Article pubs.acs.org/Biomac

Monolithic Silica Rods Grafted with Thermoresponsive Anionic Polymer Brushes for High-Speed Separation of Basic Biomolecules and Peptides Kenichi Nagase,† Jun Kobayashi,† Akihiko Kikuchi,‡ Yoshikatsu Akiyama,† Hideko Kanazawa,§ and Teruo Okano*,† †

Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, TWIns, 8-1 Kawadacho, Shinjuku, Tokyo 162-8666, Japan ‡ Department of Materials Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika, Tokyo 125-8585, Japan § Faculty of Pharmacy, Keio University, 1-5-30 Shibakoen, Minato, Tokyo 105-8512, Japan S Supporting Information *

ABSTRACT: Thermoresponsive anionic copolymer brushes, poly(N-isopropylacrylamide-co-acrylic acid-co-tert-butylacrylamide) [P(IPAAm-co-AAc-co-tBAAm)], were grafted onto a monolithic silica rod column through surface-initiated atom-transfer radical polymerization (ATRP) to prepare an effective thermoresponsive anionic chromatography matrix. An ATRP initiator was attached to the rod surface. N-Isopropylacrylamide (IPAAm), tert-butyl acrylate (tBA), tert-butylacrylamide (tBAAm), and the ATRP catalyst CuCl/CuCl2/tris[2-(N,N-dimethylamino)ethyl]amine were dissolved in 2-propanol, and the reaction mixture was pumped into the initiator-modified column. After grafting P(IPAAm-co-tBA-co-tBAAm) on the monolithic silica surfaces, deprotection of the tert-butyl group of tBA was performed. Chromatographic analysis showed that the prepared column was able to separate catecholamine derivatives and angiotensin subtypes within a shorter analysis time (5 min) than a silica-bead-packed column modified with the same copolymer brush could. These results indicated that the prepared copolymer-modified monolithic silica rod column may be a promising bioanalytical and bioseparation tool for rapid analysis of basic bioactive compounds and peptides.

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modulated by changing the external temperature.9 Confluently cultured cells on the substrate can be recovered as a “cell sheet” by simply reducing the temperature. Various types of cell sheets fabricated using thermoresponsive cell culture substrates have been used for tissue engineering and regenerative medicine. Recently, a temperature-responsive chromatography system has been investigated, using a PIPAAm-modified stationary phase, as a new type of analytical tool.2 The modification of a stationary phase by PIPAAm enables the surface hydrophobicity to be adjusted according to the column temperature, modulating the hydrophobic interactions between the sta-

INTRODUCTION Stimuli-responsive surfaces play an important role in the development of various bioapplications. These surfaces are mainly prepared by attaching functional groups or polymers to substrates; for example, temperature-responsive surfaces have been prepared by modifying substrates with poly(N-isopropylacrylamide) (PIPAAm). The thermoresponsive polymer exhibits a reversible phase transition at a lower critical solution temperature (LCST) of 32 °C.1 These PIPAAm-modified substrates show temperature-dependent hydrophilicity/hydrophobicity changes,2 and their properties have been used in various biomedical applications.3−8 Thermoresponsive cell culture substrates are one of the most successful applications of PIPAAm-grafted surfaces; the interaction between the surface of the culture substrate and cultured cells can be © 2014 American Chemical Society

Received: December 4, 2013 Revised: February 18, 2014 Published: February 19, 2014 1204

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Figure 1. (A) Scheme for preparation of P(IPAAm-co-AAc-co-tBAAm)-brush-grafted monolithic silica rod surfaces using surface-initiated ATRP. (B) Schematic illustration of thermally controlled interactions between anionic copolymer brushes and basic biomolecules.

interaction efficiency between the silica surface and analytes and higher permeability of the mobile phase than in conventional bead-packed columns.24 In our previous study, a PIPAAm-brush-modified monolithic silica rod column was prepared by modifying the rod surface with PIPAAm brushes through ATRP. The column separated a mixture of five hydrophobic steroids in a very short analysis time with high resolution.31 This result suggested that monolithic silica rods could be used as substitutes for silica beads as base materials for thermoresponsive-chromatography stationary phases. A monolithic silica rod grafted with brushes of a temperature-responsive cationic copolymer, P[IPAAm-co-2-(dimethylamino)ethyl methacrylate-co-N-tert-butylacrylamide (tBAAm)], has also been used to separate a mixture of three adenosine nucleotides in a very short analysis time.32 Monolithic silica rods modified with thermoresponsive anionic copolymer brushes would therefore be effective temperature-responsive cation-exchange chromatography matrices for the separation of basic biomolecules in a short analysis time. However, the preparation of densely packed anionic polymer brushes through surfaceinitiated ATRP remains challenging because the acidic monomers can deactivate the ATRP catalysts during polymerization.33 This article describes the grafting of temperature-responsive anionic copolymer brushes comprising poly(IPAAm-co-acrylic acid-co-tBAAm) [P(IPAAm-co-AAc-co-tBAAm)] on monolithic silica rod surfaces using surface-initiated ATRP. Acrylic acid (AAc) was used as the anionic comonomer because, as

tionary phase and bioactive analytes. This proposed temperature-responsive chromatographic system does not require any organic solvent as the mobile phase; this simultaneously preserves the biological activities of the analytes and reduces the environmental load. Several modification methods have been investigated for optimizing the performance of the PIPAAm-modified stationary phase.2 PIPAAm grafting on stationary phases by surface-initiated atom-transfer radical polymerization (ATRP) gives a high separation efficiency because of strong interactions between the grafted PIPAAm and analytes.10 These interactions result from the formation of densely packed polymer brushes on the surfaces,11−21 leading to a significant amount of PIPAAm on the stationary phases. A temperature-responsive ion-exchange chromatography system that uses a thermoresponsive ionic-copolymer-grafted stationary phase has also been investigated for analyzing ionic biomolecules.22,23 In this system, changes in the column temperature modulate the electrostatic interactions between the copolymer and the analytes because external temperature variations alter the acidities or basicities of these copolymers. The modified stationary phase, prepared through surfaceinitiated ATRP, shows a high separation efficiency, attributed to strong electrostatic interactions between the dense copolymer brushes and analytes.22,23 Monolithic silica rods have attracted attention as alternative chromatographic stationary phases to silica-bead-packed columns.24−30 A monolithic silica rod has an interconnected silica structure with large through-pores, leading to higher 1205

