Functionalization of Silica Microparticles with Multiple-Responsive

Dec 12, 2017 - College of Innovation and Entrepreneurship Education, Chongqing University of Post and Telecommunications, Chongqing 400065, China ... ...
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Article Cite This: Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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Functionalization of Silica Microparticles with Multiple-Responsive Copolymers for Smart Controlled Chromatograph Zong-Jian Liu,†,‡,§ Shuo Huang,†,‡,§ Yuan-Yuan Ran,†,‡ Jie Chen,∥ Xiao-ming Hu,† Hui-shan Du,*,† and Jin Wang*,⊥ †

China-America Institute of Neuroscience, Beijing Luhe Hospital, and ‡Central Laboratory, Beijing Luhe Hospital, Capital Medical University, Beijing 101100, China ∥ College of Innovation and Entrepreneurship Education, Chongqing University of Post and Telecommunications, Chongqing 400065, China ⊥ Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123, China S Supporting Information *

ABSTRACT: Since its first discovery more than a century ago, chromatographic separation has become increasingly essential to organic chemistry, pharmaceuticals, proteomics, the environment, and industry. However, recent developments in basic science indicate that increasing numbers of small molecules and proteins are extremely hard to separate successfully by the traditional chromatography. Herein, we propose an intelligent chromatograph that can be controlled by various conditions such as temperature, pH, and ionic strength at the same time, which is fulfilled by the functionalization of silica microparticles with multiple stimuli-responsive copolymers, poly(N-isopropylacrylamide-co-2-(diethylamino)ethyl methacrylate-co-N-tert-butylacrylamide), by surface-initiated atom transfer radical polymerization. The intelligent chromatographic properties were evaluated in a controlled manner based on adenosine nucleotides in the aqueous mobile phase. The results indicate that the influences of temperature, pH, and mobile-phase ionic strength could be well regulated by the chain length and composition of the copolymers. Moreover, the present findings can be directed toward the design and application of a multiply controlled chromatograph.

1. INTRODUCTION

Temperature-responsive chromatography is performed with an aqueous mobile phase instead of an organic mobile phase, such as methanol and acetonitrile. The application of an aqueous mobile phase is critical for maintaining the biological activities of biomolecules and reducing the environmental pollution.29 Elution is achieved by temperature-modulated changes in the hydrophobicity of the stationary phase, which is similar to the use of gradients in composition for the elution of mobile phases. In recent years, we have developed “thermally switchable chromatographic materials” for the selective capture and rapid release of biomolecules including proteins, nucleotides, and adenosine. The process is simple because the separation of biomolecules can be achieved simply by the adjustment of the temperature, whereas for the conventional method, changes in the mobile phase are needed.30−32 Silica modified with PNIPAAm containing ionizable groups is a type of thermoresponsive stationary phase that is suitable for the analysis and separation of ionic compounds. Typically, copolymers of PNIPAAm with ionizable monomers such as

Intelligent materials, which can change their structures and functions in response to surrounding conditions, such as electric field,1 pH,2 and temperature,3 have received a great deal of attention in recent years. Poly(N-isopropylacrylamide) (PNIPAAm) is a well-known thermoresponsive polymer that exhibits a reversible phase transition in water when a change in the external temperature crosses its lower critical solution temperature (LCST) at 32 °C.4 PNIPAAm is hydrophilic and expanded below the LCST, whereas it is dehydrated and forms a compact and hydrophobic conformation above the LCST. Thus, PNIPAAm has been widely used in drug and gene delivery systems,5,6 cell culture substrates,7,8 bioconjugates,9 and microfluidics,10 among other applications. Moreover, PNIPAAm-modified surfaces can alter their hydrophilic/ hydrophobic properties in response to temperature changes.11 As a result, silica microparticles have been modified with PNIPAAm and were employed as the stationary phase for temperature-responsive chromatography.12 In addition, various functional copolymers have been grafted onto silica microparticles for the separation of benzene derivatives, 13 steroids,14−20 amino acids,21,22 peptides,23−25 and other analytes.26−28 © XXXX American Chemical Society

