Preparation of core–shell molecular imprinting polymer for lincomycin A and its application in chromatographic column

Process Biochemistry 50 (2015) 1136–1145

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Preparation of core–shell molecular imprinting polymer for lincomycin A and its application in chromatographic column Zi Wang, Xuejun Cao ∗ State Key Laboratory of Bioreactor Engineering, Department of Bioengineering, East China University of Science and Technology, 130 Meilong Rd, Shanghai 200237, China

a r t i c l e

i n f o

Article history: Received 8 January 2015 Received in revised form 30 March 2015 Accepted 15 April 2015 Available online 29 April 2015 Keywords: Molecular imprinting Separation Core–shell Chromatography Purification Lincomycin

a b s t r a c t Lincomycin A is one main component of lincomycin, and the separation of lincomycin A from analogues B, C, D by organic solvent extraction has the problems of environmental pollution and multi-step process. Molecular imprinting polymer (MIP) could separate the target product from their structure analogues. In this paper, methyl methacrylate (MMA) was used as monomer in seed core and acrylamide (AM) was used as shell functional monomer. Core–shell MIP for lincomycin A was prepared using lincomycin A as template molecule, ethylene glycol dimethacrylate (EGDMA) as cross-linker and the ammonium persulfate (APS) as the initiator. The structure of MIP was characterized. Lincomycin A purification condition was explored and optimized. The result revealed that the maximal adsorption capacity was 62.66 mg/g, which reached equilibrium within 6 h. The selectivity coefficients of MIP and non-imprinted polymer (NIP) for lincomycin A related to lincomycin B were 3.14 and 1.08. In addition, MIP obtained a good purification result by column chromatography, and the recovery and purity of lincomycin A reached 97.57% and 93.3%, respectively. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Lincomycin is an antibiotic first reported by Maston in 1962, which is generated from bacteria of the genus Streptomyces [1]. It is effective against Gram-positive organisms. It acts on ribosome and competes binding site with macrolides and is widely used in all kinds of animal diseases [2]. In the process of production of lincomycin, there are several analogues such as lincomycin A, B, C and D. Lincomycin A is the main interesting product, while the others are impurities. Lincomycin C is very little and has the same pharmacological effects with lincomycin A. Lincomycin B is the typical impurity component with lower activity and higher toxicity than lincomycin A [3,4]. Thus its content in the product must be strictly restricted. It is very important to decrease the content of lincomycin B in order to improve the product quality. The elimination of analogues B, C, D is difficult along with high cost, low yield, and organic solvents. So it is necessary to develop new separation technology with high selectivity. The paper [5] has researched about lkmcomycin. In the structure of lincomycin A, there is a propyl group on 4 position in the pyrrole ring. The structures [6,7] of those compounds are shown in Fig. 1.

∗ Corresponding author. Tel.: +86 21 64252695. E-mail address: [email protected] (X. Cao). http://dx.doi.org/10.1016/j.procbio.2015.04.013 1359-5113/© 2015 Elsevier Ltd. All rights reserved.

Molecular imprinting technology shows a promising application due to the preparation of specific polymers of owning recognition sites for the target molecule [8,9]. The imprinting polymers have been widely developed in many fields, such as separations, sensors, catalysis and pseudo antibody [10,11]. The methods of molecular imprinting polymerization are involved in surface molecular imprinting [12–14], suspension polymerization, core–shell polymerization [15], and magnetic molecular imprinting [16]. Although MIP owns many significant advantages, it still exists some problems such as low binding capacity, slow mass transfer and difficult template removal. Core–shell imprinting technology can overcome above drawbacks. The binding sites of MIP are situated in the surface layer, which can recognize template molecules rapidly and remove them from the binding sites easily. Many experiments have acquired good results. Zhao et al. used core–shell MIP to selectively separate sulfonamides from chicken breast muscle [17]. Hu et al. developed a novel core–shell magnetic nano-sorbent with surface molecularly imprinted polymer separate dimetridazole [18]. Liu et al. prepared core–shell MIP using silica as supporting medium to recognize immunoglobulin heavy chain binding protein [19]. Hajizadeh et al. developed a new macroporous MIPs by utilizing PVA particles to remove ␤-blockers from complex samples [20]. So based on the current molecularly imprinting technnology, MIPs were prepared by core–shell polymerization