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IPAAm (9.48 g, 83.8 mmol, 65 mol %), tBAAm (3.28 g, 25.8 mmol, 20 mol %), and tBA (2.48 g, 19.3 mmol, 15 mol %) were dissolved in 2propanol (85.6 mL). The prepared monomer solution was deoxygenated by argon gas bubbling for 60 min. CuCl (168 mg, 1.70 mmol), CuCl2 (23.0 mg, 0.171 mmol), and Me6TREN (0.44 g, 1.91 mmol) were added to the monomer solution under an argon atmosphere and the reaction mixture was stirred for 20 min to generate the CuCl/CuCl2/Me6TREN catalyst system. The initiatormodified column and monomer solution were both placed in a glovebox. The reaction solution was pumped into the column using an HPLC pump (PU-980, JASCO) at a flow rate of 0.05 mL/min for 4 h. The P(IPAAm-co-tBA-co-tBAAm)-copolymer-modified monolithic silica rod column was rinsed with acetone, methanol, and water. Then the rinsed column was dried in a vacuum oven (DP33, Yamato, Tokyo, Japan) at 50 °C for 5 h. The tBA group was deprotected using 5% methanesulfonic acid solution in dichloromethane at a flow rate of 0.5 mL/min for 1 h (Figure 1A). The copolymer-brush-modified monolithic silica rod column was rinsed with dichloromethane and acetone. The rinsed column was dried in a vacuum oven at 50 °C for 5 h. For comparison, silica beads modified with the same copolymer were also prepared through surface-initiated ATRP, as previously reported.23 Briefly, IPAAm, tBA, and tBAAm (feed molar composition: 6.5:1.5:2) were dissolved in 2-propanol (42.8 mL) to give a total monomer concentration of 1.0 mol/L. The monomer solution was reacted with the prepared ATRP-initiator-modified silica beads in the presence of CuCl/CuCl2/Me6TREN as a catalyst, using a shaker (SNM40S, NISSIN, Tokyo, Japan), for 4 h at 25 °C. After polymerization, the copolymer-brush-grafted silica beads were rinsed with acetone, methanol, EDTA aqueous solution, and Milli-Q water. The rinsed beads were dried in a vacuum oven at 50 °C for 5 h. The tBA group was deprotected by immersing the copolymer-grafted beads in 5% methanesulfonic acid solution in dichloromethane at 25 °C for 1 h. The resulting copolymer-brush-grafted silica beads were rinsed with dichloromethane and acetone. The rinsed beads were dried in a vacuum oven at 50 °C for 5 h. Characterization of Initiator-Modified and CopolymerGrafted Silica Matrices. In order to determine the amounts of initiator and copolymer on the silica surfaces, the prepared monolithic silica rod and silica beads were analyzed using a PE 2400 series II CHNS/O analyzer (Perkin Elmer, Waltham, MA, U.S.A.). The amount of ATRP initiator on the matrix (g/m2) was calculated as follows:

previously reported, the pKa of AAc in IPAAm copolymers is ≈4.9 and the acidity of the IPAAm copolymer tends to decrease with increasing temperature at neutral pH.34 The hydrophobic tBAAm comonomer was used to suppress and balance the excessive hydrophilic properties of AAc, because it was thought to be more suitable for ATRP than other hydrophobic comonomers such as n-butyl methacrylate.35 The synthesized thermoresponsive anionic copolymer and the grafted monolithic silica rod were characterized using CHN elemental analysis, X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and gel permeation chromatography (GPC). The separation efficiency of the prepared monolithic silica rod column was investigated by observing the temperature-dependent elution profiles of catecholamine derivatives and angiotensin subtypes.

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EXPERIMENTAL SECTION

Materials. IPAAm was provided by Kohjin (Tokyo, Japan) and purified by recrystallization from n-hexane. tBAAm was purchased from Tokyo Chemical Industry (Tokyo, Japan) and purified by recrystallization from acetone. tert-Butyl acrylate (tBA), obtained from Wako Pure Chemical Industries Ltd. (Osaka, Japan), was purified by distillation at 51 °C and 58 mmHg. Tris(2-aminoethyl)amine (TREN) was obtained from Acros Organics (Pittsburgh, PA, U.S.A.). CuCl, CuCl 2 , NaOH, formic acid, formaldehyde, ethylenediamineN,N,N′,N′-tetraacetic acid disodium salt dehydrate (EDTA·2Na), hydrochloric acid, dehydrated toluene, dichloromethane, 2-propanol (HPLC grade), catecholamine derivatives, and steroids were obtained from Wako Pure Chemical Industries Ltd. Tris[2-(N,Ndimethylamino)ethyl]amine (Me6TREN), the ligand for the ATRP catalyst, was synthesized from TREN according to a previous report.36 The cellulose dialysis membrane [molecular weight cutoff (MWCO): 1000] was purchased from Spectrum Laboratories (Rancho Dominguez, CA, U.S.A.). Silica beads with an average diameter of 5 μm, a pore size of 300 Å, and a specific surface area of 100 m2/g were obtained from Chemco Scientific (Osaka, Japan). A monolithic silica rod column with a surface area of 71.8 m2/g and a mesopore diameter of 30 nm (MonoBis column, 50 mm × 3.2 mm i.d.) was obtained from Kyoto Monotech (Kyoto, Japan). A stainless-steel column (50 mm × 4.6 mm i.d.) was purchased from GL Science (Tokyo). The ATRP initiator, that is, [(chloromethyl)phenylethyl]trimethoxysilane (mixed meta and para isomers) was purchased from Gelest (Morrisville, PA, U.S.A.). Angiotensin peptides were obtained from Sigma (St. Louis, MO, U.S.A.). Initiator Modification of Monolithic Silica Rod Surfaces. A silane layer of ATRP initiator was first formed to modify the monolithic silica rod surface (Figure 1A). The unmodified monolithic silica rod column was placed in a container, where the relative humidity was ≈75%, for 18 h. The ATRP initiator solution was prepared by dissolving [(chloromethyl)phenylethyl]trimethoxysilane (6 mL) in dried toluene (14 mL). The initiator solution was circulated into the monolithic silica rod column using a pump (PU-980; JASCO) at flow rate of 0.1 mL/min for 16 h at room temperature. After the reaction, the reacted silica column was rinsed by flowing toluene and acetone using the same HPLC pump. The initiator-modified column was then dried in a vacuum oven at 110 °C for 2 h. ATRP-initiator-modified silica beads were also prepared using a previously reported silanization reaction.16 Briefly, silica beads (21.0 g) were placed in a 500 mL round-bottomed glass flask at a relative humidity of 60% for 4 h. After addition of ATRP initiator solution in toluene (53.4 mmol/L) to the flask, the reaction mixture was stirred for 16 h. The reacted silica beads were then rinsed with toluene and acetone. The ATRP-initiator-modified beads were obtained after drying at 110 °C for 2 h. Copolymer Modification of Monolithic Silica Rod Column by ATRP. Thermoresponsive anionic copolymer brushes comprising IPAAm, tBAAm, and AAc were prepared on the modified monolithic silica rod column through surface-initiated ATRP (Figure 1A). First,

