Molecularly imprinted polymers based on methacrylic acid and ethyleneglycol dimethacrylate for l -lysine recognition

Reactive and Functional Polymers 130 (2018) 98–110

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Molecularly imprinted polymers based on methacrylic acid and ethyleneglycol dimethacrylate for L-lysine recognition

T

⁎

Pisarev O.A.a,b, , Polyakova I.V.a a b

Federal State Budgetary Institution of Sciences of Russian Academy of Sciences Institute of Macromolecular Compounds, RAS, Saint-Petersburg 199004, Russia Peter the Great Saint-Petersburg Polytechnic University, Saint-Petersburg 195251, Russia

A R T I C LE I N FO

A B S T R A C T

Keywords: L-lysine Molecularly imprinted polymers Enantiomers separation Equilibrium Kinetics and dynamics of sorption

The enantiomers separation is a subject of a fundamental importance in various application fields such a pharmaceutical industry, biomedicine, food, etc. New molecularly imprinted sorbents based on methacrylic acid (MMA) as a functional monomer and ethyleneglycol dimethacrylate (EGDMA) as a crosslinking agent have been synthesized, characterized and evaluated as a selective materials for L-lysine recognition. The conditions for synthesis of optimal control non-imprinted polymer (NIP) have been found. The greatest sorption capacity and the best structural stability have been occurred on polymer containing 88 mol% MMA and 12 mol% EGDMA, which synthesized in medium of 45% water solution of isopropyl alcohol at 20% comonomers concentration. Samples of polymers molecularly imprinted with 3, 6, 9 and 12 mol% of L-lysine template (lysMIPs) were synthesized. The lysMIP-9 and lysMIP-12 possessed low structural stability therefore it was decided that these sorbents are unsuitable for use in preparative sorption processes and they were not studied further. Batch adsorption experiments were carried out as a function of pH (6.6, 8.3 and 11.0), ionic strength (0.1 M, 0.2 M and 0.4 M) and temperature (293 K, 310 K). Examination of distribution coefficients and thermodynamic functions of the L-lysine sorption on the NIP and lysMIPs indicated conversion from ion-ion interactions with non-imprinted sorbents to mainly nonionic interactions with molecularly imprinted sorbents. The sorption isotherms of L-lysine were analyzed using the generalized Langmuir and Freundlich equations. In the case of the lysMIP containing a greater number of imprint sites (lysMIP-6), the sorption of L-lysine occurred on the energetically homogeneous binding centers, forming one monolayer, while the nonspecific sorption of L-lysine on the NIP occurred with energetically heterogeneous binding of the sorbate. The best kinetic sorption properties were obtained on lysMIP-6. This could be due to the distribution of the sorbate in imprint sites, which were easily accessible in a narrow surface layer of sorbent particles. The obtained equilibrium and kinetic sorption data allowed developing an effective method of separation of D and L forms of lysine on molecularly imprinted sorbent lysMIP-6.

1. Introduction In recent years, molecular imprinting has become an important technique in preparing robust artificial recognition materials; it is widely described in the literature [1, 2]. Molecularly imprinted polymers (MIPs) are obtained by polymerization of different functional monomers and crosslinkers in the presence of a template [3–7]. Therefore, this process can be defined as a method for the construction of recognition sites in synthetic polymers, where a template is introduced during covalent assembly of the bulk phase in the course of polymerization or polycondensation. After the template molecules are removed, highly specific cavities remain within rigid three-dimensional polymer matrix. Shape and functionalities of these cavities are

complementary to those of the used templates (print species) and maintain “molecular memory” about the print templates. MIPs exhibit not only thermal, chemical, and mechanical stability, but are also able to mimic properties of natural systems (such as enzymes or antibodies) and serve as artificial receptors [8]. One of important applications of MIPs is their use as chiral stationary phases for enantiomeric separation of racemic solutions, such as amino acid derivatives and drugs [9–11]. Rational choice of copolymerization components and physicochemical conditions for the formation of a pre-polymerization complex allows improving molecular recognition ability of MIPs. If the pre-polymerization complex is highly stable during synthesis, it provides complementary interactions between template and functional monomer [3]. The presence of charged

⁎ Corresponding author at: Federal State Budgetary Institution of Sciences of Russian Academy of Sciences Institute of Macromolecular Compounds, RAS, Saint-Petersburg 199004, Russia. E-mail address: [email protected] (O.A. Pisarev).

https://doi.org/10.1016/j.reactfunctpolym.2018.06.002 Received 17 January 2018; Received in revised form 31 May 2018; Accepted 5 June 2018

Available online 15 June 2018 1381-5148/ © 2018 Elsevier B.V. All rights reserved.

