Preparation of Thermoresponsive Anionic Copolymer Brush Surfaces

Biomacromolecules 2010, 11, 215–223

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Preparation of Thermoresponsive Anionic Copolymer Brush Surfaces for Separating Basic Biomolecules Kenichi Nagase,† Jun Kobayashi,† Akihiko Kikuchi,‡ Yoshikatsu Akiyama,† Hideko Kanazawa,§ Masahiko Annaka,| 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, 2641 Yamazaki, Noda, Chiba 278-8510, Japan, Faculty of Pharmacy, Keio University, 1-5-30 Shibakoen, Minato, Tokyo 105-8512, Japan, and Department of Chemistry, Kyushu University, 6-10-1 Hakozaki, Higashiku, Fukuoka 812-8581, Japan Received September 21, 2009; Revised Manuscript Received October 28, 2009

Poly(N-isopropylacrylamide-co-acrylic acid-co-N-tert-butylacrylamide) (poly(IPAAm-co-AAc-co-tBAAm) brush grafted silica beads were prepared through a surface-initiated atom transfer radical polymerization (ATRP) with CuCl/CuCl2/Me6TREN catalytic system in 2-propanol at 25 °C for 4 h. The prepared beads were characterized by chromatographic analysis. Basic analytes, catecholamine derivatives, and angiotensin peptides could be separated by a short column length containing the beads because of its high densely grafted copolymer structure. Chromatograms for catecholamine derivatives were obtained with high resolution peaks due to their electrostatic and hydrophobic interactions to the densely grafted anionic copolymers on the beads. Effective separation of angiotensin peptides was performed near the lower critical solution temperature of copolymers, because the total electrostatic and hydrophobic interactions between the copolymer and the analytes become strong at the temperature. These results indicated that the copolymer brush grafted surfaces prepared by ATRP was an effective tool for separating basic biomolecules by modulating the electrostatic and hydrophobic interactions.

Introduction In a few decades, stimuli-responsive polymers, exhibiting dramatic property changes in response to external stimuli, have been developed and widely used in biomedical fields.1-10 One of the most attractive stimuli-responsive polymers is poly(Nisopropylacrylamide) (PIPAAm) and its derivatives.11,12 PIPAAm exhibits reversible temperature-dependent phase transition in aqueous solutions at its lower critical solution temperature (LCST) of 32 °C,13 and this intrinsic thermoresponsive property is widely used in biomedical applications, such as controlled drug and gene delivery systems,14-16 enzyme bioconjugates,17,18 microfluidics,19 cell culture substrates,20,21 and tissue engineering for regenerative medicine.22-26 Furthermore, temperature-responsive chromatography utilizing PIPAAm as a stationary phase has been developed for thermally induced separation of bioactive compounds in aqueous mobile phase without organic phase.27-29 For separating bioactive compounds by conventional chromatography, addition of organic solvents or large amount of salt were required to modulate the interaction between stationary phase and analytes. However, the use of organic solvents and large amount of salt may result in the loss of bioactivity and limit further applications of the separated biomolecules.28 On the contrary, the temperatureresponsive chromatography system is highly useful to control the properties of stationary phase for high performance liquid chromatography (HPLC) by only changing the column temperature. Additionally, this system requires no organic solvents as * To whom correspondence should be addressed. Phone: +81-3-53679945, ext. 6201. Fax: +81-3-3359-6046. E-mail: [email protected]. † Tokyo Women’s Medical University. ‡ Tokyo University of Science. § Keio University. | Kyushu University.

mobile phase for separation, preserves the biological activity of analytes, and minimizes the environmental loads. To improve the performance of PIPAAm grafted silica beads, the grafting method of PIPAAm on silica bead surfaces and the elution behavior of analytes from them were investigated.29-33 As a result, chromatographic matrices prepared by a surface-initiated atom transfer radical polymerization (ATRP) exhibit a strong interaction with analytes, because the polymerization procedure forms a densely packed polymer, called a polymer brush, on the surfaces. ATRP is an attractive polymer grafting method allowing to prepare a surface with well-defined polymer brushes by surface-immobilized ATRP initiator.34-36 The methodology can control the graft chain length by varying the duration of polymerization33 and the graft density by varying the concentration of ATRP initiator.37 We demonstrated that PIPAAm and a significant amount of PIPAAm brush-grafted silica beads exhibited a strong interaction with hydrophobic steroids and peptides.33 Additionally, we also prepared a high-density thermoresponsive cationic copolymer brush comprising P(IPAAm-co-2-(dimethylamino)ethylmethacrylate (DMAEMA)) on silica bead surfaces using the surface-initiated ATRP and used them as a temperature-responsive anion-exchange chromatography.38 The chromatography system exhibited a significant strong electrostatic interaction with acidic analytes through the densely grafted cationic copolymers on the silica bead surfaces, and their electrostatic interaction was modulated by temperature, because the basicity of the copolymer decreased by temperature alteration at neutral pH.38-40 From these results, anionic copolymer brush surfaces prepared surface-initiated ATRP would be a candidate as an effective chromatography stationary phase for the effective separation of basic bioactive compounds. However, the preparation of densely grafted anionic copolymer brush has a challenging theme, because acidic

