Separation on the Basis of Molecular Size

Gel permeation chromatography (GPC), sometimes called size-exclusion chromatography (SEC), is based on a very simple principle a gel is constructed with a narrow range of pore sizes and packed into a chromatography column. If a polymer of infinite molecular weight is applied at the top of the column, it cannot fit into any of the pores and is eluted in volume Vq, the excluded volume. A small molecule, on the other hand, can fit into all the pores and is this eluted in volume Ft, the total volume of the column. Fractionation according to molecular size thus occurs between elution volumes Fq and Ft with molecules eluted in order of decreasing size. 

The first SEC material to become available commercially was Sephadex, made by cross-linking a bacterial p-(1 6)glucan with epichlorohydrin (l-chloro-2,3-epoxypropane) which was confined to aqueous solution. A wide range of SEC materials are now available, based on various polymer chemistries and compatible with a range of organic solvents. 

Over the fractionation range of a GPC column, the relationship between elution volume Fe and molecular weight is approximately logarithmic, so eqn. (4.9) applies  

The problem, of course, comes from the implicit assumption that the gel matrix has no specific interactions with the soluble polymer, and that the relationship between effective volume and molecular weight is the same for the polysaccharide of interest and the standards. A recent development has been to place instruments which measure molecular weight at the exit of a GPC column, so that the column is used only for fractionation, and a full molecular weight distribution of a polydisperse polymer can be obtained. Viscometers and light-scattering monitors can be so employed, as can on-line electrospray mass spectrometers. The last technique is particularly powerful, since the masses determined by the mass spectrometer are absolute. 

Undecorated linear (l- 4) diequatorial polysaccharides such as cellulose tend to be insoluble in water, so any attempt at determining a molecular weight distribution first has to solubilise them. A common method is complete reaction with phenyl isocyanate (Ph-N=C=0) in warm ( 80 °C) pyridine which converts all the hydroxyl groups of cellulose into carbanilate (cell-O-CO-NH-Ph) groups. SEC in an organic solvent such as tetrahydrofuran can then, with appropriate standards, give the molecular weight distribution. 

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In analysis of homopolymers the critical interpretation problems are calibration of retention time for molecular weight and allowance for the imperfect re >lution of the GPC. In copolymer analysis these interpretation problems remain but are ven added dimensions by the simultaneous presence of molecular weight distribution, copolymer composition distribution and monomer sequence length distribution. Since, the GPC usu y separates on the basis of "molecular size" in solution and not on the basB of any one of these particular properties, this means that at any retention time there can be distributions of all three. The usual GPC chromatogram then represents a r onse to the concentration of some avera of e h of these properties at each retention time. 

This determines the size of molecules that can be admitted and the rate at which different molecules diffuse towards the surface. Molecular sieves, with their precise pore sizes, are uniquely capable of separating on the basis of molecular size. In addition, it is sometimes possible to exploit the different rates of diffusion of molecules to bring about their separation. A particularly important example referred to earlier, concerns the production of oxygen and nitrogen from air. 

In general, partition of components from a solution at a solid surface provides the principle that is most often exploited (adsorption is used only very rarely), but for amino acids and peptides, which can exist in charged forms in aqueous solutions, ion-exchange and electrophoresis separation are also available. Separation on the basis of molecular size is also used. 

The biogenic components that interfere with the GC analysis of aromatic hydrocarbons are primarily long-chain components. Such compounds are much larger molecules than the compact aromatic hydrocarbons. Because of this major difference, gel permeation chromatography (GPC), which separates on the basis of molecular size, can be used very effectively to separate the two classes of compounds (9). The GPC materials that can be used for the separation include modified dextrans, for example, Sephadex LH-20, and styrene-divinylbenzene copolymers, for example, BioBeads, Styragel, or /x-Styragel. 

Two-Dimensional Gel Electmphoresis 2-DE is the most powerful and robust technique available to separate a complex mixture of proteins from biological specimens [13-15]. It is a two-step process in which each dimension makes use of complementary properties of proteins for their separation. The first dimension is usually performed in the lEF mode, and the second in the conventional, size-based separation mode. The lEF mode separates proteins on the basis of their inherent charge or isoelectric point (pi values). In the second dimension, proteins are separated on the basis of molecular size, typically by SDS-PAGE. With this orthogonal separation scheme, a mixture that contains thousands of proteins can be separated in a single experiment to provide a two-dimensional image. The following key steps are used in a typical 2-DE separation of proteins ... 