Received: Revised: Accepted: Published: A

November 3, 2017 December 12, 2017 December 12, 2017 December 12, 2017 DOI: 10.1021/acs.iecr.7b04570 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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Industrial & Engineering Chemistry Research

pyltriethoxysilane (3-APS) were obtained from J&K Chemical Ltd. (Shanghai, China). Silica (average diameter, 5 μm; pore size, 300 Å; specific surface area, 60 m2/g) was kindly provided by Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences (Lanzhou, China). The adenosine nucleotides adenosine monophosphate (AMP), adenosine diphosphate (ADP), and adenosine triphosphate (ATP) were purchased from Beijing Bio-LAB Materials Institute. Other chemicals including cuprous chloride (CuCl), triethylamine (TEA), tetrahydrofuran (THF), 2-propanol, calcium hydride (CaH2), hydrofluoric acid (HF), sodium carbonate (Na2CO3), and sodium chloride (NaCl) were all obtained from Beijing Chemical Reagent Corp. (Beijing, China) and were of analytical grade. Cuprous chloride (CuCl) was purified in acetic acid under the protection of nitrogen. Triethylamine (TEA), tetrahydrofuran (THF), and 2-propanol were all dried over CaH2. 2.2. Apparatus. A Spectrum BX FT-IR system from Perkin Elmer was used to determine the Fourier transform infrared (FTIR) spectra of the copolymers. Elemental analyses were carried out using a Vario MICRO CUBE analyzer (Elementar, Langenselbold, Germany). To determine the grafting ratios of the copolymers on the silica surface, thermogravimetric analysis (TGA) was performed on a Mettler Toledo TGA/ DSC STARe system at a heating rate of 10 °C/min from ambient temperature to 800 °C under a nitrogen atmosphere. The grafting ratio was calculated as

cationic (N,N-dimethylamino)propylacrylamide (DMAPAAm) and 2-(dimethylamino)ethyl methacrylate (DMAEMA) respond to both temperature and pH.33−35 The copolymermodified silica surface can be switched between hydrophilicity and hydrophobicity through variations in the charge density and temperature. Thus, the separation of ionic bioactive compounds can be achieved on a column packed with silica grafted with these copolymers by adjusting the column temperature rather than changing the composition of the mobile phase. Previously, we also reported a thermoresponsive stationary phase containing cationic poly{[2(methacryloyloxy)ethyl]trimethylammonium chloride} for the separation of lactic acid and creatine phosphate disodium salt.27 Poly[2-(diethylamino)ethyl methacrylate] (PDEAEMA) is a pH-responsive polymer with a pKb value of 7.3.36 The copolymer is hydrophilic below pH 7.3, but it is hydrophobic above pH 7.3 because of the neutralization of the charged groups. The basicity of PDEAEMA in its copolymer with PNIPAAm strongly decreased with increasing temperature, whereas the pKb of PDEAEMA was found to be independent of temperature in the absence of PNIPAAm.37 Thus, the charge density of PNIPAAm-co-PDEAEMA can be regulated by changing the temperature. The copolymer is hydrophilic and positively charged at temperatures below the LCST, whereas it becomes hydrophobic and uncharged at temperatures above the LCST. There have been several reports on ternary copolymers grafted ont silica microparticles including PNIPAAm copolymerized with PDMAPAAm34,38 and PDMAEMA.39 However, the relatively high phase-transition temperatures of PNIPAAm copolymers with PDMAPAAm or PDMAEMA are not very suitable for temperature-responsive chromatography because temperature has to be elevated to modulate the properties of the stationary phase, which can result in the deactivation of biomolecules and proteins. In a previous report, the copolymer of NIPAAm and DEAEMA exhibited thermally switchable properties for the selective capture and rapid release of proteins.32 However, the influences of the mobile-phase conditions, including pH and ionic strength, and the DEAEMA content in copolymers on separation efficiency remain unknown. Therefore, in the present work, multiple-stimuliresponsive chromatography that can be controlled by temperature, pH, and ionic strength at the same time is proposed. To achieve this aim, silica microparticles grafted with poly(NIPAAm-co-DEAEMA-co-tBAAm) (where tBAAm = N-tertbutylacrylamide) were designed and synthesized by surfaceinitiated atom transfer radical polymerization (SI-ATRP). The effects of the temperature, pH value, and ionic strength of the mobile phase and the DEAEMA content in the copolymer on the separation efficiency were investigated by evaluating the elution behavior of adenosine nucleotides. The results indicate that the above-mentioned factors significantly affect the properties of the stationary-phase surface and further influence the separation efficiency.

