Preparation of polyhedral oligomeric silsesquioxane based imprinted monolith

Journal of Chromatography A, 1425 (2015) 180–188

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Preparation of polyhedral oligomeric silsesquioxane based imprinted monolith Fang Li, Xiu-Xiu Chen, Yan-Ping Huang ∗ , Zhao-Sheng Liu ∗ Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics (Theranostics), School of Pharmacy, Tianjin Medical University, Tianjin 300070, China

a r t i c l e

i n f o

Article history: Received 6 July 2015 Received in revised form 27 October 2015 Accepted 10 November 2015 Keywords: Molecularly imprinted polymers Monolith Polyhedral oligomeric silsesquioxane Molecular recognition Naproxen

a b s t r a c t Polyhedral oligomeric silsesquioxane (POSS) was successfully applied, for the first time, to prepare imprinted monolithic column with high porosity and good permeability. The imprinted monolithic column was synthesized with a mixture of PSS-(1-Propylmethacrylate)-heptaisobutyl substituted (MA 0702), naproxon (template), 4-vinylpyridine, and ethylene glycol dimethacrylate, in ionic liquid 1butyl-3-methylimidazolium tetrafluoroborate ([BMIM]BF4 ). The influence of synthesis parameters on the retention factor and imprinting effect, including the amount of MA 0702, the ratio of template to monomer, and the ratio of monomer to crosslinker, was investigated. The greatest imprinting factor on the imprinted monolithic column prepared with MA 0702 was 22, about 10 times higher than that prepared in absence of POSS. The comparisons between MIP monoliths synthesized with POSS and without POSS were made in terms of permeability, column efficiency, surface morphology and pore size distribution. In addition, thermodynamic and Van Deemter analysis were used to evaluate the POSS-based MIP monolith. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Molecular imprinting has become a powerful method for the preparation of polymeric materials that have the ability to bind a specific chemical species [1–4], which typically involves copolymerization of functional and crosslinking monomers in the presence of a template molecule and suitable porogenic solvent. Subsequent removal of the template molecules results in molecularly imprinted polymers (MIPs) with recognition sites that can interact with the template molecule specifically. The advantages of the resultant polymers are low cost, good physical and chemical stabilities. Recent reviews have summarized the current status of MIPs as alternatives to enzyme-like catalysis [5], bio-mimetic sensors [6], drug delivery system [7], solid-phase extraction [8], chromatographic stationary phase [9] and smart polymers [10]. Historically, there are two major approaches to MIPs, i.e., organic and inorganic MIPs. The former, polymeric MIPs, have the advantage of simple polymerization procedure and easy tuning of porosity and surface chemistry, but may suffer from shrinking or swelling when exposed to different solvents, leading to lack

∗ Corresponding authors. Fax: +86 022 23536746. E-mail addresses: [email protected] (Y.-P. Huang), [email protected] (Z.-S. Liu). http://dx.doi.org/10.1016/j.chroma.2015.11.039 0021-9673/© 2015 Elsevier B.V. All rights reserved.

of mechanical stability [3]. In contrast, inorganic materials, e.g., silica-based MIPs, can offer excellent mechanical strength and good solvent resistance. However, it was observed that the silica-based MIP often lost its memory for the template over time (low stability), and reproducibility in preparation may vary by 30% between batches [1,2]. Thus, organic–inorganic hybrid MIPs are supposed to combine the merits of the organic polymer and inorganic-based materials. Earlier works on the hybrid MIPs focused on the MIP film anchored covalently on the surface of silica [11–13]. An alternative approach to the preparation of the hybrid MIP is using a room temperature ionic liquid (RTIL)-mediated nonhydrolytic sol–gel protocol [14,15]. In additional, several research groups have attempted to prepare MIPs doping metal-organic framework (MOF) [16]. Recently, the hybrid MIPs grafted on iniferter-modified carbon nanotube (CNT) [17] and GO [18] were also reported. Polyhedral oligomeric silsesquioxanes (POSS) are a class of nanofillers for polymers [19,20], which embody truly inorganicorganic cagelike architecture containing an inner inorganic framework made up of silicon and oxygen. The study of POSSpolymers over the last 20 years has yielded conclusive proof that incorporation of the POSS into a polymer matrix can result in significant improvements in a variety of physical and mechanical properties due to the reinforcement at the molecular level and the inorganic framework’s ceramic-like properties [21–23]. Their unique star-shaped nanostructures and chemical properties, such

