Size exclusion effect

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. 

To ensure a better separation, molecular sieving will act much better This size exclusion effect will require an ultramicroporous (i.e pore size D < 0.7 nm) membrane Such materials should be of course not only defect-free, but also present a very narrow pore size distribution. Indeed if it is not the case, the large (less separative and even non separative, if Poiseuille flow occurs) pores will play a major role in the transmembrane flux (Poiseuille and Knudsen fluxes vary as and D respectively). The presence of large pores will therefore cancel any sieving effect... 

Figure 2. Theoretically predicted size exclusion effects of different solute types (cylindrical pores of single pore size). (O) random coil (Rg) (%) hard sphere (R, = r VIJJ) CA rod (R, = L, VIT)...

Silica gels with mean pore diameters of 5-15 nm and surface areas of 150-600 m /g have been preferred for the separation of low molecular weight samples, while silica gels with pore diameters greater than 30 nm are preferred for the separation, of biopolymers to avoid restricting the accessibility of the solutes to the stationary phase [15,16,29,34]. Ideally, the pore size distribution should be narrow and symmetrical about the mean value. Micropores are particularly undesirable as they may give rise to size-exclusion effects or irreversible adsorption due to... 

AP Sassi, HW Blanch, JM Prausnitz. Characterization of size-exclusion effects in highly swollen hydrogels Correlation and prediction. J Appl Polym Sci 59 1337-1346, 1996. 

Non-silica-based RP-HPLC stationary phases have also been developed and their separation capacity has been compared with those of silica-based ones. The porous structure of crosslinked polymer gels may be responsible for the markedly different selectivity and retention characteristics. Up till now, the mode of separation on polymer stationary phases is not entirely understood at the molecular level. It has been established that the size-exclusion effect may influence the retention of analyses on polymer gels. 

So you can observe ion exchange and hydrophobic interactions in GPC, and size-exclusion effects are not negligible if macroporous ion exchange supports are used. 

Figure 13.3. Appearance of non-size-exclusion effects on SEC-elution curves of polyelectrolytes and other charged analytes including low-molecular-weight organic acids. Kd is the distribution coefficient and Ve, V0, and Vt are elution volume of the analyte, column void volume, and total column volume, respectively.

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. 

Molecular weights appear to range from 0.3 to 700 kDa however, this range is more a reflection of differences among experimental conditions than the real differences in MW caused by different sources. Problems of SEC application for the analysis of HS were addressed in a special issue of Soil Science (1999, Vol. 164, No. 11). In addition to non-size exclusion effects, another source of MW variability is associated with a lack of proper standards (De Nobili et al., 1989). This problem was the subject of attention in Perminova et al. (1998). 

However, due to uncertainties among criteria used to define complete compensation for non-size-exclusion effects and inadequate calibration standards, molecular weight determinations can be inaccurate. Hence, estimates should be treated as apparent molecular weights. 

It is also important that the mobile phase be chosen to prevent interaction of the sample components with the surface of the packing by adsorption or other unwanted effects. Non-size-exclusion effects in GFC, such as those shown in Table 2.6, can usually be avoided by selecting proper combinations of stationary and mobile phases.55 Similarly, in GPC solvents that reduce these interactions, such as toluene or tetrahydrofuran, are commonly used. When these solvents cannot be used, salts such as lithium bromide may be added. 

Ion exchange or sorption can also occur on colloids, since colloids possess an electric surface charge, at which ions can be exchanged or sorptively bound. The proportion of colloids not caught in small pores preferentially utilizes larger pores, thus sometimes travelling faster than some of the water in groundwater (size-exclusion effect). That is why the colloid-bound contaminant transport is of such special importance. 

Using SEC, most accomplishments of the commonly used RP-LC technique can be employed, such as various detector options, high sample loading capacity, variability in stationary phases and up-scaling option. Often in SEC, non-size-exclusion effects such as electrostatic and hydrophobic interactions between the analyte and stationary phase may be observed. The separation efficiency can be improved by optimizing the mobile phase, flow rate, column length, and sample volume. Practical guidelines for SEC method development have been described [42]. 

Provided ionic adsorption and size-exclusion effects are kept minimal, the most polar peptide in a mixture will have the shortest chromatographic retention on nonpolar stationary phases with the remainder eluting... 

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... 

One of the disadvantages of the synthesis was that the silicas had pores in the low mesopore size range between 2 and 4 nm. In adsorption and chromatographic processes however larger mesopores are needed to avoid size exclusion effects. 

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