Reactive and Functional Polymers 130 (2018) 98–110

O.A. Pisarev, I.V. Polyakova

Abbreviations APS AsA EGDMA IF lysMIP M MIP MMA NIP T X

Kswell pKα

ammonium persulfate ascorbic acid ethyleneglycol dimethacrylate imprint factor polymer imprinted with L-lysine functional monomer molecularly imprinted polymer methacrylic acid non-imprinted polymer template crosslinking monomer

l ms qe qmax qt Q r t t T V Vi V0 W W0 ΔG ΔН ΔS

Notation С Сe Cs d dp D F Kd KdNIP KdNIP

initial concentration (mg/mL) equilibrium concentration (mg/mL) concentration of sorbate in the sorbent (mg/mL) sorbents bulk density (g/cm3) diameter of sorbent particle (cm) sorption diffusion coefficient (cm2/s) degree of saturation distribution coefficient distribution coefficients of L-lysine on the lysMIP distribution coefficients of L-lysine on the NIP

swelling coefficient an apparent dissociation constant of empirical modified Henderson-Hasselbalch equation thickness of the sorptive layer (cm) sorbent weight (g) equilibrium sorption capacity (mg/g) maximum sorption capacity (mg/g) sorption capacity at moment of time (t) (mg/g) total ion-exchange capacity (mg/g) radius of a sorbent particle (cm) time (s) average sorption time (s) temperature (К) volume (mL) eluent volume (mL) column empty volume (mL) weight of polymer (g) weight of dry polymer (g) free energy change of sorption (J/mol) enthalpy change of sorption (J/mol) entropy change of sorption (J/mol·К)

Greek symbols α ρ

degree of ionization of functional groups relative non-sorbing radius of the “core”

MMA-EGDMA was found. In order to optimize the efficiency of enantioselective separation of L- and D-lysines, the competitive studies of sorption equlibrium, kinetics and dynamics on molecularly imprinted polymers and on the non-imprinted polymers were carried out using batch and frontal sorption technique.

functional groups and hydrophobic/hydrophilic parts in amino acids can contribute to more specific and stronger interactions between template imprint and functional monomer. In the general case, the presence of excess amount of functional monomer can lead to the formation of non-specific binding sites [12]. Often, the template excess can worsen the MIP morphology [13]. Thus, sorption centers of MIPs are heterogeneous, so specific binding of solute can proceed together with non-specific sorption. Due to the heterogeneity of MIP networks, dilution of the concentration front of a target substance occurs during dynamic sorption [14–16]. Thus, physicochemical conditions (such as charge density of a network, pH and ionic strength of a solvent) that are used in both molecular imprinting and sorption of template should have a strong effect on the experimentally determined imprint specificity [17]. In the present work, we have used the copolymers based on methacrylic acid (MMA) and ethylene glycol dimethacrylate (EGDMA) to prepare sorbents imprinted with L-lysine. The MMA-EGDMA copolymers are very often used for molecular imprinting, but in the majority papers, the used amount of EGDMA exceeded 50 mol%, since the aim of researchers was to create macroporous MIPs with increased availability of imprint sites [18–20]. Our research group has also investigated sorption of many biologically active substances (BAS) on MMA-EGDMA sorbents [21–23]. In these studies, it was shown how the structural parameters and sorption properties of MMA-EGDMA copolymers can vary significantly with the change in the MMA to EGDMA ratio. The prominent feature of MMAEGDMA copolymers was the formation of structural segregation of polymer networks with increasing EGDMA concentration. The excess of hydrophobic crosslinking agent deteriorated thermodynamic quality of a solvent. As a result, MMA was copolymerized primarily with the formation of dense hard permeable microspheres which included the main amount of carboxyl groups (90–95%); subsequently, these microspheres were crosslinked with EGDMA forming transport macropores [24]. The aim of this work was on the basis MMA-EGDMA copolymers to synthesize molecularly imprinted polymers for the selective recognition of L-lysine. For this task, the most suitable non-imprinted copolymer

2. Experimental 2.1. Materials MMA (Vekton, St. Petersburg, Russia) was used as a functional monomer (M). EGDMA (Acros Organics, Geel, Belgium) was used as a crosslinking agent (X). Propanol-2 (Vekton, St. Petersburg, Russia) was introduced as a porogen. Chemically pure L-lysine (Sigma, USA) was employed as a template (T). Recrystallized ascorbic acid (AsA) and ammonium persulfate (APS) (Vekton, St. Petersburg, Russia) were used as initiators. All other chemicals and reagents used were of analytical grade. 2.2. Instruments Optical density measurements were conducted with the use of a SF256 UVI spectrophotometer (“LOMO”, St.-Petersburg, Russia). 2.3. Synthesis of polymers The MMA-EGDMA copolymers were prepared by radical copolymerization. The MMA and EGDMA were placed into 250 mL roundbottom three-neck flask equipped with argon inlet, dropping funnel, the Liebig condenser, and a stirring device. Comonomers (20% or 30%) were dissolved in water/propanol-2 (5.5: 4.5 v/v) solvent. L-lysine templates were introduced into polymerization mixture; the template/ comonomer molar ratios were 3, 6, 9 and 12 mol%. AsA-APS (1: 1.5) initiator (1 wt% to comonomers mixture). The reactive mixture was purged with argon to remove oxygen. Process was controlled by exothermic self-heating of the mixture up to 37 °C. After the reaction mixture became to converse into gel, the reactor was placed in water 99