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Fig. 1. The chemical structure of lincomycin. (a) The structure of lincomycin A. (b) The structure of lincomycin B.

to enrich lincomycin A from complex components in this study. From the present study in our lab, we have previous backgrounds in core–shell molecular imprinting polymers [21,22]. These were core–shell MIP for ursodeoxycholic acid and possessed excellent recognition and binding sites. In this paper, the emulsion polymerization was used to synthesis core medium, and then core–shell microspheres were prepared on its surface so that it has a bigger surface area which can provide more sites with specific adsorption for lincomycin A in aqueous solution. Column chromatography of MIPs has been applied to obtain single component of lincomycin A.

2. Experiment 2.1. Materials Lincomycin was obtained from Henan topfond pharmaceutical Co. EGDMA was supplied from Sigma. Methanol for HPLC analysis was supplied from Cinc High Purity Solvents (Shanghai) Co., Ltd. Dimethyl sulfoxide (DMSO), APS, NaHCO3 , sodium dodecyl sulfate (SDS), dodecyl alcohol, methanol and cyclohexanol, MMA and AM were all provided by Shanghai Lingfeng Chemical Reagent Co., Ltd.

2.2. Instrumentations Transmission electron microscope (TEM) (75 kV, 20 × 1000 magnification) was used for characterization of MIP. The samples

were disposed by ethanol dispersion and uniformly distributed in copper wire mesh. The FT-IR spectrum was used to detect the interaction between lincomycinA and polymers (KBr tablet). The polymer thermo-stability was determined by using Thermo-gravimetric Analyzer (TGA) with temperature range 0–800 ◦ C and temperature increasing rate 10 ◦ C/min. Energy dispersive spectrometry (EDS) (QUANTAX 40) was a part of SEM and served for slemental analysis. It can distinguish the object on the polymers by comparing with MIPs and the seed emulsion such as C, N element. The HPLC consisted of two Shimadzu LC-20AD pumps and a Shimadzu SIL-20A UV/V detector (Japan) that was used for detecting the concentration of lincomycin. The HPLC column (C18 ) size was 4.6 × 150 mm. The determination conditions were wavelength 214 nm, injection volume 20 ␮l. the mobile phase was consisted of 0.05 mol/l sodium borate solution (pH = 6) and methanol with a volume ratio of 40:60 and the flow rate was 1.0 ml/min.

2.3. Preparation of MIP for lincomycin A and NIP 2.3.1. Preparation of seed particles The core particles were prepared by emulsion polymerization method. The method is similar to that described in our previous papers. 0.238 g NaHCO3 and 0.228 g SDS and 130 ml water were added into conical flask in the water bath. Then the temperature rose to 90 ◦ C, 9.27 ml MMA and 0.866 ml EDMA were added into the flask. Nitrogen atmosphere was kept 10 min. Finally, 0.12 g APS was added into the flask and

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the reaction was carried out at 250 rpm in a shaker for 24 h (80 ◦ C). 2.3.2. Preparation of MIP and NIP A total of 50.0 ml DMSO solvent contained 0.365 g template lincomycin A, 50.0 mg functional monomer AM, 5.43 ml crosslinking agent EDMA, 29.04 ml seed (the particle solid content was 0.368 g/ml) and 3.0 ml porogenic agent with ratio of dodecyl alcohol to cyclohexanol = 2:1 ml (v/v) was mixed and added into the flask with 18.0 ml water and 0.10 g SDS. Nitrogen atmosphere was kept 10 min. Then the 0.10 g APS was added into the flask and the reaction was carried out at 250 rpm in a shaker for 24 h (60 ◦ C). The mole ratio of template:functional monomer:cross-linking agent was equal to 1:10:40. The NIP was synthesized by the same way without lincomycin A. Once the reaction was completed, the polymers were cooled to room temperature, and then were centrifuged. The product was washed by acetone and methanol to remove the self-polymers formed by monomers and the unreacted reagents, respectively. The imprinted template lincomycin A was eluted with the mixture of methanol:acetic acid (9:1, v/v) until no lincomycin A was detected by UV. Then wash with water to remove the methanol and acetic acid.

