Separation of Biopolymers

The classical version of LLC uses combinations of an organic and an aqueous or two organic liquid phases, which limits its application to small molecules without defined secondary or tertiary structure. However, the use of LLC for separation of large active bio-molecules such as nucleic acids and proteins became possible (at least in theory) when P.-A. Albertsson succeeded in developing aqueous-aqueous two-phase systems made up of two 'incompatible' water-soluble polymers such as PEG and dextran. Finding suitable supports for these systems was for a long time a major problem, however. Several attempts were made to adsorb the bottom phase onto supports made of agarose beads, polyethers immobilized on Sepharose, silicates and cellulose. The problem was solved by W. Miiller in 1986 by combining the affinity of polyac-rylamide for the dextran-rich bottom phase of the PEG/dextran system with the mechanical strength of hydrophilic vinyl (LiParGel) or silica (LiChrospher Diol) particles (Table 2). LLC, or LLPC (liquidliquid partition chromatography), was then intro-

Table 4 Separation of nucleic acids by LLPC Separation according to: Examples duced as a method for the separation of biopolymers. This technique was later developed further with respect to sensitivity, selectivity and reproducibility by U.-B. Hansson and co-workers, turning it into a powerful tool for protein analysis in particular. It has recently also been shown that dextran-grafted agarose beads (Superdex) can be used as a support for LLPC. LiParGel 650 has been used for the analysis of small as well as of large proteins while LiParGel 750 has mostly been used for the analysis of nucleic acids. LiChrospher-Diol 100 has been used for the analysis of small proteins ( < 100 000 Da) while proteins of a wide range of sizes have been analysed on LiChrospher-Diol 1000 or 4000 columns. Superdex is suitable for the analysis of small proteins ( < 100 000 Da).

Buffered phase systems formed by polyvinyl pyrrolidone (PVP)/dextran, polyvinyl alcohol (PVA)/ dextran and PEG/salt solutions have been used for LLPC, but the PEG/dextran systems are by far the most frequently used (Table 1). A serious drawback of the PEG/dextran systems is, however, the incompatibility between PEG and some proteins such as immunoglobulins. Concentrations of PEG greater than 3% can be used to precipitate antibodies. However, this incompatibility can be partly overcome by adding appropriate salts. IgG can, for example, be solubilized in PEG/dextran systems at pH 7.0, i.e. near its isoelectric point, in the presence of 100mmolL~1 betaine (<0.8mgmL~1) or 0.1 mol L_1 glycine and 0.1 mol L_1 sodium chloride (<2mgmL~1). Hence, the PEG/dextran systems are, for the moment, the obvious choice of two-phase system for LLPC of biopolymers, whereas the usefulness of other phase systems remains to be elucidated.

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Solar Panel Basics

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