Food Chemistry 145 (2014) 687–693

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Analytical Methods

Novel molecularly imprinted polymers with carbon nanotube as matrix for selective solid-phase extraction of emodin from kiwi fruit root Xiao Yang a,b, Zhaohui Zhang a,b,c,⇑, Jiaxing Li a, Xing Chen a, Minlei Zhang a, Lijuan Luo a, Shouzhuo Yao c a

Key Laboratory of Hunan Forest Products and Chemical Industry Engineering, Jishou University, Zhangjiajie 427000, China College of Chemistry and Chemical Engineering, Jishou University, Jishou 416000, China c State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082, China b

a r t i c l e

i n f o

Article history: Received 9 August 2011 Received in revised form 14 February 2012 Accepted 28 August 2013 Available online 5 September 2013 Keywords: Multi-walled carbon nanotubes Solid-phase extraction Molecularly imprinted polymers Emodin Kiwi fruit root

a b s t r a c t In this paper, we present a novel surface imprinting technique for the preparation of molecularly imprinted polymers/multi-walled carbon nanotubes (MIPs/MWNTs) for extraction of emodin from kiwi fruit root. The MIPs/MWNTs were characterised by scanning electron microscopy (SEM) and Fourier transform-infrared spectroscopy (FT-IR). The properties involving adsorption dynamics, static adsorption, and selective recognition capacity were evaluated. The MIPs/MWNTs exhibited good site accessibility in which it only took 60 min to achieve adsorption equilibrium and highly selective recognition for the template emodin. Furthermore, the performance of the MIPs/MWNTs as solid phase extraction (SPE) material was investigated in detail. The proposed MIPs/MWNTs-SPE procedure for emodin exhibited satisfactory recoveries ranging from 89.2% to 93.8% for real samples. It was used for the purification and enrichment of emodin from kiwi fruit root successfully. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Emodin is a main effective ingredient in kiwi fruit root, which shows many pharmacological activities including anti-bacterial, anti-oxidative action and protection of the damaged liver (Roberto, Laura, Anna, Salvatore, & Maria, 2011). In the past decades, emodin has been extensively studied for its traditional pharmacological activities. Recent studies reported that emodin exhibited a limelight with its anti-cancer activities against several types of cancer cells (Yi et al., 2004). Emodin has an inhibitory effect on cancer cellmigration and invasion (Huang, Shen, & Ong, 2005). Due to its biological activities, many researches on separation and purification of emodin from complex matrix have been explored using thin layer chromatography (TLC) (Lee, Kim, & Kim, 2003) and high performance liquid chromatography (HPLC) (Subhalakshmi, Abhijit, & Banasri, 2005) etc. However, these separation procedures are time-consuming and inefficient because those traditional separation sorbents are of poor affinity and lack of selectivity. Thus it is necessary to develop an efficient method to separate and enrich emodin from complex samples. Molecularly imprinted polymers (MIPs) are man-made polymers with predetermined selectivity toward a given analyte or a group of structurally related species (Xu, Fang, & Wang, ⇑ Corresponding author at: College of Chemistry and Chemical Engineering, Jishou University, Jishou 416000, China. Tel./fax: +86 743 8563911. E-mail address: [email protected] (Z. Zhang). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.08.114

2010). Recently, MIPs have attracted increasing attentions due to their outstanding advantages involving predetermined recognition ability, mechanical and chemical stability, relative ease and simple preparation. MIPs often were packed into solid-phase extraction (SPE) column as sorbents to separate and concentrate environmental samples (Shi et al., 2011). Also it was used to extract and separate the active ingredients from traditional Chinese herb (Zhai et al., 2009). Studies showed coupling MIPs with SPE is an efficient approach for purification and preconcentration of analyte from complex matrices, which has been gained considerable interest in environmental, clinical and food analysis (Arbab-Zavar, Chamsaz, Zohuri, & Darroudi, 2011; Cirillo et al., 2011; Zhao, Ji, Shao, Jiang, & Zhang, 2009). However, after crushing, grinding and sieving, only 30–40% of imprinted polymers are recovered as usable material for conventional imprinting preparation process. Up to now, more attentions have been paid to surface imprinting technique, which is to ensure almost all the imprinted sites on the surface, and then facilitates the removal and rebinding of the template molecule. Studies on surface molecularly imprinted materials including MIPs nanoparticles (Lu et al., 2007), MIPs nanocapsule (Ki & Chang, 2006) and MIPs nanotubes (Wang et al., 2006) have been bring forward. Since they have small dimension with high surface-to-volume ratio, it is expected to ease the removal of template molecules, improve the binding capacity and fast binding kinetics over conventional imprinted materials. Multi-wall carbon nanotubes (MWNTs) with unique mechanical properties and extremely large surface area can be an excellent candidate as