10.1021/bm9010744 CCC: $40.75  2010 American Chemical Society Published on Web 11/17/2009

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Figure 1. Scheme of the preparation of P(IPAAm-co-AAc-co-tBAAm) grafted silica bead surfaces using a surface-initiated atom transfer radical polymerization (ATRP).

monomers used in the reaction can deactivate ATRP catalysts by coordinating to the transition metal, and nitrogen containing ligands protonated, which interfere the metal complexation ability.41 Thus, to our best knowledge, no temperatureresponsive anionic polymer brush surfaces as chromatographic stationary phase was founded. In this study, we describe the preparation of high-density thermo-responsive anionic copolymer brush comprising P(IPAAm-co-acrylic acid (AAc)-co-tert-butyacrylamide (tBAAm)) on silica bead surfaces using a surface-initiated ATRP. Characterization of the dense cationic copolymer brush surfaces on silica beads was also investigated by chromatographic analysis using catecholamine derivatives and anigotensin peptides as model basic analytes.

Experimental Section Materials. N-Isopropylacrylamide (IPAAm) was kindly provided by Kohjin (Tokyo, Japan) and recrystallized from n-hexane. N-tertButylacrylamide (tBAAm) was obtained from Wako Pure Chemicals Industries (Osaka) and recrystallized from acetone. tert-Butylacrylate (tBA), purchased from Tokyo Kasei Kogyo (Tokyo), was purified by distillation at 51 °C (58 mmHg). CuCl and CuCl2 were purchased from Wako Pure Chemicals. Tris(2-aminoethyl)amine (TREN) was purchased from Acros Organics (Pittsburgh, PA). Formaldehyde, formic acid, and sodium hydroxide were purchased from Wako Pure Chemicals. Tris(2(N,N-dimethylamino)ethyl)amine (Me6TREN) was synthesized from TREN, according to the previous reports.42 Silica beads (the average diameter, 5 µm; the pore size, 300 Å; the specific surface area, 100 m2/g) were purchased from Chemco Scientific (Osaka). Hydrochloric acid, methanesulfonic acid, hydrofluoric acid, and ethylenediamineN,N,N′,N′-tetraacetic acid disodium salt dehydrate (EDTA · 2Na) were purchased from Wako Pure Chemicals. 2-(m/p-Chloromethylphenyl)ethyltrichlorosilane was obtained from ShinEtsu Chemical Industry (Tokyo). 2-Propanol (HPLC grade), dichloromethane, and toluene

(dehydrate) were purchased from Wako Pure Chemicals. Catecholamines were purchased from Wako Pure Chemicals, and angiotensin peptides were purchased from Sigma Chemicals (St. Louis, MO). Water used in this study was Milli-Q water prepared by an ultrapure water purification system, synthesis A10, Millipore (Billerica, MA)) unless otherwise mentioned. Preparation of ATRP Initiator Immobilized Silica Beads. 2(m/p-Chloromethylphenyl) ethyltrichlorosilane as an ATRP-initiator modified silica were prepared as shown in the first step in Figure 1, according to the previous reports.34,37 First, silica beads were washed with concentrated hydrochloric acid for 3 h at 90 °C, then rinsed with a large amount of distilled water repeatedly until the washing water pH became neutral, followed by thorough drying in a vacuum oven at 110 °C for 18 h. Formation of silane layers comprising the ATRP initiator on silica surfaces was performed as follows: silica beads (15.1 g) were placed into a round-bottom flask and humidified at 60% relative humidity for 4.0 h, followed by the addition of 3.53 mL of 2-(m/pchloromethylphenyl)ethyltrichlorosilane in 302 mL of dried toluene. Nitrogen gas was flowed over the reaction mixture for the first 5 min as HCl gas evolved and then sealed. The reaction proceeded at room temperature for overnight with continuous stirring. ATRP initiatorimmobilized silica beads were collected by filtration and extensively rinsed with toluene, methanol, dichloromethane, and acetone, and dried in a vacuum oven at 110 °C. Surface Modification of Silica Beads with Anionic Copolymer by ATRP. Anionic copolymer brushes composed of IPAAm, AAc, and tBAAm were prepared on the ATRP-initiator immobilized silica beads by ATRP, as shown in the second step in Figure 1. First, the copolymer brushes composed of IPAAm, tBA, and tBAAm were prepared by the surface initiated ATRP from silica. Typical preparation procedure was as follows: the total monomer concentration was set at 1 mol/L with the following monomer composition in feed: IPAAm (4.28 g, 37.8 mmol), tBA (0.11 g, 0.86 mmol), and tBAAm (0.55 g, 4.3 mmol; the monomer composition: tBA 2 mol % and tBAAm 10 mol %) were dissolved in 42.8 mL of 2-propanol, and deoxygenated by nitrogen gas bubbling for 30 min. The feed composition of tBA

