ARTICLE pubs.acs.org/Langmuir
Thermoresponsive Polymer Brush on Monolithic-Silica-Rod for the High-Speed Separation of Bioactive Compounds 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, 2641 Yamazaki, Noda, Chiba 278-8510, Japan § Faculty of Pharmacy, Keio University, 1-5-30 Shibakoen, Minato, Tokyo 105-8512, Japan
bS Supporting Information ABSTRACT: Poly(N-isopropylacrylamide), one of the most utilized thermoresponsive polymers, brush-grafted monolithicsilica columns were prepared through surface-initiated atom transfer radical polymerization (ATRP) for effective thermoresponsive-chromatography matrices. ATRP initiator was grafted on monolithic silica-rod surfaces by flowing a toluene solution containing ATRP initiator into monolithic silica-rod columns. N-Isopropylacrylamide (IPAAm) monomer and CuCl/CuCl2/ Me6TREN, an ATRP catalytic system, were dissolved in 2-propanol, and the reaction solution was pumped into the preprepared initiator-modified columns at 25 °C for 16 h. The constructed PIPAAm-brush structure on the monolithic silica-rod surface was confirmed by XPS, elemental analysis, SEM observation, and GPC measurement of grafted PIPAAm. The prepared monolithic silica-rod columns were also characterized by chromatographic analysis. PIPAAm-brush-modified monolithic silica-rod columns were able to separate hydrophobic steroids with a short analysis time (10 min), compared to PIPAAm-brush-modified silica-beads-packed columns, because of the horizontally limited diffusion path length of monolithic supporting materials. Additionally, diluted PIPAAm-brush monolithic silica-rod column gave a further shorting analysis time (5 min). These results indicated (1) surface-initiated ATRP constructed PIPAAm-brush structures on monolithic silica-rod surfaces and (2) PIPAAm-brush grafted monolithic silica-rod column prepared by ATRP was a promising tool for analyzing hydrophobic-bioactive compounds with a short analysis time.
’ INTRODUCTION Stimuli-responsive polymers, exhibit dramatically property change in response to external stimuli, have been developed and widely used in biomedical fields.15 One of the most attractive stimuli-responsive polymer is poly(N-isopropylacrylamide) (PIPAAm) and its derivatives. PIPAAm exhibits a reversible temperature-dependent phase transition in aqueous solutions at its lower critical solution temperature (LCST) of 32 °C,6 and this intrinsic thermoresponsive property is widely used in biomedical applications, such as controlled drug and gene delivery systems,5,7,8 bioconjugates,2,9,10 sensors,11 microfluidics,12 cell culture substrates,13,14 and tissue engineering for regenerative medicine.1518 Furthermore, temperature-responsive chromatography utilizing PIPAAm as a stationary phase has been developed for the thermally induced separation of bioactive compounds in aqueous mobile phase without organic phase.1921 This chromatography system is highly useful to control the properties of stationary phase for high performance liquid chromatography (HPLC) by only changing the column temperature. Modified PIPAAm on stationary phase alter their hydrophobicity by r 2011 American Chemical Society
changing temperature, leading to the modulation of the hydrophobic interaction between PIPAAm and analytes. Additionally, this system requires no organic solvents as a 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.2224 As the results, chromatographic matrices prepared by 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.24,25 ATRP is an attractive polymer grafting method allowing surface to obtain well-defined polymer brushes by a surface-immobilized ATRP initiator.2631 The methodology can control the graft chain length by varying the duration of polymerization24 and the Received: April 13, 2011 Revised: July 9, 2011 Published: July 09, 2011 10830
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Langmuir graft density by varying the concentration of ATRP initiator.32 Especially, PIPAAm brushes prepared by ATRP have attracted attention as novel intelligent interfaces, and these intrinsic thermoresponsive properties were investigated by various methods.29,33 These reports provided that the brush thickness gradually decreased broad temperature range, probably due to the intrinsic dehydration properties of dense PIPAAm-brush. Also, our laboratory investigated PIPAAm-brush properties by chromatographic analysis. In the temperature-dependent elution profiles of hydrophobic steroids, the longer retention times of steroids were observed because of the strong hydrophobic interactions between enhanced dehydrated PIPAAm-brush and hydrophobic steroids. Retention time of steroids gradually increased broad at a temperature range of 1040 °C, which is consistent with the shrink properties in a previous report.33 On the contrary, dilute PIPAAm-brush was prepared by diluting ATRP-initiator density with reacting other silane agents as comodifier on silica beads surfaces, and followed by surface-initiated ATRP. The effects of PIPAAm-brush density on the thermoresponsive elution behavior of bioactive compounds were investigated.32,34 Especially, diluted PIPAAm-brushs on silica beads prepared by grafting relatively hydrophilic silane to the surface exhibit (1) a remarkable thermoresponsive hydrophilic/hydrophobic alteration of grafted PIPAAm-brush and (2) the relatively shorter retention time of analyte, probably attributed to the enhanced hydration of the grafted PIPAAm-brush. On the contrary, in the fields of analytical science, monolithic silica-rod columns have attracted attention as a new chromatographic support, an alternative to silica beads packed column.3539 Monolithic silica-rod column is a single piece of silica rod having a three-dimensionally interconnected skeleton structure, which provides through-pores to the column. The three-dimensional (3D) skeleton structure and large through-pores reduce the diffusion path length and flow resistance, leading to a higher column efficiency and column permeability, compared to conventional particle-packed columns.35 From these previous studies, a PIPAAm-brush-modified monolithic silica-rod column is thought to be a good candidate as an effective thermoresponsive chromatography stationary phase for the effective separation of bioactive compounds. In this study, we described the preparation of PIPAAm-brush on monolithic silica-rod surface using surface-initiated ATRP. Separation efficiency of the prepared column was investigated by observing the temperature-responsive elution profiles of hydrophobic steroids.