(1)

where Δw1 and Δw2 are the weight losses of initiator- and copolymer-modified silica, respectively, in the range from 100 to 750 °C. The LCSTs of the copolymers were determined on a UV−vis spectrometer (TU-1810, Purkinje General, Beijing, China). All chromatographic measurements were carried out using a high-performance liquid chromatography (HPLC) system (PU-230, DAD230, Dalian Elite Analytical Instrumental Co., Ltd.). A Yataikelong YT-15A thermostated water bath (Beijing, China) with a deviation of ±0.1 °C was used to control the temperature of the column and the UV−vis spectrometer. Scanning electron microscopy (SEM) images were obtained on a field-emission scanning electron microscope (Quanta 400 FEG). The samples were coated with Au nanopowder under a current of 20 mA for 2 min. 2.3. Preparation of Amino-Functionalized Silica. Silica particles were treated with 0.1 mol/L hydrochloric acid solution for 8 h at ambient temperature, repeatedly washed with large amounts of distilled water, and then dried at 110 °C for 20 h. Next, 5.0 g of the prepared silica was placed into 50 mL of anhydrous toluene. The mixture was degassed at 0 °C, and then 5 mL of 3-aminopropyltriethoxysilane was added under nitrogen atmosphere. The reaction was allowed to proceed at 90 °C for 10 h with continuous stirring. The 3aminopropyl-functionalized silica was rinsed with toluene, methanol, and distilled water and dried under a vacuum at 80 °C for 12 h. Anal. Found (%): C, 2.11; H, 0.46; N, 0.50. The density of immobilized amino groups was calculated to be 3.57 per nm2 using the equation amino density = (N% × NA) /(MN × S × 1000), where N% is the elemental composition of nitrogen, NA is Avogadro’s number, MN is the molar mass of nitrogen (g/mol), and S is the specific surface area of the silica support (m2/mg).

2. EXPERIMENTAL SECTION 2.1. Materials. Both NIPAAm and 2-(diethylamino)ethyl methacrylate (DEAEMA) were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). NIPAAm was purified by recrystallization from n-hexane. N-tert-Butylacrylamide (tBAAm), 2-bromoisobutyryl bromide, and acetyl chloride were all provided by Acros Organics. N,N,N′,N′,N″Pentamethyldiethylenetriamine (PMDETA) and 3-aminoproB

DOI: 10.1021/acs.iecr.7b04570 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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Figure 1. Preparation of poly(NIPAAm-co-DEAEMA-co-tBAAm)-modified silica by surface-initiated atom transfer radical polymerization (SIATRP).

2.4. Preparation of 2-Bromoisobutyrate-Functionalized Silica. Precisely 3.0 g of 3-aminopropyl silica was dispersed in 30 mL of anhydrous tetrahydrofuran mixed with 1.2 mL of triethylamine under nitrogen at 0 °C. A solution of 2-bromoisobutyryl bromide (300 μL, 2.4 mmol) and acetyl chloride (1.56 mL, 21.6 mmol) (mole ratio = 10:90) in 10 mL of anhydrous tetrahydrofuran was added dropwise to the solution at 0 °C under nitrogen. The reaction was allowed to proceed at ambient temperature for 12 h. The 2-bromoisobutyrate-functionalized silica was collected by centrifugation; rinsed sequentially with ethanol, distilled water, and acetone; and dried at 50 °C for 8 h. The grafting ratio was determined to be 1.35%. Anal. Found (%): C, 2.54; H, 0.49; N, 0.48. 2.5. Surface Modification of Silica with Cationic Copolymers by SI-ATRP. Preparation of the copolymermodified silica with the 5 mol % feed ratio of DEAEMA to NIPAAm was carried out as follows: 2-Bromoisobutyratefunctionalized silica (1.0 g), NIPAAm (4.33 g, 38.2 mmol), DEAEMA (385 μL, 1.92 mmol), N-tert-butylacrylamide (0.486 g, 3.82 mmol), and PMDETA (608 μL, 2.91 mmol) were placed in 32 mL of a mixture of water and 2-propanol (20:80, v/v). The mixture was deoxygenated under a vacuum and flushed with nitrogen three times at 0 °C. CuCl (70 mg, 0.71 mmol) was added under nitrogen, and the reaction was allowed to proceed for 12 h at ambient temperature under continuous stirring. The silica was separated by centrifugation; washed using distilled water, 50 mmol/L ethylenediaminetetraacetic acid (EDTA) solution, distilled water, and ethanol; and then dried at 50 °C for 8 h. The prepared silica is denoted as SID-5, where 5 represents the feed composition of DEAEMA. For the preparation of copolymer-modified silica with a 10 mol % feed ratio of DEAEMA to NIPAAm, the reaction was performed in the same manner as described above except that 770 μL (3.83 mmol) of DEAEMA was used. The resulting silica is denoted as SID-10. FTIR analysis of the copolymers was performed on samples cleaved from the silica surface. IR (KBr, cm−1): 3298 (υN−H), 1728 (υCO) (COO), 1650

