Preparation of thermo-responsive polymer brushes on hydrophilic polymeric beads by surface-initiated atom transfer radical polymerization for a highly resolutive separation of peptides

doi:10.1016/j.chroma.2010.07.067

Journal of Chromatography A

Volume 1217, Issue 38, 17 September 2010, Pages 5978-5985

https://doi.org/10.1016/j.chroma.2010.07.067 Get rights and content

Abstract

Poly(N-isopropylacrylamide-co-N-tert-butylacrylamide) [P(IPAAm-co-tBAAm)] brushes were prepared on poly(hydroxy methacrylate) (PHMA) [hydrolyzed poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate)] beads having large pores by surface-initiated atom transfer radical polymerization (ATRP) and applied to the stationary phases of thermo-responsive chromatography. Optimized amount of copolymer brushes grafted PHMA beads were able to separate peptides and proteins with narrow peaks and a high resolution. The beads were found to have a specific surface area of 43.0 m²/g by nitrogen gas adsorption method. Copolymer brush of P(IPAAm-co-tBAAm) grafted PHMA beads improved the stationary phase of thermo-responsive chromatography for the all-aqueous separation of peptides and proteins.

Introduction

Thermo-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm) has been widely applied to biomedical field such as controlled drug and gene delivery systems [1], [2], enzyme bioconjugates [3], [4], affinity precipitation [5], microfluidics [6], [7], cell culture substrates [8], [9], and tissue engineering for regenerative medicine [10], [11], [12], [13]. Furthermore, column separation method in all-aqueous system using PIPAAm grafted surfaces as stationary phases have been developed and became a powerful tool for biomedical separations [14], [15], [16], [17], [18], [19]. PIPAAm is well-known to exhibit thermo-responsive soluble–insoluble change across its lower critical solution temperature (LCST) at 32 °C in aqueous solution [20]. Its solubility change can be explained by the reversible hydration/dehydration property of its isopropyl side chains. The hydrating allows the polymer to expand its chains in water below the LCST and become hydrophilic, while the dehydrating allows the polymer to become compact and hydrophobic above the LCST. Thermo-responsive chromatography using PIPAAm grafted silica or polystyrene beads with all-aqueous mobile phase are highly useful for controlling both stationary phase function and properties for high performance liquid chromatography (HPLC) by changing only column temperature, with the advantages of preserving the biological activity of peptides and proteins and eliminating the use of organic mobile phases commonly used in reversed-phase chromatography for reducing environmental load.

For the preparation of PIPAAm grafted stationary phases, silica beads [14], [15], [16], [17] and polystyrene beads [18], [19] have been used as starting materials. Silica-based stationary phases are widely used in HPLC systems because of their good performance as chromatographic stationary phase. However, silica beads become instable under aqueous alkaline conditions (pH higher than 8.0) due to the hydrolysis of silica [21]. Therefore, thermo-responsive chromatography using polystyrene beads as starting materials have developed for improving stability under all-aqueous mobile phase [18], [19]. Using PIPAAm grafted polystyrene beads, the thermally induced all-aqueous separation of peptides were achieved [18], [19]. However, there are several issues in the use of polystyrene as a starting material for a PIPAAm grafted stationary phase. Since polystyrene has a strong hydrophobicity [22], [23], [24], peak tailing was observed at high temperature. In addition, the specific surface area of PIPAAm grafted polystyrene beads remarkably decreased after PIPAAm grafting, because the previously used beads has relatively small pore size (the average pore size: 250 Å) [18], [19]. This factor reduces the resolution of peptide separation.

For improving the resolution of peptide separation and the stability of the stationary phase, hydrophilic beads, poly(hydroxy methacrylate) (PHMA) [hydrolyzed poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate)] [25] having larger pore (600 Å) was chosen as a starting material. Additionally, polymer grafting on the beads was performed using surface-initiated atom transfer radical polymerization (ATRP). Surface-initiated ATRP is an attractive polymer grafting method allowing the surface of beads to receive well-defined polymer brushes by surface immobilized ATRP initiators [16], [26], [27], [28]. Control of PIPAAm graft amounts and densities on porous polystyrene beads by ATRP is found to be a key factor in facilitating the separation of hydrophobic peptides in aqueous mobile phase [18].

In the present study, P(IPAAm-co-N-tert-butylacrylamide) (tBAAm), a thermo-responsive copolymer with hydrophobic group, brushes were prepared on hydrophilic PHMA beads having large pores by surface-initiated ATRP and applied to the stationary phases of thermo-responsive chromatography.

Section snippets

Materials

N-Isopropylacrylamide (IPAAm) was kindly provided by Kohjin (Tokyo, Japan) and purified by recrystallization from n-hexane, followed by thorough drying in vacuo at 25 °C. N-tert-Butylacrylamide (tBAAm) was obtained from Wako Pure Chemicals (Osaka) and purified by recrystallization from acetone, followed by through drying in vacuo at 25 °C. [Hydrolyzed poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate)] (poly(hydroxy methacrylate)) beads (CQP30 of MCI GEL, the average diameter, 10 μm;

Elemental analyses

ATRP initiator-immobilized PHMA beads were characterized by Br elemental analysis. Amount of immobilized –OCOC(CH₃)₂Br moiety, an ATRP initiator, on PHMA beads were summarized in Table 1. Elemental analyses of CHN were performed on ATRP initiator-immobilized and P(IPAAm-co-tBAAm) brush-grafted PHMA beads. CHN compositions and the amount of grafted copolymer on PHMA beads were summarized in Table 1. The amount of grafted copolymer increased with increasing the initial monomer concentration of

Conclusions

P(IPAAm-co-tBAAm) brushes were prepared on hydrophilic PHMA bead having large pores by surface-initiated ATRP for improving thermo-responsive chromatography matrices. Using the proper amount of copolymer grafted PHMA beads, peptides and proteins were effectively separated with high-resolution. Possible factors for high resolutive analyses were the hydrophilicity of the starting material with large surface area where copolymer grafted bead. The prepared thermo-responsive copolymer grafted PHMA

Acknowledgments

Present research was supported in part by the Development of New Environmental Technology Using Nanotechnology Project of the National Institute of Environmental Science (NIES), commissioned from the Ministry of Environment, Japan, and Grants-in-Aid for Scientific Research (B) No. 20300169 from the Japan Society for the Promotion of Science. The authors are grateful to Dr. Norio Ueno, Tokyo Women's Medical University for his English editing.

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¹: Present address: RIKEN (The Institute of Physical and Chemical Research), 2-1, Hirosawa, Wako-shi, Saitama 351-0198, Japan.

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Preparation of thermo-responsive polymer brushes on hydrophilic polymeric beads by surface-initiated atom transfer radical polymerization for a highly resolutive separation of peptides

Abstract

Introduction

Section snippets

Materials

Elemental analyses

Conclusions

Acknowledgments

J. Control. Release

J. Chromatogr. B

Anal. Chim. Acta

J. Chromatogr. B

Anal. Chim. Acta

Biomaterials

J. Chromatogr. A

J. Chromatogr. B

J. Chromatogr.

J. Colloid Interface Sci.

J. Chromatogr.

Acta Biomater.

Polymer

J. Chromatogr. A

J. Chromatogr. A

J. Chromatogr. A

J. Chromatogr. A

Colloids Surf. A

Colloids Surf. A

Bioconjugate Chem.

Langmuir