2.4.1. Adsorption capacity Adsorption isotherm was carried out according to following steps: 50.0 mg MIPs and NIPs were separately put into 10.0 ml lincomycin A aqueous solution with initial concentration varied from 0.1 to 1.0 mg/ml in test tubes. The test tubes were placed in a shaker and shaken at 200 rpm for 24 h (room temperature). When the adsorption finished, centrifugation was conducted, and 1.0 ml supernatant was taken out. Then the residual concentration was determined by HPLC. The adsorption capacity was calculated according to the following equation: (C0 − Ce ) V M

(1)

where Q (mg/ml) is the amount of lincomycin A adsorbed in the MIP (NIP), C0 (mg/ml) is the initial concentration of lincomycin A, Ce (mg/ml) is the equilibrium concentration of lincomycin A, V (ml) is the volume of lincomycin A aqueous solution, and M is the weight of polymers. 2.4.2. Adsorption kinetics The kinetic experiment was carried out by following steps: 50.0 mg MIP was added into 10.0 ml aqueous solution containing 1.0 mg/ml lincomycin A (all experiments were done three times). The test tubes were placed in a shaker and shaken at 200 rpm at room temperature. Then 800 ␮l of the supernatant was taken out at different time intervals (10–360 min) to detect concentrations by HPLC. The adsorption capacity was calculated based on: Q =

(C1 − C2 ) V M

Kd =

Qe Ce

(3)

where Kd (ml/g) is the distribution coefficient, Qe (mg/g) is the equilibrium adsorption amount, and Ce (mg/ml) is the equilibrium concentration. The selectivity coefficients of MIP and NIP for lincomycin A with respect to lincomycin B were derived from the following equation: ˛=

Kd (lincomycin A) Kd (lincomycin B)

(4)

where ˛ is the selectivity coefficient. The relative selectivity coefficient was obtained from the following equation:

2.4. Static adsorption for lincomycin A of MIPs and NIPs

Q =

selective adsorption capacity of lincomycin A imprinted in MIP and NIP. 50.0 mg MIPs and NIPs were separately added into 10.0 ml aqueous solution containing 1.0 mg/ml lincomycin A and 1.0 mg/ml lincomycin B. The adsorption was conducted in a shaker and shaken at 200 rpm for 6 h at room temperature. When the reaction finished, centrifugation was conducted, and 1.0 ml supernatant was taken out. Then residual concentration was determined by HPLC. The selective adsorption capacity was evaluated according to distribution coefficient, selectivity coefficient and relative selectivity coefficient. The distribution coefficients of lincomycin A and lincomycin B were calculated based on:

ˇ=

˛MIP ˛NIP

(5)

where ˇ is the relative selectivity coefficient, and ˛MIP and ˛NIP is selectivity coefficients of MIP and NIP, respectively. 2.6. Dynamic adsorption of MIPs Dynamic adsorption was evaluated by the separation of lincomycin A and lincomycin B in chromatography column. 5.0 g MIP was dispersed in 50 ml acetone and washed with 50.0 ml methanol and then equilibrated with water. After pretreatment, MIP was packed in the column (150 × 12 mm). Then 1 mg/ml lincomycin aqueous solution flowed into the column at flow rate 0.15 ml/min at room temperature. When outlet concentration reaches 90% of the inlet concentration, adsorption was stopped. Different sampling concentrations and flow rates were used to optimize adsorption conditions. The dynamic adsorption capacity was calculated according to the following equation: Q =

(C0 − Ce ) V0 V1

(6)

where C0 (mg/ml) is the concentration of lincomycin loaded into the column, Ce (mg/ml) is the equilibrium concentration. V0 (mg/ml) is the volume of lincomycin and V1 is the volume of MIP. The elution of lincomycin was carried out by methanol and acetic acid, all the chromatography experiments were carried by collecting 4.0 ml eluate per tube.

