Temperature-Responsive Chromatographic Separation of Amino Acid Phenylthiohydantoins Using Aqueous Media as the Mobile Phase Hideko Kanazawa,* ,† Tastuo Sunamoto, † and Yoshikazu Matsushima † Kyoritsu College of Pharmacy, 1-5-30 Shiba-koen, Minato, Tokyo 105-8512, Japan Akihiko Kikuchi ‡ and Teruo Okano ‡ Institute of Biomedical Engineering, Tokyo Women's Medical University, 8-1 Kawadacho, Shinjuku, Tokyo 162-8666, Japan Recently, green chemistry has become one of the most important subjects of science for environmental pollution prevention. Here, we report development of a novel chromatographic technology for phenylthiohydantoin (PTH)-amino acid analyses in which only aqueous solu- tion is used as the mobile phase. We have devised HPLC adsorbents (stationary phase) by modifying the surfaces of microparticulate silica gel using functional polymers. The thermoresponsive copolymer, poly(N-isopropylacryl- amide-co-n-butyl methacrylate) (IBc) was used to modify the silica stationary phase surfaces. This polymer-grafted surface exhibits temperature-regulated hydrophilic/ hy- drophobic property changes in water. PTH-amino acid interactions with this surface are readily modulated by changing the column temperature using an isocratic aqueous mobile phase. Increasing hydrophobic interac- tions between more hydrophobic PTH-amino acids with hydrophobized polymer-grafted surfaces at elevated mo- bile phase temperatures is used for the effective separa- tion of PTH-amino acids in aqueous solution. This study is aimed at the development of novel separation pro- cesses, which are also environmentally benign, for use with biochemical substances in order to meet the growing needs of the life sciences and biotechnology. The method is useful for various separations in life science so that proteins can maintain their biological activity and en- zymes, their enzymatic activity. Polymers that respond to various applied stimuli are widely utilized for drug delivery systems, 1,2,3 cell culture substrates, 4,5 and bioconjugates. 6,7 The stimuli studied to date include changes in concentrations of chemical species and changes in temperature, pH, and electric field. Poly( N-isopropylacrylamide) (PIPAAm) is a well-investigated thermosensitive polymer. PIPAAm exhibits thermally reversible soluble-insoluble changes in aqueous solu- tion in response to temperature changes across a prominent lower critical solution temperature (LCST) at 32 °C. 8 Polymer chains of IPAAm hydrate to expand in water below the LCST, while dehydrating to form compact, insoluble conformations above the LCST. We have previously reported dramatic, reversible surface hydrophilic -hydrophobic property alterations for PIPAAm ter- minally grafted surfaces due to rapid changes in grafted polymer hydration near the polymer’s transition temperature. 9 Observed temperature-responsive surface property changes for these ter- minally grafted surfaces were rapid and significant, which suggests that PIPAAm graft-chain conformational freedom 10 facilitates polymer dehydration and hydrogen bonding with water molecules. We have recently developed a new chromatography system based on grafted PIPAAm. 11-15 Partitioning properties of the stationary- phase surface change in response to external temperature changes (hydrophilic -hydrophobic property). 11 Solute interactions with the PIPAAm-modified stationary phase are regulated by changing the temperature of the chromatography system. Amino acid sequence analysis of proteins has been widely used for the accumulation of protein primary sequence and structural data by applying protein samples to automated systems for Edman- based phenylisothiocyanate degradation. 16 Conventional analysis uses the reversed-phase columns in which mixtures of organic solvent and buffers are required as a mobile phase to analyze degraded amino acids. Such systems exhibit difficulties with reproducibility when using these organic solvent mixtures and gradient elutions. * To whom correspondence should be addressed. E-mail: kanazawa-hd@ kyoritsu-ph.ac.jp. † Phone: +81-3-5400-2657. Fax: +81-3-5400-1378. ‡ Phone: +81-3-3353-8111. Fax: +81-3-3359-6046. (1) Bae, Y. H.; Okano, T.; Kim, S. W. J. Polym. Sci. Polym. Phys. 1990 , 28, 923-936. (2) Yoshida, R.; Sakai, K.; Okano, T.; Sakurai, Y.; Bae, Y. H.; Kim, S. W. J. Biomater. Sci., Polym. Ed. 1991 , 3, 155-162. (3) Yoshida, R.; Sakai, K.; Okano, T.; Sakurai, Y. J. Biomater. Sci., Polym. 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