Temperature-responsive molecular recognition chromatography using phenylalanine and tryptophan derived polymer modified silica beads

Abstract

Temperature-responsive polymers incorporating molecular-recognition sites were developed as stationary phases for high-performance liquid chromatography (HPLC). The grafted stationary phases consisted of functional copolymers composed of N-isopropylacrylamide (NIPAAm) and N-acryloyl aromatic amino acid methyl esters, i.e., phenylalanine and tryptophan methyl esters (Phe-OMe and Trp-OMe). Three novel temperature-responsive polymers, P(NIPAAm-co-Phe-OMe5), P(NIPAAm-co-Phe-OMe10), and P(NIPAAm-co-Trp-OMe5), were synthesized. These copolymers exhibited a reversible hydrophilic/hydrophobic phase transition at their lower critical solution temperatures (LCSTs). The polymers were grafted onto aminopropyl silica using an activated ester-amine coupling method, and were packed into a stainless steel column, which was connected to an HPLC system. Temperature-responsive chromatography was conducted using water as the sole mobile phase. More hydrophobic analytes were retained longer, and the retention times of aromatic steroids and aromatic amino acids were dramatically increased. This indicated that π-π interactions occurred between the phenyl or indole moieties of phenylalanine or tryptophan, respectively, and the aromatic compounds. Furthermore, the retention times of compounds with hydrogen bond acceptors were higher with P(NIPAAm-co-Trp-OMe5), which contained indole as a hydrogen bond donor, than with P(NIPAAm-co-Phe-OMe5). This indicated that hydrogen bonding occurred between the stationary phase and the analytes. These results indicate that hydrophobic, π-π, and hydrogen bonding interactions all affected the separation mode of the temperature-responsive chromatography, and led to selective separation with molecular recognition. Both temperature-response and molecular recognition characteristics are present in the proposed separation system that utilizes a temperature-responsive polymer bearing aromatic amino acid derivatives.

Analysis of Psychoactive Drugs by Temperature-Responsive Chromatography

“…Table 1 summarizes the polymer characterization data. The molecular weights and LCSTs determined in this study corresponded well with those reported previously [16]. These results indicated that the objective polymers were successfully synthesized.…”

“…For example, using n-butyl methacrylate (BMA) as a hydrophobic comonomer leads to increased hydrophobicity [8]. Recently, Hiruta et al reported that aromatic amino acids like L-phenylalanine and L-tryptophan were introduced to PNIPAAm as a comonomer and a new separation mode featuring molecular recognition was successfully introduced to the stationary phase, due to π-π interactions and hydrogen bonding [16].…”

“…We have been investigating temperature-responsive chromatography, in which a thermo-responsive polymer such as poly(N-isopropylacrylamide) (PNIPAAm) is used as the stationary phase [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16]. PNIPAAm exhibits thermally reversible solubility changes in aqueous solutions in response to temperature changes across its lower critical solution temperature (LCST).…”

Enhancement of Thermal Stability and Selectivity by Introducing Aminotriazine Comonomer to Poly(Octadecyl Acrylate)-Grafted Silica as Chromatography Matrix

“…The selectivity of all phase was increased with decrease of temperature. The selectivity of Sil-ODA 10 was enhanced below 40 • C, which was closed to the phase transition temperature, 38 • C (Figure 3). On the other hand, for Sil-ODA 10 -AT 1 and Sil-ODA 5 -AT 5 , the enhancement of their selectivities was observed in the higher range than 40 • C. This suggests due to higher phase transition temperature (44 and 46 • C, respectively, Figure 3) than Sil-ODA 10 .…”

“…Surface modification with a copolymer system is another important issue for the preparation of materials with tailor-made properties that are composed of two different monomers having diverse chemical and/or physical properties [4][5][6]. Inorganic carrier particles with polymeric/copolymeric phases on their surface have long been used as the stationary phase for HPLC [7][8][9][10][11] because such tailor-made surface properties directly influence the retention and separation efficiency. However, molecular ordering of copolymeric phases results in supramolecular functionality.…”