Size exclusion ionic strength

Garcia, R., Porcar, I., Campos, A., Soria, V. and Figueruelo, J. E., Solution properties of polyelectrolytes. X. Influence of ionic strength on the electrostatic secondary effects in aqueous size-exclusion chromatography, /. Chromatogr. A, 662, 61, 1994. 

In order to be exploitable for extraction and purification of proteins/enzymes, RMs should exhibit two characteristic features. First, they should be capable of solubilizing proteins selectively. This protein uptake is referred to as forward extraction. Second, they should be able to release these proteins into aqueous phase so that a quantitative recovery of the purified protein can be obtained, which is referred to as back extraction. A schematic representation of protein solubilization in RMs from aqueous phase is shown in Fig. 2. In a number of recent publications, extraction and purification of proteins (both forward and back extraction) has been demonstrated using various reverse micellar systems [44,46-48]. In Table 2, exclusively various enzymes/proteins that are extracted using RMs as well as the stability and conformational studies of various enzymes in RMs are summarized. The studies revealed that the extraction process is generally controlled by various factors such as concentration and type of surfactant, pH and ionic strength of the aqueous phase, concentration and type of CO-surfactants, salts, charge of the protein, temperature, water content, size and shape of reverse micelles, etc. By manipulating these parameters selective sepa-... 

Application of SEC for HS Analysis and Fractionation. SEC was first applied to the analysis of HS by Posner in 1963 (Posner, 1963). Since then, a vast amount of experimental data has been gathered which show that elution conditions such as pH and ionic strength are crucial for the results of this analysis (De Haan et al., 1987 Frimmel et al., 1992 Gjessing, 1973 Mori et al., 1987 Pershina et al., 1989 Piccolo, 1997 Piccolo et al., 1996 Swift, 1999 Town and Powell, 1992 Varga et al., 2000). The acidic nature of HS gives rise to non-size-exclusion effects that depend not just on molecular size but also on electrostatic and/or sorptive interactions between ionogenic analytes and hydrophilic gel matrices (Belyaeva et al., 2006 De Nobili et al., 1989 Perminova, 1999). Hence, proper interpretation of the SEC results must consider possible non-size-exclusion effects not related to molecular size but intrinsic to experimental conditions. 

In previous work [15,17,28] we have evaluated the relation between the flow ratio co, the pore size, and the ionic strength of the solution experimentally, by means of size-exclusion electrochromatography (SEEC). In SEEC the transport rates of the (neutral) macromolecules depend direcdy on co. As in conventional, pressure-driven SEC, the separation in SEEC is based on the differential accessibility of the (stagnant) mobile phase in the pores of the particles for macromolecules of different sizes. However, with increasing pore flow ratio in SEEC, the velocity difference between the mobile-phase fractions inside and outside the particles decreases. The retention ratio x (the retention time relative to a low-molecular-mass marker) for a probe molecule in SEEC is given by... 

Viscotoxin, a basic peptide of molecular weight ca. 9000 (Samulsson, 1961), moves unretarded in strongly cross-linked gels such as Sephadex G-25 (Fig. 4a) (in phosphate buffer, ionic strength 0.05, pH 6.8. When filtered under similar conditions in weakly cross-linked dextran, viscotoxin behaves quite differently (Fig. 4b). In fact it moves behind isoleucine. The gel of the first kind can be used to remove solutes of lower molecular size, the purification being based on molecular exclusion. Filtration in the second kind of gel may be used not only for separating solutes of different molecular size but also to separate peptides and other substances of similar molecular size when they differ in certain structural features. 

The calibration of the size-exclusion column with standard proteins demonstrated that the L-Ser toxin has an apparent molecular mass of 6 kDa, which is close to the real molecular mass of the toxin. The apparent molecular mass of the D-Ser toxin was too small to evaluate accurately from the calibration data. These results indicated that both (o-[D-Ser46]Aga-TK and (o-[L-Ser ]Aga-TK take monomeric form at physiological pH and ionic strength, but the two toxins are significantly different in apparent molecular mass. 

The present results suggest that PL-cellulose spherical particles can reduce the concentrations of natural LPS to 1 EU/mL or lower in drugs and fluids used for intravenous injection, at a neutral pH and ionic strengths of fi=0.05-0.4. These processes did not affect the recovery, even of acidic proteins such as BSA. The high LPS-adsorbing activity of the PL cellulose is possibly due to the cationic properties of the ligand and its suitable hydrophobic properties. The high LPS selectivity of the particles with small pore size is due to the size-exclusion effects on protein molecules. By contrast, that of the particles with... 

Due to Dorman exclusion principle [29] charged membranes can reject inorganic salts even though they have pores much larger than the salts and this ion rejection is known to decrease with increasing feed ionic strength. The example of 1.1 electrolyte filtration through different pore sizes at a pH far from the... 

The interpretation of the above results is made more difficult by a supplementary phenomenon due to the aqueous phase ionic strength. Increasing the density of charges in the vicinity of the surfactant headgroups causes a screening effect not only between surfactant and protein but also in the repulsive interaction between surfactant polar heads they come closer and the micellar size decreases (43). As a consequence, Goklen and Hatton (48) noted a decrease in the amount of water in AOT system when increasing KCl concentration. However, it is difficult from these results to state precisely whether the decrease in protein extraction is due to the decrease of electrostatic interactions or to a size exclusion effect. 

Chromatography. Purified pectins and their degradation products were studied by gel-permeation chromatography on Sepharose CL-2B or Sepharose CL-6B (Pharmacia), under conditions as described by Bar-bier and Thibault (7). High-performance size exclusion chromatography was done with a series of Biogel TSK columns types 6000, 5000, 4000 and 3000 PW (Bio-Rad Labs., Richmond, Ca., USA). The solvent was 0.1 M sodium sulfate in sodium acetate, pH 3.7, ionic strength 0.34 (16). 

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