Thermoresponsive Anionic Copolymer Brush Surfaces


(anionic monomer with protective group) was changed to 2, 5, or 10 mol %. Hydrophobic monomer, tBAAm, composition in feed was set at 10% regardless of tBA composition. CuCl (84.7 mg, 0.86 mmol), CuCl2 (11.5 mg, 0.086 mmol), and Me6TREN (0.22 g, 0.959 mmol) were added under a nitrogen atmosphere, and the solution was stirred for 20 min to form CuCl/CuCl2/Me6TREN catalyst system. ATRP initiator-immobilized silica beads (1.0 g) were placed into a clean dry 50 mL glass vessel. Both monomer solution and the silica beads were placed into a glovebag purged with dry nitrogen gas by repeated vacuum and nitrogen flush (three times). The monomer solution was then poured into the glass vessel containing the silica beads and sealed under nitrogen. The ATRP reaction proceeded for 4 h at 25 °C under continuous shaking on a shaker (SN-M40S; Nissin, Tokyo). Copolymergrafted silica beads were washed by ultrasonication in acetone for 30 min followed by centrifugation to remove unreacted monomers and ungrafted copolymers. This washing process by ultrasonication was repeated twice. Copolymer-grafted silica beads were further washed by sequential centrifugation and resuspension in methanol, 50 mM EDTA solution, and finally with Milli-Q water. Modified silica beads were filtered and rinsed with Milli-Q water and acetone and dried in a high vacuum oven at 50 °C for 5 h. After drying, the deprotection of tert-butylacrylate to acrylic acid was performed by immersing the silica beads into 5% methanesulfonic acid in dichloromethane at 25 °C for 1 h. The anionic copolymer brush grafted silica beads were filtered and rinsed with dichloromethane and acetone and dried in a high vacuum oven at 50 °C for 5 h. Characterization of Initiator Immobilized Silica and Grafted Copolymer. To determine the amount of ATRP-initiator and grafted copolymer on the silica beads, the silica beads were subject to elemental analysis using a CHN elemental analyzer VarioEL (Elementar, Hanau, Germany). ATRP-initiator and copolymer (milligrams per square meter) on silica beads was calculated by the following equations:

ATRP-initiator )

%CI %CI(calcd) × (1 - %CI /%CI(calcd)) × S (1)

grafted copolymer ) %CC %CC(calcd) × (1 - %CC /%CC(calcd) - %CI /%CI(calcd)) × S (2) where %C is the percent carbon increase as determined by elemental analysis, %C(calcd) is the calculated weight percent of carbon in initiator or copolymers, S is the specific surface area of the silica beads in square meters per gram (the manufacture’s data: 100 m2/g), and the subscripts I and C denote initiator and copolymer, respectively. Grafted copolymer on the silica bead surfaces was also retrieved and analyzed by gel permeation chromatography (GPC) for determining both the molecular weight and the polydispersity index (PDI). Copolymer grafted silica bead surfaces were treated with concentrated hydrofluoric acid for 3 h, and the solution was neutralized by the addition of sodium carbonate. The solution was filtered and dialyzed against Milli-Q water using a dialysis membrane (Spectra/Por standard regenerated cellulose dialysis membrane, molecular weight cut off (MWCO): 1000; Spectrum Laboratories, Rancho Dominguez, CA) for 3 days with daily water changed, and the copolymer was recovered by freeze-drying. Number-average molecular weights and PDI values of the polymer were determined using a GPC system (the columns: TSKgel G3000H and TSKgel G4000H; Tosoh, Tokyo) controlled with an SC8020 software. A calibration curve was obtained using poly(ethylene glycol) standards. The flow rate was 1.0 mL/min. The mobile phase was N,N-dimethylformamide (DMF) containing 100 mmol/L LiCl, and the column temperature was controlled at 45 °C using a column oven (CO-8020; Tosoh), and the elution profiles were monitored by a

