and acetonitrile were acs reagent grade water for hplc refers to in house deionized water from a barnstead nanopure ii system methanol and acetonitrile for hplc use were b amp j brand hplc grade solvents ammonium acetate was fisher hplc grade deae cellulose was schleicher and schuell anion exchange cellulose lots 2932 and 2893 tris buffer tris hydroxymethyl amminomethane was enzyme grade ultra pure bethesda research laboratories 0 05m ph 7 4 dicalite was speed plus grade filter aid grefco minerals normal phase thin layer chromatograph tlc was carried out on silica gel 60 f 254 plates em reagents cat 5765 5 u00d7 10 cm by 0 25 mm thick reversed phase tlc was accomplished with whatman mkc 18 plates cat 4803 110 0 2 mm thick plates were developed in whatman cylindrical jars with caps and 10 ml of eluant chromophores were visualized as uv quenching zones with 254 nm ultraviolet light preparative layer chromatography plc was carried out on silica gel 60 f 254 plates em reagents cat 5766 20 u00d7 20 cm by 2 mm thick plates were developed in glass tanks with covers and 100 ml of eluant the vacuum liquid chromatography vlc apparatus consisted of buchner funnel kontes art k 954100 containing a sealed in sintered glass disc m porosity a side hose connection for vacuum and a lower 24 40 joint for attachment of receiving flasks the funnels were equilibrated by pulling the initial eluant through under vacuum to form tightly packed 5 cm adsorbent bed heights samples were preadsorbed onto adsorbent and applied to funnels as slurries in the starting eluant step gradients were carried out using predetermined volumes of increasingly more polar eluant the funnel was sucked dry after each volume fractions were.