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the support material. Thus, the binding sites in the outer layer of the composite will improve the accessibility of the template molecule and reduce the binding time. The preparation of MIPs using MWNTs as the support matrix has attracted great attentions in recent years. Theophylline-imprinted polymers were immobilised on MWNTs and grafted onto iniferter-modified MWNTs by (Lee & Kim, 2009; Lee et al., 2008). An electrochemical sensor fabricated by modifying MWNTs-MIPs on a glassy carbon electrode surface to recognise dopamine has been reported by (Kan, Zhao, Geng, Wang, & Zhu, 2008). Zhang, Zhang, Hu, Yang, and Yao (2010) have demonstrated a surface imprinting technique based on MWNTs to selective adsorb melamine from milk powder. To best of our knowledge, the molecularly imprinted polymers/multi-walled carbon nanotubes (MIPs/MWNTs) applied in purification and enrichment of active ingredients from kiwi fruit root have not been reported. Herein,we prepared highly selective core–shell emodin imprinted polymers on the surface of MWNTs. The high capacity of the MIPs/MWNTs toward emodin showed that the MIPs exhibited special adsorption toward the template molecule. The results of dynamic adsorption indicated that the MIPs/MWNTs offered fast adsorption kinetics. Meanwhile, the MIPs/MWNTs were successfully employed as SPE materials coupled with HPLC to enrich trace emodin from the complex matrix samples successfully. 2. Materials and methods 2.1. Materials and reagents Multi-walled carbon nanotubes (MWNTs, diameters ranging from 10–20 nm) were obtained from Shenzhen Bill Corporation. Methacrylic acid (MAA) and ethylene glycol dimethacrylate (EGDMA) were purchased from Sigma (USA). Emodin, rhein, chrysophanol and physcion were purchased from Shanxi Xuhuang Botanical Science and Technology Development Company (Xi’an, China). N,N0 -dimethylformamide (DMF), acetonitrile, tetrahydrofuran, acetone, dimethyl formamide, aniline hydrochloride, sodium hydroxide (NaOH), hydrochloric acid (HCl), ethanol and methanol were obtained from Changsha Chemical Reagent Company (Hunan, China). All the chemicals were of analytical reagent unless otherwise stated and used directly. Ultra pure water used throughout the experiment. 2.2. Pretreatment of multi-walled carbon nanotubes Five hundred milligram of MWNTs was dispersed in 50 mL of nitric acid solution under sonication for 10 min. Then the mixture was stirred continuously at 80 °C for 8 h. Cooled to room temperature, the mixture was diluted to ten fold with ultra pure water and filtrated through a 0.22 lm polytetrafluoroethylene (PTFE) membrane. The filtered solid was rinsed with ultra pure water until the pH was neutral. Finally, the filtered solid was dried under vacuum at 80 °C for 24 h. 2.3. Polymerisation of polyaniline (PANI) on MWNTs Multi-walled carbon nanotubes-polyaniline (MWNTs-PANI) composite was synthesized according to the studies (Huang, Vanhaecke, & Chen, 2010). Briefly, 400 mg of MWNTs was dispersed in 20 mL of ethanol under sonication for 1 h. Then, aniline hydrochloride (0.72 g, 5.4 mmol) was added into the dispersion and stirred for 1 h. Ammonium peroxydisulfate (1.57 g, 6.75 mmol) dissolving in the mixture solution of ultra pure water (16 mL) and hydrochloric acid (37%, 4 mL) was drop-wise added into the mixture in succession. After the mixture was maintained at 0 °C

at pH 1 for 4 h, the product was washed with 0.2 mol/L hydrochloric acid and acetone, respectively. Finally, the product was dried under a vacuum oven at room temperature for 24 h. 2.4. Preparation of MIPs/MWNTs The MIPs/MWNTs were synthesized as follow: 0.4 mmol of emodin, 200 mg of MWNTs and 2.0 mmol of MAA were added into a 100 mL round flask containing 50 mL of ethanol. The mixture was incubated for 1 h at room temperature for pre-polymerisation. Then, 10 mmol of EGDMA was added into this mixture. Purged with nitrogen to remove oxygen, the polymerisation was initiated by the addition of 30 mg of AIBN. The reaction was allowed to proceed at 60 °C for 24 h. After the polymerisation, the polymers were washed with the mixture solution of ethanol and acetic acid (9:1, v/v) for several times until the template molecule could not be detected by HPLC. Finally, the MIPs/MWNTs were dried in vacuum at 60 °C. For comparison, non-molecularly imprinted polymers/multiwalled carbon nanotubes (NIPs/MWNTs) were prepared by the same procedure, only without addition of emodin in the polymerisation process. MIPs were prepared by the same procedure, only without using MWNTs in the polymerisation process. HPLCmeasurement was carried out with LC-2010AHT solution system. HPLC conditions were as follow: mobile phase is methanol/0.2% sodium phosphate mixture solution (85:15, v/v); flow rate is 1.0 mL/min; column temperature is 35 °C; UV detection wavelength is set at 254 nm. 2.5. Adsorption experiment The kinetic adsorption of the MIPs/MWNTs toward emodin was investigated. In a centrifuge tube, 10 mg of the MIPs/MWNTs, NIPs/MWNTs or MIPs was suspended in 10 mL of 1.0 mmol/L emodin. These tubes were incubated at room temperature under shaking. Eight samples were taken out at defined time intervals. The amount of emodin adsorbed by the MIPs/MWNTs, NIPs/ MWNTs or MIPs was determined by HPLC. Staticadsorption experiment was carried out as following procedure. A series of 10 mg of the MIPs/MWNTs, NIPs/MWNTs or MIPs were added into 10 mL tube and suspended in 10 mL of emodin ethanol solutions with initial concentrations ranging from 0.5 to.4.0 mmol/L. After shaking at 25 °C for 2 h, the mixture was centrifuged at 15,000 rpm for 10 min. The concentration of free emodin was measured by HPLC. The amount of emodin bound to the MIPs/MWNTs was calculated by subtracting the amount of free emodin in the supernate from the amount of emodin initially added. The adsorption capacity Q (lmol/g) of the polymers bound with the template emodin was calculated according to the following formula (Zhang, Qin, He, Li, & Zhang, 2008):

Q ¼ ðC  C t ÞV=m where C (mmol/L) and Ct (mmol/L) represent the initial and final emodin concentration, respectively. V (mL) is the sample volume and m (g) is the polymers mass. 2.6. Selectivity of the MIPs/MWNTs The selectivity of the MIPs/MWNTs toward emodin was estimated using similar structurally compounds involving rhein, chrysophanol and physcion as interfering substances. Twenty-five milligram of the MIPs/MWNTs or the NIPs/MWNTs was added into 25 mL flask containing 10 mL of mixture of rhein, chrysophanol and physcion with concentration of 2.0 mmol/L. After shaking for 2 h, the mixture was centrifuged at 15,000 rpm for 10 min. The

X. Yang et al. / Food Chemistry 145 (2014) 687–693

concentration of the template molecule compounds were detected by HPLC. All the performed in triplicate. The specific recognition property of the evaluated by imprinting factor (a) which is (Zhang, Suleiman, He, & Hu, 2008):

and competitive experiments were MIPs/MWNTs was defined as follows

a ¼ Q ðAÞ=QðBÞ where Q(A) and Q(B) are the adsorption capacities of the template emodin or the competitive compounds on the MIPs/MWNTs and the NIPs/MWNTs, respectively. The selectivity factor (b) is defined as follows (Zhang et al., 2008):

b ¼ a1 =a2 where a1 is the imprinting factor towards the template emodin and a2 is the imprinting factor towards the structurally similar compounds.

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Suitable ratio of template molecule, functional monomers and cross-linkers plays a key role affecting the characteristics of the MIPs/MWNTs. To achieve a good recognition characteristic, a series of adsorption experiments of the MIPs/MWNTs prepared with different amounts of emodin, MAA and EGDMA were carried out following the Section 2.5. As shown in Supplementary Material Fig. 1, the Q increased with the increase amount of functional monomers or cross-linkers, which was due to the effect of the increase in the recognition cavity amount on the MIPs/MWNTs. However, agglomeration of the MIPs/MWNTs would happen if excessive amounts of monomers and cross-linkers were used in the preparation (Zhang, Huang, Yu, & Chen, 2010). Studies showed when the molar ratio was over 1:6:20 and 1:5:25, the agglomeration of the MIPs/MWNTs would happen. Thus the adsorption experiment was not carried out because of agglomeration of MIPs/MWNTs. The results revealed the optimum molar ratio of template molecule, functional monomers and cross-linkers was 1:5:20. Therefore, 1:5:20 was selected in this work. 3.2. Characterisation

2.7. Real sample anaylsis

3. Results and discussion

Scanning electron microscopy (SEM) was used to characterise the morphologies of the crude MWNTs and MIPs/MWNTs. As shown in Fig. 2a, the crude MWNTs were in the form of small bundles or individual tube with the diameter of 10–20 nm. Coated with the MIPs, the diameter of the MWNTs increased drastically with homogeneous polymers layer (Fig. 2b). The diameter of the MIPs/MWNTs was about 45–50 nm. Thus, it could calculate that the MIPs layer was with average thickness of 10–20 nm. FT-IR spectra were applied to characterise the chemical structure of crude MWNTs, MWNTs/PANI and MIPs/MWNTs (see Supplementary Material Fig. 2). Compared with the infrared spectrogram of MWNTs, the characteristic peaks at 3428 cm1 (N–H stretching peak), 3237 cm1, 2927 cm1 and 1493 cm1 (benzenoid ring), 1304 cm1 (C–N stretching peak of secondary aromatic amine) shown in the infrared spectrogram of MWNTs/ PANI were similar with that of PANI sample (Lu et al., 2004), which verified the successful introduction of PANI on the surface of MWNTs. In the infrared spectrogram of MIPs/MWNTs, the peak at 1720 cm1 is ascribed to COOH of MAA stretching vibration. The obvious stretching vibration C–O (1720 cm1) and increase of C–O–C (1020 cm1) peak intensity revealed the existence of EGDMA (Liu et al., 2008). It confirmed that the MIPs/MWNTs composite was constructed successfully.

3.1. Preparation of MIPs/MWNTs

3.3. Adsorption performance of MIPs/MWNTs

The whole procedure for the grafting of MIPs on the MWNTs surface was shown in Fig. 1. Because of its tensile strength, chemical stability, large surface area and ultra-small size, the MWNTs were selected as attractive structural materials for the development of novel analytical devices (Iijima, 1991). In order to prepare an excellent MIPs/MWNTs composite, the good interfacial bond and interaction between the MWNTs and MIPs are very important. Prior to polymerisation, the MWNTs were functionalised with long carbon chain by treating with aniline in this study. Thus, the long carbon chain on the MWNTs surface could interact with acrylate by the covalent bonding in the polymerisation process and it could direct selective copolymerisation with functional monomers and cross-linkers in the presence of template molecule by radical polymerisation (Lu et al., 2007). Then the MIPs/MWNTs composite material was fabricated with a thermal polymerisation using emodin as the template molecule. After polymerisation, the polymers were washed with the mixture solution of ethanol and acetic acid (9:1, v/v) to remove the template emodin. Thus, a novel MIPs/MWNTs composite was synthesized successfully.

Adsorption kinetics studies were carried out to investigate the adsorption process. In the first 30 min, the adsorption rate of MIPs/MWNTs increased rapidly and reached equilibrium after 60 min. The rapid adsorption of emodin at first 30 min was attributed to a large amount of binding sites on the surface of MWNTs. When most of binding sites were filled up, the adsorption rate decreased obviously. Meanwhile, the adsorption capacities of the MIPs/MWNTs were better than that of the corresponding NIPs/MWNTs. It indicated that the template-monomer complexes of the MIPs/MWNTs were preserved in the imprinted polymers, thus it exhibits ‘‘memory’’ effect for the template molecule. The differences of the adsorption amount were mainly due to the ‘‘memory’’ sites. Since MWNTs have extremely large surface area, it can be an excellent candidate as the support material. Thus, the binding sites in the outer layer of the composite will improve the accessibility of the template molecule and reduce the binding time. Traditional MIPs in our paper took about 120 min to reach adsorption equilibrium. In our case, emodin reached the surface imprinting cavities of MIPs/MWNTs easily and it took less time

The MIPs/MWNTs column was prepared as reported as the literature (Jiang, Zhao, Jiang, Zhang, & Liu, 2008). Briefly, 100 mg of the MIPs/MWNTs or the NIPs/MWNTs was packed into an empty SPE column (100 mm  4.6 mm i.d.). The sample was prepared as following procedure. First, 0.1 g of kiwi fruit root powder and 25 mL of mixture solution of ethanol:water (5:5, v/v) was added to a 50 mL round bottom flask. The mixture was then sonicated for 40 min. Next, the supernatant was filtrated through a 0.45 lm PTFE membrane. The extraction solution was passed through the MIPs/MWNTs-SPE column at flow rate of 1.0 mL/ min. Finally, the column was eluted with 2.0 mL of the mixture solution of ethanol and acetic acid (9:1, v/v) at flow rate of 0.5 mL/min. The eluted solution was analysed by HPLC. The recovery of emodin was calculated according to the formula (Zhang et al., 2008):

Recovery % ¼ ðC  C t Þ=C where C (mmol/L) and Ct (mmol/L) represent the initial and final emodin concentration, respectively.

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O

HNO3

COOH COOH

C6H5NH2HCl

NH

NH

NH

NH

NH

NH

n

O

(NH4)2S2O8

n

EGDMA

crude MWNTs

MAA Emodin extraction

rebinding

MIPs/MWNTs Fig. 1. The protocol for synthesis of MIPs/MWNTs.

Fig. 2. Scanning electron micrographs of crude MWNTs (a) and MIPs/MWNTs (b).

(60 min) to reach adsorption saturation, which implied that the nanosized and uniform structures of MIPs/MWNTs allowed efficient mass transport (Gai, Qu, Liu, Dai, & Zhang, 2010). As a novel composite imprinted material, the key property of the MIPs/MWNTs is special adsorption capacity. The adsorption isotherm experiments for the MIPs/MWNTs, NIPs/MWNTs and MIPs were carried out in emodin concentration ranging of 0.5– 4.0 mmol/L. At lower emodin concentration, the amount of emodin was not enough to saturate the specific binding cavities. The adsorption capacity of the MIPs/MWNTs toward emodin increased with the increasing emodin initial concentration. When the concentration of emodin reached up to 3 mmol/L, the adsorption capacities remain constant with the maximum adsorption capacity of 65 lmol/g, which was higher than that of the NIPs/MWNTs (21 lmol/g) and MIPs (46 lmol/g). The high adsorption affinity should attribute to the hydrogen bonding between the MIPs/ MWNTs and emodin (Gu et al., 2010). However, the NIPs/MWNTs had not generated specific recognition sites due to the absence of emodin during the preparation process. When the MWNTs were used as backbone for the polymerisation of MIPs, the binding capacity of the MIPs/MWNTs toward emodin increased.

3.4. Selectivity The selectivity of the MIPs/MWNTs was investigated in the mixture solution of emodin, rhein, chrysophanol and physcion. The adsorption amounts onto the MIPs/MWNTs or the NIPs/MWNTs were investigated. As shown in Fig. 3, the MIPs/ MWNTs exhibited higher adsorption capacities toward emodin than that toward other molecules. Even more, the adsorption capacity of the MIPs/MWNTs toward emodin was much higher than that of the NIPs/MWNTs. Additionally, the imprinted factor (a) and selectivity factor (b) were used to evaluate the specific recognition property of the MIPs/MWNTs and the results were listed in Table 1. The imprinted factor (a) toward the template emodin is calculated as 2.55, which is greater than that toward similar structurally compounds rhein (1.04), chrysophanol (1.07), physcion (1.18). The b values for similar structurally compounds are rhein (2.45), chrysophanol (2.38), physcion (2.16), which indicated that the MIPs/MWNTs have not specific adsorption toward similar structurally compound. The MIPs/MWNTs exhibited an excellent recognition and selective toward the template molecule due to the existence of imprinted cavities with fixed size, shape