(υCO) (CONH). Grafting ratios (%): SID-5, 11.31; SID-10, 22.72. Anal. Found for SID-5 (%): C, 8.20; H, 1.43; N, 1.65. Found for SID-10 (%): C, 11.65; H, 1.95; N, 2.12. 2.6. Gel Permeation Chromatography Measurements. The molecular weight and polydispersity index (PDI) of the grafting copolymers were determined by gel permeation chromatography (GPC) measurements. The copolymers were cleaved from the silica surface using hydrofluoric acid and neutralized with sodium carbonate. The polymer was purified by dialysis in distilled water using a cellulose membrane (molecular weight cutoff = 1000) for 7 days, and the water was changed every 12 h. The polymer was finally obtained by freeze-drying under a vacuum. GPC was performed with a system consisting of a Waters 1515 pump and a Waters 2414 differential refractive index detector. Three linear Styragel columns including HT3, HT4, and HT5 were used at an oven temperature of 50 °C. Dimethylformamide (DMF) containing 0.06 wt % LiBr at a flow rate of 1.0 mL/min was used as the eluent. Polystyrene (PS) standards were used for GPC calibration. The amount of grafted copolymer (mc, mg/m2) was calculated according to the equation32

where ΔWSI and ΔWSC denote the weight loss ratios of the initiator- and copolymer-grafted silica, respectively, in the range from 100 to 750 °C; W750 is the residual weight ratio of the thermoresponsive silica at 750 °C; and S is the specific surface area of the silica (m2/mg). The grafting density of the copolymer was calculated using the equation32,34,35

where mc denotes the amount of grafted copolymer, Mn represents the number-average molecular weight of the grafted copolymer (g/mol), and NA is Avogadro’s number. C

DOI: 10.1021/acs.iecr.7b04570 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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Industrial & Engineering Chemistry Research 2.7. Transmittance Measurements. The thermoresponsive properties of the cationic copolymers were evaluated by determining the optical transmittance at various temperatures. The copolymers were cleaved from the silica surface using hydrofluoric acid and neutralized with sodium carbonate. The polymer was purified by dialysis in distilled water using a cellulose membrane (molecular weight cutoff = 1000) for 7 days, and the water was changed every 12 h. The polymer was finally obtained by freeze-drying under a vacuum. The cleaved copolymers are denoted as ID-x, where x represents the feed composition of DEAEMA. The optical transmittance of the copolymer solution (5 mg/mL) was determined at 450 nm under various mobile-phase conditions. LCST is defined as the temperature where 50% optical transmittance of the aqueous copolymer solutions was observed. 2.8. Temperature-Responsive Chromatography. The copolymer-grafted silica was dispersed in methanol and packed into stainless steel columns (50 mm × 1.0 mm ii.d.) under a maximum pressure of 40 MPa. The column was connected to an HPLC system. The temperature-responsive elution behaviors of adenosine nucleotides were monitored at 260 nm at a flow rate of 0.1 mL/min with an injection volume of 5 μL. Phosphate buffer solutions (10 mmol/L) with various pH values (6.7, 7.0, and 7.3) and phosphate buffer solutions (10 mmol/L, pH 7.0) with different concentrations of NaCl (0.02 and 0.04 mmol/L) were used as the mobile phases. The column temperature was controlled with a water bath.

Figure 2. TGA curves of modified silica: (1) 3-aminopropyl silica, (2) 2-bromoisobutyrate-modified silica, (3) SID-5, (4) SID-10.