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refractometer (RI-8022; Tosoh). Graft density of the coplymer on the silica bead surfaces was estimated using the following equation:

graft density )

mC·NA Mn

(3)

where mc is the amount of grafted copolymer on the silica bead surfaces per square meter (g/m2), NA is Avogadro’s number, and Mn is the number average molecular weight of the grafted copolymer. Synthesis of Anionic Copolymer by ATRP. To characterize the anionic copolymer, P(IPAAm-co-AAc-co-tBAAm) products with various feed ratios (the monomer composition in feed: tBA 2, 5, or 10 mol % with tBAAm 10%) were synthesized by solution-phase ATRP in the similar conditions as the copolymer grafting onto silica bead surfaces. Copolymerization was performed by the same protocol as grafting copolymer onto silica, except that R-chloro-p-xylene (53.4 mg, 380 µmol) was added as an initiator in the reaction solution instead of silica beads. After the copolymerization, the solution was dialyzed against EDTA solution using the dialysis membrane for 3 days with changing EDTA solution every day, followed by dialysis against Milli-Q water for 2 days, and the copolymer was obtained by lyophilization. Deprotection of tert-butyl acrylate to acrylic acid was performed by immersing the silica beads into 5% methanesulfonic acid in dichloromethane at 25 °C for 1 h. The anionic copolymer was obtained by reprecipitation through dropping the solution including anionic copolymer into diethylether, and the precipitate was dried in vacuum at 25 °C for 3 h. The obtained copolymer was dialyzed and lyophilized for removing methanesulfonic acid. Characterization of Anionic Copolymer. Prepared copolymer, P(IPAAm-co-tBA-co-tBAAm), was analyzed by the GPC system to determine both the molecular weight and the PDI. Phase transitions of the anionic copolymer solutions in pure water were observed by optical transmittance changes. Solutions of P(IPAAm-co-AAc-co-tBAAm) containing various amounts of AAc were prepared using 66.7 mmol/L phosphate buffer at pH 7.0 (10 mg/mL). Optical transmittance changes of the copolymer solutions were monitored at 600 nm by a UV/visible spectrometer (V-530; JASCO, Tokyo). The sample cuvette was thermostatted with a Peltier-effect cell holder (EHC-477; JASCO) with a heating rate of 0.10 °C/min. The LCST was defined as the temperature at 90% transmittance of solution. tBAAm content in the copolymers was determined by 1H NMR (UNITYINOVA 400 MHz spectrometer; Varian, CA) using chloroform-d containing 0.03 v/v % tetramethylsilane as a solvent. Mole fraction of tBAAm in the copolymer was calculated from the peak areal ratio of the singlet of methyl protons of tert-butyl side chains at 1.3 ppm and the singlet of the resonance of the methine proton of isopropyl groups in IPAAm units at 4.0 ppm. AAc content in the copolymer was determined by acid-base titration in water at 4 °C with N2 gas bubbling.43 Apparent dissociation constants pK′a of the copolymers in a 66.7 mmol/L KCl solution were determined by titration using the following Henderson-Hasselbalch equation.44

pKa ) pH - log

R 1-R

(4)

where R is the degree of dissociation for carboxyl groups. Experimental details of pK′a measurement procedure was as follows: Copolymer (100 mg) was dissolved in 20 mL of distilled water containing 66.7 mmol/L KCl. A half of carboxyl groups in copolymer were dissociated stoichiometrically by adding 0.05 mmol/L NaOH aq, resulting in R of 0.5. According to eq 4, the relationship between pH and pK′a at R ) 0.5 represents

pKa ) pH

(5)

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Nagase et al. Table 3. Characterization of P(IPAAm-co-AAc-co-tBAAm) Anionic Copolymer IPAAm/tBA/tBAAm (molar ratio) codea

in feed

in copolymerb

Mnc

IAB-3.1 IAB-7.7 IAB-11.1 IP IPB

88.0/2.0/10.0 85.0/5.0/10.0 80.0/10.0/10.0 100/0/0 100/0/10.0

83.1/3.09/13.8 79.9/7.73/12.4 76.4/11.1/12.5

9500 9200 8100 8200 7700

Mw/Mnc LCSTd 1.31 1.37 1.33 1.13 1.14

26.4 30.0 35.4 30.7 22.4

a Abbreviated as IAB-x, where x represents the mole fraction of AAc in the copolymer. b Determined by acid-base titration (n ) 3) and 1H NMR measurement. c Measured by GPC using DMF containing 50 mmol/L LiCl with PEG standards. Mw/Mn and Mn of IAB copolymer were measured before the deprotection for avoiding the peak tailing (see Figure S.1). d Defined as the temperature at 90% transmittance in 66.7 mmol/L phosphate buffer solution (pH 7.0).

