM columns as it is for other types of SEC columns. As the mobile phase flows through the column, large molecules are forced down the column at faster rates than small molecules because the large molecules have less access to the column volume inside the pores. Consequently, molecules that are too large to permeate any of the pore... 

II. USING AND SELECTING ZORBAX PSM COLUMNS AND MOBILE PHASES... 

A. Conducting HPSEC Experiments with Zorbax PSM Columns... 

Running an HPSEC experiment with Zorbax PSM columns is similar to running an experiment with any other GPC or HPSEC column. The process is summarized as follows ... 

Eigure 3.3 shows the calibration plots for Zorbax PSM columns. (The calibration plots for silanized and unsilanized columns are comparable.) These calibration plots allow the chromatographer to select the appropriate columns for samples. Eor example, the Zorbax PSM 60 column provides resolution of... 

Zorbax PSM Bimodal and Trimodal columns are packed with mixed pore-size packing to achieve linear size separations over a broad molecular weight range (Table 3.3). Zorbax PSM Bimodal columns are packed with PSM 60 and PSM 1000 particles, and Trimodal columns contain PSM 60, PSM 300, and PSM 3000 particles (Fig. 3.4). Carefully selecting and mixing different pore-size particles in columns provide much better linearity than coupling columns that are each packed with single pore-size particles. 

FIGURE 3.4 Calibration plots for Zorbax PSM Bimodal and Trimodal columns. 

Select mobile phases for HPSEC based on their ability to dissolve the sample and their compatibility with the column. Zorbax PSM columns are compatible with a wide variety of organic and aqueous mobile phases (Table 3.4), but analysts should avoid aqueous mobile phases with a pH greater than 8.5. As mentioned earlier, select mobile phases that minimize adsorption between samples and silica-based packings. Sample elution from the column after the permeation volume indicates that adsorption has occurred. If adsorption is observed or suspected, select a mobile phase that will be more strongly adsorbed onto the silica surface than the sample. For example, N,N-dimethyl-formamide (DMF) is often used for polyurethanes and polyacrylonitrile because it eliminates adsorption and dissolves the polymers. When aqueous mobile phases are required, highly polar macromolecules such as Carbowax can be used to coat the silica surface and eliminate adsorption. Table 3.5 provides a list of recommended mobile-phase conditions for some common polymers. 

Column dispersity (band spreading) causes the measured molecular weight distribution to be broader than the true molecular weight distribution (Fig. 3.5). Because Zorbax PSM columns exhibit very low band-spreading characteristics, these columns have historically provided better molecular weight distribution accuracy than many gel-type columns. 

Many high molecular weight synthetic polymers, such as polyethylene and polypropylene, have a large percentage of their molecules in the crystalline state. Prior to dissolution, these polymers must usually be heated almost to their melting points to break up the crystalline forces. Orthodichlorobenzene (ODCB) is a typical mobile phase for these polymers at 150°C. The accuracy and stability of the Zorbax PSM columns under such harsh conditions make them ideal for these analyses (Fig. 3.8). 

The size separation of proteins has been routinely called gel filtration because of the historic use of cross-linked gels for this application. Specially modified Zorbax PSM columns, Zorbax GF-250 and Zorbax GE-450, are used for separating proteins by size. These columns are packed with porous silica micro-... 

FIGURE 3.8 The stability of Zorbax PSM columns makes them ideal for applications that require extremely harsh conditions, such as high temperature HPSEC of polyolefins. 

TABLE 3.8 Recovery of Purified Proteins on Zorbax GF-250 Columns... 

Proteins are separated on Zorbax GF columns based on their hydrodynamic size, which may be related to the proteins molecular weights (Fig. 3.10). Under ideal conditions, two proteins whose molecular sizes differ by a factor of 2 can be baseline separated. 

Zorbax GF columns can be used for size-separation applications such as estimating molecular weight purifying complex mixtures monitoring reac-... 

FIGURE 3.10 Under ideal conditions, proteins are separated on Zorbax GF columns based on their hydrodynamic size. 

As with other size-exclusion techniques, the pore size of the selected Zorbax GF column should provide resolution over the molecular size range of the proteins that are to be separated. The Zorbax GF-250 column separates proteins in the range of 4000 to 400,000 Da. The Zorbax GF-450 provides separation over the range of 10,000 to 1,000,000 Da. When these two columns are coupled, they can be used to separate proteins with molecular weights of 4000 to 1,000,000. 

FIGURE 3.11 Zorbax GF columns are routinely used to quantitate or isolate different forms (monomer, dimer, aggregates) of biomolecules. 

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