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bath (40 °C) for 1 h to complete the reaction. Then, MMA-EGDMA copolymers were pre-washed with distilled water to remove non-reacted monomer, dried and ground; thus, the particles with a size of 160÷315 μm were obtained. To synthesize the lysMIPs, 3 mol%, 6 mol%, 9 mol% or 12 mol% of L-lysine were introduced into the polymerization mixture. The lysMIPs were pre-washed with distilled water to remove non-reacted monomer, dried and ground. Then they were washed with 0.5 N NaOH, water, 0.5 N HCl and then again with water until pH of the extract became neutral in order to remove L-lysine template molecules from the polymers networks. 2.4. Characterization of sorbents Surface morphology was examined using scanning electron microscopy (SEM). SEM studies of copolymers samples were performed using the Zeiss SUPRA–55VP microscope (Carl Zeiss AG, Germany). In order to remove surface charge and shield the incident beam from the charge accumulated in the bulk of the material, copolymer particles were deposited onto NEM TAPE carbon scotch, and thin conducting layer of carbon (~10 nm) was formed above. Prior to analysis, samples were dried in air at 25 °C for 7 days. 2.5. Swelling coefficient and density of the polymers To find swelling coefficient (Kswell), polymer samples (50 mg) were immersed in water solution. The swollen polymer sample (W) that was carefully wiped with a filter paper and weighed. The values of Kswell were calculated as:

Kswell =

W − W0 W0

(1)

where W is the weight of swollen polymer sample (g), W0 is the weight of dry polymer sample. The bulk density d (g/cm3) was found by weighing portion of a sorbent (1 cm3). 2.6. Determination of total ion exchange capacity and potentiometric titration of polymers The total ion exchange capacity (Q) was found experimentally using “the method of one weigh portion” [25]. Distilled water was added to weighed portions of the adsorbents (~0.1 g), and the mixture was stirred until the complete swelling was achieved. The excess of water was removed by centrifugation. Portions of 0.1 N NaOH (50 mL) were added to the swollen sorbents, and the mixture was left under stirring until equilibrium was reached. To suppress hydrolysis of the sodium form of weak acid ion exchangers, determination of total ion exchange capacity was performed in the presence of 0.1 N NaCl. A fixed volume of the equilibrated solution was titrated with 0.1 N HCl, and the amount of NaOH necessary for neutralization of ionogenic groups in the cation exchangers was determined. The Q value was calculated as an amount of functional groups (mg-eq) in a given portion of the air-dry sorbent (g):

− CNaOH ) VNaOH (C 0 Q = NaOH m

Fig. 1. The effect of the amount of EGDMA and the concentration of comonomers on the swelling coefficients (a) and the structural stability (b) of the MMAEGDMA copolymers.

where VNaOH is the titrant volume (mL); CNaOH is the concentration of titrant (mg-eq·mL−1); and ms is the sorbent mass (g). The pKa is an apparent dissociation constant of empirical modified HendersonHasselbalch equation:

pH = pKa − n ⎛log10 ⎡ ⎣ ⎝

(2)

and CNaOH are the initial and the final concentrations of where NaOH (mg-eq mL−1), respectively; VNaOH is the solution volume (mL); and m is the sorbent mass (g). For the potentiometric titration, various volumes of the titrant were added to weighed portions of polymers to obtain different degrees of ionization (α) of functional groups. The α value was calculated as:

CNaOHl × VNaOH Q × ms

(4)

Parameter n is a characteristic indicating the change in the apparent dissociation constant with increasing degree of dissociation of ionogenic groups. The more n deviates from 1, the greater the ionization effect of each previous carboxyl group on ionization of the subsequent one (cooperative nature of ionization process). When n = 1, the modified Henderson-Hasselbalch equation becomes the logarithmic form of true dissociation constant and the equation becomes the true Henderson-Hasselbalch equation.

C0NaOH

α=

1−α ⎞ ⎤ α ⎦⎠

(3) 100

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Fig. 2. SEM images of the surfaces of the MMA-EGDMA copolymers synthesized with 6 mol% of EGDMA (a) and with 12 mol% of EGDMA (b).

Fig. 3. Potentiometric titration of MMA-EGDMA copolymers synthesized at 20% (a) and 30% (b) comonomers concentration and at various amounts of EGDMA.

2.7. Sorption equilibrium experiments

The equilibrium sorption capacity (qe) was calculated as.