’ EXPERIMENTAL SECTION Materials. N-Isopropylacrylamide (IPAAm) was kindly provided by Kohjin (Tokyo, Japan) and recrystallized from n-hexane. CuCl and CuCl2 were purchased from Wako Pure Chemicals (Osaka). Tris(2-aminoethyl)amine (TREN) was purchased from Acros Organics (Pittsburgh, PA, U.S.A.). Formaldehyde, formic acid, and sodium hydroxide were purchased from Wako Pure Chemicals. Tris(2-(N,Ndimethylamino)ethyl)amine (Me6TREN) was synthesized from TREN, according to the previous reports.40 Monolithic silica column (MonoBis column, 50 3.2 mm i.d., the meso pore diameter: 30 nm, the specific surface area: 84 m2/g) was purchased from Kyoto Monotech (Kyoto). 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). Stainless steel column (50 4.6 mm i.d.) was obtained from GL Science (Tokyo). Hydrochloric acid, hydrofluoric acid, and
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ethylenediamine-N,N,N0 ,N0 -tetraacetic acid disodium salt dehydrate (EDTA 3 2Na) were purchased from Wako Pure Chemicals. ((Chloromethyl)phenylethyl)trimethoxysilane (mixed m,p isomers) as an ATRP initiator was obtained from Gelest (Morrisville, PA). (3Glycidoxypropyl)trimethoxysilane (GPTMS) was obtained from ShinEtsu Chemical Industry (Tokyo). 2-Propanol (HPLC grade), dichloromethane, and toluene (dehydrate) were purchased from Wako Pure Chemicals. Steroids and uracil were purchased from Wako Pure Chemicals. Water used in this study was Milli-Q water prepared by an ultrapure water purification system, synthesis A10, Millipore (Billerica, MA)), unless otherwise mentioned.
Surface Modification of Monolithic Silica Column with Silane Agents. Formation of silane layer of ATRP initiator on monolithic silica-rod surfaces was performed as follows: monolithic silica-rod columns were placed into a humidified container for 18 h at approximately 75% relative humidity. ATRP initiator solution was prepared by adding 6.0 mL ((chloromethyl)phenylethyl)trimethoxysilane in 14 mL dried toluene, followed by circulating the solution into the column using an HPLC pump (PU-980) (JASCO) with a flow rate of 0.1 mL/min for 16 h. The reaction proceeded at room temperature for overnight with continuous circulating the solution. The ATRP initiatormodified column was extensively rinsed with flowing toluene and acetone, and dried in a vacuum oven at 110 °C. For the preparation of both ATRP initiator and GPTMS-modified monolithic silica-rod surfaces, which was used for preparing dilute PIPAAm-brush layer,34 a mixture solution of ATRP initiator and GPTMS solution was prepared by adding 4.5 mL of ((chloromethyl)phenylethyl) trimethoxysilane and 1.36 mL of GPTMS in 14 mL of dried toluene. Then, the reaction of mixed silane with a monolithic silica-rod column was performed in a manner similar to a reaction using only ATRP initiator. The ATRP initiator-modified silica beads was also prepared according to the previous report.32 Briefly, silica beads were humidified at 60% relative humidity for 4 h, followed by adding toluene solution of ATRP initiator (53.4 mmol/L) into silica beads in a round-bottom flask and stirring for 16 h. The ATRP initiator immobilized silica beads were rinsed with toluene and acetone and dried in a vacuum oven at 110 °C. PIPAAm Modification of Monolithic Silica by ATRP. PIPAAm-brushes were prepared on the ATRP-initiator immobilized monolithic silica-rod column by ATRP as shown in the second step in Figure 1. First, IPAAm (14.6 g, 129 mmol) was dissolved in 85.6 mL of 2-propanol, and the solution was deoxygenated by nitrogen 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 into the solution under a nitrogen atmosphere, and the solution was stirred for 20 min for allowing a CuCl/CuCl2/Me6TREN catalyst system to appear. Both monomer solution and the initiator-modified monolithic silica-rod column were placed into a glovebox purged with dry nitrogen gas by repeated vacuum and nitrogen flush three times. The monomer solution was pumped into the column by the HPLC pump with a flow rate of 0.05 mL/min for 16 h. The PIPAAm grafted column was extensively rinsed with flowing acetone, methanol, EDTA aqueous solution, and water, and dried in a vacuum oven at 50 °C. The PIPAAm-brush grafted silica beads was also prepared, according to the previous reports.34 Briefly, IPAAm 2-propanol solution (1.0 mol/L) containing CuCl/ CuCl2/Me6TREN was reacted with the silica beads with continuous shaking on a shaker for 16 h at 25 °C. Then, the ATRP initiator immobilized silica beads was rinsed with acetone, methanol, EDTA aqueous solution and water, and dried in a vacuum oven at 50 °C.