Figure 2. Chemical structure of catecholamine derivatives. Table 1. Properties of Catecholamine Derivatives analyte

molecular weight

pKa

log Pa

DOPA adrenalin dopamin tyramine

197.2 183.2 153.2 137.2

8.72 9.8-9.9 10.6 10.4

-2.740 -0.685 0.019 0.616

a

Partition coefficient of n-octanol/water system.43,45

Table 2. Amino Acid Sequences and Properties of Angiotensin Peptides analyte (abbreviation)

amino acid sequence

pI

C log Pa

Asp-Arg-Val-Tyr-Ile-His-Pro-Phe- 7.7 -3.34 His-Leu angiotensin II (AII) Asp-Arg-Val-Tyr-Ile-His-Pro-Phe 7.75 -4.71 angiotensin III (AIII) Arg-Val-Tyr-Ile-His-Pro-Phe 9.85 -1.13

angiotensin I (AI)

a

C log P values were calculated by Mac LogP, version 4.0, for the Macintosh (Biobyte Corp.).46

pH values of the copolymer solution as pK′a was measured by pH meter with vigorous stirring and at various temperatures. Temperature Modulated Elution of Bioactive Compounds. P(IPAAm-co-AAc-co-tBAAm) grafted silica beads were packed into a stainless steel column (4.6 mm i.d. × 50 mm). A slurry of copolymergrafted silica beads in water/methanol mixed solvents (1:1) was poured into a slurry reservoir (TOSOH, Tokyo) connected to a stainless steel column. Water/methanol mixed solvent (1:1) was flowed through the slurry reservoir using an HPLC pump (PU-980; JASCO) at 350 kg/ cm2 for 1 h, followed by equilibration with Milli-Q water for at least 12 h. Copolymer-grafted bead-packed columns were connected to an HPLC system (PU-980 and UV-970; JASCO) controlled by a personal computer with a Borwin analysis software version 1.21 (JASCO). Four model analytes having amino groups, catecholamine derivatives (D,LDOPA, adrenaline, dopamine hydrochloride, and tyramine), were used for obtaining chromatograms at a concentrations of 1.0 mg/mL with 2.7 mg/mL Na2SO3 for preventing sample oxidization. Chemical structure and properties of these analytes were shown in Figure 2 and Table 1, respectively. PBS (66.7 mM, pH 7.0) was used as a mobile phase. Thermoresponsive elution behavior for catecholamine derivatives was monitored at 254 nm with a flow rate of 1.0 mL/min. Column temperature was controlled with a deviation of (0.1 °C using a thermostatted water bath (RE206; Lauda, Lauda-Ko¨nigshofen). Basic bioactive peptides, angiotensin subtypes I, II, and III, were also used to evaluate the property of prepared surfaces. Amino acid sequence and properties of these peptides were shown in Table 2. Each peptides were dissolved with PBS at a concentration of 0.1 mg/mL. PBS (66.7 mmol/L, pH 7.0) was used as a mobile phase. Thermoresponsive elution behavior for angiotensin subtypes was monitored at 220 nm with a flow rate of 1.0 mL/min.

Figure 3. Phase transition profiles of P(IPAAm-co-AAc-co-tBAAm) in 66.7 mmol/L phosphate buffer solution (pH 7.0). The closed circles represent PIPAAm homopolymer; the closed diamonds, P(IPAAmco-tBAAm); the open circles, IAB-3.1; the open triangles, IAB-7.7; and the open diamonds, IAB-11.1.

Results and Discussion Characterization of Thermoresponsive Anionic Copolymer. Characteristics of anionic copolymers of P(IPAAm-co-AAcco-tBAAm) are summarized in Table 3. Prepared copolymers are abbreviated as IAB-x, where x represents the mole fraction of AAc in the copolymer. Mole fraction of AAc and tBAAm in the copolymer was larger than the feed composition, which is probably due to the higher reactivity ratio of tBA and tBAAm compared to IPAAm in ATRP procedure using CuCl/CuCl2/ Me6TREN catalyst system with 2-propanol as the solvent. Polydispersity of prepared copolymers were approximately 1.3, indicating that the polymerization was controlled compared to a conventional radical polymerization. Furthermore, the polydispersity of the copolymers were relatively large compared to that of PIPAAm and P(IPAAm-co-tBAAm) copolymers, because the ATRP catalytic system CuCl/CuCl2/Me6TREN is ordinarily used for acrylamide derivatives, IPAAm and tBAAm, except for tBA.47,48 LCST values for the copolymers in PBS increased with the increase of AAc content in the copolymer (Figure 3). Incorporation of hydrophilic comonomer increases LCST due to increasing the hydrophilicity of the random copolymer.49 Additionally, the temperature range of the transmittance changes became wider with the increase of AAc contents, which is attributed to the disruption of the hydrophobic aggregation of PIPAAm sequences by the incorporation of ionic AAc units. However, the observed phase transition temperatures of the copolymers,