F. Li et al. / J. Chromatogr. A 1425 (2015) 180–188

as facile chemical modification, good pH tolerance, high temperature and oxidation resistance properties, make POSS excellent building block for constructing multi-functional materials. In addition, POSS chemical reagents are thought to be the smallest silica particles with sizes of 1–3 nm, which can be easily incorporated into common polymers via copolymerization, grafting, or blending [24–26]. In addition, the end-capping by POSS can drastically enhance the rigidity of the polymers [27]. For example, it was found from dynamic mechanical analysis (DMA) that a large increase of the modulus dramatically by 2-fold was observed with only 5 wt% vinyl-POSS [28]. In spite of this, so far there are no reports on the incorporation of the POSS into MIPs matrix. This paper described a novel method for the preparation of MIP monolith based on POSS. The objective of the work was to take advantage of rigidity reinforcement at the molecular level from POSS to improve the performance of MIP monoliths. Such MIP monoliths are expected to have higher imprinting efficiency than conventional POSS-free MIP since the POSS unit is introduced to control the pore structure and wettability of the obtained MIP. In contrast to the role of POSS as functional monomer [26] or crosslinker [24] used in previous POSS-based monoliths, the function of POSS unit here is co-monomer to supress the non-selective binding sites. Naproxen was selected as model template, arising from very high solubility in the polymerization system. By our knowledge, this was the first work to prepare MIP with POSS. The influence of polymerization parameters on the imprinting performance of the resultant MIP was investigated in the present study. The textural and morphological parameters of the MIP monoliths, such as mode pore diameter and pore size distributions, were also measured.

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2,2-Azobis (2-isobutyronitrile) (AIBN) (analytical grade) was supplied by Kermel Chemical Reagent (Tianjin, China). 1-Butyl3-methylimidazolium tetrafluoroborate ([BMIM]BF4 ) (98%) was obtained from Shanghai Chengjie Chemical Reagent (Shanghai, China). PSS-(1-Propylmethacrylate)-heptaisobutyl substituted (MA 0702) was purchased from Sigma (St. Louis, MO, USA). Methyl methacrylate (MMA) (analytical grade) was from Tianjin Kewei Chemical Reagent (Tianjin, China). HPLC-grade acetonitrile (ACN) was purchased from Tianjin Biaoshiqi Chemical Reagent (Tianjin, China). Kermel Chemical Reagent (Tianjin, China) supplied dimethyl sulfoxide (DMSO) (analytical grade). Other analytical reagents were from Tianjin Chemical Reagent Ltd. Co. (Tianjin, China). 2.2. Preparation of POSS-based imprinted monoliths The preparation of NAP-imprinted monolith was carried out as follows. A pre-polymerization mixture was prepared by mixing 4-VP, MA 0702 or MMA, EDMA, [BMIM]BF4 , DMSO, NAP, and AIBN (15 mg), as shown in Table 1. The pre-polymerization mixture was sonicated for 15 min and introduced into a stainless steel column (100 mm × 4.6 mm). With the ends sealed, the column was submerged in a 60 ◦ C water bath for 90 min. The resulting column was connected to a HPLC pump and flushed with acetonitrile to elute any unreacted reagents. The template molecules were removed by washing with a mixture of methanol and acetic acid (9:1, v/v) until no template molecules were detected in the extraction solvent. The blank polymer in the absence of NAP was synthesized in same manner. 2.3. Chromatographic evaluation

2. Experimental 2.1. Materials and instruments Naproxen (NAP) (98%) was obtained from Zhejiang Xianju Chemical Reagent (Zhejiang, China). Ibuprofen (IBU), ketoprofen (KET), flurbiprofen (FLU) and fenbifen (FENBI), were from Wuhan Yuancheng Chemical Reagent (analytical grade, Wuhan, China). 4-Vinylpyridine (4-VP) (98%) and ethylene glycol dimethacrylate (EDMA) (98%) were purchased from Sigma (St. Louis, MO, USA).

High performance liquid chromatography was performed with an Agilent 1100 series chromatographic system. Data processing was carried out by a HPCORE workstation and the samples were monitored at 254 nm. All of mobile phases were filtered through a 0.22 ␮m membrane from Millipore before use. The injection volume was 20 ␮L and each sample was analyzed three times. Imprinting factor (IF) was calculated as IF = kMIP /kNIP

(1)

Table 1 The compositions of imprinted polymers (MIPs) and non-imprinted polymers (NIPs). Monolithic column

NAP (mmol)

POSS (mmol)

4-VP (mmol)

EDMA (mmol)

DMSO (mL)

[BMIM]BF4 (mL)

Time (h)