Characterization of Initiator Immobilized Silica and Grafted PIPAAm. To determine the amount of ATRP-initiator on the monolithic silica-rod and silica beads were subject to halogen analysis using a organic halogens and sulfur analyzer (AQF-100, Mitsubishi Chemical, Tokyo) and with an ion-chromatography system ICS1500 (Dionex, Sunnyvale). Immobilized ATRP initiator on the silica supports 10831
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Figure 1. Scheme of the preparation of poly(N-isopropylacrylamide) (PIPAAm) brush grafted monolithic silica-rod surfaces using surface-initiated atom transfer radical polymerization (ATRP); (A) dense PIPAAm brush surface and (B) dilute PIPAAm brush surface. (g/m2) was calculated from the chloride composition of initiator immobilized silica beads as in the following equation: immobilized ATRP initiator %Cl ¼ %ClðcalcdÞ ð1 %Cl=%ClðcalcdÞÞ S
modified silane layers %Cs %Cs ðcalcdÞ ð1 %Cs =%Cs ðcalcdÞÞ S
ð2Þ
where %Cs is the carbon composition of silane layers-modified silica beads as determined by elemental analysis, %Cs(calcd) is the calculated weight percent of carbon in the silane layers. %Cs(calcd) of mixed silane comprising initiator and GPTMS was calculated using the feed composition. Amount of grafted PIPAAm on silica bead surfaces (g/m2) was calculated using the following equation:
molecular weight
log Pa
hydrocortisone prednisolone
362.46 360.44
1.61 1.62
dexamethasone
392.46
1.83
hydrocortisone acetate
404.50
2.30
testosterone
288.42
3.32
analyte
ð1Þ
where %Cl is the percent chloride, as determined by elemental analysis, %Cl(calcd) is the calculated weight percent of chloride in initiator, and S is the specific surface area of the monolithic silica-rod (the manufacture’s data: 84 m2/g) and silica beads in square meters per gram (the manufacture’s data: 100 m2/g). To determine the amount of grafted PIPAAm, silica beads were analyzed using a PE 2400 series II CHNS/O analyzer (PerkinElmer, Waltham, MA). Amount of modified silane layers on silica supports (g/m2) was calculated using the following equation:
¼
Table 1. Properties of Hydrophobic Steroids
a
Partition coefficient in an n-octanol/water system.41
(MWCO): 1000] (Spectrum Laboratories, Rancho Dominguez, CA) for 1 week with daily water changed, and PIPAAm was recovered by freeze-drying. Number-average molecular weights and PDI values of the polymer were determined using a GPC system (the columns: TSKgel SuperAW2500, TSKgel SuperAW3000, and TSKgel SuperAW4000; Tosoh, Tokyo) controlled with GPC-8020 model II ver. 5.0 (Tosoh). A calibration curve was obtained using poly(N-isopropylacrylamide) standards (Polymer Source, Dorval, Canada). The flow rate was 1.0 mL/ min. The mobile phase was N,N-dimethylformamide (DMF) containing 50 mmol/L LiCl, and the column temperature was controlled at 45 °C using an equipped column oven, and the elution profiles were monitored by a refractometer. Graft density of PIPAAm on the monolithic silica and silica bead surfaces was estimated using the follow equation: graft density ¼
grafted PIPAAm ¼
%Cp %Cp ðcalcdÞ ð1 %Cp =%Cp ðcalcdÞ %Cs =%Cs ðcalcdÞÞ S ð3Þ
where %Cp is the percent carbon increase from that of the modified silane layers, as determined by elemental analysis and %Cp(calcd) is the calculated weight percent of carbon in the IPAAm monomer. Grafted PIPAAm on the monolithic silica-rod surfaces was also retrieved and analyzed by gel permeation chromatography (GPC) for determining both the molecular weight and polydispersity index (PDI). PIPAAm grafted monolithic silica-rod 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
mc 3 NA Mn
ð4Þ
