Size-exclusion column

Ni-NTA superflow resin column, size exclusion column. 

Alizarin Red S (3,4-dihydroxy-9,10-dioxo-2-anthracene sulfonic acid, Na salt. H2O) [130-22-3] M 360.4, pKj <1, pK 5.49, pK 10.85 (11.01). Commercial samples contain large amounts of sodium and potassium chlorides and sulfates. It is purified by passing through a Sephadex G-10 column (size exclusion column), followed by elution with water, then 50% aqueous EtOH [King Pmden Analyst (London) 93 601 1968]. Finally dissolve it in EtOH and precipitate it with Et20 several times [Sacconi J Phys Chem 54 829 1950, polarography Fumam Stone J Am Chem Soc 70 3055 1948, Beilstein 11 IV 682.]... 

The principles causing retention behaviour, separation variables, molecular weight calibration and associated terminology such as interparticle and intraparticle volume, selective permeation, fractionation range and molecular hydrodynamic radius are as for open column size exclusion (Chapter 4). 

Clean-up steps such as SPE, liquid adsorption chromatography in columns, size exclusion chromatography, SPME, solvent exchange, reduction of solvent volume... are absolutely necessary when dealing with food samples. Given the complexity of the food samples, as well as the necessity of a number of clean-up steps to purify the extract, the utilization of IS and/or RS, as well as adequate certified materials, becomes an inherent necessity. These clean-up steps have been described in other sections of this book chapter. 

Two classes of micron-sized stationary phases have been encountered in this section silica particles and cross-linked polymer resin beads. Both materials are porous, with pore sizes ranging from approximately 50 to 4000 A for silica particles and from 50 to 1,000,000 A for divinylbenzene cross-linked polystyrene resins. In size-exclusion chromatography, also called molecular-exclusion or gel-permeation chromatography, separation is based on the solute s ability to enter into the pores of the column packing. Smaller solutes spend proportionally more time within the pores and, consequently, take longer to elute from the column. 

Examples of the application of size-exclusion chromatography to the analysis of proteins. The separation in (a) uses a single column that in (b) uses three columns, providing a wider range of size selectivity. (Chromatograms courtesy of Alltech Associates, Inc. Deerfield, IL). 

Size-exclusion chromatography can be carried out using conventional HPLC instrumentation, replacing the HPLC column with an appropriate size-exclusion column. A UV/Vis detector is the most common means for obtaining the chromatogram. 

An example of a size-exclusion chromatogram is given in Figure 7 for both a bench-scale (23.5 mL column) separation and a large-scale (86,000 mL column) mn. The stationary phase is Sepharose CL-6B, a cross-linked agarose with a nominal molecular weight range of 5000-2 x 10 (see Fig. 6) (31). 

Fig. 7. Chromatograms of size-exclusion separation of IgM (mol wt = 800,000) from albumin (69,000) where A—D correspond to IgM aggregates, IgM, monomer units, and albumin, respectively, using (a) FPLC Superose 6 in a 1 x 30 — cm long column, and (b) Sepharose CL-6B in a 37-cm column.

Column Si. Size-exclusion chromatography columns are generally the largest column on a process scale. Separation is based strictly on diffusion rates of the molecules inside the gel particles. No proteins or other solutes are adsorbed or otherwise retained owing to adsorption, thus, significant dilution of the sample of volume can occur, particularly for small sample volumes. The volumetric capacity of this type of chromatography is determined by the concentration of the proteins for a given volume of the feed placed on the column. 

The total stationary-phase volume required to process a given feed stream is proportional to the inlet concentration and volume of the feed. For example, for a typical inlet concentration of protein of 10 g/L, in a 100 L volume of feed, a column volume of at least 100 L is needed for size-exclusion chromatography. In comparison, an ion-exchange column having an adsorption capacity of 50 g/L would only require 20 L of column volume for the same feed. 

C-S. Wu (Ed.), Column Handbook for Size Exclusion Chromatography, Academic Press, NY, 1998. ISBN 0127655557... 

Interleukin (from human source). Purified using lyophilisation and desalting on a Bio-Rad P-6DC desalting gel, then two steps of HPLC, first with hydroxylapatite, followed by a TSK-125 size exclusion column. [Kock and Luger J Chromatogr 296 293 7984 ]... 

A trend in chromatography has been to use monosized particles as supports for ion-exchange and size-exclusion chromatography and to minimize the column size, such as using a 15 X 4.6-mm column packed with 3-/rm polymer particles for size exclusion chromatography. The more efficient and lower back pressure of monosized particles is applied in the separation. 

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