Sorption experiments were carried out using the batch method. Weighed portions of the sorbents (0.01 g) were suspended in 10 mL of Llysine solution in sodium acetate buffer with a certain initial concentration. The suspension was stirred for 24 h at constant temperature, and then the sorbent particles were filtered off. The experiments were carried out at various values of ionic strength (0.1 M; 0.2 M; 0.4 M), pH (6.3; 8.3; 11.0) and different temperatures (293 and 310 K). Concentration of the amino acid after sorption was determined spectrophotometrically. Before measurements, 0.2 mL of 10% aqueous solution of pyridine and 0.2 mL of 2% aqueous solution of ninhydrin were added into test tubes containing 0.2 mL of L-lysine solution. The tubes were covered and heated in a water bath for 5 min until solutions turned dark violet; then 10 mL of distilled water was added to the cooled solutions. The UV absorbency was registered at λ = 570 nm.

qe =

(C − Ce )⋅V ms

(5) −1

where С and Сe (mg·mL ) are the initial and the equilibrium concentrations of L-lysine, respectively; V (mL) is the volume of the aqueous amino acid solution; ms (g) is the sorbent mass. Frontal dynamic sorption experiments were carried out on laboratory column (D × H = 10 mm × 50 mm) packed with the corresponding polymer. The samples of solution were taken at the outlet of the column at regular intervals, and concentration of the amino acid in these samples was determined spectrophotometrically. Dynamic curves were plotted in the Сi against (Vi– V0) coordinates using the obtained data; here, Сi (mg·mL−1) is the amino acid concentration in the sample; 101

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Table 1 Physicochemical properties of MMA-EGDMA copolymers synthesized at different monomers concentrations and at various relative content of EGDMA. Comonomers concentration in polymerization mixture, %

EGDMA, mol %

d, g/cm3

Q, mgeq/g

pKα

n

20

3 6 9 12 15 3 6 9 12 15

0.71 0.69 0.71 0.71 0.71 0.70 0.70 0.70 0.69 0.68

9.9 8.9 8.7 8.6 7.6 10.2 9.5 9.4 9.1 9.0

6.0 6.0 6.2 6.3 6.4 6.1 6.0 5.8 6.1 5.6

1.9 1.7 1.7 1.5 2.0 1.1 1.2 1.3 1.3 1.3

30

Vi and V0 (mL) are the eluent volume and the empty column volume, respectively. 2.8. Thermodynamic function of L-lysine sorption The change of Gibbs free energy (ΔG) of sorption is calculated as: (6)

ΔG = −RT ln K d −1

where R is the gas constant (8.31 kJ·mol ); T is the absolute temperature (K), Kd is the integral distribution coefficient of sorption. Unlike the differential values of the distribution coefficients characterizing sorption at a fixed concentration of sorbtive, the integral value characterizes “overall” the sorption process in the investigated concentration range. Experimentally, the Kd value can be obtained by integrating lnKdi against γ plots: 1

ln K d =

∫ ln Kdi dγ

(7)

0

where Kdi is the differential distribution coefficient that corresponds to the ith-point on the sorption isotherm. The Kdi value was calculated as:

K di =

Cs Ce

(8)

where Cs is the concentration of sorbate in the sorbent (mg·mL was found as:

CS = (C − Ce )

−1

V VS

) and

(9)

where Vs is the volume sorbent (mL), respectively. In (7), 0 ≤ γ ≤ 1 is calculated as

γ=

qe qmax

(10) Fig. 4. The effect of EGDMA relative content on the sorption of L-lysine by MMA-EGDMA copolymers synthesized at 20% (a) and 30% (b) comonomer concentration. Sorption occurred at different concentration of sodium acetic buffer: 1–0.05 M; 2–0.1 M; 3–0.2 M.

where qmax corresponds to plateau on isotherms qe against Ce. The enthalpy change ΔH (kJ·mol−1) was calculated as [26]:

ln

K d1 ΔH ⎛ 1 1 = − ⎞ K d2 R ⎝ T2 T1 ⎠ ⎜

⎟

(11)

where Kd1 and Kd2 are the distribution coefficient calculated at T1 and T2, respectively. Entropy change ΔS (kJ·(mol·K)−1) was calculated from the equation:

T ΔS = ΔH − ΔG

Table 2 Physicochemical properties of the lysMIPs and of the reference NIP.

(12)

The imprinting factor (IF) was calculated as:

IF =

K d (MIP ) K d (NIP )

(13)

where Kd(MIP) and Kd(NIP) are the values of distribution coefficients obtained for a MIP and the NIP, respectively. 102

Sorbent

d, mg/g

Q, mg-eq/g

pKα

n

NIP lysMIP-3 lysMIP-6 lysMIP-9 lysMIP-12

0.71 0.71 0.70 0.69 0.69

8.6 9.0 8.8 9.1 9.2

6.3 6.4 6.3 6.3 6.2

1.7 1.5 1.4 1.2 1.2

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Fig. 5. Potentiometric titration of the NIP and the lysMIPs.

