Gel filtration column

Figure 1. Column gel filtration chromatography using Sephadex G-75 for the...

Coelution of procyanidins with cinnamics and flavonoids presented a major problem in quantitating the procyanidins. (Cinnamics and flavonoids could be measured in the presence of procyanidins by monitoring at 320 nm where procyanidins are nonabsorbing.) This was overcome with preliminary separation of compound classes with mini column gel filtration (4). Figure 4B shows an HPLC separation of apple juice procyanidins. Compounds absorbing at 320 nm which coeluted with catechin, epicatechin and the procyanidins have been eliminated by the cleanup procedure. The HPLC conditions (column, solvent gradient, etc.) are identical to those used for separation of cinnamics and flavonoids, the separation taking 60 min. 

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. 

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... 

Size exclusion was first noted in the late fifties when separations of proteins on columns packed with swollen maize starch were observed (Lindqvist and Storgards, 1955 Lathe and Ruthven, 1956). The run time was typically 48 hr. With the advent of a commercial material for size separation of molecules, a gel of cross-linked dextran, researchers were given a purposely made material for size exclusion, or gel filtration, of solutes as described in the classical work by Porath and Flodin (1959). The material, named Sephadex, was made available commercially by Pharmacia in 1959. This promoted a rapid development of the technique and it was soon applied to the separation of proteins and aqueous polymers. The work by Porath and Flodin promoted Moore (1964) to apply the technique to size separation, gel permeation chromatography of organic molecules on gels of lightly cross-linked polystyrene (i.e., Styragel). 

Irrespective of the development of media, many of the traditional media are successfully defending their position. This is due to their hydrophilic nature, preserving biological function of the separated molecules, but also the fact that columns may be prepared easily and, finally, some of the classical media, e.g., Sephadex, have a selectivity that is so far unsurpassed and therefore very fit for use. Intersting enough, Sephadex is still the premiere gel filtration medium for desalting due to the optimal pore size and particle size of this medium (see Section II,C). 

Numerous application examples are related to gel filtration (e.g., see Hagel, 1989 Hagel and Janson, 1992 Pharmacia, 1991). A selected number of applications are discussed with respect to their goals and to types of methods and SEC media attributes, which impact the selection and or construction of a suitable SEC column. Specific examples of these various applications types are given later under Sections II,C and III. The optimization of running conditions to achieve the desired results are discussed in Section VI. 

FIGURE 2.4 Calibration curve of dextran on Sephacryi S-300 SF. Calibration curves were calculated from one chromatogram of a broad MWD reference sample using data for the molecular mass distribution as obtained by a calibrated gel filtration column ( , upper curve) and on-line MALLS ( ). The calibration curve was found useful for estimating the size of globular proteins. [Reproduced from Hagel et al. (1993), with permission.]... 

Based on the requirements of the separation, media of suitable pore size, particle size, and surface properties are selected as well as column dimensions and column material. In some cases a suitable combination of media type and column dimensions may be available as a prepacked column. In most cases, this is a more expensive alternative to preparing the column yourself but will provide a consistent quality as assured by the manufacturing and testing procedures of the vendor. The consistent quality may be critical in obtaining reproducible results and may thus be a cost-effective solution. Also, the fact that smaller particle-sized media are more difficult to pack and require special, and expensive, equipment has resulted in that gel filtration media of small particle size, e.g. smaller than 15 /zm, are predominantly supplied as prepacked columns. 

Often, media of similar characteristics as the one used for prepacked columns (i.e., except for particle size) are obtainable, e.g., as preparative grade material, having particle sizes of typically 30-50 /j,m. This is required for large-scale gel filtration where the sample capacity of the prepacked columns is often insufficient. 

The selection of column characteristics is determined by solvent resistance, the need to visually inspect the bed, the pressure rating of the system, and the dimensions [column inner diameter (i.d.) and length (L)] required from productivity considerations. Productivity considerations will vary if the requirement is based on the amount of information per unit time (analytical gel filtration) or the amount of substance per unit time (preparative gel filtration). 

Gel filtration separations are performed in an aqueous environment and the requirement on chemical resistance is therefore modest. Most column tubes and accessories (e.g., support net, sealings) withstand the solvent mixtures sometimes used in aqueous SEC such as 20% ethanol, 3 M guanidinium hydrochloride, 6 M urea, or 0.1 M HCl and 1 M NaOH, the last two being used for cleaning the packed column (see Section V). 

The diameter of the column is selected from the volume of sample that is to be processed. As a rule of thumb the maximum productivity is obtained at a sample volume of 2-6% of the bed volume in preparative gel filtration on a 50-/rm chromatographic medium (Hagel et al., 1989). Thus, the required column diameter is calculated from the bed volume needed to cope with the sample volume and the column length needed to give the resolution desired. 

The packing method supplied by the manufacturer of the gel filtration medium may need to be revised according to the column being selected. It is therefore important to have an understanding about the basic principles governing the packing of chromatographic beds. 

Gel filtration is very suitable for the purity check of protein preparations, especially if these have been purified by adsorptive techniques. It can be expected that high-resolution gel filtration columns will easily separate dimeric forms from monomeric forms to reveal heterogeneities of the preparations. However, a size difference of less than 20% will not result in total resolution of the peaks (although the chromatogram may be used for a qualitative judgment of the... 

The size separation of proteins has been routinely called gel filtration because of the historic use of cross-linked gels for this application. Specially modified Zorbax PSM columns, Zorbax GF-250 and Zorbax GE-450, are used for separating proteins by size. These columns are packed with porous silica micro-... 

I. SILICA-BASED TSK-GEL SW/SWxl COLUMNS FOR GEL-FILTRATION CHROMATOGRAPHY (GFC)... 

TABLE 4.11 Recommended Column Selection Guide for High-Performance Gel-Filtration Chromatography... 

FIGURE 5A.1 A gel filtration chromatogr phy column. Larger molecules are excluded from the gel beads and emerge from the column sooner than smaller molecules, whose migration is retarded because they can enter the beads. 

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