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Previous results have shown the possibility for obtaining high-resolution separations of glycosaminoglycans by partition in butanol/aqueous two-phase systems containing quaternary ammonium salts. In this paper, the effects on partition behavior of both polymer molecular weight and sulfation were examined. Two series of fractionated chondroitin sulfate polymers were isolated in which the molecular weight and sulfation varied systematically. In the molecular weight series the six samples, spanned the range from 3200 +/- 300 to 19 700 +/- 500 and each sample carried 0.8 sulfate groups per uronic acid residue. In the sulfation series, each sample had an essentially constant molecular weight of 13 000, but the sulfation varied from 0.58 to 0.88 sulfate groups per uronic acid. The C50 of each of these samples was determined in the 1-butanol/aqueous NaCl phase system containing 1% hexadecylpyridinium chloride. In the series wherein the molecular weight varied, the C50 increased with molecular weight up to 12 000 where a limiting value was reached. In the series wherein the sulfation varied, a linear relationship was found between the C50 and the square of the number of anionic substituents per disaccharide. These results show that fractionation by partition techniques will be sensitive to the anionic nature of the polymer, but for the common connective tissue glycosaminoglycan, there will be no fractionation according to molecular weight.

The complexes formed between quaternary ammonium cations and polyanionic glycosaminoglycans can be partitioned between partially miscible aqueous inorganic salt and alcohol phases. Small changes in salt concentration can completely shift the complex from one phase to the other. The effect of the phase composition variables: the type of inorganic salt, the type of quaternary ammonium salt, and the alcohol used, were systematically investigated. The sharp transition from solubility in the upper non-aqueous phase to solubility in the lower, aqueous phase was found to be strongly affected by the type of inorganic salt. This transition occurred at higher salt concentrations when NaCl, KCl, or LiCl were used than when CaCl2 or MgCl2 were used. Differences in behavior among glycosaminoglycans were larger for NaCl than for CaCl2. The complex is stabilized to dissociation by salt by increasing hydrophobicity of the non-aqueous phase. However, aggregation of the complex into an insoluble form is also favored by an increasingly hydrophobic environment. The most consistent partition was observed with 1- and 2-butanol. The partition isotherm of chondroitin 4-sulfate was investigated at constant salt concentration. It was found that the partition coefficient varies with the concentration of chondroitin 4-sulfate, although the magnitude of this effect could be diminished by increasing the quaternary ammonium salt concentration.