Gel-filtration

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. 

Sephasorb HP (ultrafine, prepared by hydroxypropylation of crossed-linked dextran) can also be used for the separation of small molecules in organic solvents and water, and in addition it can withstand pressures up to 1400 psi making it useful in HPLC. These gels are best operated at pH values between 2 and 12, because solutions with high and low pH values slowly decompose them (see further in Chapter 6). 

Sephasorb HP (ultrafine, prepared by hydroxypropylation of crossed-linked dextran) can also be used for the separation of small molecules in organic solvents and water, and in addition it can withstand pressures up to 

1400 psi making it useful in HPLC. These gels are best operated at pH values between 2 and 12, because solutions with high and low pH values slowly decompose them (see further in Chapter 7). 

Vesterberg (50) studied the separation of carrier ampholytes from proteins by dialysis and by gel filtration. He used ampholytes containing 

The affinity method may be biospecific, for example as an antibody-antigen interaction chemical as in the chelation of boronate by cij-diols, or of unknown origin as in the binding of certain dyes to albumin. 

The mobile phase in vapour phase chromatography is a gas (e.g. hydrogen, helium, nitrogen or argon) and the stationary phase is a non-volatile liquid impregnated onto a porous material. The mixture to be purified is injected into a heated inlet whereby it is vaporised and taken into the column by the carrier gas. It is separated into its components by partition between the liquid on the porous support and the gas. For this reason vapour-phase chromatography is sometimes referred to as gas-liquid chromatography. 

The choice of carrier gas is largely determined by the type of detection system that is available (see below). Column 

The coating material (about 75ml per l(X)ml of column packing) is applied as a solution in a suitable solvent such as methylene chloride, acetone, methanol or pentane, which is then allowed to evaporate in air, over a steam-bath, or in a vacuum oven (provided the adsorbed substance is sufficiently non-volatile). The order in which a mixture of substances travels through such columns depends on their relative solubilities in the materials making up the stationary phases. 

Diethylene glycol adipate Methyl esters of fatty acids up to C24 

Where substances are sufficiently stable, removal of solvent from recrystallised materials presents no problems. The crystals, after filtering at the pump (and perhaps air-drying by suction), are heated in an oven above the boiling point of the solvent (but below this melting point of the crystals), followed by cooling in a desiccator. Where this treatment is inadvisable, it is still often possible to heat to a lower temperature under reduced pressure, for example in an Abderhalden pistol. This device consists of a small chamber which is heated externally by the vapour of a boiling solvent. Inside this chamber, which can be evacuated by a water pump or some other vacuum pump, is 

Methods for removing water from solids depends on the thermal stability of the solids or the time available. The safest way is to dry in a vacuum desiccator over concentrated sulfuric acid, phosphorus pentoxide, silica gel, calcium chloride, or some other desiccant. Where substances are stable in air and melt above lOO , drying in an air oven may be adequate. In other cases, use of an Abderhalden pistol may be satisfactory. 

in drying inorganic salts, the final material that is required is a hydrate. In such cases, the purified substance is left in a desiccator to equilibrate above an aqueous solution having a suitable water-vapour pressure. A convenient range of solutions used in this way is given in Table 17. 

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Gel permeation chromatography, exclusion chromatography. gel filtration chromatography. A technique for separating the components of a mixture according to molecular volume differences. A porous solid phase (a polymer, molecular sieve) is used which can physically entrap small molecules in the pores whilst large molecules pass down the column more rapidly. A solvent pressure up to 1000 psi may be used. 

Sephadex A trade name for an insoluble hydrophilic substance prepared by cross-linking dextran, and used in gel filtration. It can also be linked to acidic or basic groups for ion exchange or to alkanes for the chromatography of lipophilic compounds. 

The resulting oligonucleotide is often of surprising purity as judged by analytic HPLC or electrophoresis, and up to 30 mg of a deoxyeicosanucleotide (20-base DNA) can be routinely obtained. Nevertheless small amounts of short sequences, resulting from capping and from base-catalysed hydrolysis, must always be removed by quick gel filtration, repeated ethanol precipitation from water (desalting), reverse-phase HPLC, gel electrophoresis, and other standard methods. 

Gel filtration chromatography (GFC) is the name used to describe this method of separation in the biochemical literature. Under this heading, the method is primarily applied to aqueous solutions of solutes of biological origin. 