Table 1. Characterization of Initiator and Poly(NIPAAm-coDEAEMA-co-tBAAm)-Modified Silica elemental compositiona (%) codeb 3-aminopropyl silica 2-bromoisobutyratefunctionalized silica SID-5 SID-10

3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of CopolymerModified Silica Particles. The preparation of cationic temperature-responsive copolymer-modified silica microparticles by SI-ATRP is illustrated in Figure 1. The bromo groups in 2-bromoisobutyryl on the silica surface were the ATRP initiation sites. 3-Aminopropyl was used for the immobilization of 2-bromoisobutyryl bromide on the silica surface. To obtain a proper grafting density of the copolymers on silica surface, only a fraction of the amino groups were reacted with 2bromoisobutyryl bromide, whereas the other amino groups were reacted with acetic chloride. The preparation of the cationic temperature-responsive stationary phase by SI-ATRP was carried out with NIPAAm as the temperature-responsive component, DEAEMA as the pH-sensitive component, and tBAAm as a hydrophobic component in 2-propanol/H2O with CuCl/PMDETA as the catalyst. Previous reports suggested that the introduction of a small amount of ionic monomer into PNIPAAm could greatly affect the collapse of the polymer chains, resulting in an increase of its LCST.37,40 However, higher LCSTs are not suitable for temperature-responsive chromatography because the column temperature must be elevated to modulate the electrostatic properties. Therefore, DEAEMA was employed, and the hydrophobic tBAAm was incorporated to synthesize the poly(NIPAAm-co-DEAEMA-cotBAAm) copolymers. The contents of the copolymers grafted onto the silica surface were quantitatively determined by TGA, as presented in Figure 2. From 100 to 750 °C, the weight losses of ATRPinitiator immobilized silica, SID-5, and SID-10 were found to be 2.46%, 13.77%, and 25.18%, respectively. Based on eq 1, the grafting ratios of the copolymer on SID-5 and SID-10 were calculated as 11.31% and 22.72%, respectively (see Table 1 for details).

C

H

N

grafting ratioc (%)

2.11 2.54

0.46 0.49

0.50 0.48

− 1.35

8.20 11.65

1.43 1.95

1.65 2.12

11.31 22.72

a Determined by CHN elemental analyses (n = 2). bSID-x represents copolymer-modified silica with the x% molar feed ratio of DEAEMA to NIPAAm. cDetermined by thermogravimetric analysis.

The results of elemental analysis (C, H, and N) are also summarized in Table 1. Elemental compositions of C, H, and N increased as a result of the polymerization process for SID-5 and SID-10. Furthermore, the grafting ratio and elemental compositions for SID-10 were higher than those for SID-5. These results indicate that the feed ratio of DEAEMA affected the amount of PDEAEMA in the grafted polymers. The greater the feed ratio, the larger the amount of DEAEMA grafted onto the silica surface. These results provide evidence that poly(NIPAAm-co-DEAEMA-co-tBAAm) copolymers containing different DEAEMA contents were successfully grafted onto the silica surface. GPC analysis showed a number-average molecular weight (Mn) of 55033 and a PDI of 1.48 for SID-5. (The GPC curves are shown in Figure S1 of the Supporting Information.) The grafting density of the copolymer was calculated to be 0.024 chains/nm2. The Mn value of the polymer cleaved from SID-10 was 72075, and the PDI was 1.39. The corresponding grafting density of the copolymer was calculated to be 0.035 chains/ nm2. Figure 3 shows SEM images of the polymer-grafted silica and unmodified silica surfaces. Very distinctive surface morphologies can be observed. The polymer-grafted silica was covered with smooth copolymer layers, whereas a porous surface was observed for the unmodified silica particles. The cationic copolymers were cleaved from the modified silica surface, and their temperature-responsive phase-transition temperatures were measured in solutions of different pH values and different ionic strengths at various temperatures, as shown in Figure 4 and Table 2. As can been seen in Figure 4, D

DOI: 10.1021/acs.iecr.7b04570 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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Figure 3. SEM images of the silica microparticles: (a) unmodified silica microparticle, (b) SID-5, (c) SID-10.