Eiectrophoresis. Electrophoresis is widely practiced on biopolymers (266-268) for the determination of molecular weights of proteins and their subunits. Typically, a polyacrylamide bed is used to separate or characterize these solutes under the influence of an electric field, on the basis of charge and molecular size. The proteins are frequently denatured with sodium dodecyl sulfonate (SDS), and these subunit-SDS complexes are separated on the basis of molecular size. A densitometer employing a soft laser scanning device is commercially available to automate the data-reduction process (269). 

Metal nanotube membranes with electrochemically suitable ion-transport selectivity, which can be reversibly switched between cation-permeable and anion-permselective states, have been reported. These membranes can be viewed as universal ion-exchange membranes. Gold nanotube molecular filtration membranes have been made for the separation of small molecules (< 400 Da) on the basis of molecular size, eg. separation of pyridine from quinine (Jirage and Martin, 1999). 

Two-dimensional electrophoresis is normally run so that proteins are separated from each other on the basis of a different molecular property in each dimension. The most commonly utilized method entails separation of proteins by isoelectric focusing (see below) in the first dimension, with separation in the second dimension being undertaken in the presence of SDS, thus promoting band separation on the basis of protein size. Modified electrophoresis equipment that renders two-dimensional electrophoretic separation routine is freely available. Application of biopharmaceuti-cal finished products to such systems allows rigorous analysis of purity. 

Molecular sieves are synthetic zeolites that can be manufactured with extremely close control of pore size. Therefore, they can be tailored to suit specific applications. In addition to gas drying applications, molecular sieves are used for the separation of gases and vapors on the basis of molecular size and shape. Surface areas range from 350 to 1000 ft2/ft3. 

Gel-filtration chromatography materials such as Ultrogel AcA44 separate molecules on the basis of molecular size. Larger protein molecules are unable to penetrate the small pores in this material and pass through the column more rapidly than the smaller protein molecules. 

Gel permeation chromatography (GPC), also called size-exclusion chromatography, is the most widely used cleanup technique for pesticides in fatty foods. It is the method of choice for rapid cleanup of biological extracts, especially from high-fat samples, to determine pesticide residues, since separation occurs on the basis of molecular size (7). 

Miiller et al., 2000 Her et al., 2003). Emphasis has been placed on (a) the use of gel chromatography or gel permeation chromatography for the fractionation of DOM on the basis of molecular size differences and (b) the application of electrophoretic separation methods (Perminova et al., 1998,2003 Specht and Frimmel, 2000), including electrophoresis, capillary electrophoresis (CE), isotachophoresis, isolelectric focusing,polyacrylamide gel electrophoresis (PAGE), and capillary zone electrophoresis (CEZ) (De Nobili et al., 1989,1998 Schmitt-Kopplin et al., 1998). 

One of the most common low-temperature methods for removing impurities involves the use of selective solid adsorbents. Such materials as silica gel, carbon, and synthetic zeolites (molecular sieves) are widely used as adsorbents because of their extremely large effective surface areas. Most of the gels and carbons have pores of various sizes in a given sample, but the synthetic zeolites can be manufactured with closely controlled pore size openings ranging from 0.4 to 1.3 nm. This additional selectivity is useful because it permits separation of gases on the basis of molecular size. 

Size-exclusion chromatography (SEC), or gel permeation chromatography (GPC), separates species on the basis of molecular size. Molecular interactions between... 

What does exist, however, is GPC, which separates the molecules in a polymer on the basis of molecular size (weight). The large molecules elute from the column earlier than small molecules and from this elution profile both the weight-average molecular weight (size) and the molecular weight (size) distribution can be calculated. 

Chromatographic separation of proteins on the basis of molecular size (or, more precisely, ease of diffusion) is readily accomplished by size-exclusion chromatography on dextran, agarose, or polyacrylamide beads. 

Gel-permeation chromatography [on a modified dextran, poly (acrylamide), or agarose gel] separates proteins on the basis of molecular size, alpha- and hefa-Amylases have been purified on Sephadex, a gel of cross-linked dextran, but anomalous behavior is observed with alpha-amylases on such gels these enzymes are eluted from the gel less readily than would be expected on the basis of their molecular size, and it is thought that a weak, protein—gel complex is formed, similar in nature to an enzyme—substrate complex. 

Carbon molecular sieve (CMS) is useful in air separation processes because of its ability to selectively discriminate on the basis of molecular size and hence adsorb the smaller oxygen molecule over nitrogen. The difference in the adsorption kinetics of various gases allows the separation of gas mixtures into pure components using pressure swing adsorption (PSA). 

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