2.9. Kinetic sorption experiments Sorption kinetics of L-lysine was studied in batch experiments, too. The 50 mL of L-lysine solution with the initial concentration of 1 mg·mL−1 in 0.2 M sodium acetate buffer (pH 8.3) was poured into the flask containing 50 mg of the swollen adsorbent. The flask was shaken. Samples (0.2 mL) were taken from the flask at regular intervals t (s), and concentration of L-lysine in these samples was determined. On the basis of the experimental data, the F against t0.5 dependences were plotted; the F value was defined as follows:

F=

qt qe

(14)

where F is a saturation degree of the sorbent with the sorbate, qt and qe (mg·g−1) are the sorption capacity at a moment of time (t) and the equilibrium sorption capacity, respectively. 3. Results and discussion 3.1. Characterization data of sorbents The most important parameter in the synthesis of MIPs is the M/X ratio that provides obtaining MIPs with sufficient permeability of the polymeric matrix, charge density, and accessibility of imprint sites [27]. At the same time one of the most important characteristics, affecting exploitable properties of sorbents used in preparative sorption of organic ions, is sorbents structural stability or the ability slightly to change their swelling upon changing sorption conditions. Therefore, the premature stage for synthesis of lysMIPs was to find conditions for synthesis of optimal non-imprinted MMA-EGDMA copolymers and to study their physicochemical and sorption properties. Two series of MMA-EGDMA copolymers with various content of crosslinking agent were synthesized with 20% and 30% comonomers concentrations in polymerization mixture. The abnormal dependence of the swelling coefficients on the relative content of EGDMA was observed for sorbents in hydrogen form for both series (Fig. 1a). The decrease in the swelling coefficients with increasing content of EGDMA from 3 to 9 mol% was caused by the increase in crosslinking of polymer chains and their mobility limitation, which is typical for gel type sorbents [28]. The increase in the swelling coefficients with further increasing content of EGDMA from 9 to 15 mol% was conditioned by conversion of polymer network from the gel structure to the heterogeneous structure. The abnormal increase in polymer swelling is

Fig. 6. The effect of the template concentration on the swelling coefficients (a) and structural stability of the lysMIPs (b).

associated with an increase in the number of intermolecular contacts in the microgels inside the polymer network. As a result, the microgels are “compressed” due to physical interactions forming “secondary” porosity. In the case of MMA-EGDMA copolymers, such effect may be caused by hydrophobic interactions between MAA [16, 24, 29]. The structural stability of sorbents of both series increased with increasing amount of the crosslinking agent. At the same time, the structural stability was higher for copolymer containing 12 mol% of EGDMA synthesized at 20% monomers concentration in polymer mixture (Fig. 1b). As shown in Fig. 2b, MMA-EGDMA copolymer with 6 mol% of EGDMA had the homogeneous surface. The polymer network of the MMA-EGDMA copolymer with 12 mol% of EGDMA was structurally segregated; both firmly crosslinked domains (microglobules) and transport channels were observed in it (Fig. 2b). The acidity of sorbents changed at varying the M/X ratio (Fig. 3). A decrease in the value of Q with the increase in the content of EGDMA 103

Reactive and Functional Polymers 130 (2018) 98–110

O.A. Pisarev, I.V. Polyakova

Fig. 7. The influence of pH and ionic strength on the sorption of L-lysine on the NIP (a), on the lysMIP-3 (b) and on the lysMIP-6 (c). Sorption was carried out at amino acid initial concentration of 1 mg/ml.

Fig. 8. Isotherms of L-lysine sorption from 0.2 M sodium acetic buffer at pH 8.3 and 0.2 M ionic strength.T1 = 293 K (a); T2 = 310 K (b). 1– the NIP; 2 – the lysMIP-3; 3 – the lysMIP-6.

was observed for both series of sorbents (Table 1). The parameter n also pointed to some structural differences of copolymers synthesized at different M/X ratio. The more deviation of n from 1 pointed to the greater effect of the ionization of each previous carboxylic group on ionization of the subsequent one. The n values were higher for copolymers synthesized at 20% of comonomers. This could be due to the microheterogeneity in diluted polymerization mixture appeared at an earlier stage of the synthesis and the formation of the heterogeneous network was carried out with a lower amount of crosslinking agent. As a result, the copolymers had better structural stability than those ones synthesized with 30% of comonomers. The effect of the content of EGDMA on the sorption properties of MMA-EGDMA copolymers was the same for both series of sorbents. The values of q decreased with increasing ionic strength (Fig. 4). This confirmed the significant contribution of ion-ionic interactions to the sorption of L-lysine by non-imprinted MMA-EGDMA copolymers. The 104