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molecule from the NIPs/MWNTs-SPE column, while the template molecule should be retained by the MIPs/MWNTs-SPE column. The results showed that best effect was obtained by using acetone as the washing solution. The purpose of elution step is to remove the binding emodin as much as possible. The mixture solutions with different percentage of ethanol and acetic acid were investigated and the results were shown in Table 2. With the increment of acetic acid in the eluting solution, the recoveries of emodin increased steadily, and reached the highest when the amount of acetic acid was up to 10%. However, when the amount of acetic acid was over 10%, the recoveries of emodin decreased. The most likely explanation was that acetic acid could compete with emodin for the functional groups in the binding sites, but too much acetic acid did not benefit the eluting (Feng, Zhao, Yan, Lin, & Zheng, 2009). Therefore, 2 mL of ethanol/ acetic acid (90:10, v/v) was chosen as the eluting solvent in the following studies.

50

MIPs/MWNTs NIPs/MWNTs

Q(µmol/g)

40 30 20 10 0 emodin

rhein

chrysophanol

physcion

Fig. 3. The selectivity of MIPs/MWNTs and NIPs/MWNTs.

Table 1 Imprinting factor (a) and selectivity factor (b) of MIPs/MWNTs and NIPs/MWNTs. Target

QMIPs

QNIPs

a

b

Emodin Rhein Chrysophanol Physcion

47.3 18.2 17.3 20.1

18.5 17.4 16.1 16.9

2.55 1.04 1.07 1.18

– 2.45 2.38 2.16

3.6. Method validation

and binding sites toward the template molecule. The high selectivity of the MIPs/MWNTs provides an effective way for eliminating interferences of other relevant compounds.

To evaluate the accuracy and application of the developed method, the samples spiked with four levels (1, 2, 3 and 4 lg/L) of emodin were analysed. At each concentration, five measurements were performed. The recoveries of emodin in kiwi fruit root and pure ethanol were ranged from 89.2% to 93.8% and 92.3% to 98.1%, respectively. The relative standard deviation (RSD) was less than 4.0%. The results revealed that the MIPs/MWNTs-SPE column could be directly used for selective adsorption and determination of emodin in real samples coupled with HPLC.

3.5. Optimised of SPE

3.7. Application of MIPs/MWNTs-SPE column in real samples

Loading solvent plays an important role in the enrichment of the analyte. In order to evaluate the effect of loading solvent in SPE procedure, loading solvents ethanol/H2O with different ratio (30%, 50%, 100%, v/v) were investigated on the molecularly imprinted polymers/multi-walled carbon nanotubes-solid phase extraction (MIPs/MWNTs-SPE) column. The results were shown in Table 2. With the increment of ethanol in the loading solvent, the recoveries of emodin decreased steadily. When 100% ethanol was employed, most of the loading analyte was retained by the MIPs/MWNTs-SPE column. Thus 100% ethanol solution was selected as the sample loading solution in subsequent experiments. The aim of the washing step is to minimise the interferences for the analysis step and activate the binding sites of the MIPs/MWNTs for maximising their interactions with the target analyte. Washing procedure was assessed using various mixtures including acetone, tetrahydrofuran, dimethyl formamide and acetonitrile (shown in Table 2). The employed solvent is required to remove the template

The validated method was applied for selective enrichment of emodin in kiwi fruit root samples. According to optimum condition of the MIPs/MWNTs-SPE procedure, the extraction solution spiked with emodin concentration of 4 lg/L was passed through the MIPs/ MWNTs-SPE column. Finally, the column was eluted with 2.0 mL of ethanol and acetic acid (9:1, v/v). A volume of 10.0 lL of sample solution was analysed by HPLC. The chromatograms of samples spiked with emodin concentration of 4 lg/L adsorbed by the MIPs/MWNTs-SPE column or the NIPs/MWNTs-SPE column were displayed in Fig. 4. The peak of emodin could not be observed from chromatogram of kiwi fruit root spiked with 4 lg/L emodin (Fig. 4a). After the enrichment of kiwi fruit root with the MIPs/ MWNTs-SPE column, and washing by ethanol/acetic acid (9:1, v/v), the peak of emodin appeared distinctly and other irrelevant compounds in the real samples were nearly eliminated (Fig. 4b). The chromatograms confirmed that emodin in kiwi fruit root samples was selectively enriched by the MIPs/MWNTs-SPE column