C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 C19 C20

0.25 0.33 0.50 0.67 1.00 0.50 0.50 0.50 0.50 0.50 – – – – 0.50 0.50 0.50c 0.50d 0.50c 0.50d

0.15 0.15 0.15 0.15 0.15 1.60a 0.05 0.10 0.15 0.15 0.15 1.60a 0.05 0.10 0.20 0.15 0.15 0.15 0.15a 0.15a

2.01 2.01 2.01 2.01 2.01 2.01 2.01 2.01 2.01 2.01 2.01 2.01 2.01 2.01 2.01 2.01 2.01 2.01 2.01 2.01

15.61 15.61 15.61 15.61 15.61 15.61 15.61 15.61 15.61 15.61 15.61 15.61 15.61 15.61 15.61 11.71 15.61 15.61 15.61 15.61

0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20

0.66 0.66 0.66 0.66 0.66 0.66 0.66 0.66 0.66 0.66 0.66 0.66 0.66 0.66 0.66 0.66 0.66 0.66 0.66 0.66

2 2 2 2 2 1.5 1.5 1.5 1.5 2.5 1b 1.5 1.5 1.5 – – 2 2 2 2

a b c d

MMA Polymerization time beyond 1 h led to NIP monolith with very high column pressure IBU KET

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where kMIP is the retention factor of the template molecule eluted from the imprinted polymer and kNIP is the retention factor of the template molecule eluted from the non-imprinted polymer [29]. 2.4. Frontal analysis In order to investigate the interactions between solutes and MIP stationary phase, frontal analysis was carried out using different solutions of NAP (0.001–0.24 mg L−1 ) in ACN/acetate (pH 3.6, 50 mmol L−1 ) (99/1, v/v) as mobile phase at a flow rate of 1.0 mL min−1 . The experiments data was processed with the following equation [30]: 1/{[A0 ](V − V0 )} = Kdiss /{[A0 ] Bt } + 1/Bt

(2)

where [A0 ] is the concentration of NAP, V and V0 are the elution volume of the template and void volume (marked with acetone) of the monolithic MIPs, respectively. 1/{[A0 ](V − V0 )} was plotted versus 1/[A0 ] and the number of affinity sites (Bt ) and dissociation constant (Kdiss ) were calculated from the intercepts (1/Bt ) and the slope (Kdiss /Bt ). 3. Results and discussion 3.1. Preparation of POSS-based imprinted monolith with NAP imprints 3.1.1. Demonstration of enhanced imprinting effect of POSS-based MIP This work is an effort to improve the imprinting effect of MIPs with POSS monomer. The enhanced molecular recognition towards the imprint species is expected to be achieved by reducing nonspecific interactions using POSS. To investigate the effect of POSS monomer on enhancing the affinity of the resulting MIPs, we prepared POSS-based MIPs in a monolithic format (Table 1). In the present work, porogen formulation is crucial to prepare POSS-based NAP MIP. First, the porogenic solvent can dissolve POSS. Next, the porogen should produce large pores to assure good flow-through properties of the resultant MIP. It was found that a previously developed porogenic system, a mixture of DMSO and [BMIM]BF4 , can solve the problems above [31]. [BMIM]BF4 was used as porogenic solvent because of its unique ability to create MIP monolith with high column permeability. Other imidazolium-based ILs containing the same anion (BF4 − ) but different cation alkyl chain length (butyl to hexadecyl) led to the POSS-based MIP monoliths with very high back pressure, which can not be evaluated further. [BMIM]+ -type ionic liquids with fixed cations were also used as a component of the porogenic solvent. However, the miscibility of the [BMIM]+ -type ionic liquid ([BMIM]PF6 or [BMIM]HSO4 ) with the pre-polymerization mixture limited the use of the cations-type ILs in this study. The selectivity of the resultant MIP monolith was evaluated in acetonitrile with acetate buffer (50 mmol L−1 , pH 3.6) as modifiers (99:1, v/v). Greater difference of retention factor between the POSSbased MIP C3 (k = 11.5) and POSS-based NIP C11 (k = 0.78) can be observed and the imprinting factor of NAP was 14.6 (Fig. 1). Other analogues of NAP were also retained on the POSS-based MIP, but to a lesser extent less retained than the template (Fig. S1). Selectivity factor (S), defined by the ratio of retention factor of the analogues of NAP (IBU and FENBI) between imprinted (C9) and non-imprinted column (C11) was 1.94 and 1.28, respectively. For comparison, a non-hybrid MIPs monolith (C6) was prepared in absence of POSS. In the recipe of preparing the POSS-free MIPs monolith, POSS was replaced with an equal weight of nonfunctional monomer MMA. This substitution attempts to ensure that the total weight of the prepolymerization mixture remains constant. The retention factor of POSS-free MIP C6 and POSS-free

Fig. 1. Chromatograms for NAP on the POSS-based imprinted (C3) and non-imprinted monoliths (C11) to illustrate imprinting effect. Mobile phase, acetonitrile-acetate buffer (50 mmol L−1 , pH 3.6) (99:1, v/v); flow rate, 0.5 mL min−1 ; detection wavelength, 254 nm; temperature, 25 ◦ C.