immobilized ATRP initiator %C I = %C I(calcd) × (1 − %C I/%C I(calcd)) × S

(1)

where %CI is the weight percentage of carbon determined by elemental analysis, %CI (calcd) is the calculated weight percentage of carbon in the initiator, and S is the specific surface area of the matrix in square meters per gram. The specific surface area was found to be 71.8 m2/g by nitrogen adsorption for the monolithic silica rod and provided as 100 m2/g by the manufacturer for the silica beads. The amount of grafted copolymer on the monolithic silica rod or silica beads (g/m2) was calculated using the following equation:

grafted copolymer = %C p/{%C p(calcd) × [1 − %C p/% C p(calcd) − %C I/%C I(calcd)] × S}

(2)

where %Cp is the percentage carbon increase compared with the initiator-modified layers, determined by elemental analysis, and % Cp(calcd) is the calculated weight percentage of carbon in the copolymer. The grafted thermoresponsive copolymers were cleaved from the silica matrices by treatment with concentrated NaOH solution (10 mol/L) at room temperature overnight. The solution was neutralized with hydrochloric acid, and the copolymer was purified using a dialysis membrane (MWCO 1 kD) for 7 d against pure water. The copolymer was obtained by lyophilization.37 The number-average molecular 1206

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weights (Mn) and polydispersity indices (PDIs) of the copolymers were measured using a GPC system (GPC-8020 model II, Tosoh, Tokyo, Japan). Three GPC columns (TSKgel SuperAW2500, TSKgel SuperAW3000, and TSKgel SuperAW4000) were serially connected to the GPC system and calibrated using poly(ethylene glycol) standards. The column temperature was maintained at 45 °C using a column oven. N,N-Dimethylformamide (DMF) containing LiCl (50 mmol/L) was used as the mobile phase for GPC, with a flow rate of 1.0 mL/min. The elution profiles of the copolymers were monitored using a refractometer. The copolymer graft density on the monolithic silica rod and silica bead surfaces was estimated using the following equation:

graft density =

changed daily, and the purified copolymer was obtained by lyophilization. The tBA group was deprotected by immersing the copolymer in methanesulfonic acid solution in dichloromethane (5%) at 25 °C for 1 h with continuous stirring. The dichloromethane was evaporated and the methanesulfonic acid was removed by dialysis to produce the thermoresponsive anionic copolymer. Characterization of Anionic Copolymer P(IPAAm-co-AAc-cotBAAm). The molecular weight and PDI of the anionic copolymer, P(IPAAm-co-AAc-co-tBAAm), were determined using GPC. The measurements were performed prior to deprotection to avoid peak tailing, which is probably caused by interactions between carboxylic acid groups and the GPC column matrices (Figure S2). The temperature-dependent phase-transition profile of P(IPAAm-co-AAcco-tBAAm) was examined in PB solution by monitoring thermally induced optical transmittance changes. Copolymer solutions (10 mg/ mL) were prepared with 33.7 and 66.7 mmol/L PB solutions at pH 7.0. The pH value of the P(IPAAm-co-AAc-co-tBAAm) solution in 33.3 mmol/L PB was lower than 7.0 because of the relatively weak buffering capacity of PB and was adjusted to 7.0 by adding NaOH. Temperature-dependent optical transmittance changes were monitored at 600 nm using a UV/visible spectrometer (V-530, JASCO, Tokyo, Japan). The sample cuvette in the spectrometer was heated at a rate of 0.20 °C/min with a Peltier-effect cell holder (EHC-477, JASCO). The LCST of the copolymer was defined as the temperature at which the solution had a transmittance of 90%. The tBAAm content of the copolymer was determined by 1H NMR (UNITYINOVA 400 MHz spectrometer, Varian, Palo Alto, CA, U.S.A.), using DMF-d7 as the NMR solvent. The tBAAm content of the copolymer was estimated from the ratio between the peak area of the tert-butyl side chain methyl protons (δ = 1.4 ppm) to that of the IPAAm isopropyl methine proton (δ = 3.9 ppm). The AAc content of the copolymer was determined by acid−base titration. The copolymer (100 mg) was dissolved in 10 mL of Milli-Q water and the titration was performed at 4 °C with argon gas bubbling. The apparent dissociation constants, pK′a, of the copolymer in KCl solutions of various concentrations were determined using the Henderson−Hasselbalch equation:34,40

mp · NA Mn

(3)

where mp is the amount of grafted copolymer per square meter (g/ m2), NA is Avogadro’s number, and Mn is the number-average molecular weight of the grafted copolymer. The surface elemental compositions of the modified monolithic silica rod and silica beads were determined using XPS (K-Alpha, Thermo Fisher Scientific, Waltham, MA, U.S.A.). Excitation X-rays were produced by a monochromatic Al Kα1,2 source, with a takeoff angle of 90°. Wide scans were performed to analyze all existing elements on the monolithic silica rod and silica bead surfaces and highresolution narrow scan analysis was performed for peak deconvolution of carbon C1s signals. All binding energies were referenced to the C1s hydrocarbon peak at 285.0 eV. ATRP-initiator-bearing and copolymer-brush-grafted matrices were stained with osmium tetroxide and their surface morphologies were examined using SEM (S-4300, Hitachi, Tokyo, Japan). Temperature-Induced Wettability Changes of Thermoresponsive Anionic Copolymer. Glass beads grafted with thermoresponsive anionic copolymer brushes were prepared to evaluate the temperature-induced wettability changes of the brushes because the porosities of monolithic silica rods and silica beads make it difficult to measure contact angles. More specifically, ATRP-initiator-modified glass beads of diameter of 212−250 μm were prepared according to a previously reported procedure.38 The copolymer was then grafted onto the modified glass surfaces under the same polymerization conditions as were used for the silica beads by adding glass beads (20 g) to the reaction solution rather than silica beads. The thermoresponsive wettability of the P(IPAAm-co-AAc-co-tBAAm) brushes was evaluated as previously reported.39 Briefly, 5 μL of phosphate buffer (PB; 66.7 mmol/L, pH 7.0) or glycine buffer solution (66.7 mmol/L, pH 3.6) was deposited on a microscope slide (24.0 mm × 50 mm; Matsunami Glass, Osaka, Japan), and a small amount of modified glass beads was placed around the buffer solution. A microscope cover slide (24 mm × 24 mm; Matsunami Glass) was pressed over the solution, and then the glass beads were squeezed at the liquid−air boundary. The contact angles of the beads were measured using photographs captured with a phase-contrast microscope (ECLIPS TE2000-U, Nikon, Tokyo, Japan) equipped with a digital camera (FinePix, S2Pro, Nikon). Photographs of the copolymer-grafted glass beads are shown in Figure S1 (Supporting Information). The glass bead temperature was regulated within ±0.1 °C on the microscope slide using a microcool plate (Kitazato Supply, Shizuoka, Japan). Data are expressed as the mean of five measurements with a standard deviation. Synthesis of Thermoresponsive Anionic Copolymer by ATRP. For characterization of P(IPAAm-co-AAc-co-tBAAm), the copolymer was prepared by solution-phase ATRP, because the amounts of copolymer recovered from the silica rod and silica bead surfaces by treatment with NaOH solution were insufficient. The copolymerization was performed according to the procedure used for the grafted copolymer, with α-chloro-p-xylene (53.4 mg, 380 μmol) as the solution-phase ATRP initiator rather than the silica-supported initiator. After copolymerization, the solution was dialyzed against an EDTA solution for 3 d and then Milli-Q water for 2 d using a dialysis membrane (MWCO 1 kDa); the EDTA solution or water was