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Na-form significantly swelled as compared to the H-form, and the lowest structural stability was observed for lysMIP-9 and lysMIP-12 (Fig. 6a, b). So it was decided that these sorbents are unsuitable for use in preparative sorption processes and they were not studied further. 3.2. Influence of pH and ionic strength on the equilibrium sorption On the NIP, the non-specific sorption was much affected by the ionic strength and pH (Fig. 7a). The effect of the ionic strength decreased when L-lysine was bound with lysMIPs. (Fig. 7b, c). The sorption of Llysine by lysMIPs differed from that by the NIP. The least dependence on ionic strength and the significant increase in sorption capacities were observed for the sorption by lysMIP-6. 3.3. Study of the sorption thermodynamics The thermodynamic functions were calculated by using the integral values of the Kd that were calculated from isotherms obtained under various pH (6.6, 8.3 and 11.0), ionic strength (0.1 M, 0.2 M and 0.4 M), and temperature (293 K, 310 K). First, the differential values of Kdi were calculated for each concentration point on the isotherms, and then the Kd values were calculated as the area under the lnKdi against γ curves. Because of a large number of graphs were made, the article provides, as an example, only some of the isotherms and the lnKdi against γ plots (Figs. 8, 9). All calculated values of Kd and thermodynamic functions are presented in Table 2. In the case of the non-specific sorption of L-lysine on the NIP at pH 6.5, the sorption proceeded as an entropy-controlled process. Hydrophobic interactions prevailed in binding of L-lysine with the NIP and the increase in temperature led to the increase of the Kd value. At pH 8.3 and 11.0, when the NIP was completely ionized, sorption proceeded as an enthalpy-controlled process due to ion-ionic interactions and the increase in temperature led to reduction of the Kd values (Table 3). Thermodynamic functions of sorption on lysMIPs differed from those on the NIP. On the lysMIP-6, a positive entropy change was observed for all sorption conditions. The growth in entropy change with the temperature increase was probably caused by hydrophobic interactions, which could be a consequence of intensive disordering of solvent molecules on the sorbent surface when L-lysine was bound with imprint sites specifically. The effect of imprinting on the sorption of L-lysine by the lysMIPs was defined by the IF (Table 4). The better values of IF were observed for the sorption of the amino acid by the lysMIP-6, especially at 0.2 M ionic strength. 3.4. Sorption isotherm modelling Successful implementation of the process of dynamic sorption depends on the correct description of the equilibrium separation between the two phases. In order to optimize the sorption system for isolating the amino acid from the aqueous solution, it is necessary to establish the most accurate parameters for the equilibrium sorption curve. In our work we used the Langmuir model [30] and the empirical Freundlich Eq. [31] to study mechanisms of the L-lysine distribution and calculate constants for L-lysine sorption at pH 8.3 and 0.2 M ionic strength, which are presented in Fig. 8 a, b. The generalized Langmuir model takes into account the binding of substances in the independent energetically homogeneous sorption regions:

Fig. 9. The integral values of the lnKd calculated for L-lysine sorption on the NIP (white area), on the lysMIP-3 (grey area) and on the lysMIP-6 (hatched area). The sorption was carried out from 0.2 M sodium acetic buffer at pH 8.3.T1 = 293 K (a); T2 = 310 K (b).

best sorption occurred on sorbent containing 12 mol% EGDMA synthesized at 20% comonomer concentration. Therefore, this sorbent was chosen as the reference non-imprinted polymer or the NIP. Based on the conditions for the synthesis of this NIP, the lysMIPs were further synthesized. Then samples of MIPs with 3, 6, 9 and 12 mol% of L-lysine were synthesized. The lysMIPs had close values of d, Q and pKα (Table 2, Fig. 5). The structure of lysMIPs became sparser with increasing concentration of the template and the n values decreased. Sorbents in the

m

qe =

∑ i=1

qmax i KLi Ce 1 + KLi Ce

(15)

where qmaxi is the limiting amount of the boun sorbate in the ith layer, mg/g; KLi is the Langmuir constant in the ith monolayer, L/mg. 105

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Table 3 Influence of pH and ionic strength on the distribution coefficients and thermodynamic functions of the L-lysine sorption on the NIP and lysMIPs. Sorbent

NIP

pH

6.3

8.3

11.0

lysMIP-3

6.3

8.3

11.0

lysMIP-6

ΔH, kJ mol−1

I, M

6.3

8.3

11.0

0.1 0.2 0.4 0.1 0.2 0.4 0.1 0.2 0.4 0.1 0.2 0.4 0.1 0.2 0.4 0.1 0.2 0.4 0.1 0.2 0.4 0.1 0.2 0.4 0.1 0.2 0.4

61.8 47.7 35.0 −81.3 −78.0 −29.0 −37.4 −34.3 −21.9 −32.4 46.4 35.8 −7.1 16.1 −21.1 38.8 31.6 6.4 −12.5 −1.6 −8.7 −4.3 −6.2 0.5 76.2 67.0 46.2

293 К

310 К

Kd1

ΔG, kJmol

76.7 48.8 85.5 122.0 109.5 124.9 32.4 25.0 15.1 256.9 66.5 100.1 67.9 64.9 57.5 12.3 10.3 21.4 200.3 150.0 218.2 102.6 144.9 113.0 8.8 17.6 21.0

