Dextrans, cross-linked, as molecular

Porath, J. Cross-linked dextrans as molecular sieves. Advances in Protein Chcmistiy 17, 209—226 (1962). 

Even when the stationary phase is of the same composition as the mobile phase, the porous support for the immobilized fluid is customarily referred to as stationary phase. Porath and Flodin " used cross-linked dextrans as a molecular sieve for carrying out separations, and this material has been unusually effective. It... 

The timely adoption of the cross-linked dextran gels (i.e., Sephadex) in late-fifties as a packing material for column chromatography opened an altogether new horizon of chromatographic separation whereby substances, in general, undergo separation more or less as per their molecular size. 

Procedure The chromatographic procedure may be carried out at room temperature using (a) a column (1 M x 25 mm) packed with a cross-linked dextran suitable for fractionation of globular proteins in the range of molecular weights from 5,000 to 350,000 (Sephadex G-150 is suitable), (b) mixed phosphate buffer pH 7.0 with azide as the mobile-phase with a flow rate of about 20 ml (4 ml per square centimetre) of column cross-sectional area) per hour, and (c) a detection wavelength of 280 nm. 

A good separation of L-rhamnose, 2-acetamido-2-deoxy-D-glucose, D-glucose, and 2-amino-2-deoxy-D-glucose, eluted in that order, was obtained by Zeleznick80 on a small column packed with the highly cross-linked, dextran gel Sephadex G-25, with 62 15 25 (v/v) butyl alcohol-M acetic acid-water as the eluant however, the gel functioned more as a support for partition chromatography than as a molecular sieve. 

Viscotoxin, a basic peptide of molecular weight ca. 9000 (Samulsson, 1961), moves unretarded in strongly cross-linked gels such as Sephadex G-25 (Fig. 4a) (in phosphate buffer, ionic strength 0.05, pH 6.8. When filtered under similar conditions in weakly cross-linked dextran, viscotoxin behaves quite differently (Fig. 4b). In fact it moves behind isoleucine. The gel of the first kind can be used to remove solutes of lower molecular size, the purification being based on molecular exclusion. Filtration in the second kind of gel may be used not only for separating solutes of different molecular size but also to separate peptides and other substances of similar molecular size when they differ in certain structural features. 

The molecular-sieving effect on proteins has been very clearly demonstrated by Pedersen (1962). Bovine serum albumin, obtained by fractionated ammonium sulfate precipitation, was filtered through a column of cross-linked dextran of water regain 15 gm/gm (Sephadex G-200). Monomer and dimer forms were easily separated from each other and from other components of larger sedimentation constants (Fig. 8). Heterogeneity in the monomer as well as in the dimer preparation was indicated... 

These bacterial polysaccharides have been considered to be slimes they are often in reality loose capsules that are produced extracellularly by the bacteria. It was found that low molecular weight L. mesenteroides NRRL B-512F dextran could be used as a blood plasma extender and was produced on a relatively large scale during the cold war , but also found uses as a gel-filtration material when cross-linked by epichlorohydrin to give a family of cross-linked dextrans [41]. 

The column packing in SEC comprises porous, spherical gel beads with a defined pore size distribution. Most often, these beads are made from poly(styr-ene-divinylbenzene). (For GFC, cross-linked dextran and agarose gels are often used. ) The sample is dissolved in a suitable solvent that is often used as the mobile phase as well. Separation occurs as a result of differences in accessibility of pore volume. Small molecules can freely access the whole pore volume as a result, the column retards these molecules the greatest. As molecular volume increases, less and less pore volume is accessible for molecules to sample, and elution times decrease. For all molecules with hydrodynamic volumes that are too large to penetrate into the pores of the packing, elution occurs at the (interstitial) void volume of the column. The retention volume for each solute can be described mathematically as ... 

The free and complexed Cd (II) are separated by two 25 cm HPLC columns of Sephadex G-10 (a cross-linked dextran gel of 40-120 p bead diameter). The mobile phase was distilled deionized water. Sephadex G-10 xerogel has an exclusion limit 700, that is, it can be used to fractionate species of molecular weight less than 700. The larger Cd-fulvic acid complex is unretained and elutes before hydrated Cd (II). As with the phosphorus esters above, SEC is a viable method not only for separating these complexes for analysis but also for purification. 

Newer 2-diethylaminoethyl ethers, such as 0-( 2-diethylaminoethyl )-agarose gels," are useful as media for electrophoresis and immunoelec-trophoresis, and the 2-diethylaminoethyl derivative of O-(2-hydroxy-propyl ) cross-linked dextran (Sephadex LH20) is an effective, lipophilic anion-exchanger. The relative stiffness of the molecular chain of 0-(2-diethylaminoethyl) dextran has been estimated from measurements of viscosity at various ionic strengths, and compared with that of other polysaccharide polycations and polyanions. 0-(2-Diethylaminoethyl) dextran itself is claimed to be toxic." ... 

Aqueous SEC was first reported in 1959 by Porath and Flodin [1]. They separated proteins and salts according to molecular size by using cross-linked dextran gels. Since then it has been widely employed, especially in the field of biochemistry, for various purposes such as purification of proteins and nucleic acids, estimation of molecular masses of proteins and determination of molecular mass distributions of polysaccharides. In addition, it has been a powerful tool for the determination of molecular mass distributions of water-soluble synthetic polymers since high-performance aqueous SEC was realized in 1978 by the development of semirigid microparticulate macroporous supports based on hydrophilic synthetic polymers [2-4]. 

Molecular sieves had been in use since the mid-1920s for chromatographic separations of small molecules. The application to water-soluble fragile materials of biological origin was the subject of intense activity after the discovery of cross-linked dextran gels by Porath and Flodin in 1957 in what became known as gel filtration or size exclusion chromatography. 

Dextran of molecular weight = 80,000 is used as a blood plasma extender. Cross-linked dextran is employed as a column filler in gel permeation chromatography. 

Polymeric, cross-linked dextran gels, called Sephadex , are used in size-exclusion TLC. Sephadex gels, which are available in coarse (100-300 p,m), medium (50-150 p-m), fine (20-80 pm), and superfine (10-40 pm) particle size distributions, must be applied in a total swollen condition as chromatographic sorbents and eluted with the aid of continuous development techniques. A typical application in TLC is the determination of molecular weights of proteins. " ... 

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