Aqueous SEC

Although size separation was pioneered by the use of Sephadex (Pharmacia Fine Chemicals) columns in aqueous systems (gel filtration), the development of high-performance SEC for water-soluble low-molecular-mass compounds (see also Chapter 7) has been disappointing. Most columns for aqueous use have been designed for separation of large biochemical molecules such as proteins and peptides. Complex interactions, such as adsorption, or ionic effects which lead to a non-SEC mechanism, may occur. The essential simplicity of SEC is therefore lost. 

An example of an aqueous separation is that of low-molecular-mass peptides [57] (MM 240-6700). Using a TSK SW 2000 column with a 0.05 M phosphate eluent adjusted to pH 5.0-8.0, the separation showed poor correlation between molecular mass and elution volume. The addition of 35% methanol and 1% trifluoroacetic acid to the eluent gave a satisfactory size-related separation, by minimizing interactions with the gel. Many water-soluble compounds are also soluble in organic solvents such as tetrahydro-furan, and may therefore be analysed more easily under these conditions. 

Krishen, in Size Exclusion Chromatography, ed. T. Provder, ACS Symp. Ser. 245, American Chemical Society, Washington DC, Chapter 16 (1984). 

Kirkland and D.D. Bly, Modern Size-Exclusion Liquid Chromatography, Wiley-Interscience, New York (1979). 

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Gel filtration separations are performed in an aqueous environment and the requirement on chemical resistance is therefore modest. Most column tubes and accessories (e.g., support net, sealings) withstand the solvent mixtures sometimes used in aqueous SEC such as 20% ethanol, 3 M guanidinium hydrochloride, 6 M urea, or 0.1 M HCl and 1 M NaOH, the last two being used for cleaning the packed column (see Section V). 

Small peptides are among the most difficult compounds to analyze by aqueous SEC, although conditions for good separations have been developed for both TSK-GEL SW and TSK-GEL PW column types (15,16). Pigure 4.27 (page 124) compares the results of separating two mixtures of peptides on... 

Column sorbents for aqueous media show )ust average properties. This is due to the different copolymerization process, which does not allow easy formation of macroporous beads with proper pore topology. This fact also reflects many experiences of SEC users, who have to input much more effort to get good aqueous SEC work accomplished. 

The field of aqueous SEC is of growing interest and also represents an area of active development by several companies, with Polymer Laboratories included. In this area, contrary to the organic SEC scenario, a variety of polymer chemistries are employed in the preparation of the column packing materials. This fact alone makes the practice of aqueous SEC more difficult than its organic counterpart and requires the suppliers of such columns to offer a good level of technical support to the column user. This chapter outlines the characteristics and applications of PL aquagel-OH aqueous SEC columns. 

Whereas for organic SEC column technology a particular type of bead (PS/ DVB) is used almost universally, in the field of aqueous SEC there have been a variety of approaches to derive polymeric beads suitable for the application. For this reason there is more secrecy about the chemical composition of the packing materials and columns produced by different manufacturers. 

The basic requirements for an aqueous SEC column are (1) the beads must exhibit an extremely hydrophilic surface chemistry, (2) the beads should exhibit... 

Based on this background, a selected number of preparative, semipreparative, and analytical aqueous SEC systems for the analysis of polysaccharides, summarized in Table 16.1, are presented and discussed briefly in this chapter. 

TSK PW columns and Shodex OH-pak columns are made respectively by Toyo Soda and Showa Denko and have become very popular aqueous SEC columns for synthetic water-soluble polymers since the early 1980s. Linear or... 

The purpose of this study is twofold to compare four linear aqueous SEC columns made by Tosoh and Showa Denko in terms of composition and performance and to evaluate the effect of commercial PEO standards on the accuracy and precision of the MW and MWD of polyvinylpyrrolidone (PVP) and NIST PEO standards. In terms of performance, emphasis will be placed on factors not commonly covered by column manufacturers. Successful SEC conditions for PVP in water, in water/methanol, and in dimethylformamide can be found in the literature (8,9,10). This study deals mainly with the effects of column, mobile phase, and PEO standards on the MW and MWD of PVP. 

E. Comparison of the Separation Efficiency of Commercial Aqueous SEC Columns in Water and in Water/Methanol... 

In summary, methanol as a mobile-phase modifier has a significant effect on the separation of PVP in aqueous SEC with these four linear columns. The best separation of all PVP grades can be achieved with the SB-806M column in 50 50 water/methanol with 0.1 M lithium nitrate. It is interesting to note that despite the improvements reported by the manufacturers for the newer columns (SB-806MHQ and PWxl), the newer columns do not necessarily perform better than the older columns (SB-806 and PW) for aqueous SEC of PVP. 

A summary of typical experimental conditions used with TSK-PW columns for nonionic polymers is described in Table 20.3. A common mobile phase is an aqueous solution of 0.05 N sodium nitrate. A salt solution of sodium nitrate is a good choice because it is not as corrosive as a solution of sodium chloride. For the descriptions and examples that follow, a bank of either five or six TSK-PW columns in series (G1000-G5000 or G1000-G6000) was used for the aqueous SEC work. These configurations allow for molecular mass characterization from less than 1,000 Da to 1,000,000 Da or greater. 

Molecular weights for fully hydrolyzed and partially hydrolyzed grades of PVA (expressed in terms of 4% solution viscosity) from aqueous SEC-MALLS are summarized in Table 20.6. M values range from less than 20,000 Da for a 3 centipoise (cP) viscosity grade to over 230,000 Da for a super-high viscosity... 

TABLE 20.6 Molecular Weight of PVA from Aqueous SEC-MALLS Using TSK-PW Columns... 

The second method assumes that the molecular weight calibration curve is linear on a semilog plot and should be employed where universal calibration is not practical as with aqueous SEC. Several variants of these methods involving different molecular weight data for the standards are discussed. The proposed methods have been evaluated using aqueous SEC and polydextran standards and nonaqueous SEC with polyvinylchloride standards. 

Methods based on universal calibration will be illustrated using nonaqueous SEC and broad MWD PVC standards. The and of these standards is known. Methods based on the linear molecular weight calibration will be illustrated using aqueous SEC and broad MTO polydextran standards for which M and My are known. 

We now move on to aqueous SEC and the use of the method of calibration based on a linear molecular weight calibration curve and two broad MWD polydext ran s with known and M. ... 

Non-ideal behavior in SEC, due to the contribution of nonsize effects to retention, particularly in the case of the separation of polyelectrolytes in aqueous SEC, can render erroneous the calculation of molecular weight and molecular weight... 

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