Figure 4. Carboxylate pK′a of P(IPAAm-co-AAc-co-tBAAm) in 66.7 mmol/L KCl solution as a function of temperature. The open circles represents IAB-3.1; the closed triangles, IAB-7.7; the closed diamonds, IAB-11.1.

below 40 °C, were suitable for temperature-responsive chromatography, because the controlling column temperature for the modulating interaction with analytes could be performed at a specific temperature region for avoiding possible deactivations of bioactive compounds. Temperature-dependent apparent dissociation constants (pK′a) of the copolymers were observed (Figure 4). Although the pK′a value is slightly increased below the LCSTs, a drastic change in the copolymer pK′a was observed above their LCST because of the increase of the copolymer’s hydrophobicity with an increase in the temperatures. Incorporation of hydrophobic comonomers into polyelectrolytes was reported to result in the decreased acidity or basicity of weak acid and the base moieities in the polyelectrolytes.50,51 Above the LCST of copolymers, their hydrophobicity dramatically increases due to the dehydration of them, leading to increasing the local hydrophobicity in the vicinity of carboxyl groups. Additionally, the intra- and inter aggregations of compact globules of dehydrated copolymer chains reduced the interface between the copolymer molecules and the solution environment. Thus, the dissociation of carboxyl group in the copolymer was modulated by changing temperature. Increment of pK′a value increased with the decrease of the acrylic acid ratio in the copolymer. This was probably due to the local hydrophobicity increase of the copolymers. As mentioned above, the hydrophobicity of copolymers increased with the decrease of the acrylic acid group composition due to the hydrophilic property of AAc. At the larger composition ratio of AAc in the copolymer, the most of carboxyl groups dissociate due to the weak hydrophobicity in the vicinity of carboxyl groups. On the other hand, at the smaller composition ratio of AAc, the copolymer is relatively hydropobic compared to that of the larger AAc composition copolymer. Additionally, the copolymers having the small AAc composition tended to aggregate and reduced the interface between the solution and the polymer chains, leading to the reduce of the apparent carboxyl groups in the solution. Thus, the high AAc composition ratio in the copolymer kept the anionic property of the copolymer with the changing temperature compared to the small AAc composition. Characterization of Initiator Surfaces and Copolymer Grafted Silica Beads. Initiator immobilized silica beads and copolymer-grafted silica beads were characterized by elemental analysis. Detected elements (C, H, and N) and the amounts of immobilized initiator are summarized in Table 4. Thermore-


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sponsive copolymer grafted silica bead surfaces via surface initiated ATRP was prepared. These surfaces are abbreviated as IAB-xB, where x represents the feed composition of AAc. B in xB denotes the “brush”. Amounts of grafted copolymer were greater than that of polymer hydrogel-modified silica beads prepared by the conventional radical polymerization we reported previously.46 This was due to the graft configuration of polymer brush prepared by the surface initiated ATRP. Polymer brushes prepared by the surface-initiated ATRP formed densely packed configurations compared to that by other radical polymerizations, because the initiation efficiency of ATRP was quite high. Thus, P(IPAAm-co-tBA/(AAc)-co-tBAAm) was densely grafted on silica bead surfaces, leading to the significantly large amount of grafted copolymer on these surfaces. To characterize the chain lengths and graft densities of copolymers on the silica surfaces, the molecular weight of grafted copolymer was determined by GPC after the chains was cleaved from silica beads with hydrofluoric acid. These data are also summarized in Table 4 (GPC charts of cleaved polymer are shown in Figure S.2). The polydispersity index of the cleaved copolymer was larger than that of copolymers prepared in solution. The larger polydispersity was suggested to be attributed to the porous geometry of silica beads.33,37,38 Polymerization reaction inside the pores was limited by the insufficient of monomer supply compared to outer exposed surfaces. In addition, the propagation of the polymer chains from the initiator inside the pores was also restricted to the pore diameter. These factors led to the large polydispersity of grafted copolymers on porous silica bead surfaces. Elution Behavior of Catecholamine Derivatives from P(IPAAm-co-AAc-co-tBAAm) Brush Surfaces. To investigate the interfacial electrostatic properties of the P(IPAAm-co-AAcco-tBAAm) brush surfaces, the elution behavior of the catecholamine derivatives from the copolymer grafted silica beads used as chromatographic stationary phases was investigated. Figure 5a-c shows the chromatograms of catecholamine derivatives at various temperatures on IAB-2B, IAB-5B, and IAB-10B bead-packed columns using PBS (66.7 mmol/L, pH 7.0) as a mobile phase, respectively. Figure 6a-c shows changes in the retention times with various temperatures on these columns. Four catecholamine derivatives were successfully separated by the prepared beads as a chromatographic stationary phase, although the column length is one-third of the previously reported anionic hydrogel modified column.43,45 This was attributed to the high holding capacity of the grafted structure of copolymer on silica bead surface. Preparation of polymer grafted surfaces made by surface-initiated ATRP gave the densely packed structure of polymer brush. The dense copolymer grafted structure led to strong interactions with analytes, due to the large amount of grafted copolymers. Catecholamine derivatives, except zwitterionic DOPA, were all retained on the prepared copolymer modified columns, and the retention time for catecholamine increased with the increase of the feed composition of AAc. These results indicated that the copolymer brush modified surfaces remained its anionic property, and basic catecholeamines were also separated with electrostatic interaction with the copolymer brush grafted on the silica beads. Additionally, the retention times increased in the following order: DOPA < adrenaline < dopamine < tyramine, which is agreed with the hydrophobicity (polarity) of analytes (Table 1),43 and the retention times of tyramine increased with the increase of the column temperature. These results suggested that the retention of catecholamine were also caused by the hydrophobic interaction. Thus, the phase transition of the grafted copolymer

