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In general, silica-based packings are less hydrophobic and less expensive than polymeric packings. Furthermore, silica packings are more robust to high-temperature applications and can be used with virtually any organic mobile phase, without swelling or shrinking; however, the pore volume of silica columns tend to be lower than those of polymeric packings.
Particle sizes range from 3 to 20 fin³, and corresponding column efficiencies go from about 20,000 to 4,000 plates per 25-cm column. Actual pore sizes of high-performance SEC packings extend from close to 30 A to as high as 3000 A, which corresponds to an effective separation range of approximately 100 to 106 g mol ', or higher, for linear, random coils. Pore sizes larger than 3000 A are too fragile for use in high-performance SEC.
The “pore size” designations used for polystyrene packings are based on the extended chain length of a totally excluded polystyrene calibrant. To obtain the equivalent molecular weight of an excluded polystyrene calibrant, the column designation is multiplied by 40 g mol-1 A-1. For example, a “104-A” column has an exclusion limit of approximately 400,000 g mol-1, which corresponds to an actual pore size of near 500 A.
IX. COLUMN SELECTION
For random coils, a single pore-sized packing can separate 1.5-2 decades of molecular weight . Thus, in principle, a polydisperse sample containing a molecular weight range from 103 to 105 g mol-1, would require, as a minimum, just a single “104-A” polystyrene column. For
Size Exclusion Chromatography
increased resolution and molecular weight accuracy, as shown in Eqs. (17) and (18), multiple columns of the same pore size should be used.
For samples that have a molecular weight distribution greater than two decades, multiple columns of different pore size are needed. For best calibration curve linearity, pore volumes of the columns should be matched. Alternatively, linear or mixed-bed columns, which contain packings of different pore sizes, can be employed, rather than a series of different pore-sized columns.
X. MOBILE SELECTION
A. Organosoluble Polymers
For organosoluble polymers, a good solvent is required that completely dissolves the polymer of interest at the SEC-operating temperature. To prevent adsorption of the polymer onto the packing, the solubility parameter of the mobile phase should be close to that of the packing. For cross-linked polystyrene, mobile phases, such as tetrahydrofuran, toluene, or chloroform, are good choices. If the polymer contains amino functionality, triethylamine or trifluoroacetic acid (typically about 0.1%) can be added to the mobile phase to prevent adsorption. For silica packings, a large range of mobile phases can be used; however, basic polymers will adsorb because of acidic silanol groups, and may require the addition of triethylamine or trifluoroacetic acid to the mobile phase.
B. Water-Soluble Polymers
Mobile-phase development for water-soluble polymers is oftentimes difficult because of hydrophobic or electrostatic interactions that can take place between the polymer and packing. The three most common types of interactions are ion-exchange, ion-exclusion, and hydrophobic . As discussed in the following, mobile-phase composition must be fine-tuned to set Äß = 0, by adjusting mobile phase pH, ionic strength, and organic moderator content.
Most polymeric packings contain residual ionic groups, predominantly carboxylate, inadver-tently introduced during synthesis. For silica, residual silanol groups are the problem. These cationic exchange sites can ion-exchange with polymers containing a cationic functionality, resulting in either a highly tailed or irreversible adsorbed peak. The basic approaches to eliminate this effect are (1) reduce mobile phase pH to less than 4 to prevent dissociation of silanol or carboxylate groups; (2) increase the ionic strength of the mobile phase to help shield electrostatic interactions; (3) add a competitive compound to the mobile phase (e.g., triethylamine); (4) use a surface-modified silica packing to reduce the concentration of silanols; (5) use a polyethyleneimine-coated silica (commercially available through Micra Scientific, see Appendix B).
2. Ion Exclusion
Because of the presence of residual antionic groups on packings, anionically charged polymers tend to be excluded from the pores, owing to electrostatic repulsive forces. Furthermore, an electric double layer is formed that decreases the effective pore volume of the packing. As a result, anionic polyelectrolytes elute earlier than expected, causing an overestimation in mo-lecular weight. As described earlier, the same approaches are employed as those used to eliminate on exchange, except for the use of polyethyleneimine-coated packings, to prevent ion exclusion.
Adsorption is a general term used to describe polymer elution in which enthalpic interactions play a role. The most commonly encountered adsorption mechanism in aqueous SEC is based