NIP C12 was 4.98 and 2.07, respectively. In contrast to the POSSbased MIPs monolith, the non-hybrid imprinted monolith showed smaller imprinting factor (IF = 2.41). Furthermore, selectivity factor of the analogues of NAP (IBU and FENBI) between imprinted (C6) and non-imprinted column (C12) was 1.57 and 1.21, respectively, which was lower than POSS-based MIP C3. It seemed that the POSS-free MIP had smaller affinity and greater non-selective retention than the POSS-based MIP by comparing the retention factor of two MIPs. The sample load capacity of the POSS-based MIP was determined by successive injections of NAP at different concentrations. A decrease of the retention factor for the template was observed (Fig. S2). This result was different from to the work of Andersson et al. [32], suggesting that higher order complexes were not formed in the POSS-based MIP. The reason may be attributed to unfavourable conformation induced by the larger rotation diameter of the higher order complexes for NAP, limiting its access to the binding sites at highly crosslinking network formed by POSS. 3.1.2. Effect of the content of organic modifier and pH on imprinting The selectivity of the POSS-based MIP can be tailored by changing the content of organic modifier. The mixture of acetonitrile-acetate buffer solution (50 mmol L−1 , pH 3.6) was used as the mobile phase, with the acetonitrile content ranging from 70% to 99% (Fig. 2a). The results suggested that the contribution of hydrophobic interactions for recognition in this system studied is not mainly specific in character and hydrogen bonding and/or ionic interaction is significant in the defining of imprint recognition sites during the pre-organization of the template and functional monomer. For example, the retention factors of NAP and its analogues all decreased when acetonitrile content changed from 70% to 90% (hydrophobic interactions-controlled recognition). In contrast, the retention factors of all the analytes increased rapidly with the increase in acetonitrile content from 90% to 99% (hydrogen bonding-controlled recognition). At 90% ACN, both hydrophobic and hydrogen bonding interactions are weak and the retention factor is the smallest. Little difference in retention properties for NAP and its analogues on the POSS-based NIP demonstrated that the shift in retention factors on the POSS-based MIPs is due to imprinting effect. The dependence of NAP and its analogues on pH was investigated over the pH range of 3.0 to 5.0 on the POSS-based MIP (C3) (Fig. 2b). The retention factor and imprinting fact were all decreased with the increase in pH value. Apparently, the retention

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Fig. 3. Imprinting factors of MIPs with the different amount of POSS. HPLC conditions: column temperature, 28 ◦ C; mobile phase, acetonitrile–acetate buffer (50 mmol L−1 , pH 3.6) (99:1, v/v); detection wavelength, 254 nm; flow rate, 0.5 mL min−1 .

Fig. 2. Influence of organic phase composition (a) and pH (b) on imprinting factor and retention factors of NAP and its structural analogues. Mobile phase, acetonitrile/acetate buffer (50 mM) (95:5, v/v); flow rate, 0.2 mL min−1 ; detection wavelength, 254 nm; injected volume, 20 ␮L; temperature, 28 ◦ C.

behavior is dependent on the degree of ionization of the vinylpyridine residues. In looking at the non-imprinted control (C6), little change of the retention for NAP as increasing pH was observed. This result could be explained by the protonation level of the 4VP and the degree of deprotonation of NAP in the different mobile phase pH (pKa 4.2 for NAP). At higher pH (5.0), ionic exchange will be diminished, thus the decrease in the retention of the template and discriminating ability of the polymer was observed. In contrast, NAP and 4-VP are either positively charged or undissociated at pH below 4.2, increased retention factor are due to strong hydrophobic interactions at this mobile phase. This result is in contrast to the previously reported 4-VP-based MIP, in which constant retention were observed with eluent pH between 2.4 and 4.8 [4]. The change of affinity in this investigation also support the fact that imprinting effect derived from hydrophobic interactions on the POSS-based MIP is stronger than that on the 4-VP-based MIP. 3.2. Role of POSS on selectivity of MIP Higher mechanical reinforcement of copolymers containing POSS may arise from aggregates of these units into larger POSS clusters. The degree of aggregation may be expected to depend on the mole fraction of POSS monomer, or the degree of compatibility of the POSS with the host polymer component. Thus, the effect of MA0702 amount on the affinity of the resulting NAP MIPs

was further investigated to understand the function of POSS. As shown in Fig. 3, an increased trend of retention for NAP on the POSS-based MIPs was observed. In contrast, a decreased trend of retention for NAP on the POSS-based NIPs was found. Obviously, the introduce of POSS into MIP matrix caused the decreases in nonselective retention. Further increase in the amount of POSS led to an insoluble mixture and the preparation of MIP monolith was impossible. It seemed that the interaction between POSS monomer and the template was not involved in the recognition of the resulting MIP from an NMR study with a pseudo-pre-polymerization mixture consisting of MA0702 and NAP (Fig. S3). The polymerization time provides a control over the pore property of the resulting MIP monolith depending on the conversion. Generally, the specific surface area of MIP monolith decreases with the polymerization time. Although the conversion of monomers to polymer is close to quantitative after about 1.5 h, some additional structural changes still occur within the MIP monolith if the system is kept longer at the polymerization temperature. Table 2 shows that the imprinting factor reaches a maximum after about 2 h of polymerization (IF = 14.6). Further increase of polymerization time led to the decline of imprinting factor (IF = 13.6), along with decreased retention. A possible reason of the decrease in imprinting effect is that the specific surface area of the MIP prepared at 2.5 h was too low to provide adequate imprinted cavities. 3.3. Influence of template-to-monomer ratio on the imprinting effect Since the cavities formed in MIPs depended on the template molecule content, further experiments were performed to determine the optimum molar ratio of template-to-monomer (T/M). In this work, a pronounced influence of T/M ratio on the column permeability and efficiency was found for the POSS-based MIPs. As shown in Fig. 4, an increased trend of imprinting factor with the increase in amounts of NAP was observed on the POSS-based MIP. This may be attributed to more affinity sites on Table 2 Retention factor and imprinted factor of NAP and its structural analogues with the different synthesis time. MIP

Time (h)

kNAP

kIBU

kFLU

kFENBI

IF

C9 C3 C10

1.5 2 2.5

7.94 11.43 10.64

4.09 6.03 7.12

4.15 6.03 6.99

6.19 7.86 10.15

10.2 14.6 13.6

Mobile phase, acetonitrile–acetate buffer (50 mmol L−1 , pH 3.6) (99:1, v/v); flow rate, 0.5 mL min−1 ; detection wavelength, 254 nm; temperature, 25 ◦ C.

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Fig. 4. Imprinting factor of POSS based MIPs with the different ratio of functional monomer to template. Mobile phase, acetonitrile-acetate buffer (50 mmol L−1 , pH 3.6) (99:1, v/v); flow rate, 0.5 mL min−1 ; detection wavelength, 254 nm; temperature, 25 ◦ C.

the resulting MIP with an increase in amounts of the template in the pre-polymerization mixture [32]. Compared with the reported NAP-MIP containing 4-VP [4], higher T/M ratio (1:4) in this case caused less functional monomer unassociated in the prepolymerization mixture. This leads to a decrease in the random integration of the functional monomer into the polymer matrix and produces a smaller population of low-affinity binding sites. Thus, a large fraction of functional monomer forms higher order complexes that presumably yield the high-affinity binding sites on polymerization [1]. Further increase in the amount of the template caused NAP insoluble in the pre-polymeriztion system. Thus, T/M ratio of 1:2 (C5) was found to be the optimum in terms of imprinting factor (IF = 22). Considering shorter retention time, following experiments were performed with the MIP at T/M ratio of 1/4.

H and S can be calculated form the slopes and the intercepts of linear portion of corresponding Eq. (3). This approach can offer an insight into the nature of the interaction between the template and the MIP and help to elucidate the chemical forces underlying the interactions. The plots for the POSS-based MIP and NIP indicated two intersected linear parts with different slopes. The linear portions were fitted by linear regression analysis, giving values assigned as apparent H and S (Table 3). The correlation coefficients for the fits were at least equal to 0.998. The major change in the slopes of Van’t Hoff plots observed at 25–35 ◦ C is thought to be associated with a change in mechanism of binding at this temperature interval. The value of apparent H at 25–35 ◦ C was ca. two times larger than that at 40–50 ◦ C. This may be attributed to be related to the aggregation properties of POSS-polymer composite at lower temperatures (300 K) [33]. In contrast, the plots for the POSS-free MIP and NIP (data not shown) were linear, which suggested that no change in mechanism of binding occurred at the range of temperature studied here. Thus, non-linear Van’t Hoff plots may be attributed to the individual POSS cages of POSS-based MIP. 3.5. Comparison of POSS-based and POSS-free MIP monoliths