−10.5 −9.5 −10.8 −11.7 −11.4 −11.7 −8.5 −7.8 −6.6 −13.5 −10.2 −11.2 −10.3 −10.2 −9.9 −6.1 −5.7 −7.4 −12.9 −12.2 −13.1 −11.3 −12.1 −11.5 −5.3 −7.0 −7.4

−1

72.3 57.2 45.8 −69.6 −66.6 −17.3 −29.0 −26.5 −15.3 −18.9 56.7 47.0 3.2 26.3 −11.3 44.9 37.2 13.8 0.4 10.6 4.4 7.0 5.9 12.0 81.5 74.0 53.6

lysMIP-3

pH

6.3

8.3

11.0

lysMIP-6

6.3

8.3

11.0

I, M

0.1 0.2 0.4 0.1 0.2 0.4 0.1 0.2 0.4 0.1 0.2 0.4 0.1 0.2 0.4 0.1 0.2 0.4

IF T = 293 К

T = 310 К

3.3 1.4 1.2 0.6 0.6 0.5 0.4 0.4 1.4 2.6 3.1 2.6 0.8 1.3 0.9 0.3 0.7 1.4

0.4 1.3 1.2 3.0 5.0 0.9 2.1 1.8 2.7 0.5 1.1 0.9 4.8 6.7 2.5 3.5 6.9 6.5

qmax KL Ce 1 + KL Ce

ΔG, kJ mol−1

TΔS, kJ (Kmol−1)

308.8 143.2 188.1 19.5 18.9 56.9 13.9 11.5 9.2 123.6 189.6 224.5 57.9 93.5 35.7 29.5 21.0 24.7 151.1 144.7 179.5 93.2 126 114.4 48.8 79.9 59.6

−14.8 −12.8 −13.5 −7.6 −7.6 −10.7 −67.8 −62.8 −5.7 −12.4 −13.5 −13.9 −10.4 −11.7 −9.2 −8.7 −7.8 −8.3 −12.9 −12.8 −13.4 −11.7 −12.4 −12.2 −10.0 −11.3 −10.5

76.5 60.5 48.5 −73.7 −70.4 −18.3 −30.9 −28.1 −16.2 −20.0 59.9 49.7 3.4 27.8 −11.9 47.5 38.4 14.6 0.4 11.2 4.7 7.4 6.2 12.7 86.2 78.3 56.7

(18)

where K1 = 1/Сmax; K2 = 1/Сmin. Best-fitting isotherm model estimation was determined based on the use of the correlation coefficient, R2 to calculate the error deviation between experimental and predicted equilibrium sorption data after non-linear analysis [33]: np

np

⎡⎛ ∑ (qi,exp − qi,exp )2 − ∑ (qi,exp − qi,mod el )2 ⎞ ⎢⎜ ⎟ i R2 = 1 − ⎢ 1 − i np ⎟ ⎢⎜ 2 ∑ (qi,exp − qi,exp ) ⎟ ⎢⎜ i ⎠ ⎣⎝ ⎤ np − 1 ⎤ ⎥ ⎡ × ⎢ np − p ⎥ ⎥ ⎣ ⎦⎥ ⎥ ⎦

(19)

where qi,model is each value of q predicted by the fitted model; qi,exp is each value of q measured experimentally; qexp is the average of q experimentally measured; np is the number of experiments performed and p is the number of parameters of the fitted model. The calculated constants are given in Table 5. The values of R2 indicated that the sorption of L-lysine by the lysMIP-6 showed good agreement with the Langmuir isotherm. Thus, the sorption conditions provided an increase in the contribution of binding the amino acid to the thermodynamically homogenous imprint sites. On the lysMIP-3, only the first step of the bi-Langmuir isotherm showed good agreement with experimental isotherm. In the wide concentrations range, the sorption on the lysMIP-3 better described with the Freundlich equation. Thus, L-lysine bound with both with imprint sites and non-specific

(16)

The empirical Freundlich equation describes the distribution of sorbates on heterogeneous sorption surfaces:

qe = KF Ce1/ n

Kd2

qmax = KF (1 − 1/ n2)(K1−1/ n − K2−1/ n )

The summing is performed over all types of sorption center, each of which has its limiting capacity of monolayer and is characterized by its own adsorption constant. At m = 1, the equation of the generalized Langmuir constant is transformed into the model that describes the sorption of the substance in a monomolecular layer on the energetically equivalent sorption sites:

qe =

)

capacity; 1/n is the surface heterogeneity index; 0 ≤ 1/n ≤ 1. At 1/n → 1, the heterogeneity decreases; and at 1/n = 1, the Freundlich equation transforms into a linear isotherm. Since function (18) has no upper bound, the maximum sorption capacity qmax in the concentration range on the experimental sorption isotherms (from Cmin to Cmax) was calculated in the following way [32]:

