E Size-Exclusion Chromatography

Column Si e. Size-exclusion chromatography columns are generally the largest column on a process scale. Separation is based strictly on diffusion rates of the molecules inside the gel particles. No proteins or other solutes are adsorbed or otherwise retained owing to adsorption, thus, significant dilution of the sample of volume can occur, particularly for small sample volumes. The volumetric capacity of this type of chromatography is determined by the concentration of the proteins for a given volume of the feed placed on the column. 

Kostanski, L.K., Keller, D.M. and Hamielec, A.E., Size-exclusion chromatography—a review of calibration methodologies. J. Biochem. Biophys. Meth., 58,159-186 (2004). 

Hydroxyl number and molecular weight are normally determined by end-group analysis, by titration with acetic, phthaUc, or pyromellitic anhydride (264). Eor lower molecular weights (higher hydroxyl numbers), E- and C-nmr methods have been developed (265). Molecular weight deterrninations based on coUigative properties, eg, vapor-phase osmometry, or on molecular size, eg, size exclusion chromatography, are less useful because they do not measure the hydroxyl content. 

Hagel, L., and Janson, J.-C. (1992). Size-exclusion chromatography. In Chromatography (E. Heftmann ed.), 5th Ed., pp. A267-A307. Elsevier, Amsterdam. 

Malawer, E. G. (1995). Introduction to size-exclusion chromatography. In Handbook of Size-Exclusion Chromatography (Chi-san Wu, Ed.), Dekker, New York. 

Meehan, E., Oakley, S. A., Warner, F. P., (1992). Narrow Bore Columns for Size Exclusion Chromatography. Presented at Pittcon 92, New Orleans. 

Most size exclusion chromatography (SEC) practitioners select their columns primarily to cover the molar mass area of interest and to ensure compatibility with the mobile phase(s) applied. A further parameter to judge is the column efficiency expressed, e.g., by the theoretical plate count or related values, which are measured by appropriate low molar mass probes. It follows the apparent linearity of the calibration dependence and the attainable selectivity of separation the latter parameter is in turn connected with the width of the molar mass range covered by the column and depends on both the pore size distribution and the pore volume of the packing material. Other important column parameters are the column production repeatability, availability, and price. Unfortunately, the interactive properties of SEC columns are often overlooked. 

A more complicated, but flexible, system has been reported by Blomberg et al. (46). Here, size exclusion chromatography (SEC), normal phase EC (NPLC) and GC were coupled for the characterization of restricted (according to size) and selected (according to polarity) fractions of long residues. The seemingly incompatible separation modes, i.e. SEC and NPLC, are coupled by using an on-line solvent-evaporation step. 

Stopher and Gage used size-exclusion chromatography (SEL) coupled to reversed phase HPLC for the direct injection of plasma in the analysis of an antifungal agent, voriconazole (12). Their system consisted of three columns, i.e. first a size-exclusion... 

When the full distribution is needed, it is measured by size-exclusion chromatography (also called gel permeation chromatography). This is a solution technique that requires dissolution of the polymer in a reasonable solvent such as tetrahydrofuran or tetrachlorlobenzene. For polymers that require exotic solvents or solution temperatures above about 150°C, a simple measurement of solution viscosity can be a useful surrogate for the actual molecular weight. The viscosity of the pure polymer (i.e., a polymer melt viscosity) can also be used. Such simplified techniques are often satisfactory for routine quality control, particularly for condensation polymers such as PET that vary in average molecular weight but usually have a polydispersity of 2. 

Size exclusion chromatography (SEC) separates molecules of a polymer sample on the basis of hydrodynamic volume. When the chromatograph is equipped only with a concentration-sensitive detector, i.e. conventional SEC, a molecular weight distribution (MWD) can be obtained from the chromatogram only through use of a calibration function relating molecular weight and elution volume V (2). 

Garcia-Rubio, L.H., MacGregor, J.F., Hamielec, A.E., "Copolymer Analysis Using GPC with Multiple Detectors , presented at the Symposium on Recent Developments in Size Exclusion Chromatography , 178th ACS National Meeting, Washington, D.C., September 9-14, 1979. 

Hancock, D. O. and Synovec, R. E., Refractive index gradient detection of femtomole quantities of polymers by microbore size-exclusion chromatography, Anal. Chem., 60, 1915, 1988. 

Garcia, R., Porcar, I., Campos, A., Soria, V. and Figueruelo, J. E., Solution properties of polyelectrolytes. X. Influence of ionic strength on the electrostatic secondary effects in aqueous size-exclusion chromatography, /. Chromatogr. A, 662, 61, 1994. 

Pfannkoch, E., Lu, K. C., Regnier, F. E., and Barth, H. G., Characterization of some commercial high-performance size-exclusion chromatography columns for water-soluble polymers, /. Chromatogr. Sci., 18, 430, 1980. 

Wu, C. S., Senak, L., and Malawer, E. G., Size exclusion chromatography of poly(methyl vinyl ether-co-maleic anhydride) (PMVEMA). I. The chromatographic method, J. Liq.Chromatogr., 12, 2901, 1989. 

Kirkland, J. J. and Antle, P. E., High-performance size-exclusion chromatography of small molecules with columns of porous silica microspheres, J. Chromatogr. Sci., 15,137,1977. 

Styring, M. G., Armonas, J. E., and Hamielec, A. E., An experimental evaluation of a new commercial viscometric detector for size-exclusion chromatography (SEC) using linear and branched polymers, /. Liq. Chromatogr, 10, 783, 1987. 

Meehan, E. and O Donohue, S., Characterization of block copolymers using size exclusion chromatography with multiple detectors, in Chromatographic Characterization of Polymers, Hyphenated and Multidimensional Techniques, Provder, T., Barth, H. G., and Urban, M. W., Eds., American Chemical Society, Washington, D.C., 1995, chap. 18. 

Yu, L.P. and Rollings, J. E., Quantitative branching of linear and branched polysaccharide mixtures by size exclusion chromatography and on-line low-angle laser light-scattering detection, /. Appl. Polym. Sci., 35, 1085, 1988. 

Beri, R. G., Walker, J., Reese, E. T., and Rollings, J. E., Characterization of chitosans via coupled size-exclusion chromatography and multiple-angle laser light-scattering techniques, Carb. Res., 238, 11, 1993. 

---