(2)

where Q (mg/ml) is the amount of lincomycin adsorbed on MIPs at different times, C1 (mg/ml) is the initial concentration of lincomycin, C2 (mg/ml) is the concentration of lincomycin at different times, V (ml) is the volume of lincomycin aqueous solution and M (g) is the weight of MIPs. 2.5. Selective adsorption As Lincomycin A and lincomycin B have similar structures, lincomycin B was selected as competitive agents to evaluate the

3. Results and discussion 3.1. Preparation of core–shell MIP for lincomycin A In this paper, the core–shell MIPs were prepared. The core structure as seed was prepared by emulsion polymerization [23]. Then the monomers were polymerized on the seed surface. Fig. 2 described the reaction process of the polymerization. First, MMA was used as functional monomer in the core. The smaller size of the seed particle the bigger surface area and the more imprinted sites in shell. Considering density, radius of polymer particles, MMA

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Fig. 2. Schematic diagram of the preparation of core–shell MIPs. (A) Polymerization of monomer (MMA) and cross linker. (B) Polymerization of monomers, template and cross linker. (C) Removal of the template leaves binding cavities. (D) TEM figure of seed particles of the polymer.

as a function monomer was mixed with a small amount of EGDMA as cross-linker [24]. After the seed was synthesized, the AM containing template molecules was polymerized on the seed, and EGDMA was used to cross-link the monomer-template complex. The choice of function monomer AM was based on comparing with the result of methacrylic acid (MAA) and acrylic acid (AA). This choice confirms that the hydrogen bond of amide group was stronger than that of carboxyl group [25] and the adsorption capacity of MIP with AM was better than others. The hydrogen bond interaction between AM and template molecular determines the recognition ability of MIP for template. At last, the lincomycin A template was removed by elution. 3.2. Characterization of MIP 3.2.1. Morphological characteristics MIP and NIP were observed by TEM with different magnification (75 kV, 20 × 1000 magnification, 50 nm, a/b/d/e; 200 nm, c/f). The TEM of seed particles (75 kV, 20 × 1000 magnification, 50 nm) was shown in Fig. 2. As shown in Fig. 3, both MIP and NIP look like uniformly spherical particles. The size of MIP is bigger than that of NIP with the 200 nm of particle diameter. From the TEM of seed particles, it can be seen the size of the seed which equals to 40 nm. Because there is the same size of MMA core, the size of shell is thicker than that of NIP. We can calculate the thickness of polymer layer (80 nm) through the size of MIPs minus that of seed particles. Compared Fig. 3(a and b) with Fig. 3(d–e), it can be observed that the surface of NIP is more smooth than MIP. It may be influenced by the existence of templates. The function monomers with template were polymerized on the seed particles. 3.2.2. FT-IR spectra From the FT-IR spectra in Fig. 4a and b, the change of the peak at 1150 cm−1 from MIP caused by superposition of C O C comes

from EGDMA and MAA. The C C at 1600 cm−1 from core-structure disappear in MIP which proved the interaction between EGDMA and monomers was formed by fracture of C C. At 3000 cm−1 the unsaturated C H changed by the connection of AM (Fig. 4b). From the above analysis, it can be concluded that the functional monomer AM and cross-linker EGDMA were both successfully polymerized on seed. Compared with Fig. 4b, c and e, it can be obviously seen there is no lincomycin A characteristic absorption peak in Fig. 4e. Thus it can prove there is no imprinted hole in NIP and the adsorption belongs to non-specific physical adsorption. In Fig. 4c and d, the C N characteristic peak of pyrrole at 800 cm−1 has some change may be caused by AM. The changes of shape and position of the peaks at 1100–1400 cm−1 come from pyrane were affected by the hydrogen bonds with AM. The O C NH from lincomycin A at 1600 cm−1 have some skewing in Fig. 4d. The peak at 3200–3500 cm−1 appeared some changes, and the reason is that the O H formed hydrogen bond and overlapped with N H from MIPs. From the above analysis, it can be known that the lincomycin A and AM formed hydrogen-bond. And it can be clearly obtained a molecular schematic diagram to show the interaction between AM and template (Fig. 5). 3.2.3. Energy dispersive spectrometry (EDS) The energy dispersive spectrometry is a technique used to determine the energy and to characterize the element composition of the polymers. From Fig. 6, it can know that there exists N element (1.4%) in the MIP (Fig. 6b) and there is no N element in the core structure (Fig. 6a). It indicates that the functional monomer has been grafted onto the core structure and the grafting process was successful. 3.2.4. Thermogravimetric analysis (TGA) From Fig. 7, it can be seen that the weight of polymers didn’t change up to 250 ◦ C. Then the weight began to decrease and the chemical bond start to fracture. From the Thermo-gravimetric

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Fig. 3. The TEM images of core–shell MIPs: (a) scale bar: 50 nm, (b) scale bar: 50 nm, (c) scale bar: 200 nm; TEM images of core–shell NIPs: (d) scale bar: 50 nm, (e) scale bar: 50 nm, (f) scale bar: 200 nm.