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Table 4. Characterization of P(IPAAm-co-AAc-co-tBAAm) Grafted Silica Beads elemental composition (%)b

c

codea

C

H

N

initiator-immobilized silica IAB-2B IAB-5B IAB-10B

4.7 16.5 16.6 15.3

1.1 2.6 2.6 2.3

0.3 2.3 2.3 1.9

immobilized initiator (µmol/m2)

grafted polymer (mg/m2)

Mnc

Mw/Mnc

graft density (chains/nm2)

2.52 2.58 2.61

16300 13600 11100

2.45 3.19 4.03

0.093 0.114 0.141

4.56

a Abbreviated as IAB-xB, where x represents the feed composition of AAc. B in xB denotes the “brush”. b Determined by elemental analysis (n ) 2). Determined by GPC using DMF containing 100 mmol/L LiCl.

Figure 5. Chromatograms of catecholamines separated on HPLC of which packing materials was P(IPAAm-co-AAc-co-tBAAm) grafted silica beads at various temperatures: (a) IAB-2B, (b) IAB-5B, and (c) IAB-10B columns. Mobile phase is 66.7 mmol/L phosphate buffer solution (pH 7.0). The peaks 1, 2, 3, and 4 are due to DOPA, adrenaline, dopamine, and tyramine, respectively.

Figure 6. Temperature-dependent retention time changes of catecholamines on (a) IAB-2B, (b) IAB-5B, and (c) IAB-10B columns. The open circles represents DOPA; the closed triangles, adrenaline; the open squares, dopamine; the closed diamonds, tyramine.

was considered to be important for the analysis of the retention mechanism. Figure 7 shows the van’t Hoff plots of these catecholamines, which exhibit a relationship between the analyte retention and the column temperature. The retention factor k′ value was defined as k′ ) Rt/(Rt - R0); where Rt is the retention time for a known sample at predetermined temperature and R0 is the retention time of uracil as the initial standard. A linear relationship between ln k′ values and reciprocal temperature (1/T) is commonly observed in the van’t Hoff plots of commercially available reversed phase columns in a normal chromatographic process. However, as shown in Figure 7, the slope of the retention factor decreased above the LCSTs of copolymers, indicating that the interaction with analytes weak-

ened above the LCSTs. Our laboratory has previously reported that hydrophobic steroid retention with hydrophobic interaction was dramatically increased above the LCSTs of grafted PIPAAm.30 Thus, the decreased interaction above LCSTs indicated that the hydrophobic interaction is unlikely to be considered the predominant factor for the retention of chatecholamine. Additionally, the observed significant decrease in pK′a above the LCSTs supported the decreased interaction above the LCSTs. Electrostatic interaction between the grafted copolymer and the analytes decreased above the LCSTs due to the restricted dissociation of carboxyl groups with increase of hydrophobicity. These results suggested that the electrostatic interaction pre-



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Figure 7. The van’t Hoff plots of catecholamines on (a) IAB-2B, (b) IAB-5B, and (c) IAB-10B columns. The closed triangles represents adrenaline; the open squares, dopamine; the closed diamonds, tyramine.

