Cyclodextrins (CDs) are generally considered to form inclusion complexes which mimic biological interactions, e.g., the complex between an enzyme and substrate'. These molecular adducts of CDs have been extensively studied in solution2 and by X-ray crystallography3-'. Matrix-bound CDs have manifold chromatographic applications in the purification of low-molecular-weight substance@. In contrast, the chromatography of CDs on chemically modified supports has not been studied. Formally this system closely resembles affinity chromatography and hence could serve as a model of it. Since CDs are small, non-ionic, commercially available pure substances, whose concentration can be varied over large ranges, experiments which cannot be carried out with other biomolecules are possible. The existence of three homologous forms of CDs, a-, /I-and y-, with variable complexing properties further facilitates analysis of this model.
We describe here an affinity system for CDs involving naphthoxyacetic acid bound to aminated Bio-Gel P-6 or to cellulose. These supports separated the three CD-forms. The effects of concentration, ligand, temperature and binding capacity were studied. Interactions of CDs between free and support-bound ligands are discussed.
EXPERIMENTAL
Preparation of the modified sorbents
Bio-Gel P-6 (20&400 mesh; Bio-Rad Labs., Richmond, CA, U.S.A.) was aminated and the concentration of the amino groups determined by normal acid-base titration'. (ZNaphthoxy)acetic acid (1-5 g; Aldrich, Milwaukee, WI, U.S.A.) was dissolved in aqueous 20% dimethyl sulphoxide and then 50 ml (a settled volume) of aqueous aminated Bio-Gel P-6 were added and the pH was adjusted to 4.7 with 1 M sodium hydroxide. The suspension was stirred at 20°C while N-ethyl-N-(3-dimethylaminopropyl)carbodiimide hydrochloride (4 g in 20 ml of water at 0°C; Sigma, St. Louis, MO, U.S.A.) was added dropwise during 30 min while the pH was maintained at 4.7. Gentle stirring was continued for 4 h. The gel was washed with water, 50% ethanol, ethanol, 50% ethanol, water, 0.5 A4 sodium chloride and water, 500 ml of each.
A 10-g amount of cellulose MN 300 (Sigma) was suspended in water and swelled overnight. It was washed with 50 ml of 2 M sodium hydroxide at &5"C on a glass filter