Polysaccharides Sephadex

Between 1950 and 1960 Size Exclusion Chromatography (SEC) became a popular technique in two branches the fractionation of synthetic polymers, described as gel permeation chromatography, and in the resolution of biopolymers, termed as gelfil-tration. The former was performed on cross-linked porous synthetic polymers, the latter on cross-linked polysaccharides (Sephadex). 

Polysaccharides Sephadex G-50, G-lOO, G-200, Bio-Gel P-6 P-100, styrene divinyl-benzene-based size-exclusion resins (e.g., Bio-Gel SEC), and their DEAE-bonded material... 

Sephadex. Other carbohydrate matrices such as Sephadex (based on dextran) have more uniform particle sizes. Their advantages over the celluloses include faster and more reproducible flow rates and they can be used directly without removal of fines . Sephadex, which can also be obtained in a variety of ion-exchange forms (see Table 15) consists of beads of a cross-linked dextran gel which swells in water and aqueous salt solutions. The smaller the bead size, the higher the resolution that is possible but the slower the flow rate. Typical applications of Sephadex gels are the fractionation of mixtures of polypeptides, proteins, nucleic acids, polysaccharides and for desalting solutions. 

The gel-like, bead nature of wet Sephadex enables small molecules such as inorganic salts to diffuse freely into it while, at the same time, protein molecules are unable to do so. Hence, passage through a Sephadex column can be used for complete removal of salts from protein solutions. Polysaccharides can be freed from monosaccharides and other small molecules because of their differential retardation. Similarly, amino acids can be separated from proteins and large peptides. 

Dextran gels have been utilized since the late 1950s (1) for the separation of biopolymers. First attempts on Sephadex (2-5) and Sephadex/Sepharose (6-8) systems are documented for hydrolyzed and native starch glucans. Up until now, particularly for the preparative and semipreparative separation of polysaccharides, a range of efficient and mechanically stable Sephacryl gels (9-14) have been developped. 

Gel chromatography on Sephadex GlOO (2.8x50cm) of polysaccharide fraction I (Sample 2). The polysaccharide fraction was dissolved in a phosphate buffer at pH 6. After centrifugation, the supernatant was applied to the column at a flow rate of 0.8 ml/min. The elution was performed with a phosphate buffer and fractions of 10 ml each were collected. The refraction of each fraction was measured interferometrically. Fractions with coincident peaks were collected and analyzed for content of galacrturonic acid, neutral sugars and protein. 

Light scattering (nephelometry) was used as a detection system for gly-cosaminoglycans from urine, eluted from a DEAE Sephadex (Pharmacia Biotechnology Uppsala, Sweden) A-25 column.68 This technique has been more recently applied to protein characterization.69 Interferometry was used for analysis of dextran eluted from a size exclusion column.70 One of the problems of electrochemical detection is that it is relatively insensitive to polymers. Because many of the materials discussed below (DNA, proteins, and polysaccharides) are polymeric, a brief mention of some alternative... 

Many natural materials are porous but also proton-rich such as wood or other plant products. Relaxation of liquids in these materials has features in common with both inorganic matrices and the protein systems discussed above. The class of porous polysaccharide materials used for size exclusion chromatography provides an example one commercial product is Sephadex. The material swells on solvation to form a controlled pore gel. The main application involves excess liquid, generally water, which flows through the gel bed carrying solutes of various size. The large solutes are excluded from the pore interior and elute rapidly while the smaller ones equilibrate with the pore interior and elute later. The solvent generally samples the pore interior as well as the bulk phase. 

Barker and coworkers have applied gel chromatography in studies of pneumococcal polysaccharides.121 Purification of the type-specific polysaccharide of Pneumococcus Type II was effected by chromatography on Sephadex G-200 in M sodium chloride in this way, the ribonucleic acid, a persistent impurity in preparations of this polysaccharide, was almost completely removed. The complex formed between the polysaccharide and the nucleic acid is largely dissociated in M sodium chloride, so that the two are free in this solvent and may be separated on the basis of their differing molecular size. 

Dextrans are bacterial and yeast polysaccharides made up of (a 1—>6)-linked poly-D-glucose all have (al—>3) branches, and some also have (al—>2) or (al—>4) branches. Dental plaque, formed by bacteria growing on the surface of teeth, is rich in dextrans. Synthetic dextrans are used in several commercial products (for example, Sephadex) that serve in the fractionation of proteins by size-exclusion chromatography (see Fig. 3-18b). The dextrans in these products are chemically cross-linked to form insoluble materials of various porosities, admitting macromolecules of various sizes. 

Dextran gels used as molecular sieves are formed by crosslinking dextrans with epichlorhydrin to give a semisynthetic polysaccharide with a well-defined pore size (Sephadex ). 

Figure 3-9 Elution volume of various proteins on a column of Sephadex G-200 as a function of molecular mass. The right-hand vertical axis shows the ratio of the elution volumes to that of blue dextran, a high-molecular-mass polysaccharide that is excluded from the internal volume. After Andrews.193...

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