Sample pretreatment is important for biological analysis and for purification of a target material from impurities. Solid-phase extraction (SPE) is widely used for sample pretreatment, because of its usability, time shortening and economy of solvent compared to other methods. While SPE is also used to purify biomacromolecules, it causes a problem on the loss of their bioactivity because using organic solvent or high salt concentration solvent as eluent. Therefore, SPE using an eluent that doesn’t deactivate biomaterials is required to purify the bioactive molecules.

We have developed a all-aqueous chromatography utilizing a temperature-responsive polymer, poly(N-isopropylacrylamide) (PNIPAAm).  PNIPAAm exhibits reversible hydrophobic-hydrophilic change in the vicinity of its lower critical solution temperature (LCST).  Using this chromatographic system, bioseparation of synthetic pharmaceutical agents, peptide and proteins were well accomplished until now. In this study, we developed the novel SPE utilizing temperature-responsive polymer modified stationary phase, with only aqueous solution as eluent.

Silica sationary phases were coated with thermo-responsive hydrogels based on PNIPAAm introduced N, N-dimethylaminopropyl acrylamide as cationic monomer, and butyl methacrylate as hydrophobic monomer. The PNIPAAm-modified silica-gel was packed into cartridge devices.

Well temperature-dependent separation was achieved using only aqueous eluent on the temperature-responsive polymer based SPE cartridge. This SPE procedure is simple and not need to prepare complicated wash or eluents that are contained organic solvent.  Now, we focus on the separation of bioactive proteins without impairing their activities.