Size-exclusion chromatography applications

The application of UV spectrophotometers to the analysis of styrene containing copolymers has been extensively reported in the literature. However, hypochromic effects and band shifts which result in deviations from Beer s Law have limited the use of UV detectors as mass or composition detectors in size exclusion chromatography applications. Deviations from Beer s Law for low conversion styrene-acrylonitrile copolymers in tetrahydrofuran have been experimentally investigated and compared with results previously reported in the literature. The behaviour of the extinction coefficient as a function of the copolymer composition is discussed in view of the information obtained from infrared and nuclear magnetic resonance measurements on the same polymers. As a result of this investigation, a quantitative correlation of the extinction coefficients of styrene-acrylonitrile copolymers with the length of the styrene sequences has been obtained which, in turn, allows for the use of UV spectrophotometers as sequence length detectors. 

Refractive index and specific refractive index increments - (k = dn/dc) of polymers in solution have been studied extensively in connection with light scattering measurements and size exclusion chromatography applications to polymer characterization for which refractometers are used as standard concentration detectors. Contrary to the observations made in the infrared region (12), refractive index increments have been shown to be a function of the molecular weight of the polymers (2) and, in some cases, of the copolymer composition (17). Therefore, the assumptions of linearity and additivity (Eq. 1 to 4) have to be verified for each particular polymer system. In the case of styrene/acrylonitrile copolymers, there is an additional uncertainty due to the... 

Beer M. U., Wood P. J., Weisz J. 1999. A simple and rapid method for evaluation of Mark-Houwink-Sakurada constants of linear random coil polysaccharides using molecular weight and intrinsic viscosity determined by high performance size exclusion chromatography Application to guar galactomannan. Carbohvdr. Polymers. 39, 377-380. 

Examples of the application of size-exclusion chromatography to the analysis of proteins. The separation in (a) uses a single column that in (b) uses three columns, providing a wider range of size selectivity. (Chromatograms courtesy of Alltech Associates, Inc. Deerfield, IL). 

PREPARATION OF BEADED ORGANIC POLYMERS AND THEIR APPLICATIONS IN SIZE EXCLUSION CHROMATOGRAPHY... 

Microspherical polymer beads are widely used as packing materials for chromatography and a variety of other applications. Size exclusion chromatography is based on pore size and pore-size distribution of microbeads to separate... 

VI. APPLICATION OF POLYMERIC BEADS AS SIZE EXCLUSION CHROMATOGRAPHY SUPPORTS... 

D. Application of Monosized Polymeric Particles in Size Exclusion Chromatography... 

The evolution of media covering aqueous and nonaqueous systems on the one hand and analytical as well as microscale and macroscale preparative applications on the other hand has resulted in an arbitrarily nomenclature within the field. Thus the current practice is to refer to the separation principle based on solute size as size exclusion chromatography (SEC) whereas the application in aqueous systems is traditionally referred to as gel filtration (GF) and the application in nonaqueous systems is designated gel-permeation... 

Toyopearl HW size exclusion chromatography resins are macroporous packings for bioprocessing chromatography. They are applicable for process-scale... 

Size exclusion chromatography (SEC, also known as GPC and GFC) has become a very well accepted separation method since its introduction in the late-1950s by works of Porath and Flodin (1) and Moore (2). Polymers Standards Service (PSS) packings for SEC/SEC columns share this long-standing tradition as universal and stable sorbents for all types of polymer applications. In general, PSS SEC columns are filled with spherical, macroporous cross-linked, pressure-stable, and pH-resistant polymeric gels. 

For the classical form of size exclusion chromatography in organic solvents, packings based on highly cross-linked styrene-divinylbenzene are used. For SEC of polar polymers using polar or aqueous solvents, packings based on a polar methacrylate polymer are used. Diol-derivatized silica is used for the separation of proteins and other polar polymers. The different packings will be discussed in sections dedicated to their different application areas. 

Protein-Pak packings are designed for the size exclusion chromatography of proteins and related compounds. They are based on silica, which is deactivated with glycidylpropylsilane. The diol function prevents the interaction of the target analytes with the silica surface. However, because coverage of the silica surface is always incomplete, residual acidic silanols can interact with the analytes. For this reason, most applications are carried out with a salt concentration above 0.2 mol/liter, which eliminates the interaction of analytes with surface silanols. Protein-Pak packings are stable from pH 2 to pH 8. 

Generally, size exclusion chromatography is carried out using columns with an internal diameter of 7.8 mm. However, some SEC applications require the use of expensive solvents. For this purpose, size exclusion columns with a smaller internal diameter (4.6 mm) have been developed. Of course one should use proportionally lower flow rates with narrow-bore columns. If the standard column size uses a flow rate of 1 ml/min, then the smaller 4.6-mm columns should be used at a flow rate of 0.35 ml/min. This provides the same linear velocity as 1 ml/min on 7.8-mm columns. The decreased flow rate reduces solvent consumption and solvent disposal cost. The performance of the smaller diameter columns is not compromised if properly optimized instrumentation is used. 

Styragel columns are compatible with most solvents commonly used in size exclusion chromatography. Exceptions are found on both sides of the polarity scale the use of standard general-purpose Styragel columns with aliphatic hydrocarbons or with alcohols (except hexafluoroisopropanol) and water is generally not recommended. However, it is possible to pack columns in special solvents for special-purpose applications. The interested user should contact Waters for additional information. 

For the size exclusion chromatography of proteins on silica-hased diol packings, it is generally recommended to use fully aqueous mobile phases with a salt concentration between 0.1 and 0.3 M. In general, a phosphate buffer around pH 7 is used as the mobile phase. Under these circumstances, the tertiary structure of most proteins is preserved without difficulty and the interaction of proteins with each other is minimized. However, other inorganic buffers or combinations of buffers with organic solvents can be used without difficulties for special applications. 

APPLICATIONS AND USES OF COLUMNS FOR AQUEOUS SIZE EXCLUSION CHROMATOGRAPHY OF WATER-SOLUBLE POLYMERS... 

The instrumentation of HdC, including a pump, an injector, a column (set), a detector, and a recorder or computer, is very similar to size exclusion chromatography SEC). The essence of this technique is the column. There are two types of HdC columns open microcapillary tubes and a nonporous gel-packed column. This chapter emphasizes column technology and selection and the applications of this technique on the molecular weight analysis of macromolecules. 

A number of analytical techniques such as FTIR spectroscopy,65-66 13C NMR,67,68 solid-state 13 C NMR,69 GPC or size exclusion chromatography (SEC),67-72 HPLC,73 mass spectrometric analysis,74 differential scanning calorimetry (DSC),67 75 76 and dynamic mechanical analysis (DMA)77 78 have been utilized to characterize resole syntheses and crosslinking reactions. Packed-column supercritical fluid chromatography with a negative-ion atmospheric pressure chemical ionization mass spectrometric detector has also been used to separate and characterize resoles resins.79 This section provides some examples of how these techniques are used in practical applications. 

In addition to the insoluble polymers described above, soluble polymers, such as non-cross-linked PS and PEG have proven useful for synthetic applications. However, since synthesis on soluble supports is more difficult to automate, these polymers are not used as extensively as insoluble beads. Soluble polymers offer most of the advantages of both homogeneous-phase chemistry (lack of diffusion phenomena and easy monitoring) and solid-phase techniques (use of excess reagents and ease of isolation and purification of products). Separation of the functionalized matrix is achieved by either precipitation (solvent or heat), membrane filtration, or size-exclusion chromatography [98,99]. 

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