of PDEAEMA through the shielding of the polymer charges by counterions.37 Thus, the hydrophilicities of the copolymers were weakened upon the addition of NaCl to the solution. In conclusion, the temperature-sensitive properties of the copolymers were greatly affected by the DEAEMA content, the pH, and the ionic strength of the solutions. 3.2. Effects of Temperature on the Elution Behaviors of Adenosine Nucleotides. Figure 5 shows the chromatograms of three adenosine nucleotides (AMP, ADP, and ATP) at various temperatures on the SID-5 column in various mobile phases. Figure 6 shows the changes in retention time with temperature in various mobile phases. The retention times and resolution of these adenosine nucleotides decreased significantly as the column temperature was raised. Short retention times with overlapping peaks at elevated temperature resulted in poor resolution. The three adenosine nucleotides have similar chemical structures (adenosine nucleotide chemistry) but different numbers of anionic phosphate units, which led to different strengths of their electrostatic interactions with the cationic copolymer-modified surface. The elution times increased in the order AMP < ADP < ATP, in agreement with the number of anionic phosphate units. The changes in the temperature-dependent retention behaviors can be attributed to changes in the charging properties of the cationic thermoresponsive copolymer on the stationary-phase surface. Feil et al. reported that the basicity of DEAEMA in its copolymer with NIPAAm decreased strongly as the temperature was increased and attributed this change to the enhanced hydrophobicity of the copolymer sequence and the subsequent increase in th dehydration temperature.37,41 The cationic copolymer gradually became hydrophobic as the temperature was increased, which led to a decrease of the degree of dissociation of the amino groups in the copolymer. Therefore, the decrease of the charge density in the copolymer with the increase in temperature resulted in a weakened electrostatic interaction between the adenosine nucleotides and the stationary phase. The weak retention of these adenosine nucleotides was observed at elevated temperature, which might due to the weak hydrophobic interactions between the hydrophilic adenosine nucleotides and the stationary phase. Therefore, their retention times decreased as the temperature

Figure 4. Temperature dependence of the optical transmittance of poly(NIPAAm-co-DEAEMA-co-tBAAm) in 10 mmol/L phosphate buffer solution.

the LCSTs of the copolymers in various solutions were higher than that of PNIPAAm (i.e., 32 °C) because DEAEMA in the copolymers prevented their dehydration. The LCSTs of the copolymers were shifted to higher temperatures with increasing DEAEMA content. The LCSTs of ID-10 and ID-5 in phosphate buffer solution (10 mmol/L, pH 7.3) were 42.8 and 35.2 °C, respectively. The LCST of poly(NIPAAm-coDEAEMA-co-tBAAm) was relatively lower than those of PNIPAAm copolymers containing PDMAEMA or PDMAPAAm.34,35 The LCST of ID-5 was increased markedly with decreasing pH value, which can be explained by the fact that PDEAEMA is uncharged at higher pH and charged at lower pH.37,41 The degree of protonation of the amino groups in the copolymers strengthened the hydrophilicity similarly to the incorporation of a hydrophilic comonomer into PNIPAAm. Therefore, a higher LCST was observed in the lower-pH solution. Ionic strength affected the LCST of the copolymers upon the addition of NaCl to the solution (Table 2). The LCSTs of the copolymers decreased with increasing the ionic strength (higher concentration of NaCl). A previous report showed that the addition of NaCl to the solution could lower the phase-transition temperature of PNIPAAm. In addition, increased ionic strength can also increase the pKb

Table 2. LCSTs of Poly(NIPAAm-co-DEAEMA-co-tBAAm) Cationic Copolymer LCSTa (°C) code

b

ID-5 ID-10

pH 6.7

pH 7.0

pH 7.0 (0.02 mol/L NaCl)

pH 7.0 (0.04 mol/L NaCl)

pH 7.3

38.2

37.4

36.8

36.2

35.2 42.8

a

Defined as the temperature at 50% transmittance in 10 mmol/L phosphate buffer solution. bID-x represents copolymers cleaved from silica surface with the x% molar feed ratio of DEAEMA to NIPAAm. E

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Figure 5. Chromatograms of adenosine nucleotides on SID-5 packed column at various temperatures. Flow rate, 0.1 mL/min; detection wavelength, 260 nm; Mobile phase, 10 mmol/L phosphate buffer solution at (a) pH 7.3, (b) pH 7.0, (c) pH 6.7, (d) pH 7.0 with 0.02 mol/L NaCl, and (e) pH 7.0 with 0.04 mol/L NaCl. Peaks: (1) AMP, (2) ADP, (3) ATP.

Figure 6. Temperature-dependent retention time changes of adenosine nucleotides on SID-5 packed column: (■) AMP, (●) ADP, (▲) ATP. Mobile phase: 10 mmol/L phosphate buffer solution at (a) pH 7.3, (b) pH 7.0, (c) pH 6.7, (d) pH 7.0 with 0.02 mmol/L NaCl, and (e) pH 7.0 with 0.04 mmol/L NaCl.