pK ′a = pH − log

α 1−α

(4)

where α is the degree of dissociation of carboxyl groups. The copolymer (100 mg) was dissolved in 6.67, 33.3, and 66.7 mmol/L KCl solutions. Half of the carboxyl groups (α = 0.5) in the copolymer were dissociated stoichiometrically by adding NaOH solution. According to eq 4, for α = 0.5 pK ′a = pH

(5)

The pH values of the thermoresponsive anionic copolymer solution therefore indicate the pK′a value of the copolymer. The pK′a value (pH of the solution) was monitored with a pH meter at various temperatures using a thermostatted bath. Temperature-Modulated Elution of Basic Biomolecules and Peptides. The copolymer-brush-modified silica beads were packed into a stainless-steel column (50 mm × 4.6 mm i.d., GL Science, Tokyo, Japan), as previously reported.10 Although two different types of columns with the same surface areas, column diameters, and column lengths would be more suitable for comparison of the performances of the packing materials, such similarity could not be achieved. The specific surface areas of the monolithic silica rod and silica beads were 71.8 and 100 m2/g, respectively, and the filling rates of these packing materials were different in each column. The column performances were therefore evaluated using two columns with the same lengths (50 mm) and similar diameters of 3.2 and 4.6 mm for the monolithic silica rod column and the silica-bead-packed column, respectively. Each column was connected to a high-performance liquid chromatography (HPLC) system (PU-980, JASCO) equipped with a UV-970 UV/ visible detector and controlled using Borwin analysis software (version 1.21, JASCO). Catecholamine derivatives, namely, DOPA, adrenaline, dopamine, and tyramine, were used as amino-group-containing model analytes to study the elution behaviors of the columns. All the sample 1207

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Table 1. Characterization of P(IPAAm-co-AAc-co-tBAAm) Anionic Copolymer LCSTd

IPAAm/AAc(tBA)/tBAAm (molar ratio) code

a

IPAtB IPA IPtB IP

b

c

in feed

in copolymer

Mn

65.0/15.0/20.0 85.0/15.0/0 80.0/0/20.0 100/0/0

64.4/16.6/19.1 82.0/18.0/0 78.8/0/21.2

6500 6450 6700 5400

Mw/Mnc

in 33.3 mmol/L PB

in 66.7 mmol/L PB

pKae

1.45 1.48 1.23 1.19

39.5 63.8 17.6 32.9

35.6 57.0 16.5 32.1

4.96 5.06

All samples were prepared through solution phase ATRP and named using abbreviated monomer names. “IP,” “A,” and “tB” represent IPAAm, AAc, and tBAAm, respectively. bDetermined by acid−base titration at 4 °C (n = 3) and 1H NMR measurement. cMeasured by GPC using a 50 mmol/L LiCl solution in DMF as a mobile phase. Mw/Mn and Mn were measured before deprotection for IPAtB and IPA copolymers to avoid peak tailing (Figure S2). dDefined as the temperature where the sample solution had a transmittance of 90%. eMeasured by acid−base titration at 4 °C.

a

solutions contained 2.7 mg/mL of Na2SO3 to prevent sample oxidation. The properties of the catecholamine derivatives are shown in Table S1. PB solution (33.3 mmol/L, pH 7.0) was used as the mobile phase, and the DOPA, adrenaline, dopamine, and tyramine concentrations were 0.20, 1.50, 2.50, and 3.50 mg/mL, respectively. Temperature-dependent elution profiles were monitored at 254 nm with a flow rate of 1.0 mL/min. The column temperature was controlled with a deviation of ±0.1 °C by immersing the column in a thermostatted water bath equipped with a constant-temperature water circulator (CTA400, Yamato, Tokyo, Japan). The elution behaviors of the basic peptides known as angiotensin subtypes I, II, and III were also investigated at concentrations of 1.33, 0.20, and 3.34 mg/mL, respectively. PB solution (66.7 mmol/L, pH 7.0) was used as the mobile phase. The temperature-dependent elution profiles of the angiotensin subtypes were monitored at 220 nm, with a flow rate of 1.0 mL/min. The angiotensin peptide properties are also shown in Table S2. Two thermostatted water baths were used to record the elution profiles with a step-temperature gradient. The first bath, which was equipped with a constant-temperature water circulator (CTA400), was set at 30 °C, and the second one (RE206, Lauda, Lauda-Königshofen, Germany) was set at 50 °C. First, angiotensins II and I were eluted at 30 °C by immersing the column in the first bath. The column temperature was then raised by immersion in the second water bath and the elution profile of angiotensin III was monitored at 50 °C. It was expected that in this procedure the column temperature would promptly equilibrate with that of the water bath because of the stainless-steel outer casing and the small diameter of the column. The hydrophobic properties of the grafted copolymer brushes on silica surfaces were also investigated by observing the elution behaviors of hydrophobic steroids. Hydrocortisone and dexamethasone were dissolved in ethanol and used to investigate the hydrophobic properties of the copolymer-grafted silica surfaces at concentrations of 0.385 mg/mL and 0.467 mg/mL, respectively. The properties of these hydrophobic steroids are shown in Table S1. The same two concentrations of PB, 33.3 and 66.7 mmol/L, were used as the mobile phases for observing the elution behaviors of hydrophobic steroids, because previous reports indicated that the phase-transition behaviors of PIPAAm and PIPAAm copolymers are influenced by the salt concentration in solution.41−43