where mc is the amount of grafted PIPAAm on the monolithic silica-rod and silica bead surfaces per square meter (g/m2), NA is Avogadro’s number, and Mn is the number average molecular weight of the grafted PIPAAm. For obtaining surface elemental composition, X-ray photoelectron spectroscopy (XPS) measurement was performed for ATRP-initiatormodified and PIPAAm-brush grafted silica-rod and silica bead surfaces by an XPS (K-Alpha, Thermo Fisher Scientific, Waltham, MA). Excitation X-rays were produced from a monochromatic Al KR1,2 source and a takeoff angle of 90°. Wide scans were recorded to analyze all existing elements on the surfaces, and high resolution narrow scan analysis was performed for the peak deconvolution of carbon C1s signals. All binding energies were referenced to a C1s hydrocarbon peak at 285.0 eV. Surface morphology of osmium tetroxide (OsO4)-stained ATRP initiator-immobilized and PIPAAm-brush-grafted monolithic silica-rod 10832
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Table 2. Elemental Analyses of PIPAAm-Brush Grafted Silica-Rod and Silica Beads by an XPS Take-Off Angle of 90° atomc (%) codea
C
N
O
Si
Cl
IM100
21.4 ( 3.23
0.00 ( 0.00
57.6 ( 2.95
19.9 ( 5.94
1.15 ( 0.56
IM100-IP
38.9 ( 1.06
5.48 ( 0.35
38.1 ( 1.16
17.0 ( 2.13
0.46 ( 0.06
IGM75
7.11 ( 3.70
0.18 ( 0.19
62.4 ( 0.43
30.2 ( 3.84
0.16 ( 0.18
IGM75-IP
33.4 ( 5.82
1.93 ( 0.48
52.6 ( 1.20
11.8 ( 6.00
0.25 ( 0.21
IB100
21.2 ( 2.22
0.53 ( 0.27
52.8 ( 0.69
25.0 ( 3.12
0.41 ( 0.27
IB100-IP
59.3 ( 0.82
7.70 ( 1.00
26.7 ( 0.32
5.60 ( 0.50
0.71 ( 0.25
calcdb
75.0
12.5
12.5
N/C ratio
0.141 0.057 0.130 0.17
a
All samples were named using silane components, silica structure, and the feed composition of ATRP initiator. Initiator only and a mixture of initiator and GPTMS were denoted as I and IG, respectively. M and B denote “monolith” and “beads”, respectively. IMx, IGMx, and IBx, where x represents the feed composition of ATRP initiator. IP represents PIPAAm modified. b Theoretical atomic composition of (N-isoporpylacrylamide) monomer. c Data from three separate experiments are shown as mean ( SD. and silica beads were observed using a scanning electron microscopy (SEM) S-4300 (Hitachi, Tokyo).
Temperature-Modulated Elution of Bioactive Compounds. PIPAAm grafted silica beads were packed into a stainless steel column (4.6 mm i.d. 50 mm), according to the previous report.24 PIPAAmbrush grafted monolithic silica-rod column or beads packed column was connected to an HPLC system (PU-980 and UV-970; JASCO) controlled by a personal computer with Borwin analysis software version 1.21 (JASCO). Hydrophobic steroids were used for obtaining chromatograms at a concentration of 0.217 mg/mL. The properties of steroids were summarized in Table 1.41 Milli-Q water was used as a mobile phase. Thermoresponsive elution behaviors of steroids were 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 constant temperature water circulator (CTA400; Yamato, Tokyo). For observing elution profiles using step-temperature gradient,42 two thermostatted water baths (RE206, Lauda, Lauda-Konigshofen), set at 35 and 50 °C, respectively, were used. First, relatively hydrophilic steroids were eluted at 50 °C. Then, column temperature was reduced by immersing the columns into another thermostatted water bath set at 35 °C, and elution behavior at 35 °C was observed. Because the outer casing of the column was stainless steel with a small size diameter, the temperature of the column was speculated to be promptly equilibrated with that of water bath. To observe analytes' retention behavior on the prepared columns, van’t Hoff plots for these analytes were obtained. The retention factor k0 value was defined using the follow equation: retention factor ¼
tR t0 t0
ð5Þ
where tR is the retention time of known sample at a specific temperatures and t0 is the retention time of uracil as an initial standard,34 because there is no interaction between uracil and PIPAAm, confirmed by observing the same as deuterium oxide. Resolution between two hydrophobic steroids was calculated according to the following equation:43 resolution ¼ 2:0
tR 2 tR 1 W1 þ W2
ð6Þ
where tR1 and tR2 (tR1 < tR2) are the retention times of analytes and W1 and W2 are the peak widths of analytes at the baseline.
’ RESULTS AND DISCUSSION Characterization of Initiator and PIPAAm-Brush Grafted Monolithic Silica Surfaces. For obtaining the information of
elemental composition of the prepared monolithic silica-rod and silica bead surfaces, XPS measurement was performed (Table 2). All samples are named using silane components, silica structure, and the feed composition of ATRP initiator. Initiator only or a mixture of initiator and GPTMS are denoted as I and IG, respectively. M and B denote the “monolith” and “beads”, respectively. IMx, IGMx, and IBx, where x represents the feed composition of ATRP initiator. IP represents PIPAAm modified. The peak deconvolution was performed according to the previous reports.44,45 The peak deconvolution of XPS carbon C1s peaks for the surfaces were shown in Figure S.1 in Supporting Information. The different peaks were observed between ATRP-initiator-modified surfaces (IM100 and IGM75) and PIPAAm grafted monolithic silica-rod surfaces (IM100-IP and IGM75-IP). In the spectrum of PIPAAm grafted monolithic silica-rod surfaces, an additional peak was observed at 288 eV (Figure S.1B and D), corresponding to the CdO bond of PIPAAm, while there were no peaks in the spectrum of ATRPinitiator-modified monolithic silica-rod surfaces (Figure S.1A and C). Additionally, nitrogen contents increased and silicon contents decreased after grafting PIPAAm on the surfaces, because PIPAAm-brush layer covered onto monolithic silicarod surfaces. These results indicated that PIPAAm was successfully grafted on monolithic silica-rod surfaces through surfaceinitiated ATRP. For measuring the amount of initiator immobilized and grafted PIPAAm on monolithic silica surfaces, CHN and chloride elemental analyses were performed. Elemental composition of carbon, nitrogen, and chlorine were summarized in Table 3. Initiator density of silica beads, estimated from the chlorine composition, was relatively smaller than that estimated from the carbon contents, due to the difference of detection sensitivity between these elemental analyzers. The amount of immobilized initiator on monolithic silica-rod estimated from carbon composition was almost the same as that on silica beads and that reported in the previous studies,32 indicating that silane coupling reaction was successfully performed with monolithic silica-rod surface in column through the reaction condition. Various conditions of silane coupling reaction have been investigated, such as humidifying condition, silane agent concentration, and the flow velocity of reaction solution. As a result, 75% relative humidity, 30% silane agent volume concentration, and 0.1 mL/min circulation rate were found to be suitable for silanizing monolithic silica surface in column. The chlorine composition of IGM75 was 10833
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Table 3. Characterization of PIPAAm Brush Grafted Monolithic Silica-Rod and Silica Beads elemental composition (%) immobilized initiatord a
code
b
C
b
c
immobilized silanee
grafted polymer
(μmol/m )
(μmol/m2)
(mg/m2)
Mnf
Mn/Mwf
(chains/nm2)
1.50
9600
2.46
0.094
1.16
9700
2.23
0.072
2.75
13300
3.13
0.125
2
N
Cl
0.60
2.03
4.97
0.19
0.69
2.48
1.04
2.96
4.86
IM100
4.32 ( 0.04
0.23 ( 0.03
IM100-IP
10.9 ( 0.08
2.33 ( 0.03
IGM75
2.01 ( 0.03
0.03 ( 0.02
IGM75-IP
7.46 ( 0.18
1.83 ( 0.13
IB100
4.99 ( 0.16
0.45 ( 0.08
IB100-IP
17.6 ( 0.13
3.34 ( 0.10
graft density
a
All samples were named using silane components, silica structure, and the feed composition of ATRP initiator. Initiator only and a mixture of initiator and GPTMS were denoted as I and IG, respectively. M and B denote “monolith” and “beads”, respectively. IMx, IGMx, and IBx, where x represents the feed composition of ATRP initiator. IP represents PIPAAm modified. b Determined by elemental analysis (n = 3). c Determined by organic halogens and sulfur analyzer (n = 2). d Estimated from chlorine composition. e Estimated from carbon composition. f Determined by GPC using DMF containing 50 mmol/L LiCl.
smaller than that of IM100, indicating that ATRP initiator density on IGM75 was small than that compared on IM100. This was able to be explained by the relative bulk and steric hindrance of GPTMS.34 PIPAAm-brush was grafted from these initiator-modified monolithic silica-rod and silica beads via surface-initiated ATRP. These PIPAAm-brush-modified silica rods and silica beads were named as IM100-IP, IGM75-IP, and IB100-IP. Amounts of grafted PIPAAm on these surfaces including silica-rod and -bead 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 PIPAAmbrush prepared by surface initiated ATRP. Polymer brushes prepared by surface-initiated ATRP formed densely packed configurations, compared to that by other radical polymerizations, since the initiation efficiency of ATRP was quite high.47 Thus, PIPAAm was densely grafted on monolithic silica-rod surfaces, leading to the significantly large amount of grafted PIPAAm on the surfaces. In addition, the grafted amount of PIPAAm on IB100-IP was slightly larger than that on IM100-IP, attributed to the different reaction conditions of ATRP. To characterize the chain lengths and graft densities of PIPAAm on the silica surfaces, the molecular weight of grafted PIPAAm was determined by GPC after PIPAAm chains was cleaved from silica-rod and silica beads with hydrofluoric acid. These data were also summarized in Table 3 (GPC charts of cleaved polymer are shown in Figure S.2, Supporting Information). The polydispersity index of the cleaved PIPAAm was larger than that of PIPAAm prepared in solution,48 and two narrow peaks overlapped in GPC chromatograms. The larger polydispersity was suggested to be attributed to the porous geometry of monolithic silica-rod and silica beads.24 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 polymer on porous silica substrates. Graft density of IGM75-IP was predictably smaller than those of IM100-IP and IB100-IP, due to the smaller initiator density. Thus, the incorporation of GPTMS into initiator silane layer was able to be dilute PIPAAm-brush on monolithic silica-rod surfaces like silica beads surfaces previously reported.34