Figure 7. van’t Hoff plots of adenosine nucleotides on SID-5 packed column: (■) AMP, (●) ADP, (▲) ATP. Mobile phase: 10 mmol/L phosphate buffer solution at (a) pH 7.3, (b) pH 7.0, (c) pH 6.7, (d) pH 7.0 with 0.02 mmol/L NaCl, and (e) pH 7.0 with 0.04 mmol/L NaCl.

was raised, even though the surface of the stationary phase became hydrophobic at higher temperatures. The van’t Hoff plots for these adenosine nucleotides are shown in Figure 7 and exhibit a relationship between the column temperature and the reciprocal temperature (1/T). The value of the retention factor k′ is defined as k′= (tR − t0)/ t0, where tR is the retention time of the adenosine nucleotide and t0 is the retention time of adenosine as the initial standard. The slope of the retention factor decreased above the LCSTs of the copolymers, indicating that the interaction between the adenosine nucleotides and the stationary phase was weakened above the LCSTs. The data also illustrate that the hydrophobic interaction is not the predominant factor for the retention of the adenosine nucleotides, whereas the temperature-dependent

electrostatic interaction predominantly influences the separation of the adenosine nucleotides. 3.3. Effects of the pH of the Mobile Phase on the Retention Behaviors of Adenosine Nucleotides. Conventional ion-exchange chromatography is carried out with the gradient-elution technique by changing the mobile-phase composition. Here, the charging properties of the novel temperature-responsive stationary-phase surface were modulated by adjusting the external temperature rather than changing the mobile-phase composition. The influence of the pH value of the mobile phase on the separation efficiency of the adenosine nucleotides was investigated. As shown in Figure 5, excellent resolution was achieved at temperatures below the LCST, and the separation efficiency decreased with increasing pH at a given temperature above the LCST. For instance, F

DOI: 10.1021/acs.iecr.7b04570 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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graphic separation adenosine nucleotides was performed on SID-10 packed column in 10 mmol/L phosphate buffer solution (pH 7.3). Figures 8 and 9 show chromatograms, retention times, and van’t Hoff plots of the adenosine nucleotides at various temperatures.

AMP, ADP, and ATP were completely separated in phosphate buffer solution (pH 6.7) at 50 °C. They were separated partially at pH 7.0 and were unresolved at pH 7.3. The retention times of the adenosine nucleotides increased with decreasing pH of the mobile phase. The results illustrate that the pH value of the mobile phase has a strong effect on the separation efficiency of adenosine nucleotides. Similarly to the influence of the pH on the LCST, the degree of dissociation of the amino groups in the copolymer decreased, which resulted in a reduced charge density and subsequent weakened electrostatic interaction when the pH was raised. The electrostatic interaction was the dominant driving force during the separation process. Therefore, the retention times of the adenosine nucleotides decreased, and the resolution became poor with increasing pH of the mobile phase, particularly at temperatures above the LCST. Meanwhile, the hydrophilicity of the copolymer was stronger at lower pH owing to the increasing ionization of the amino groups. As shown in Table 2, the phase transition occurred at a much higher temperature in the lower-pH solution. The electrostatic interactions of analytes with the stationary phase at lower pH are stronger than those at higher pH at the same temperature. It should be noted that the peak width was also broadened at lower pH values, which was not suitable for separation even though the retention time was prolonged. 3.4. Effects of the Ionic Strength of Solutions on the Retention Behaviors of Adenosine Nucleotides. To investigate the effects of ionic strength on the separation properties of the cationic temperature-dependent column, we evaluated the retention behaviors of the three adenosine nucleotides on the SID-5 packed column at different NaCl concentrations (as shown in Figure 5). The retention times decreased dramatically upon the addition of NaCl to the mobile phase. A shorter retention time was observed at higher ionic strength (higher concentration of NaCl). These results indicate that the ionic strength of the mobile phase has a significant effect on the elution behavior of the adenosine nucleotides. A previous study showed that increasing the ionic strength causes an increase in the pKb of amino group, because if the shielding of the polymer charges by counterions.37 Therefore, the electrostatic interactions between the analytes and the stationary phase were weakened by the addition of NaCl. As shown in Table 2, the addition of salt enhanced the hydrophobicity of the copolymers and resulted in a lower phase-transition temperature. The data are in agreement with previous reports.12 Thus, the charge density of the grafted copolymers decreased, and subsequently, the electrostatic interactions were weakened. In addition, Cl− in the salt solutions could interact with the cationic surface, which generated a competition effects on the electrostatic interactions between the adenosine nucleotides and the cationic surface. The electrostatic interactions were weakened in the presence of the competition. Consequently, the retention times of the adenosine nucleotides were shortened. The adenosine nucleotides were eluted much more easily in the mobile phases with higher ionic strengths. 3.5. Effects of the DEAEMA Content in the Copolymers on the Elution Behaviors of Adenosine Nucleotides. To investigate the influence of DEAEMA contents in the copolymers on the interfacial electrostatic properties of the Poly(NIPAAm-co-DMAEMA-co-tBAAm) surface, chromato-