3.3. Adsorption of MIP and NIP

Fig. 4. The FT-IR spectra of different samples: (a) core-structure, (b) MIPs, (c) the spectra of lincomycin A, (d) the MIPs after adsorption for lincomycin A, (e) the NIPs after adsorption for lincomycin A.

curve, it can be concluded that the polymer decomposes rapidly at 294 ◦ C. With the temperature increasing, the weight decrease slowly and the rate of decomposition gradually decrease to 0%/min until the temperature is risen to 540 ◦ C. From 250 to 390 ◦ C, the weight of the polymers decreased rapidly. Then the weight decreased slowly. From this conclusion, the curve of TGA divided into two parts. The reason may be that the shell of the core–shell MIPs first broke down, and then the core began to decompose. And it can be seen that the chemical bond is strong and the polymers have good thermal stability.

3.3.1. Adsorption isotherm To investigate the adsorption capacity of lincomycin binding on MIP and NIP, the adsorption isotherm experiment was conducted at different initial concentrations ranged from 0.1 to 1.0 mg/ml. As shown in Fig. 8a, the capacity of lincomycin adsorbed on MIP and NIP increases with concentration of lincomycin from 0.1 to 0.5 mg/ml. When the concentration reaches 0.6 mg/ml, the adsorption trends saturation. The maximum adsorption capacity of MIP reaches 62.66 mg/g. And the MIP has a higher adsorption capacity than NIP, which can explain the MIP has more specific binding sites than NIP. It also can be calculated that the imprinting factor (IF) [26] is 1.73 based on the binding ratio of lincomycin A on MIP and NIP, which indicates the MIP for lincomycin A has a better recognition than NIP. From Mieczyslaw Jaroniec’s paper [27], Eq. (7) is often used in liquid adsorption. The Langmuir–Freundlich isotherm is utilized to analyze the adsorption data and can better fit the data. The equation is written as follows: Qe =



Qm (KCe )m 1 + (KCe )m



(7)

where Qe (mg/g) is the equilibrium adsorption quantity, Ce (mg/ml) is the equilibrium concentration of lincomycin A, Qm (mg/g) is the maximum adsorption capacity, K is a binding constant and m is the heterogeneity parameter. The fitting plot of adsorption isotherm is shown in Fig. 8b and the fitting parameters are shown in Table 1. From the fitting parameters, the maximum adsorption capacity of MIP for lincomycin A reaches 63.07 mg/g. This number is almost same as that (62.66 mg/g) at an initial concentration of lincomycin A (1.0 mg/ml). It can be concluded that there is no much difference between experimental value and theoretic value with Langmuir–Freundlich

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Fig. 5. The formation process of MIPs. (A) The mixture of AM (functional monomer), EGDMA (cross linker) and lincomycin A (template); (B) the binding process of AM, EGDMA and lincomycin A; (C) the elution of template.

Fig. 6. The EDS spectra of (a) organic polymer core and (b) MIPs.

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Table 1 The parameters of fitting plot of adsorption isotherm. Qm MIPs NIPs

(mg/g)

63.09 ± 2.45 43.15 ± 1.03

K

m

4.66 ± 0.09 4.28 ± 0.14

5.9 ± 1.16 8.06 ± 1.24

in the internal structure of the MIP resulted in a slow adsorption. From the result, the adsorption reaches equilibrium quickly. The adsorption kinetic has been proved that it is complied with first-order or second-order model in many researches. The secondorder kinetic model is found in many adsorbate–adsorbent systems [28,29]. The equation is written as: t = qt



1 kq2e

+

t qe

(8)

where qt (mg/g) is the adsorption capacity at different times, t (min) is the adsorption time, qe (mg/g) is the equilibrium adsorption capacity, and k (g/(min mg) is the constant, which includes the effects of mass transfer, physical adsorption and/or chemical reaction. From the fitting plot (Fig. 9b), we can obtain the equation: t/qt = 0.4878 + 0.0152t. The qe value is calculated to be 65.74 mg/g according to slope that is close to the Qtmax in adsorption kinetic curves. The slope k is 0.000474 g/(min mg). The correlation coefficient is 0.98869. From these results, we can obtain t/qt has a better linear correlation with t. This can explain that the kinetic adsorption conforms to second-order model.

