Separation Size exclusion

The size of the pores determines the molecular weight range of the compounds that can be separated. Size exclusion often is used to clean up an extracted solution. For example, it is used to remove the fats and waxes from the pesticide residues that are extracted from a food composite. 

The smaller the particle size of the stationary phase the greater is the peak capacity of the column and the better and faster are the separations. Size-exclusion chromatography is generally subdivided further into mobile phase categories. The mobile phase is aqueous in GFC (gel filtration chromatography) and is an organic solvent in GPC (gel permeation chromatography). 

Figure 1. Purification of rat CpnIO (101 residues) using Fmoc probe 2. (A) Analytical RP-HPLC (C4 medium) of crude underivatized rat CpnIO. (B) Addition of lipophilic probe 2 increases the retention time of the protein (labelled 2) thus facilitating purification from underivatized truncated sequences (labelled 1). (C) Purified protein derivatized with 2. (D Purified protein after treatment with 5% aqueous TEA to remove 2. (E) ESI-MS of purified rat CpnIO. (R Deconvoluted mass spectrum for purified rat CpnIO. The calculated mass of target product is 10770.57 Da (average). The found mass is 10771.0. (Q) RP-HPLC (C4 medium, gradient TFA-water into 100% TFA-AcCN, 60 min) of purified rat CpnIO. The insert shows the expanded peak. (H) CZE of purified rat CpnIO. The concentration of the major peak (protein in its native, heptameric state) is 84%. Separate size-exclusion chromatography experiments showed that the majority of the flanking peaks correspond to protein with correct sequence but having an aggregation state different from the major peak.

Wei G T, Liu F K and Wang C R C 1999 Shape separation of nanometre gold particles by size-exclusion chromatography Anal. Chem. in press... 

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. 

In size-exclusion chromatography, the smallest solute that can be separated from other solutes all smaller solutes elute together. 

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). 

SI units stands for Systeme International d Unites. These are the internationally agreed on units for measurements, (p. 12) size-exclusion chromatography a separation method in which a mixture passes through a bed of porous particles, with smaller particles taking longer to pass through the bed due to their ability to move into the porous structure, (p. 206)... 

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. 

This reversed-phase chromatography method was successfully used in a production-scale system to purify recombinant insulin. The insulin purified by reversed-phase chromatography has a biological potency equal to that obtained from a conventional system employing ion-exchange and size-exclusion chromatographies (14). The reversed-phase separation was, however, followed by a size-exclusion step to remove the acetonitrile eluent from the final product (12,14). 

Aqueous-detergent solutions of appropriate concentration and temperature can phase separate to form two phases, one rich in detergents, possibly in the form of micelles, and the other depleted of the detergent (Piyde and Phillips, op. cit.). Proteins distribute between the two phases, hydrophobic (e.g., membrane) proteins reporting to the detergent-rich phase and hydrophilic proteins to the detergent-free phase. Indications are that the size-exclusion properties of these systems can also be exploited for viral separations. These systems would be handled in the same way as the aqueous two-phase systems. 

Except for the high molecular weight range, nearly all substances can be separated by reversed-phase (RP) HPLC. The many different separation mechanisms in RP HPLC, based on hydi ophobic, hydi ophilic and ion-pairing interactions, and size exclusion effects together with the availability of a lai ge number of high quality stationary phases, explain its great populai ity. At present approximately 90% of all HPLC separations are carried out by reversed-phase mode of HPLC, and an estimated 800 different stationai y phases for RP HPLC are manufactured worldwide. 

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