Figure 8. Chromatograms of adenosine nucleotides on SID-10 packed column at various temperatures. Mobile phase: 10 mmol/L phosphate buffer solution (pH 7.3); flow rate, 0.1 mL/min; detection wavelength, 260 nm. Peaks: (1) AMP, (2) ADP, (3) ATP.

Figure 9. (a) Temperature-dependent retention time changes and (b) van’t Hoff plot of adenosine nucleotides on SID-10 packed column: (■) AMP, (●) ADP, (▲) ATP. Mobile phase: 10 mmol/L phosphate buffer solution (pH 7.3).

As shown in Figure 9, the retention times of the adenosine nucleotides were longer and their peak widths were much broader on the SID-10 packed column than on the SID-5 packed column at various temperatures. These results indicate that stronger electrostatic interactions occurred between the SID-10 surface and the analytes. The reason is that more amino groups were immobilized on the SID-10 surface and interacted with the adenosine nucleotide analytes, leading to longer retention times. As mentioned above, the phasetransition temperature increased in the presence of DEAEMA in the copolymer. The LCST of ID-10 was found to be 7.6 °C higher than that of ID-5 in 10 mmol/L phosphate buffer solution (pH 7.3). This result indicates that the hydrophilicity of ID-10 was stronger than that of ID-5 in a certain range of temperatures. Therefore, the charge density of the SID-10 G

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surface was higher than that of the SID-5 surface. This led to a stronger electrostatic interaction between the SID-10 surface and the analytes. As shown in Table 1, we also found that increasing the feed composition of DEAEMA can improve the grafting ratio of the copolymer on the silica surface, probably because of the higher reactivity of the DEAEMA monomer.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.iecr.7b04570. GPC curves and results (PDF)

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REFERENCES

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4. CONCLUSIONS A chromatographic stationary phase modified with the temperature-, pH-, and ionic-responsive copolymer poly(NIPAAm-co-DEAEMA-co-tBAAm) was prepared by SIATRP, and the DEAEMA contents in the copolymers were modulated by the feed composition of the DEAEMA monomer in the ATRP process. Simultaneous changes in the charging properties and hydrophobicity of the stationary-phase surface were induced with external temperature. The chromatographic separation of three adenosine nucleotides was achieved with a single mobile phase through the control of the column temperature. The results showed that the degree of ionization of the diethylamino groups in the copolymers played a key role in achieving separation at various temperatures. The temperature-dependent charging properties were greatly influenced by the pH value and ionic strength of the mobile phase, as well as the DEAEMA content in the stationary phase. Therefore, the separation conditions, such as the column temperature, pH, and ionic strength of the mobile phase, and the DEAEMA content have significant effects on the chromatographic behavior, which suggests that we have successfully designed a system capable of multiple controlled chromatography. In addition, electrostatic interactions are the predominant driving force for the separation of the three hydrophilic analytes, although the surface hydrophobic properties were strengthened at elevated temperature. In the future, the stationary phase might become a useful means for separating biomolecules with a charged moiety, such as peptides and proteins, at low temperature and for separating hydrophobic biomolecules at high temperatures.

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Article

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Jin Wang: 0000-0003-1573-574X Author Contributions §

Z.-J.L. and S.H. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported by the National Natural Science Foundation of China (81671161, 21404117, 51773225), the Natural Science Foundation of Jiangsu Province (BK20151234), and the Suzhou Science and Technology Bureau (SYG201630). H

DOI: 10.1021/acs.iecr.7b04570 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.iecr.7b04570 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX