Size-Exclusion Chromatography SEC

Size exclusion chromatography (SEC) or gel permeation (or filtration) chromatography (GPC/GFC) uses a porous material as the stationary phase and a liquid as a mobile phase. The diameters of the pores of the porous material can range from 5 to 

If the sample is a single polymer, the chromatogram represents the molecular weight distribution. This method is very valuable for determining the molecular weight 

Schematic representation of gel-filtration chromatography. Molecules of different size in the frame are separated according to size during migration through the gel-filtration matrix as shown in the middle and right frames. 

Care must be taken in interpreting retention data to estimate molecular weights since the separation is based on the size of the molecule rather than on the actual molecular weight. This means that the shape of molecule has a significant effect on the results. For example, two molecules of the same molecular weight, one straight chained and the other highly branched, will be retained somewhat differently because each has a different 

The advent of size exclusion chromatography in the 1960 s provided an alternative to drying polymer-polymer-solvent samples to determine the equilibrium phase compositions. Size exclusion chromatography separates solvents from polymers and to a varying extent, polymers from polymers, based on the size of the molecules in solution. Ultraviolet spectrometry and refractive index detectors may be used to determine the concentrations of each of the polymers in each of the phases (Lloyd et al., 1980). 

In size exclusion chromatography (SEC), dissolved molecules are separated according to their size, which is closely related to their molecular weight. This method can be applied for the separation of polymers in non-aqueous solutions, which is sometimes referred to as gel permeation chromatography (GPC). It can also be used for the separation of biomolecules in aqueous solutions. Then the method is referred to gel filtration chromatography. 

Differentiation and separation only occurs over a certain range of molecular sizes, typically between molecular weights of 2 kDa and 200 kDa, although this can be increased up to 1,000 kDa by the use of more specialised gels. This size range is dependent on the sizes of the pores and pore size distribution in the gel matrix. Retention volumes Vr are often used in size exclusion chromatography instead of retention times tR. The total volume V, of the separation column is the sum of the volume of the gel particles Vg, the volume of the solvent inside the pores, also called the intrinsical volume V and the volume of the free solvent outside the pores, the inter particle volume Vq  

Unlike other chromatographic methods the mobile phase acts just as a solvent. Its physical properties do not influence the separation process. Solvent gradients do not alter the retention volume. As all compounds leave the column between Vq and Vo + Vi, no sample is lost on the stationary phase. The mobile phase usually consists of an aqueous buffer with an ionic strength of 50 to 100 mM. Typical flow rates are in the order of 0.1 to 1 mL min.  

Holler and T. A. Nieman, Prinicples of Instrumental Analysis, 5 edtion. Brooks Cole, 1997. 

Ballon, G. K. Ehrlich, W.-J. Fnng and W. Berthold (editors), Affinity Chromatography Methods and Protocols, Humana Press, 2000. 

Application of size-exclusion chromatography offline low-angle and multi-angle light scattering (SEC-MALLS) [74]. This technique has been used to determine long-chain 

The stationary phase consists of materials with different pore sizes and the molecules permeate the phase as they elute. The mobile phase solely serves as a carrier for the analyte as it does not induce any chemical interaction. In SEC, small molecules penetrate the porous structure more easily than large 

Since the columns are defined in terms of their molecular-weight exclusion limits, it might be necessary to connect as many as eight columns in series to perform a separation. With non-aqueous mobile phases, polystyrenes with 

Size exclusion chromatography (or gel filtration ) has a unique set of attributes which sets it apart from other methods. Its applications are limited because it has low resolution, low capacity, and is slow. However, it is the only practical separation method based on size, whidi is a relatively invariant property of immunoglobulins in contrast to charge, hydrophobidty, and other specific binding interactions. The resolution achieved by SEC may not be spectacular however separations are predictable and nearly independent of buffer conditions. so the need for method development is minimal. 

Concentrate the sample to 10-50 mg/ml by a suitable preliminaiy step, e.g. precipitation with ammonium sulfate (Protocol 3). 

Apply the sample in a final voliune of not more than 5% of the bed volume. 

Elute with 2 CV of elution buffer. Collect fractions, and identify the antibody which will normally be the first peak eluted. 

Hydrophobic interaction chromatography (HIC) is a powerful method for resolving all types of biomolecules. Immimoglobulins have strongly hydro-phobic domains compared with the majority of typical contaminants and so this technique is especially appropriate. My impression is that it is not routinely used by many investigators or companies who supply antibodies for the re- 

Hydrodynamic radius of the polymer is also dependent on the analyte interaction with the solvent. Polymer conformation and degree of the solvation varies with the variation of the solvent properties. Detailed discussion of all aspects of size exclusion chromatography is given in Chapter 6. 

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The molecular weight of SAN can be easily determined by either intrinsic viscosity or size-exclusion chromatography (sec). Relationships for both multipoint and single point viscosity methods are available (18,19). Two intrinsic viscosity and molecular weight relationships for azeotropic copolymers have been given (20,21) ... 

Poly(ethylene oxide). The synthesis and subsequent hydrolysis and condensation of alkoxysilane-terniinated macromonomers have been studied (39,40). Using Si-nmr and size-exclusion chromatography (sec) the evolution of the siUcate stmctures on the alkoxysilane-terniinated poly(ethylene oxide) (PEO) macromonomers of controlled functionahty was observed. Also, the effect of vitrification upon the network cross-link density of the developing inorganic—organic hybrid using percolation and mean-field theory was considered. 

Size-exclusion chromatography (sec) easily and rapidly gives the complete molecular weight distribution and any desired average (6). Thus, it has become the technique of choice for determining molecular weights despite its relatively high initial cost. 

Stmctural and chemical modification of urethane containing polymer matri-ces with macrocycles - calixarenes having reactive hydrazide groups have been carried out and stmcture, physico chemical and sensor properties of polyure-thanesemicarbazides (PUS) synthesised have been studied. The polymers obtained (on the base of polypropylene glycol MM 1000 and polysiloxane diol MM 860, hexamethylene diisocyanate and calixarene dihydrazide) are identified by IR-spectroscopy, size exclusion chromatography (SEC), DSC, WAXS and SAXS methods. 

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... 

OVERVIEW OF SIZE EXCLUSION CHROMATOGRAPHY (SEC) OF SMALL SOLUTES... 

DESIGN, PROPERTIES, AND TESTING OF POLYMER STANDARDS SERVICE SIZE EXCLUSION CHROMATOGRAPHY (SEC) COLUMNS AND OPTIMIZATION OF SEC SEPARATIONS... 

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. 

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. 

B. Broad Standard Size Exclusion Chromatography (SEC) Calibration... 

This chapter makes no distinction between gel-permeation chromatography (GPC) and size exclusion chromatography (SEC). We make mention of specific analysis conditions wherever possible. We have attempted to include a variety of conditions but by no means should this chapter be considered a comprehensive review of conditions for analyzing polyacrylates. We have drawn extensively from our own experience in selecting examples. 

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. 

Other groups have also used EC and CE to perform non-comprehensive multidimensional separations (15, 16). A three-dimensional separation was performed by Stromqvist in 1994, where size exclusion chromatography (SEC), reverse-phase HPLC, and CZE were used in an off-line manner to separate peptides (17). The most useful information gained from all of these non-comprehensive studies was knowledge of the orthogonality and compatibility of EC and CE. 

The premise of size exclusion chromatography (SEC) is that solute molecules are separated according to their effective molecular size in solution. SEC allows the separation of fractions according to their molecular weight and eliminates the... 

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. 

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. 

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