over, the PDI values of the prepared copolymer were from 1.2 to 1.5. These results indicated that the ATRP in the procedure was more controlled than conventional radical polymerizations.44 Furthermore, the PDI values of IPtBAtB and IPtBA, which both contain tBA, were larger than those of IP and IPtB, because the ATRP catalyst, CuCl/CuCl2/Me6TREN, is normally used for acrylamide monomers such as IPAAm and tBAAm, not for tBA. IPAtB and IPA copolymers displayed higher LCST values than IP and ItB in PB (33.3 or 66.7 mmol/L) at pH 7.0 (Figure 2), suggesting that on incorporation of ionic monomers, the

Figure 2. Phase-transition profiles of P(IPAAm-co-AAc-co-tBAAm) in PB solution (pH = 7.0). Open and closed circles represent the IPAtB copolymer (IPAtB in Table 1) in 33.3 and 66.7 mmol/L PB, respectively. Open and closed triangles represent the IPA copolymer (IPA in Table 1) in 33.3 and 66.7 mmol/L PB, respectively. Open and closed diamonds represent the PIPAAm homopolymer (IP in Table 1) in 33.3 and 66.7 mmol/L PB, respectively. Open and closed squares represent the IPtB copolymer (IPtB in Table 1) in 33.3 and 66.7 mmol/L PB, respectively.

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LCST increased with increasing copolymer hydrophilicity.45 Moreover, AAc copolymers exhibited a slightly wider temperature range for transmittance changes because the incorporation of ionic AAc units disrupted the hydrophobic aggregation of the PIPAAm sequences. The observed LCSTs of P(IPAAm-co-AAc-co-tBAAm) were 35.6 and 39.5 °C in 66.7 and 33.3 mmol/L PB, respectively, which is suitable for bioseparation via thermoresponsive chromatography because pharmacological peptide and protein analytes tend to lose their activities at relatively high temperatures. In contrast, the P(IPAAm-co-AAc) copolymer had an excessively high LCST, which makes it unsuitable for thermoresponsive chromatography. The temperature-dependent apparent dissociation constants (pK′a) of the thermoresponsive copolymers were determined at

RESULTS AND DISCUSSION Characterization of Thermoresponsive Anionic Copolymer. P(IPAAm-co-AAc-co-tBAAm), a thermoresponsive anionic copolymer, was prepared by solution-phase ATRP. The temperature-dependent ionic and hydrophobic properties of the copolymer were determined by full characterization. The data are summarized in Table 1. The copolymers are shown using abbreviated monomer names, that is, “IP,” “tBA,” “A,” and “tB” correspond to IPAAm, tBA, AAc, and tBAAm, respectively. The molar fractions of tBA and tBAAm in the copolymers were slightly larger than their feed compositions. This was probably because of the higher reactivity ratios of these monomers than that of IPAAm in the CuCl/CuCl2/ Me6TREN-catalyzed ATRP procedure in 2-propanol. More1208

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Figure 3. Temperature-induced pKa shifts of P(IPAAm-co-AAc-co-tBAAm) carboxyl groups in (A) 6.66, (B) 33.3, and (C) 66.7 mmol/L KCl solutions. Open circles and closed triangles represent apparent pKa and transmittance of P(IPAAm-co-AAc-co-tBAAm) (IPAtB in Table 1), respectively.

Table 2. Elemental Analyses of P(IPAAm-co-AAc-co-tBAAm)-Brush-Grafted Silica Rod and Silica Beads Using XPS Take-Off Angle of 90° atom (%)c codea

C

IM IM-IPtBAtB IM-IPAtB IB IB-IPtBAtB IB-IPAtB calcd of IPtBAtBb calcd of IPAtBb

13.1 ± 1.61 50.9 ± 8.57 52.17 ± 2.34 18.2 ± 1.19 52.2 ± 9.05 55.0 ± 1.96 76.0 73.0

N 1.04 7.10 7.42 0.59 6.72 6.89 10.2 10.2

± ± ± ± ± ±

O

Si

55.6 ± 1.52 31.6 ± 9.47 28.1 ± 2.03 52.4 ± 1.09 31.49 ± 11.4 27.3 ± 2.21 13.8 16.8

0.49 1.12 1.07 0.71 1.75 0.66

29.2 8.93 11.3 27.0 8.09 9.75

± ± ± ± ± ±

Cl 1.09 2.01 1.09 0.52 0.78 0.71

1.14 1.52 0.98 1.75 1.46 1.10

± ± ± ± ± ±

N/C ratio 0.68 1.57 0.34 0.55 0.35 0.42

0.139 0.142 0.128 0.125 0.134 0.139

All samples were named using silica structure and grafted copolymer component. IM and IB represent “initiator-modified monolithic silica-rod” and “initiator-modified silica beads,” respectively. “IP,” “tBA,” “A,” and “tB” represent IPAAm, tBA, AAc, and tBAAm, respectively. bEstimated atomic composition of P(IPAAm-co-AAc-co-tBAAm). cData from three separate experiments are shown as mean ± SD.

a

Table 3. Characterization of P(IPAAm-co-AAc-co-tBAAm)-Brush-Grafted Monolithic Silica Rods and Silica Beads codea IM IM-IPtBAtB IM-IPAtB IB IB- IPtBAtB IB- IPAtB

elemental composition of carbonb (%)

immobilized initiatorc (μmol/m2)

± ± ± ± ± ±

3.74

2.82 16.2 14.2 4.75 17.1 15.5

0.15 0.05 0.31 0.08 0.01 0.07

grafted copolymerc (mg/m2)

Mnd

Mw/Mnd

graft density (chains/ nm2)

3.86 3.31

13800

1.48

0.169

2.62 2.32

12100

1.53

0.131

4.61

a All samples were named using silica structure and grafted copolymer component. IM and IB represent initiator-modified “monolith silica rods” and “silica beads,” respectively. “I,” “tBA,” “A” and “tB” represent IPAAm, tBA, AAc, and tBAAm, respectively. bDetermined by elemental analysis (n = 3). cEstimated from the carbon composition. dDetermined by GPC using a 50 mmol/L LiCl solution in DMF as a mobile phase.