Fig. 7. The TGA and DSC analysis of MIPs in air atmosphere.

isotherm. Comparing the binding constant (K) of MIP with NIP, it can be concluded that MIP can easily bind on template molecules. From the curve in Fig. 8b, the tangent slope k changes with the increasing of concentration. The k increases gradually before 0.27 mg/ml, and then it reaches saturation. Comparing the heterogeneity parameter (m) of MIP and NIP, it reveals that the MIP has more homogeneous recognition sites. Because of the existence of template, it leaves imprinting holes in MIP. The correlation coefficient is 0.9805. 3.3.2. Adsorption kinetic curve Fig. 9a described the adsorption kinetic curve of MIP, which was determined by different adsorption time 10, 30, 60, 100, 150, 210, 280, 360 min, respectively. From the figure it can be seen that the adsorption quantity increases rapidly within 1 h. Then the adsorption rate of lincomycin increases slowly during 1–3.5 h and finally reaches equilibrium at 6 h. The maximum of Qt is 62.69 mg/g. The following two reasons can explain the adsorption process. First, the recognition sites located on the surface of the polymers resulted in quick adsorption. Second, the imprinted sites situated

3.3.3. The effect of pH on adsorption capacity The pH of the solution plays an important part in adsorption procedure and can influence the interaction between templates and MIPs. Adsorption capacity of lincomycin was investigated in acid and alkaline solution [30]. Fig. 10 shows the effect of pH on the adsorption capacity from pH 3 to 11. The quantity of lincomycin binding on MIP increases with pH rising from 3 to 9, and then the quantity slightly decreases from pH 9 to 11. The maximum adsorption capacity is 77 mg/g. It could conclude that the adsorption capacity in the alkaline solution is much more than that in acid solution. When pH > 9, alkaline solution can break the interactions between monomer and template and lead to the adsorption quantity decrease. From the results, it can obtain the highest adsorption capacity at pH 9.0 and the next experiments were all carried out at pH 9.0. 3.3.4. Selective adsorption In order to estimate the selectivity of lincomycin A binding on MIP and NIP, lincomycin B was selected as a analogue. Table 2 shows that the adsorption amount of lincomycin A is higher than that of lincomycin B bound to MIP. While the lincomycin A and lincomycin B almost had the same adsorption quantity on the NIP. Obviously, the distribution coefficients Kd value of lincomycin A adsorbed on

Fig. 8. (a) The adsorption isotherm of MIPs and NIPs; (b) the fitting plot of the adsorption isotherm of MIPs and NIPs.

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Fig. 9. (a) The adsorption kinetic curve of MIPs and NIPs. The initial concentration ranged from 0.1 to 1.0 mg/ml; (b) the fitting plot of the adsorption kinetic curve of MIPs and NIPs. Table 2 The results of selectivity adsorption experiments. MIPs Qe Lincomycin A Lincomycin B

(mg/g)

67.38 31.8

NIPs Kd

(ml/g)

118.37 37.75

˛MIP

Qe

3.14

49.8 46.9

(mg/g)

ˇ Kd

(ml/g)

66.38 61.56

˛NIP 1.08

2.91

Fig. 10. The effect of pH on adsorption capacity: pH (5, 7, 9 and 11).

MIP is higher than that of lincomycin B. From ˛ value, it can be seen that the selectivity coefficient of MIPs (3.14) is about three times of that of NIP (1.08). The relative selectivity coefficient is 2.91. It indicates that MIPs have a superior selectivity for lincomycin A compared with NIP. From this result, we can further investigate the separation performance of MIP by using column chromatography. 3.4. Column chromatography 3.4.1. Dynamic adsorption To estimate the dynamic adsorption of lincomycin A on MIP, the effect of flow rate and sample concentration on the adsorption capacity in column chromatography was investigated. To measure the effect of flow rate on adsorption capacity, the selected flow rate was 0.1, 0.15, 0.2 ml/min, respectively. Fig. 11a shows the adsorption quantity of lincomycin increased with the

