Non-size exclusion effects

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. 

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]. 

Although separation occurs by sieving or exclusion of the constituents by the column packing material, other so-called non-size exclusion effects are also known to occur. These effects, due mainly to the presence of charged residues on the column material, complicate the interpretation of a separation, and in the case of metal-containing molecules may lead to the alteration of the species. The non-size exclusion effects can be divided into two major groups ionic interactions and adsorption. The extent of these interferences is determined in part by the type and process used in the manufacture of the column packing material, and in part by the experimental conditions. 

Extensive discussions on non-size exclusion effects can be found in articles and reviews by various authors (Stenlund, 1976 Gelotte, 1960 Pfannkoch et al., 1980 Barth, 1980 Engelhardt, 1981 Belew, 1978 and Dubin, 1992). 

Besides non-size exclusion effects, dilutions of the sample constituents during fractionation may lead to the dissociation of the labile complexes and thus affect the distribution of the metal. In order to reduce this problem some authors (Evans et al., 1978 and Yoza, 1977) have advocated the addition of a constant level of the metal or ligand in the buffer used to elute the sample from the column. This Is not a very useful approach since even if the buffer does not form strong complexes with the free ion added new species may be introduced. The only sensible approach is to minimise dilution. 

Both the time needed for a separation and the amount of dilution of the sample constituents have been drastically reduced with the introduction of high performance SEC (HPSEC). However, non-size exclusion effects tend to be more severe when chemically bonded phases are used. Fortunately, as the problems are identified and understood new column materials have been developed to replace the older ones. In a few articles (Cassidy, 1981 Drull, 1984) the usefulness of HPSEC for the fractionation of metal che-... 

Characterization of PVA by SEC has closely followed the advances in column and detection technology. Aqueous SEC, in particular, presents a number of challenges as a result of frequently encountered secondary, non-size exclusion effects. These effects, common to aqueous SEC, have been reviewed by Barth (12). 

Silanized silica gel has also been studied for the SEC analysis of cellulose trinitrate (37,38), using THF as the eluant. It was concluded that the elution behavior of the cellulose trinitrate sample was influenced by polyelectrolytic effects and was not based strictly upon size exclusion. The addition of 0.01 mol acetic acid per liter THF suppressed the non-size exclusion effects. Under these conditions, a universal calibration curve relating cellulose trinitrate and polystyrene could be established. 

Ideally, an internal marker should be a polymeric species under the same influences as the samples. In practice, since the elution volume range available in SEC is very limited, the internal markers used normally have low molecular mass, either added solvents or system peaks, for instance those due to antioxidant [11]. However, some care is required, as these solvent or system peaks can shift as a consequence of non-size exclusion effects, for instance moisture content of solvent encouraging selective adsorption. An internal marker is run at the same time as the calibrants, and when samples are run, the elution times of the internal marker are compared and the elution times of the sample are adjusted to correspond to the calibration. 

In summary, the various non-size exclusion effects operating In SEC of proteins affect the linearity of the calibration curve unfavourably and make an assessment of reliable molecular weight data difficult. In the most favourable case, a standard deviation of 15% In the log-linear calibration plot for proteins was reported on a TSK gel 3000 SW column. ... 

Perminova, I. V. 1999. Size exclusion chromatography of humic substances Complexities of data interpretation attributable to non-size exclusion effects. Soil Science 164, no. 11 834-840. 

SEC is commonly referred as room-temperature SEC or high temperature SEC, but the separation mechanism for both techniques is the same. As the name indicates, room-temperature SEC is used for polymers that are soluble at room temperature in the mobile phase tetrahydrofuran is a commonly used solvent in this case, but many other solvents are equally adequate. High temperature SEC is mostly used for polyolefins and their copol5uners since they are only soluble at temperatures from 120 to 150°C in solvents such as trichlorobenzene. In general, solvent type does not play a very important role in SEC, provided that it does not interfere with the column pore structure (when cross-linked polymer beads are used) and with the on-line detectors at the exit of the columns. However, care must be taken to avoid non size-exclusion effects, particularly for room-temperature SEC. 

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