SEM observation of before and after PIPAAm grafting onto monolith silica-rod and PIPAAm-modified silica beads surfaces was performed (Figure 2). SEM photographs of initiator-modified monolithic silica-rod and beads are also shown in Supporting Information, Figure S.3. The macropore structure in monolithic silica-rod was remained, although the sufficient amount of PIPAAm was modified on the silica surface, which would lead to large flow paths and a low back pressure. Figure 3 shows the back pressure of these columns at various temperatures by flowing Milli-Q water at a flow rate of 1.0 mL/min. The back pressure of these PIPAAm-modified monolithic silica rod columns was found to keep a low back pressure,49 although a small increase in back pressure after PIPAAm modification was observed. These results indicated that the PIPAAm grafting method in this study gave no clogging character to the monolithic silica-rod. Additionally, the prepared PIPAAm brush-modified monolithic silica columns can be used various condition, such as lower temperature and higher flow rate of mobile phase, due to its low back pressure. Elution Behavior of Hydrophobic Steroids from PIPAAmBrush Surfaces. The elution behavior of hydrophobic steroids from PIPAAm grafted monolithic silica-rod column and silica beads used as chromatographic stationary phases was investigated. Figure 4AC shows the chromatograms of steroids at various temperatures on IM100-IP, IGM75-IP, and IB100-IP columns using Milli-Q water as a mobile phase, respectively. Figure 5AC shows changes in the retention times of analytes with various temperatures on these columns. Retention times of steroids on all columns were increased with increasing column temperature, as explained by the hydrophobic drive partitioning interactions between dehydrated PIPAAm chains and steroids. On IM100-IP column, five hydrophobic steroids were successfully separated with a short retention time (10 min), compared to that on IB100-IP, although the resembled structure of PIPAAmbrush were grafted on both silica surfaces in the same column length (50 mm). High resolution separation with a remarkable short retention time was attributed to the different structures between monolithic silica-rod and silica beads packed column. For investigating the linear velocity of mobile phase in these columns, the retention time of uracil, which fails to interact with PIPAAm, were measured. The retention times on IM100-IP and IB100-IP were 0.53 and 0.86 min, respectively. These values indicated that the linear velocity in IM100-IP was higher than 10834
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Figure 2. SEM photograph of PIPAAm brush-modified monolithic silica column. (A, B) SEM of dense PIPAAm-brush grafted monolithic silica-rod (IM100-IP in Table 2) and high-magnified SEM, respectively; (C, D) dilute PIPAAm-brush grafted silica-rod (IGM75-IP); (E, F) unmodified monolithic silica-rod; (G, H) dense PIPAAm brush grafted silica beads; and (I, J) unmodified beads. The back ground patterns in (I) and (J) are doublestick tape for fixing the sample. These photographs were obtained as 5000- and 15000-fold magnifications.
Figure 3. Temperature-dependent back pressure changes on prepared PIPAAm brush grafted monolithic silica and silica beads. The open circles represent back pressure observed on dense PIPAAm-brush grafted monolithic silica-rod column (IM100-IP in Table 2); the open triangles, dilute PIPAAm-brush grafted silica-rod column (IGM75-IP); the open square, dense PIPAAm-brush grafted silica beads column (IB100-IP); the closed circles, unmodified monolithic silica columns; closed squares, unmodified beads packed column.
that of IB100-IP. Additionally, previous reports regarding monolithic supporting materials suggested that monolithic supporting materials have a 3D fine-dendritic skeleton structure with large through-pores, reducing the diffusion path length and resistance, compared to the beads-packed column.35,36,38 In addition, as shown in the SEM photograph in Figure 2, the narrow skeleton structure of monolithic silica-rod compared to the beads diameter contributed to give the effective utilization of surface area where it interacts with the analyte. Thus, the 3D structure of PIPAAm-brush grafted monolithic silica-rod was able to allow five hydrophobic steroids to be separated rapidly.
Figure 4. Chromatograms of steroids separated on HPLC of which packing materials were PIPAAm brush grafted monolithic silica-rods and PIPAAm brush grafted silica beads at various temperatures: (A) dense PIPAAm-brush grafted monolithic silica-rod column (IM100IP in Table 2), (B) dilute PIPAAm-brush grafted silica-rod column (IGM75-IP), and (C) dense PIPAAm-brush grafted silica beads column (IB100-IP). Mobile phase is Milli-Q water. The peak No. 1 represents hydrocortisone; No. 2, prednisolone; No. 3, dexamethasone; No. 4, hydrocortisone acetate; No. 5, testosterone. Due to their high-speed separation abilities, the time scales of monolithic columns (IM100-IP and IGM75-IP) are expanded compared to those of the silica beads column (IB100-IP).
Different retention profiles, which concerned a phase transition profile on grafted PIPAAm, were observed on IM100-IP and 10835
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Figure 5. Temperature-dependent retention time changes of steroids on (A) dense PIPAAm-brush grafted monolithic silica-rod column (IM100-IP in Table 2), (B) dilute PIPAAm-brush grafted silica-rod column (IGM75-IP), and (C) dense PIPAAm-brush grafted silica beads column (IB100-IP). The open circles represent hydrocortisone; the closed triangles, prednisolone; the open squares, dexamethasone; the closed diamonds, hydrocortisone acetate; the closed circles, testosterone.