various KCl concentrations (Figure 3) to study their thermoresponsive ionic properties. The copolymers showed greater change in their pK′a values above their LCSTs because their hydrophobicities increased with increasing temperature. Previous reports regarding polyelectrolytes indicated that the copolymerization of hydrophobic monomers to polyelectrolytes reduced their acidities or basicities.46,47 Above the LCST, the copolymer hydrophobicity significantly increased as a result of dehydration, which enhanced the local hydrophobicity in the vicinity of the AAc carboxyl groups. Moreover, aggregation of the dehydrated copolymer chains reduced the interface between the copolymer and the solution environment, augmenting their hydrophobicity. Consequently, the dissocia-

tion of the carboxyl groups in the copolymer could be modulated by temperature changes. The observed pK′a increment corresponded to the increase in KCl concentration in the copolymer solution because, at higher KCl concentrations, dehydration of the IPAAm copolymer increased as a result of the salting effect.41 Moreover, in the higher ion concentration region, intra- and intermolecular repulsive forces between AAc carboxyl groups were suppressed, enhancing aggregation between copolymers. Larger changes in the pK′a were therefore observed at higher KCl concentrations. Characterization of Copolymer-Modified Silica Surfaces. XPS measurements were performed to determine the surface elemental compositions of the prepared monolithic 1209

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Figure 4. (A-1, A-2) SEM images of P(IPAAm-co-AAc-co-tBAAm)-brush-grafted monolithic silica rod (IM-IPAtB in Table 2); (B-1, B-2) SEM images of unmodified monolithic silica rod; (C-1, C-2) SEM images of p(IPAAm-co-AAc-co-tBAAm)-brush-grafted silica beads (IB-IPAtB in Table 2); (D-1, D-2) SEM images of unmodified silica beads. Background patterns in (C-1, C-2) and (D-1, D-2) are double-sided tape used to affix the samples. Images numbered (A-2), (B-2), (C-2), and (D-2) are high-resolution photographs (×15000 magnification).

efficiency of the initiator.11 Moreover, the amount of copolymer grafted on IM-IPAtB was smaller than on that on IB-IPAtB because of differences between the reaction conditions. The copolymer chain lengths and graft densities on these silica surfaces were investigated by determining the Mn and PDI values of the grafted copolymer by GPC cleaving with NaOH solution.37 These data are also summarized in Table 3, and the GPC charts of the cleaved copolymer are shown in Figure S4. The grafted copolymer molecular weights were determined for cleaved IPtBAtB copolymers that contained protective groups because the GPC charts of the AAc copolymers exhibited peak tailing, attributed to interactions between the GPC column matrices and acidic copolymers (Figure S4). The cleaved copolymers displayed slightly larger PDIs than copolymers prepared in solution. This slightly larger PDI may be attributable to the porous structures of these silica base materials.10,37 The pore diameters of the silica base materials restricted copolymer propagation from the initiator inside the pores, whereas the copolymer propagated from the initiator outside the pores without restriction. Moreover, insufficient monomer supply inside the pores compared with the outer exposed surfaces limited the polymerization reaction. High copolymer graft densities were observed on these silica surfaces, indicating that the copolymer brushes formed densely packed layers on these surfaces. The surface morphologies of the monolithic silica rod and silica beads before and after grafting with P(IPAAm-co-AAc-cotBAAm) were investigated using SEM (Figure 4). SEM images of the ATRP-initiator-modified and P(IPAAm-co-tBA-cotBAAm)-copolymer-grafted monolithic silica rod and beads are shown in Figure S5. These images showed that the monolithic silica rod retained its macroporous structure, although a sufficient amount of copolymer was attached to the silica surfaces, suggesting large flow paths and low back pressure in the column. The effect of copolymer grafting on back pressure was investigated at various temperatures by flowing PB solution into the modified columns at a rate of 1.0 mL/min (Figure 5). The back pressure of the prepared columns increased with decreasing column temperature

silica rod and silica beads. The results are summarized in Table 2; samples are named according to the structure of the base materials and copolymer component. IM and IB represent initiator-modified “monolith” and “bead,” respectively. “I,” “tBA,” “AAc,” and “tB” represent IPAAm, tBA, AAc, and tBAAm, respectively. The chemical bonds of the base material surfaces were investigated by deconvolution of the C1s carbon peaks, performed according to a previous report;38 the results are shown in Figure S3. The XPS peaks of the ATRP-initiatormodified monolithic silica rod and silica bead surfaces (IM and IB) were different from those of their copolymer-grafted counterparts (IM-IPtBAtB, IM-IPAtB, IB-IPtBAtB, and IBIPAtB). Specifically, the spectra of the copolymer-grafted surfaces displayed an additional peak at 288 eV, which corresponds to the CO bonds of the copolymers (Figure S3B,C,E,F). Furthermore, the silicon contents decreased and the carbon and nitrogen contents increased after copolymer grafting because a copolymer brush layer covered the silica surfaces. These XPS measurements suggested that the copolymer successfully modified the monolithic silica and silica bead surfaces by surface-initiated ATRP. CHN elemental analyses were performed to measure the amounts of immobilized initiator and grafted copolymer on the monolithic silica rod and silica bead surfaces. Table 3 shows the carbon contents, estimated amounts of grafted copolymer, and grafted copolymer densities. Although higher concentrations of initiator were reacted with the monolithic silica rod than with silica beads, the carbon compositions showed that slightly fewer initiator molecules were immobilized on the rod than on the beads. This unexpected result may be the result of differences between the reactivities of the monolithic silica rod and silica bead surfaces, which are related to differences in the silicawashing step that precedes the silane-coupling reaction. Greater amounts of copolymer were grafted on the silica rod and silica bead surfaces using ATRP than were grafted on similar polymer-modified silica beads prepared by conventional radical polymerization.2,48 This was because of the grafting configuration, that is, densely packed copolymer brushes, formed by surface-initiated ATRP; this arises from the high 1210

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results indicate that copolymer grafting preserved the porous structure of the monolithic silica rod but not that of the silica beads. Thermoresponsive Wettability Changes of Anionic Copolymer Brush. To investigate the temperature-dependent changes in the wettability of P(IPAAm-co-AAc-co-tBAAm) brushes, copolymer-brush-grafted glass beads were prepared using the same protocol as for silica beads, and their contact angles were measured. Nitrogen microanalysis showed that the amount of copolymer grafted on the glass beads was 3.03 mg/ m2,38 indicating that the copolymer brushes formed on the glass surfaces were similar to those on the silica surfaces. The contact angles of the P(IPAAm-co-AAc-co-tBAAm)-brush-modified glass beads were measured using the method described in previous reports.39,53 Figure 6 shows the temperature-induced

