Cross-Linked Thermoresponsive Anionic Polymer-Grafted Surfaces To Separate Bioactive Basic Peptides

Cross-linked, thermoresponsive poly(N-isopropylacrylamide-co-acrylic acid-co-N-tert-butylacrylamide) [poly(IPAAm-co-AAc-co-tBAAm)] thin hydrogel layers on silica beads were used as new column matrix modifiers for LC separation of basic bioactive peptides, angiotensin subtypes I, II, and III. Terpolymer poly(IPAAm-co-AAc-co-tBAAm) showed both phase transition and apparent carboxylate pKa shifts in water, depending on temperature. Polymer-grafted silica bead surfaces exhibited simultaneous thermally modulated changes in hydrophilic/hydrophobic properties and charge densities. More effective separation of angiotensin peptide subtypes was achieved on columns of these terpolymer thin hydrogel grafted surfaces, as compared to an uncharged control binary copolymer of IPAAm and tBAAm. Although hydrophobic interactions effect separation of angiotensin subtypes, combined electrostatic and hydrophobic interaction resulted in more pronounced retention. At temperature below the terpolymer phase transition, hydrophobic interactions predominated, and minimal changes in electrostatic interactions were supported by little shift in the apparent AAc carboxylate pKa values. Above the phase transition temperature, electrostatic interactions were dramatically reduced as a result of the decreased charge densities of the polymer grafted surfaces. Therefore, peptide retention times were also reduced, exhibiting a maximum at near 30-35 (degree)C. Interestingly, column retention behavior of angiotensins is dramatically modulated by applied step temperature gradients. Thermoresponsive surface property alteration is a very rapid, reversible phenomenon, allowing step temperature gradients on thermoresponsive columns to enable the analogous performance advantages as gradient elution in reversed-phase HPLC. More importantly, injected peptides were recovered completely from the columns from calculation of peak area. In conclusion, these anionic thermoresponsive polymer-modified surfaces are good candidates for improved separation of bioactive peptides under exclusively aqueous conditions.