(3)

3.5.1. van Deemter analysis and column permeability The column permeability of the MIP monoliths was also investigated by determining the pressure drop of monolithic column generated at varying rate from 0.2 to 3.0 mL min−1 (Fig. S4). The permeability (B0 ) measured was 3.04 × 10−8 mm2 , much higher than the ordinary silica packed columns. This further validates the formation of through pores with large pore size in the monoliths. It should be noted that the POSS-based MIP monolith (C3) indicated lower column permeability than the POSS-free MIP monolith (C6) (B0 = 2.09 × 10−7 mm2 ), probably resulting from the formation of smaller through pore in the former. In order to compare the chromatographic behaviors of the POSSbased and POSS-free imprinted monoliths, the column efficiency of the template was measured at different flow rates. As shown in Fig. 6, the theoretical plate number of NAP on the POSS-based MIP monolith C3 is lower than POSS-free MIP C6. A flat Van Deemter plot of height equivalent to a theoretical plate (HETP) for NAP vs superficial velocity was observed on the POSS-free MIP monolith. The lower efficiency for the template could be explained by higher resistance to mass transfer. In contrast, the plate height of NAP on the POSS-based MIP was highly dependent of flow-rate compared to the POSS-free MIP. The slope of the HETP curve for u values higher than 0.5 mL min−1 was 674 and 74.0 s for POSS-based MIP C3 and

Fig. 5. Van’t Hoff plots by plotting lnk vs. 1/T on monolith C3, C6 and C11. Mobile phase, acetonitrile–acetate buffer (50 mmol L−1 , pH 3.6) (99:1, v/v); detection wavelength, 254 nm.

Fig. 6. Van Deemter plots of the POSS-based (C3) and POSS-free imprinted monoliths (C6). Mobile phase, acetonitrile-acetate buffer (50 mmol L−1 , pH 3.6) (99:1, v/v); detection wavelength, 254 nm; temperature, 30 ◦ C.

3.4. Effect of temperature Recent work has shown that the physical crosslinking formed by POSS can significantly retard thermal motion, while at the same time the individual POSS cages can act as flow-aids at elevated temperatures [33]. In the present study, the effect of temperature on the retention of NAP was studied by varying the temperature from 298 K to 318 K on the POSS-based MIP monolith C3 (Fig. 5). Van’t Hoff equation was used to estimate the thermodynamic properties of the separation [34]: ln k = −H/RT + S/R + ln ϕ

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Table 3 Thermodynamic properties of NAP on monolith C3, C6 and C11. Column

C3

Temperature (K) H (KJ mol−1 ) S (J mol−1 K−1 ) R

298–308 −18.43 −39.94 0.998

C11 308–323 −8.08 −6.93 0.998

C6

301–308 21.23 62.66 0.997

313–323 −5.50 −24.88 0.998

298–323 −10.85 −33.38 0.999

Mobile phase, acetonitrile–acetate buffer (50 mmol L−1 , pH 3.6) (99:1, v/v); detection wavelength, 254 nm.

Table 4 Adsorption parameters of different MIPs.

C3 (POSS based MIP) C11 (POSS based NIP) C6 (POSS free MIP)

Bt (␮mol g−1 )

Kdiss (mmol L−1 )

R2

Kf (␮mol g−1 )

m

R2

14.91 78.31 12.60

0.90 7.87 0.48

0.999 0.999 0.999

3.74 0.78 3.82

0.714 0.535 0.557

0.993 0.994 0.990

POSS-free MIP C6, respectively. The result means higher C-term contributions to HETP on the POSS-based MIP monolith, suggesting the feature of micropores of the column.