Figure 5. Back pressure of prepared P(IPAAm-co-AAc-co-tBAAm)brush-grafted monolithic silica rod (IM-IPAtB in Table 2, open circles) and silica bead columns (IB-IPAtB in Table 2, open triangles) at various temperatures. Closed circles and triangles represent unmodified monolithic silica rod and bead columns, respectively.

because the hydrated and extended copolymer brushes on the silica surfaces increased the flow resistance of the mobile phase. The monolithic silica rod grafted with the thermoresponsive anionic copolymer had a low back pressure, similar to that of an existing monolithic column.49 These results indicate that the method for copolymer modification of monolithic silica surfaces described in this study gives clogging-free columns. Moreover, this relatively low back pressure allows the copolymer-brush-grafted monolithic silica column to be used under various conditions such as high mobile phase flow rates and low temperatures. The adsorption and desorption isotherms of the copolymerbrush-grafted monolithic silica rod and silica beads were measured using nitrogen gas, with a nitrogen adsorption measuring apparatus (BELSORP18PLUS-HT, BEL Japan, Osaka, Japan) to observe changes in the surface areas and pore diameter distributions on copolymer grafting.50−52 The nitrogen adsorption−desorption isotherms and pore size distributions are shown in Figures S6 and S7. Table 4

Figure 6. Effects of temperature on contact angles for P(IPAAm-coAAc-co-tBAAm)-brush-grafted glass beads. Open circles and closed triangles represent P(IPAAm-co-AAc-co-tBAAm)-brush-grafted glass beads in pH 7.0 PB (66.7 mmol/L) and pH 3.6 glycine buffer (66.7 mmol/L) solutions, respectively. Closed circles represent ATRPinitiator-modified glass beads in pH 7.0 phosphate buffer solution (66.7 mmol/L). Each data point represents average calculated from five separate experiments and is shown with its standard deviation.

Table 4. Characterization of P(IPAAm-co-AAc-co-tBAAm)Brush-Grafted Monolithic Silica Rod and Silica Beads by Nitrogen Adsorption−Desorption Measurements codea IM-IPAtB unmodified monolithic silicarod IB-IPAtB unmodified silica beads

surface areab (m2/g)

total pore volumec (cm3/g)

peak pore diameterc (nm)

75 77

0.24 0.19

59.0 51.1

65 95

0.18 0.42

16.0 28.3

changes in the contact angles of P(IPAAm-co-AAc-co-tBAAm) in neutral and acidic solutions. The contact angle (cosθ) of the copolymer-brush-grafted glass beads was higher than that of the ATRP-initiator-modified beads, indicating that grafting enhanced the surface hydrophilicity. In the neutral buffer solution (pH 7.0), the contact angles decreased with increasing temperature. These results indicated that the surface hydrophobicity increased with increasing temperature. The contact angles in acidic buffer solution (pH 3.6) were lower than those in the neutral buffer. This is attributed to suppression of dissociation of the copolymer carboxyl groups under acidic conditions. In the pH 3.6 buffer, the carboxyl groups scarcely dissociated because the pKa of the copolymer was ≈5.0, reducing the hydrophilicity of the copolymer brushes. Moreover, under acidic conditions, the temperature effects on the contact angle values were relatively small compared with those observed under neutral conditions. In the neutral buffer, the carboxyl groups dissociated readily at low temperature, leading to relatively strong hydrophilicity. As the temperature increased, this dissociation was suppressed because of increased local hydrophobicity in the vicinity of the carboxyl groups, leading to a larger hydrophilicity change in the copolymer brush than in PIPAAm. In contrast, in acidic solution, the carboxyl groups of the copolymer brush scarcely dissociated, regardless of the temperature, leading to poor thermoresponsive hydrophobicity.

a

All samples were named using silica structure, and the component of grafted copolymer. IM and IB represent initiator-modified “monolith silica rod” and “silica beads,” respectively. “I,” “A,” and “tB” represent IPAAm, AAc, and tBAAm, respectively. bCalculated using the Brunauer−Emmett−Teller (BET) method. cCalculated using the Barrett−Joyner−Halenda (BJH) method.

summarizes the surface areas, total pore volumes, and pore diameters of the copolymer-modified monolithic silica rod and silica beads. The surface area of the silica rod scarcely changed upon grafting with the IM-IPAtB copolymer, but the surface area of the silica beads decreased, indicating that the monolithic silica structure retained a larger surface area, facilitating analyte interactions after copolymer modification. The pore volume and diameter were unchanged by copolymer grafting for the monolithic silica rod, but decreased for the silica beads. These 1211

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These results indicated that P(IPAAm-co-AAc-co-tBAAm)brush-grafted surfaces exhibited temperature-dependent hydrophobicity under neutral conditions. A neutral buffer solution would therefore be suitable as a mobile phase for a P(IPAAmco-AAc-co-tBAAm)-brush-grafted stationary phase in chromatographic applications. Elution Profiles of Catecholamine Derivatives on Columns Grafted with Thermoresponsive Anionic Copolymer Brushes. The temperature-dependent elution profiles of catecholamine derivatives were investigated on monolithic silica rod and silica bead columns grafted with thermoresponsive anionic copolymer brushes. Figure 7A,B Figure 8. Temperature dependences of catecholamine retention times on (A) P(IPAAm-co-AAc-co-tBAAm)-brush-grafted monolithic silica rod column (IM-IPAtB in Table 2) and (B) P(IPAAm-co-AAc-cotBAAm)-brush-grafted silica-bead-packed column (IB-IPAtB in Table 2). Open circles, closed triangles, open diamonds, and closed squares represent DOPA, adrenaline, dopamine, and tyramine, respectively.

