Aqueous chromatography utilizing pH-/temperature responsive polymer stationary phases to separate ionic bioactive compounds

Cross-linked poly(N-isopropylacrylamide-co-acrylic acid) (poly(IPAAm-co-AAc ))-grafted silica bead surfaces mere prepared and applied as new column mat ric; materials that exploit temperature-responsive anionic chromatography t o separate basic bioactive compounds,specifically catecholamine derivatives ,in aqueous mobile phases. Since poly(IPAAm-co-AAc); has a well-known tempe rature-responsive phase transition and apparent pK(a) shift, polymer-grafte d silica bead surfaces are expected to exhibit simultaneous hydrophilic/hyd rophobic and charge density alterations under thermal stimuli. Elution beha vior of catecholamine derivatives from a copolymer-modified head packed col umn was monitored using aqueous mobile-phase HPLC under varying temperature and pH, Catecholamine derivatives had higher retention times on poly(IPAAm -co-AAc) columns at higher pH in comparison with those or, noncharged PIPAA m reference columns, suggesting an electrostatic interaction as a separatio n mode. Temperature also affected the retention behavior of catecholamine d erivatives. Optimal separation of four catecholamine derivatives aas achiev ed at elevated temperature, 50 degreesC, and at pH 7.0. This is due to the increased hydrophobicity of the stationary phase as evidenced by the elutio n of a nonionic hydrophobic steroid. From these results, mutual influences of both electrostatic and hydrophobic interactions between basic catecholam ine derivatives and pH-/temperature-responsive surfaces are noted. Conseque ntly, elution of weakly charged bioactive compounds is readily regulated th rough the modulation of stationary-phase thermoresponsive hydrophilic/hydro phobic and charge density changes.