3.5.2. Binding sites and heterogeneity The dissociation constant (Kdiss ) and the number of binding sites (Bt ) of the template and the imprinted polymer can be calculated with the result of frontal chromatography (Fig. 7). The Kdiss value of the POSS-based and POSS-free MIP monoliths was 0.90 (C3) and 7.87 mmol L−1 (C6), respectively (Fig. S5 and Table 4). The difference in Kdiss value of the POSS MIP and NIP was coincident with the results of imprinting factor obtained from elution chromatography. The number of binding sites for the POSS-based MIP and NIP monoliths was 14.9 and 78.3 ␮mol g−1 , respectively. This meant that non-selective sites have highly been compressed as POSS were introduced into MIP matrix. Freundlich isotherm (FI) is often used to analyze the data of adsorption on MIPs since it is able to accurately measure the heterogeneity of an MIP even though it is usually restricted to a narrow portion of the entire binding isotherm [35]. The equation of Freundlich expression is shown as [36] Qe = Kf Cem

where Qe and Ce are the concentrations of bound and free template, respectively (Fig. 8). The fitted constants, Kf , and m (heterogeneity index), determined from the linear form of equation (4) are shown in Table 4. A more quantitative assessment of the validity of applying the FI to the binding isotherm can be made from the R2 of the fit. In most cases, MIP has a higher degree of heterogeneity than its corresponding non-imprinted control and polymers with a greater imprinting factor have also been shown to be more heterogeneous [35]. However, the imprinted polymer C3 synthesized with POSS was more homogeneous (m = 0.71, R2 = 0.993) in comparison to the more weakly imprinted polymer C6 (m = 0.56, R2 = 0.990) synthesized without POSS. In addition, the heterogeneity index for the POSS MIPs was higher than the POSS NIPs (m = 0.54, R2 = 0.994). Possible reason of the abnormal phenomenon is that the POSSbased MIPs might be measured in a concentration regimes that are too narrow to allow accurate assessment of the FI coefficients than the POSS-free MIPs, in which m usually overestimates the actual heterogeneity index [37]. However, the heterogeneity index m can still be estimated with a relatively high degree of certainty for a

(4)

Fig. 7. Binding isotherms and the Freundlich model for (a) POSS-based MIP (C3); (b) POSS-based NIP (C11); (c) POSS-free MIP (C6). Mobile phase, acetonitrile-acetate buffer (50 mmol L−1 , pH 3.6) (99:1, v/v); flow rate, 0.5 mL min−1 ; detection wavelength, 254 nm; temperature, 30 ◦ C.

Fig. 8. SEM of NAP -imprinted monoliths (5000 magnification). (a) POSS-based MIP (C3); (b) POSS-free MIP (C6).

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F. Li et al. / J. Chromatogr. A 1425 (2015) 180–188 Table 5 Imprinting parameters of NAP of POSS-based MIPs with other MIPs. Polymer

Imprinting factor

Selectivitya

Saturation capacities (␮mol g−1 )

References

4-VP/EDMA 4-VP/EDMA MAA/EDMA IL-SG/TMOS 4-VP/EDMA/POSS

1.79 3 1.41 24 22

1.14 1.33 1.13 6.2 1.94

– – – 3.7 14.9

[38] [39] [40] [41] This work

a The selectivity factor (S) was evaluated from the equation S = kNAP /kIBU for comparison.

3.7. Dynamic binding capacity of POSS-based MIP

Fig. 9. Differential pore size distribution curves for (a) POSS-based MIP (C3); (b) POSS-free MIP (C6).

comparison of the POSS-based MIPs and POSS-free MIPs due to higher adsorption amount to the template. 3.5.3. Characterization of POSS-based imprinted monoliths The morphology of the POSS-based and POSS-free imprinted monoliths was observed by scanning electron microscope, respectively. As shown in Fig. 8, the formation of macropores with large pore size in the POSS-based monoliths is demonstrated. The NAPimprinted monolith C3 prepared by POSS showed fused globules similar to the corresponding POSS-free imprinted monolith C6. In contrast, larger flow-through pores and clumped globules can be seen on the POSS-free monolith C6. According to these observations, as POSS was introduced into MIP matrix, the morphology of the monoliths was transformed from fused globules to aggregated globules. Further, we characterized the macropore structure of the MIP monoliths by mercury intrusion porosimetry. As shown in Fig. 9, the mode pore size (the pore diameter at the maximum of the pore distribution curve) of the POSS-based MIP (C3) is 1153 nm. In contrast, the mode pore size of the POSS-free MIP (C6) was 1302 nm. The template effect on the decrease in mode pore size was in contrast to other MIP monoliths [29–31]. The results highlights that the macropore size distribution for the POSS-based MIP is wider and appealing more heterogenous/disordered as compared with the POSS-based NIP. The pore volume of the POSS-based MIP reached a value of 0.50 mg/mL that represents a porosity of about 38.6%. In contrast, pore volume of the POSS-free MIP reached a value of 0.77 mg mL−1 that was equivalent to a porosity of about 52.4%.