Table 2 Recoveries obtained from MIPs/MWNTs-SPE and NIPs/MWNTs-SPE column under different loading, washing and eluting solutions. Step

Fractions

1A 1B 1C 2A 2B 2C 2D 3A 3B 3C

Loading, 2 mL 30% ethanol Loading, 2 mL 50% ethanol Loading, 2 mL 100% ethanol Washing, (after step 1C) 2 mL acetone Washing, (after step 1C) 2 mL tetrahydrofuran Washing, (after step 1C) 2 mL dimethyl formamide Washing, (after step 1C) 2 mL acetonitrile Eluting, (after step 2A), 2 mL ethanol and acetic acid (85:15, v/v) Eluting, 2 mL (after step 2A), 2 mL ethanol and acetic acid (90:10, v/v) Eluting, 2 mL (after step 2A), 2 mL ethanol and acetic acid (95:5, v/v)

Recoveries (%) n = 5 MIPs/MWNTs

NIPs/MWNTs

10.7 ± 2.9 8.9 ± 3.4 6.8 ± 3.6 9.4 ± 2.9 10.1 ± 3.4 27.2 ± 3.9 25.7 ± 3.5 55.4 ± 2.8 74.8 ± 1.5 34.1 ± 4.0

15.5 ± 3.4 12.9 ± 4.4 10.6 ± 1.2 46.8 ± 3.2 35.1 ± 1.9 36.5 ± 3.2 41.7 ± 2.2 20.8 ± 3.8 22.4 ± 2.5 23.5 ± 2.4

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(a)

4. Conclusions

100

In this work, a novel emodin molecularly imprinted polymers was prepared using MWNTs as the support matrix via a novel surface imprinting technique. The results of SEM and FT-IR showed that the MIPs were successfully immobilised on the surface of MWNTs. The adsorption results of the MIPs/MWNTs showed that the adsorption equilibrium was reached in 60 min with the maximum adsorption capacity of 65 lmol/g. Moreover, the feasibility of the MIPs/MWNTs-SPE column to enrich target analyte was confirmed by real samples application. The results showed the MIPs/MWNTs-SPE column is an ideal material to enrich the active target analyte from the complex samples.

Intensity/mv

80 60 40 20 0

0

2

4

6

8

10 Acknowledgements

Time (min)

(b)

40

Project supported by the National Natural Science Foundation of China (No. 21005030), the Research Foundation of Education Bureau of Hunan Province, China (No. 10A099), the Twelfth Five-year-plan National Technology Support Program, China (No. 2011BAD10B01), Science and Technology Innovative Research Team in Higher Educational Institutions of Hunan Province and Graduate Innovation Foundation of Jishou University (No. JGY 201111).

20

Appendix A. Supplementary data

100

Emodin

Intensity/mv

80 60

0

0

2

4

6

8

10

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.foodchem.2013.08.114.

Time (min)

(c)

References

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Intensity/mv

80 60 40 Emodin 20 0 0

2

4

6

8

10

Time (min) Fig. 4. (a) Chromatogram of crude extract of kiwi fruit root (b) chromatogram of eluting solutions from MIPs/MWNTs-SPE column (c) chromatogram of eluting solutions from NIPs/MWNTs-SPE column.

and recovered by the eluting step. As shown in Fig. 4c, the extraction efficiency and selectivity of the NIPs/MWNTs-SPE column are much lower than those of the MIPs/MWNTs-SPE column. In addition, the enrichment factor (EF), which is defined as the ratio of the concentration of analyte in the concentrated phase Ccon (lg/ L) and the initial concentration of analyte within the sample C0 (lg/L), was introduced. C0 includes total emodin in sample and spiked emodin. The enrichment factor of the MIPs/MWNTs-SPE column was calculated up to 20.12, which is higher than that of the NIPs/MWNTs-SPE column (2.41). The results showed the MIPs/MWNTs-SPE column is an ideal material for enrichment the active target analyte from the complex samples.

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