temperature. These results indicated that the surface hydrophobicity increased with increasing column temperature, as previously described for columns with ionic modified PIPAAm copolymers.23 Catecholamine separation was therefore mediated by electrostatic and hydrophobic interactions between the grafted copolymers and analytes. Moreover, although the same anionic copolymer was grafted on columns of the same column length (50 mm), the IM-IPAtB silica rod column separated the catecholamine derivatives in a significantly shorter time (5 min) than the IB-IPAtB bead-packed column did. This high-speed separation at high resolution can be attributed to structural differences between monolithic silica rod and silica bead columns. Previous reports indicated that mobile phases have higher linear velocities in monolithic silica rod columns than in bead-packed columns31 because of the lower flow resistance of the monolithic silica rod columns. The three-dimensionally interconnected structure of monolithic silica provides large through-pores in the rods, reducing their diffusion path length and flow resistance.24,25,27 The SEM images (Figure 4) also suggested that the fine-skeleton structure of the rod contributed to a more effective use of the surface area than in the case of silica beads, enhancing surface−analyte interactions. This structure allowed the grafted silica rod column to separate the catecholamine mixture in a very short separation time. Elution Profiles of Basic Peptides on Columns Grafted with Thermoresponsive Anionic Copolymer Brushes. The ability of the monolithic silica rod grafted with thermoresponsive anionic copolymer brushes to separate basic catecholamine derivatives with relatively small molecular weights quickly, through electrostatic and hydrophobic interactions, implies that this column could separate larger biomolecules such as peptides in short analysis times. Figure 9A,B shows the temperature-dependent elution profiles of angiotensin peptides on IM-IPAtB-grafted monolithic silica rod and silica-bead-packed columns, respectively, using PB (66.7 mmol/L, pH 7.0) as the mobile phase. Changes in the retention times of the angiotensin peptides at various temperatures are also shown in Figure 10A,B. The peptide mixture was separated more easily with increasing temperature. Angiotensin III, which was the most basic peptide, exhibited the longest retention time, indicating that the angiotensin peptides were retained on the columns through electrostatic interactions between their basic amino acid residues and the copolymer

Figure 7. Temperature-dependent elution profiles of catecholamine derivatives separated on HPLC columns using P(IPAAm-co-AAc-cotBAAm)-brush-grafted monolithic silica rod and silica beads as packing materials: (A) P(IPAAm-co-AAc-co-tBAAm)-brush-grafted monolithic silica rod column (IM-IPAtB in Table 2) and (B) P(IPAAm-co-AAcco-tBAAm)-brush-grafted silica bead column (IB-IPAtB in Table 2). The mobile phase was 33.3 mM phosphate buffer (pH 7.0). Peak Nos. 1, 2, 3, and 4 represent DOPA, adrenaline, dopamine, and tyramine, respectively. Because of its high-speed separation abilities, the time scale for the IM-IPAtB column is expanded compared with that for the IB-IPAtB column.

show the chromatograms of catecholamine derivatives at various temperatures on IM-IPAtB-grafted silica rod and bead columns, respectively, using PB (33.3 mmol/L, pH 7.0) as the mobile phase. The changes in retention times at various temperatures are shown in Figure 8A,B for these prepared columns. Mixtures of catecholamine derivatives were successfully separated at high temperature using the copolymermodified columns as chromatographic stationary phases. Although the zwitterionic DOPA was not retained on the columns, the other catecholamine derivatives were retained through electrostatic interactions between the anionic copolymer and the basic catecholamine groups. The retention times increased in the order DOPA, adrenaline, dopamine, and tyramine, in agreement with their hydrophobic properties,34 and the tyramine retention time increased with increasing column temperature. These results indicated that catecholamine retention also relied on hydrophobic interactions. To investigate the hydrophobic properties of the copolymer-brushmodified surfaces, the effects of temperature on the elution behaviors of hydrophobic steroids were examined (Figures S8 and S9). The hydrophobic steroids were separated at high temperature and their retention times changed as a function of 1212

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hydrophobic interactions between the grafted copolymer and angiotensin peptides increased with increasing column temperature. The optimal temperature for separation of these peptides was therefore observed near the copolymer LCST. The IMIPAtB silica rod column separated the peptides in a significantly shorter time (6 min) than the IB-IPAtB column did (25 min), suggesting that the monolithic silica rod provided a reduced diffusion path length for analytes and a higher mobile-phase linear velocity compared with the silica beads. The copolymerbrush-grafted monolithic silica rod column could therefore rapidly separate basic peptides. It was speculated that the acidic and hydrophobic properties of the grafted thermoresponsive anionic copolymer brushes would change rapidly with external temperature. Step-temperature gradients were therefore assumed to shorten the total analysis time using these columns in temperature-controlled chromatography at high resolution. The effects of the steptemperature gradient on angiotensin peptide elutions using IMIPAtB and IB-IPAtB columns are shown in Figure 11. The

Figure 9. Effects of temperature on elution profiles of angiotensins separated on HPLC columns using P(IPAAm-co-AAc-co-tBAAm)brush-grafted monolithic silica rod and silica beads as packing materials. (A) P(IPAAm-co-AAc-co-tBAAm)-brush-grafted monolithic silica rod column (IM-IPAtB in Table 2) and (B) P(IPAAm-co-AAcco-tBAAm)-brush-grafted silica bead column (IB-IPAtB in Table 2). The mobile phase was 66.7 mM phosphate buffer (pH 7.0). Peak Nos. 1, 2, and 3 represent angiotensin II, I, and III, respectively. Because of its high-speed separation abilities, the time scale for the IM-IPAtB column is expanded compared with that for the IB-IPAtB column.

Figure 11. Step-temperature gradient in angiotensin elution from (A) P(IPAAm-co-AAc-co-tBAAm)-brush-grafted monolithic silica rod column (IM-IPAtB in Table 2) and (B) P(IPAAm-co-AAc-cotBAAm)-brush-grafted silica-bead-packed column (IB-IPAtB in Table 2). The mobile phase was 66.7 mmol/L PB (pH 7.0). Peak Nos. 1, 2, and 3 represent angiotensin II, I, and III, respectively. Angiotensin chromatograms at 30 and 50 °C are shown for comparison. Because of its high-speed separation abilities, the time scale for the IM-IPAtB column is expanded compared with that for the IB-IPAtB column.

Figure 10. Temperature dependences of angiotensin retention times on (A) P(IPAAm-co-AAc-co-tBAAm)-brush-grafted monolithic silica rod column (IM-IPAtB in Table 2) and (B) P(IPAAm-co-AAc-cotBAAm)-brush-grafted silica bead column (IB-IPAtB in Table 2). Open circles, closed triangles, and closed diamonds represent angiotensin I, II, and III, respectively.

relatively weak basic and hydrophobic angiotensin I and II peptides were separated through relatively strong electrostatic and hydrophobic interactions at 30 °C. When the column temperature rose to 50 °C, the retention time of angiotensin III was shortened, with a narrower peak. On bead-packed columns, the step-temperature gradient shortened the retention time of angiotensin peptides to an analysis time of ≈20 min. In contrast, the modified monolithic silica rod column reduced the total analysis time (