For preparative or semipreparative scale separations, column binding capacity is the critical factor determining the throughput. The effect of flow rate on the dynamic binding capacity of NAP was investigated by frontal analysis method. The dynamic binding capacity [43], q, was calculated as Eq. (5): q = (t50% − t0 )FC/Vc

(5)

where t50% is the time of 50% breakthrough, F the volumetric flow-rate, C the adsorbate concentration in the feed, Vc the volume of the column and t0 the elution time of the void marker. It was observed that the dynamic binding capacities of NAP on the MIP were 4.17 and 3.12 mg g−1 , respectively, by keeping the flow rate of 0.5 and 2.5 mL min−1 with the loading concentration of 0.1 mmol L−1 . Although the dynamic binding capacity was relatively lower at the high flow rates than that at the low flow rates, the dynamic binding capacity was also almost unaffected at the high flow rates (Fig. 10). This independent dynamic binding capacity on the flow rate is obviously different from the flow rate-dependence of dynamic capacity on conventional stationary phases, which can be attributed to convection effect of transport mechanism on POSSbased MIP monolith [44,45]. As s result, a potential application of the POSS-based MIP monolith in high throughput separation may be expected. 3.8. POSS-based MIP with KET and IBU imprints In order to prove the feasibility of the POSS-based MIP improving the affinity, we also synthesized POSS-based MIP using IBU (C17) and KET (C18) as templates, respectively. Good molecular affinity was achieved on the POSS-based MIP with KET or IBU imprints (Table 6). For instance, the imprinting factor of IBU and KET on

3.6. Comparison with other NAP MIPs Previously, a number of methods have been adopted to prepare NAP-imprinted polymers [38–42] with 4-VP [38,39], methacrylic acid [40] and acrylamide [42] as functional monomer by bulk polymerization, multi-step swelling and other polymerization methods. Sol–gel approach was also used to prepare NAP MIP [41]. The imprinting parameters of these NAP-imprinted polymers are compared to the POSS-based NAP MIP to further assess the performance of the POSS-based imprinted polymer (Table 5). Among all the NAP MIPs, the sol–gel-based MIP showed the greatest imprinting factor (IF = 24) but lower saturation capacities (3.7 ␮mol g−1 ) (Table 5). In other word, the performance of the POSS-based NAP MIP prepared in the present study is superior or equal to the performance of the other MIP adsorbent, documented by higher imprinting factor (IF = 22) (C5) and saturation capacities (14.9 ␮mol g−1 ).

Fig. 10. Relationship of dynamic binding capacity of NAP vs. flow rate on the POSSbased MIP monolith. Mobile phase, acetonitrile-acetate buffer (50 mmol L−1 , pH 3.6) (99:1, v/v) with NAP concentration of 0.1 mmol L−1 ; detection wavelength, 254 nm; temperature, 25 ◦ C.

F. Li et al. / J. Chromatogr. A 1425 (2015) 180–188 Table 6 Imprinting and retention parameters of IBU and KET POSS-based and POSS-free MIPs. MIP

kNAP

kKET

kIBU

kFLU

kFBU

IF

IBU POSS MIP(C17) KET POSS MIP(C18) POSS NIP IBU POSS-free MIP (C19) KET POSS-free MIP (C20) POSS-free NIP

2.79 14.33 0.78 5.60 7.08 1.82

6.65 22.99 2.12 9.23 12.07 2.36

2.53 7.21 0.65 3.53 4.28 1.18

2.49 7.63 0.67 3.86 4.36 1.13

4.67 10.34 0.96 7.95 10.07 1.97

3.87 10.83 3.08 3.89

the POSS-based MIP was 3.87 and 10.8, respectively. As a comparison, the imprinting factor of KET and IBU on the corresponding POSS-free MIP was 3.08 (C19) and 3.89 (C20), respectively. Thus, incorporation of POSS unit into MIP network does improve the affinity of the resulting MIP. 4. Conclusions In summary, POSS was first used to prepare imprinted monolithic column successfully. The approach suggested here allowed the creation of an efficient and highly selective imprinting system (IF = 22) under a relative facile condition (<2 h). It was demonstrated the superiority of the POSS-based MIP compared with the POSSfree MIP in terms of imprinting factor. Furthermore, the imprinted polymer synthesized with POSS displayed more homogeneous sites than the imprinted polymer synthesized without POSS. Further work should be considered performing other applications with the new POSS-based MIP, such as sensor, solid phase extraction or drug release system. Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant No. U1303202) and by the Hundreds Talents Program of the Chinese Academy of Science. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chroma.2015.11. 039.

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