Figure 6. van’t Hoff plots of steroids on (A) dense PIPAAm-brush grafted monolithic silica-rod column (IM100-IP in Table 2), (B) dilute PIPAAmbrush grafted silica-rod column (IGM75-IP), and (C) dense PIPAAm-brush grafted silica beads column (IB100-IP). The open circles represent hydrocortisone; closed triangles, prednisolone; open squares, dexamethasone; closed diamonds, hydrocortisone acetate; closed circles, testosterone.
IB100-IP, although similar PIPAAm brush was formed on both surfaces. This was probably attributed to a slight difference in PIPAAm brush density on both surfaces. This study demonstrated that the retention times of steroids on dense PIPAAmbrush grafted silica-beads packed column increased with increasing temperature up to 40 °C, and the retention decreased as column temperature increased beyond this point,24 because PIPAAm dehydration completed at this temperature, and no hydrophobic interactions increased further with increasing temperature. By contrast, steroid solubility in the mobile phase increases with increasing temperature. Thus, the retention times of steroids decreased above 40 °C. On the contrary, the retention times on dilute PIPAAm brush increased with increasing temperature beyond the phase transition temperature of PIPAAm (32 °C), because its dehydration failed to complete at this point, and the hydrophobicity of PIPAAm increased beyond the temperature. In the present study, PIPAAm graft density on IM100-IP was relatively lower than that on IB100-IP (Table 3). This slight difference in the graft density led to the different elution profiles between IM100-IP and IB100-IP. The analysis time of five hydrophobic steroids on the IGM75IP column was shorter than that on the IM100-IP column. This was attributed to diluted PIPAAm-brush density on the monolithic
silica-rod surfaces. Our previous report suggested that diluted PIPAAm-brush on relatively hydrophilic graft interface exhibits an enhanced hydration and temperature-responsive hydrophilic/ hydrophobic alteration, compared to dense PIPAAm-brush.34 Actually, on IGM75-IP at low temperatures, steroids except testosterone were eluted as one peak with a short retention time, although several peaks were observed on IM100-IP at low temperatures. Figure 6 show the van’t Hoff plots of these steroids, which exhibited the relationship between the analyte retention and the column temperature. The retention factor k0 value was defined as k0 = Rt/(Rt R0); where Rt is the retention time of a known sample at predetermined temperature, and R0 is the retention time of uracil as an initial standard. Change in ln k0 values of IGM75-IP was larger than that of IM100-IP, explained by the larger hydrophilic/hydrophobic change of diluted PIPAAmbrush.34 Additionally, a broader change in ln k0 with temperature was observed in IB100-IP, attributed to the intrinsic dehydration properties of dense PIPAAm-brush prepared by surface-initiated ATRP.24,29,33 Figure 7AC shows changes in the resolutions of steroids at various temperatures on these columns. Resolutions of steroids on all columns were increased with increasing column temperature 10836
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Figure 7. Temperature-dependent resolution changes of steroids on (A) dense PIPAAm-brush grafted monolithic silica-rod column (IM100-IP in Table 2), (B) dilute PIPAAm-brush grafted silica-rod column (IGM75-IP), and (C) dense PIPAAm-brush grafted silica beads column (IB100-IP). The closed triangles represent the resolution calculated between hydrocortisone and prednisolone; the open squares, hydrocortisone and dexamethasone.
Figure 8. Step-temperature gradient on steroids elution from (A) dense PIPAAm-brush grafted monolithic silica-rod column (IM100-IP in Table 2) and (B) dilute PIPAAm-brush grafted silica-rod column (IGM75-IP). Mobile phase is Milli-Q water. The peak No. 1 represents hydrocortisone; No. 2, dexamethasone; No. 3, testosterone. Chromatograms of steroids at 35 and 50 °C are represented for comparing retention times.
because of the dehydrated and shrunken PIPAAm-brushes at high temperature. Increased hydrophobic interaction between dehydrated PIPAAm and steroids enhanced the separation of hydrophobic steroids. In addition, shrunken PIPAAm-brush prevented hydrophobic steroids from penetrating into the brush, compared to that into hydrated PIPAAm-brush at lower temperature that leads to broad peaks. Additionally, the resolutions of steroids on IM100-IP were larger than those on IB100-IP. This was attributed to several factors that suppressed the diffusion path length of analyte, the higher linear velocity of mobile phase, and slight difference in PIPAAm graft configuration. In addition, different surface areas between the monolithic silica rod columns and silica beads packed column would affect the separation efficiency. Grafted PIPAAm-brush surface hydrophobicity and partitioning alterations with temperature are reversible. Thus, steptemperature gradients shorten total analysis time in temperature-responsive chromatography.42,46,50 Figure 8 shows the effects of a step-temperature gradient on steroid elution from
IM100-IP and IGM75-IP columns. First, hydrocortisone and dexamethesone, relatively hydrophilic steroids, were separated by relatively strong hydrophobic interactions at 50 °C. After the column temperature was reduced to 35 °C, the retention time of testosterone was found to be shortened. A mixture of hydrocortisone, prednisolone, and dexamethesone was also separated using step-temperature gradient, and the retention time of testosterone was effectively modulated (shown in Supporting Information, Figure S4). Our previous reports regarding thermoresponsive chromatography indicated that the step-temperature gradient shortens the retention time of analytes in a relatively longer analysis time, approximately 30 min.42,46,50 On the contrary, the PIPAAm-brush monolithic silica-rod column was able to further shorten the retention time (total analytical time < 5 min), which was speculated to be attributed to the suppressed diffusion path length and thermoresponsive properties of the PIPAAm-brush structure.48 These results demonstrated that (1) PIPAAm-brushes was successfully formed on monolithic silica-rod surfaces through surface-initiated ATRP with flowing the reaction solution into the column, and (2) PIPAAm-grafted monolithic silica column allows us to use the high-speed separation of bioactive compounds with a higher resolution than that of PIPAAm-brush packed column. Thus, PIPAAm-brush grafted monolithic silica-rod column would be an alternative to PIPAAm-brush grafted silica beads.