Fig. 11. (a) The effect of flow rate (0.1, 0.15 and 0.2 ml/min) on dynamic adsorption capacity for lincomycin A. (b) The effect of initial concentration (0.5, 1.0 and 1.5 mg/ml) on dynamic adsorption capacity for lincomycin A.

decreasing of flow rate. The adsorption quantity at slow flow rate was higher than that at fast flow rate. The maximum adsorption capacity can reach 12.47 mg/ml. However, slow flow rate can lead to longer loading time. At the flow rate of 0.15 ml/min, suitable adsorption could be obtained. The adsorption quantity at different initial concentration (0.5, 1.0, 1.5 mg/ml) was investigated. From Fig. 11b, it can be seen that the quantity increased with the increasing of concentration. From 1.0 to 1.5 mg/ml, the quantity increases at a limit extent. The concentration at 1.0 mg/ml can obtain suitable adsorption quantity.

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Fig. 12. The HPLC spectra of the eluate. (a) The initial stage, (b) the middle stage, (c) the later stage of elution of lincomycin A. Column: C18; size: 150 × 12 mm; room temperature.

From the above results, it can also obtain that the optimized column chromatography conditions are found to be: flow rate at 0.15 ml/min and the concentration at 1.0 mg/ml. 3.4.2. Separation of lincomycin A by MIP column chromatography To separate lincomycin A from lincomycin crude product, the lincomycin sample solutions (80% lincomycin A + 20% lincomycin B) were loaded in the column. Next step, the mixture of methanol and acetic acid with ratio of 9:1 (v/v) was used for elution of lincomycin A and lincomycin B. In eluate, there was no lincomycin A or lincomycin B in first 2 h. Then lincomycin appeared from 2 to 4 h (Fig. 12a). And then, the adsorption quantity reached saturation. Elution was conducted by using above elution agent. From the elution result, lincomycin A and lincomycin B increased from 5 to 7 h (Fig. 12b), and then they decreased. At 8 h, there is only lincomycin A in eluate (Fig. 12c). The concentration was detected by HPLC. The lincomycin A has the 97.57% recovery and the 93.3% purity. From this result, it can be concluded the MIP column chromatography obtained a good separation effect. Compared with other paper [31], the MIP of this paper has a good selectivity for lincomycin A. The purity was higher in column chromatograph with MIP. At last, the separation effect of MIP in lincomycin fermentation broth was verified by column chromatograph. After preprocessing fermentation broth, loaded them to column. The conditions were the same with the above ones. From the elution results, it can be observed that many impurities outflow with loading, there are only lincomycin A imprinted on MIP. That is because MIP has good imprinted sites for lincomycin A. The lincomycin B is decreased

faster than lincomycin A in eluate. MIP for lincomycin A shows a better selective adsorption than lincomycin B. From the HPLC spectrogram, it can be found that the lincomycin A has the 90.9% recovery and the 81.1% purity. The low purity may be affected on the impurities in fermentation broth. On the whole, the MIP of lincomycin A has a good separation in fermentation broth with column chromatograph. 4. Conclusion In this work, core–shell MIP was prepared successfully. TEM, FT-IR, element analysis and TGA were used to characterize MIP for lincomycin. According to the adsorption isotherm of lincomycin, the maximum capacity of lincomycin binding on MIPs could reach 62.66 mg/g. The adsorption of lincomycin reached the equilibrium within 6 h based on kinetic curve. In addition, the selectivity coefficient of MIPs for lincomycin A with regard to lincomycin B was 3.14. The relative selectivity coefficient was 2.91. The above results indicate the MIP has good selectivity for lincomycin A. The column chromatograph with MIP obtains a good application potential. The recovery of lincomycin A was found to be 97.57% and the purity was 93.3%. References [1] Argoudelis AD, Fox JA, Mason DJ, Eble TE. New lincomycin-related antibiotics. J Am Chem Soc 1964;86:5044–5. ˛ ˛ [2] Kowalski P, Konieczna L, Oledzka I, Plenis A, Baczek T. Development and validation of electromigration technique for the determination of lincomycin and clindamycin residues in poultry tissues. Food Anal Methods 2013;7:276–82.

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