Figure 8. Chromatograms of angiotensin peptides separated on HPLC of which packing materials are P(IPAAm-co-AAc-co-tBAAm) grafted silica beads at various temperatures: (a) IAB-2B, (b) IAB-5B, and (c) IAB-10B columns. Mobile phase is 66.7 mmol/L phosphate buffer solution (pH 7.0). The peaks 1, 2, and 3 are due to AII, AI, and AIII, respectively.

Figure 9. Temperature-dependent retention time changes of angiotensin peptides: (a) IAB-2B, (b) IAB-5B, and (c) IAB-10B columns. The open circles represent AI; the closed triangles, AII; the closed diamonds, AIII.

dominantly influenced the separation of catecholamines compared with hydrophobic interaction. Elution Behavior of Basic Peptides from P(IPAAm-co-AAcco-tBAAm) Brush Surfaces. The anionic copolymer brush columns separated basic catecholamines through a relatively

strong electrostatic interaction and a hydrophobic interaction. This implied that the anionic copolymer brush columns would also separate larger molecules such as peptides with strong interactions. Figure 8a, b, and c show the chromatograms of angiotensin peptides at various temperatures on IAB-2B, IAB-

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5B, and IAB-10B bead-packed columns using PBS (66.7 mmol/ L, pH 7.0) as a mobile phase, respectively. Figure 9a-c shows changes in retention times with temperatures on these columns. The increase of the composition of AAc in the copolymers improved the separation of all angiotensin subtypes. Especially the retention of angiotenisin III, which has the most basic property, was prolonged with the increase of AAc composition. These results indicated that the anigiotensin peptides retained with the electrostatic interaction between the carboxyl groups of the copolymer and the basic amino acid residues of angiotensin peptides. As shown in Figure 9, the retention times of angiotensin subtypes increased with the increase of temperature, indicating that a hydrophobic interaction between the analyte and the hydrophobic surface caused by the dehydration of the grafted copolymer brush also contributed to the retention of the angiotensin peptides. Additionally, the maximum points in the retention times of angiotensin III were observed at 30 °C on the IAB-10B column. The result would be explained by the two interactions, electrostatic and hydrophobic, which competed with increasing temperature. Above the copolymer LCSTs, the charge density of the grafted copolymer brush decreased with an increase of the hydrophobicity induced dehydration of copolymer, which leads to a weakened electrostatic interaction with the increase of temperature. On the contrary, the hydrophobic interaction with anigiotensin peptides increased with the increase of temperature, that is, the proceeding dehydration of the grafted copolymer brush. Thus, an optimal temperature of the separation of angiotensin peptides appeared near the copolymer’s LCST. Anionic copolymer brush grafted silica beads as a chromatographic stationary phase separate basic biomolecules, catecholamine derivatives, and angiotensin peptides by electrostatic and hydrophobic interactions between the analytes and the densely grafted copolymers. These interactions were varied with temperature due to the phase transition of grafted copolymers. Thus, the separation of basic compounds with anionic copolymer brushes can be facilitated by modulating the optimal column temperature, which is related to the LCST of copolymers.

Conclusions Anionic copolymer, P(IPAAm-co-AAc-co-tBAAm), was successfully grafted onto silica bead surfaces by grafting P(IPAAmco-tBA-co-tBAAm) using surface initiated ATRP and cleaving the tert-butyl group. The grafted amount of P(IPAAm-co-AAcco-tBAAm) was relatively large compared to the previously reported hydrogel grafted silica beads. Two kinds of basic analytes were separated on the copolymer grafted column. Chromatograms of catecholamine derivatives with a high resolution were obtained at a high temperature because the electrostatic interaction between the stationary phase and the analytes can predominantly contribute to the high resolution separation and the hydrophobic interaction gives a limited contribution only at high temperature. The longest retention time for angiotensin peptides was observed near the LCST of the grafted copolymer because of the two interactions, electrostatic and hydrophobic, which competed with increasing temperature. These results suggested that the prepared copolymer brush surfaces interacted with analytes both electrostatically and hydrophobically, and the modulation of column temperature allowed the surfaces to be an attractive separation tool for basic biomolecules. Acknowledgment. Part of the present research was financially supported by the Development of New Environmental Technol-

Nagase et al.

ogy Using Nanotechnology Project of the National Institute of Environmental Science (NIES), commissioned from the Ministry of Environment, Japan, Grants-in-Aid for Scientific Research (B) No. 19591568 from the Japan Society for the Promotion of Science, and Grants-in-Aid for Young Scientists (B) No. 20700399 from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. We would also like to thank Dr. Norio Ueno for the English editing. Supporting Information Available. GPC molecular weight chart for prepared copolymers in solution and cleaved copolymers from silica bead surfaces. This material is available free of charge via the Internet at http://pubs.acs.org.

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Preparation of Thermoresponsive Anionic Copolymer Brush Surfaces

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