Fig. 6. Fractionation ranges of commercially available gel-filtration matrices (D) small, ( f- ) medium, and ( . i) large (31).

L. Hagel, "Gel Filtration" in J-C. Janson and L. Ryden, ed.. Protein Purification Principles, High Resolution Methods, and Applications, VCH Pubhshers,... 

Pish protein concentrate and soy protein concentrate have been used to prepare a low phenylalanine, high tyrosine peptide for use with phenylketonuria patients (150). The process includes pepsin hydrolysis at pH 1.5 ptonase hydrolysis at pH 6.5 to Hberate aromatic amino acids gel filtration on Sephadex G-15 to remove aromatic amino acids incubation with papain and ethyl esters of L-tyrosine and L-tryptophan, ie, plastein synthesis and ultrafiltration (qv). The plastein has a bland taste and odor and does not contain free amino acids. Yields of 69.3 and 60.9% from PPG and soy protein concentrate, respectively, have been attained. 

Finally, the techniques of nmr, infrared spectroscopy, and thin-layer chromatography also can be used to assay maleic anhydride (172). The individual anhydrides may be analyzed by gas chromatography (173,174). The isomeric acids can be determined by polarography (175), thermal analysis (176), paper and thin-layer chromatographies (177), and nonaqueous titrations with an alkaU (178). Maleic and fumaric acids may be separated by both gel filtration (179) and ion-exchange techniques (180). 

Desalting and depigmentation of urine sample was carried out by gel-filtration on Sephadex G25 with cut mass 10 kDa. 

In contrast to vapour phase chromatography, the mobile phase in liquid chromatography is a liquid. In general, there are four main types of liquid chromatography adsorption, partition, ion-chromatography, and gel filtration. 

L. Fischer, Gel Filtration, 2nd Edn, Elsevier, North-Holland, Amsterdam, 1980. ISBN 0444802231. 

W.W. Yau, D.D. By and J.J. Kirkland, Modern Size Exclusion Liquid Chromatography Practice of Gel Permeation and Gel Filtration Chromatography, J. Wiley and Sons, New York, 1979. ISBN 0471033871. 

Acetoin dehydrogenase [from beef liver acetoin NAD oxidoreductase] [9028-49-3] Mr 76000, [EC 1.1.1.5]. Purified via the acetone cake then Ca-phosphate gel filtration (unabsorbed), lyophilised and then fractionated through a DEAE-22 cellulose column. The Km for diacetyl in 40pM and for... 

Bromelain (anti-inflammatory Ananase from pineapple) [37189-34-7] Mr 33 000, [EC 3.4.33.4]. This protease has been purified via the acetone powder, G-75 Sephadex gel filtration and Bio-Rex 70 ion-exchange chromatography and has Aj 20.1 at 280nm. The protease from pineapple hydrolyses benzoyl glycine ethyl ester with a Km (app) of 210mM and kcat of 0.36 sec. [Murachi Methods Enzymol 19 273 1970 Balls et al. nd Eng Chem 33 950 1941.]... 

EC 1.15.1.1]. Purified by DEAE-Sepharose and copper chelate affinity chromatography. The preparation was homogeneous by SDS-PAGE, analytical gel filtration chromatography and by isoelectric focusing [Weselake et al. Anal Biochem 155 193 1986 Fridovich J Biol Chem 244 6049 7969]. 

Corticotropin [92307-52-3] polypeptide Mr -4697. Extract separated by ion-exchange on CM-cellulose, desalted, evapd and lyophilised. Then run on gel filtration (Sephadex G-50) [Lande et al. Biochemical Preparations 13 45 1971 Esch et al. Biochem Biophys Res Commun 122 899 1984],... 

Follicle Stimulating Hormone (FSH, foilitropin) [9002-68-0] Mr 36,000. Purified by Sephadex GlOO gel filtration followed by carboxymethyl-cellulose with NH4OAC pH 5.5. The latter separates luteinising hormone from FSH. Solubility in H2O is 0.5%. It has an isoelectric point of 4.5. A soln of Img in saline (lOOmL) can be kept at 60° for 0.5h. Activity is retained in a soln at pH 7-8 for 0.5h at 75°. The activity of a 50% aq EtOH soln is destroyed at 60° in 15 min. [Bloomfield et al. Biochim Biophys Acta 533 371 1978 Hartree Biochem J100 754 1966 Pierce and Parsons Ann Rev Biochem 50 465 1981.]... 

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