’ CONCLUSIONS PIPAAm-brush was successfully grafted onto monolithic silica surfaces using surface-initiated ATRP, and the separation efficiencies of the prepared columns were investigated by observing the thermoresponsive elution profiles of the steroids. Hydrophobic steroids were successfully separated on the prepared PIPAAm-brush grafted monolithic column with a remarkable shorter retention time compared to PIPAAm-brush grafted silica beads packed column because of the suppressed diffusion path length of PIPAAm-modified monolithic silica. Additionally, dilute PIPAAm-brush grafted monolithic silica column allowed the shortening of the analysis time of these steroids to be available, attributed to the enhanced hydration of grafted PIPAAm. Thus, PIPAAm-brush grafted monolithic silica would be an effective tool for analyzing hydrophobic bioactive compounds with a short analysis time and a high resolution. 10837
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’ ASSOCIATED CONTENT
bS
Supporting Information. XPS peak deconvolution of XPS C1s peaks, GPC chart of the grafted PIPAAm on silica surfaces for obtaining the molecular weight, SEM photograph of monolithic silica and silica beads, and step-temperature gradient on steroids elution from (A) IM100-IP and (B) IGM75-IP. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Phone: +81-3-5367-9945, ext. 6201. Fax: +81-3-3359-6046. E-mail:
[email protected].
’ ACKNOWLEDGMENT Part of the present research was financially supported by the Development of New Environmental Technology Using Nanotechnology Project of the National Institute of Environmental Science (NIES), commissioned from the Ministry of Environment, Japan, Grants-in-Aid for Scientific Research (B) No. 20300169 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 appreciate Mr. Seiichi Kosaka for the assistance of SEM observation and Dr. Norio Ueno for English editing. ’ REFERENCES (1) Gil, E. S.; Hudson, S. M. Stimuli-reponsive polymers and their bioconjugates. Prog. Polym. Sci. 2004, 29 (12), 1173–1222. (2) Hoffman, A. S.; Stayton, P. S. Conjugates of stimuli-responsive polymers and proteins. Prog. Polym. Sci. 2007, 32 (89), 922–932. (3) Kikuchi, A.; Okano, T. Nanostructured designs of biomedical materials: applications of cell sheet engineering to functional regenerative tissues and organs. J. Controlled Release 2005, 101 (13), 69–84. (4) Sethuraman, V. A.; Na, K.; Bae, Y. H. pH-Responsive sulfonamide/PEI system for tumor specific gene delivery: An in vitro study. Biomacromolecules 2005, 7 (1), 64–70. (5) Meng, F.; Zhong, Z.; Feijen, J. Stimuli-responsive polymersomes for programmed drug delivery. Biomacromolecules 2009, 10 (2), 197–209. (6) Heskins, M.; Guillet, J. E. Solution properties of poly(N-isopropylacrylamide). J. Macromol. Sci., Part A 1968, 2, 1441–1455. (7) Cammas, S.; Suzuki, K.; Sone, C.; Sakurai, Y.; Kataoka, K.; Okano, T. Thermoresponsive polymer nanoparticles with a core-shell micelle structure as site-specific drug carriers. J. Controlled Release 1997, 48 (23), 157–164. (8) Bae, Y. H.; Kim, S. W. Hydrogel delivery systems based on polymer blends, block co-polymers or interpenetrating networks. Adv. Drug Delivery Rev. 1993, 11 (12), 109–135. (9) Takei, Y. G.; Aoki, T.; Sanui, K.; Ogata, N.; Okano, T.; Sakurai, Y. Temperature-responsive bioconjugates. 1. Synthesis of temperatureresponsive oligomers with reactive end groups and their coupling to biomolecules. Bioconjugate Chem. 1993, 4 (1), 42–46. (10) Chilkoti, A.; Chen, G.; Stayton, P. S.; Hoffman, A. S. Sitespecific conjugation of a temperature-sensitive polymer to a genetically engineered protein. Bioconjugate Chem. 1994, 5 (6), 504–507. (11) Mori, T.; Maeda, M. Temperature-responsive formation of colloidal nanoparticles from poly(N-isopropylacrylamide) grafted with single-stranded DNA. Langmuir 2003, 20 (2), 313–319. (12) Yu, C.; Mutlu, S.; Selvaganapathy, P.; Mastrangelo, C. H.; Svec, F.; Frechet, J. M. J. Flow control valves for analytical microfluidic chips
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