Table 4 The effect of pH and ionic strength on the IF values. Sorbent

TΔS, kJ (Kmol

−1

(17)

where KF is the Freundlich constant that characterizes the sorption 106

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O.A. Pisarev, I.V. Polyakova

Table 5 Constants of of L-lysine sorption on the NIP and the lysMIPs calculated using the Langmuir and Freundlich isotherms. The sorption was carried out from 0.2 M sodium acetic buffer at pH 8.3. Т = 293 К

Sorbent

Т = 310 К

qmax

KL

R2

qmax

KL

R2

Langmuir isotherm NIP lysMIP-3 lysMIP-6

732.95 490.50 1303.62

1.43 2.81 5.90

0.5992 0.4011 0.9561

1561.89 1591.35 1754.89

0.17 0.45 1.26

0.7620 0.6027 0.9273

bi-Langmuir isotherm NIP 1step 2step lysMIP-3 1step 2step

508.3 1977.53 320.71 824.71

2.12 0.40 10.25 0.84

0.2263 0.8644 0.9995 0.4605

41.94 1561.89 503.32 4580.81

3.27 0.17 1.50 0.18

0.5306 0.4976 0.9999 0.7303

Freundlich isotherm Sorbent NIP lysMIP-3 lysMIP-6

KF 587.96 380.88 1210.78

1/n 0.85 0.41 0.70

R2 0.9484 0.8337 0.6109

qmax 389.03 292.52 399.59

KF 184.24 608.67 1168.95

qmax 215.21 248.70 789.24

6 exp (−π 2F0) π2

F≈1−

F0 =

1/n 1.40 1.13 0.86

R2 0.9039 0.9648 0.7172

(20)

Dt r2

(21) 2 −1

where D is the effective diffusion coefficient, cm s ; t is the diffusion time, s; r is the radius of sorbent grain, cm. After the Taylor series expansion for small values of F0, the following approximation for the first term of the series (n = 1) is valid:

6 π

F=

F0 − 3F0

(22) 0.5

In the case of the initial parts of the kinetic curves F against t are linear in a sufficiently extended interval (up to F ~ 0.3–0.4), the curves are well described by the asymptotic expression for sorption in spherical grains [34]:

6 r

F≈

Dt π

(23)

where t is the average sorption time. According to the method of statistical moments [35]:

Fig. 10. The effect of the particle size on the sorption of L-lysine on MMAEGDMA copolymers at different initial concentrations. The sorption was carried out from 0.2 M sodium acetic buffer at pH 8.3. 1–0.5 mg/mL; 2–1.0 mg/mL; 3–2.0 mg/mL.

∫0

t =

1

tdF

(24)

The experimental t is found as the area above the kinetic curve in the coordinates F against t. Then D value for sorption in spherical grains is calculated from the expression:

sorption sites. When concentrations were small, the binding with thermodynamically homogenous imprint sites were preferable. The non-specific binding of the amino acid with the NIP agreed best with the Freundlich equation. This fact pointed to the heterogeneity of NIP sorption sites.

D =

r2 15t

(25)

However, the diffusion of organic ions from the periphery to the center of the particle can be carried out in a limited layer [21, 22, 36, 37]. In this case, the “core-shell” model can be used to estimate kinetic parameters:

3.5. The sorption kinetics The sorption of organic molecules in the solid phase often is limited by interparticle diffusion kinetics because of steric obstacles during movement of the sorptive inside the polymeric network [21, 22]. The use of the mathematical apparatus of the theory of thermal conductivity makes it possible to obtain a fairly accurate expression for calculating kinetic parameters. Upon contact of a sorbent spherical grain with a continuously renewing solution, the sorption kinetics is described by solving the well-known Fick equations of diffusion into a sphere with boundary conditions of the first term and with constant diffusion coefficients:

F≈

6 1 + ρ + ρ2

Dt πl 2

(26)

where ρ = 1 − l/r is the value of nondimensional radius of the nonsorbing “core”; l is the «thickness» of sorption layer, cm. The sorption diffusion coefficients for the “core-shell” model can be calculated as:

D =

(1 + 3ρ + 6ρ2 + 5ρ3) l2 × 2 (1 + ρ + ρ ) 15t

(27)

The value of the l was determined for MMA-EGDMA copolymer 107

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O.A. Pisarev, I.V. Polyakova

Table 6 Effective diffusion coefficients and average sorption time of L-lysine sorption on the NIP and the lysMIPs. Sorbent

NIP

lysMIP-3

lysMIP-6

dp, μm

130 90 75 130 90 75 130 90 75

t , min

63.2 6.9 1.1 8.6 4.3 1.3 2.5 1.2 0.7

D × 108 , cm2/s The Boyd model

The “core-shell” model

0.06 0.32 1.42 0.43 0.52 1.21 1.47 1.88 3.23

0.04 0.28 1.38 0.97 0.45 1.17 1.02 1.65 3.17

from the qe against dp dependence obtained at various initial concentrations of L-lysine (Fig. 10). As the sorbent was grinded, the particles size decreased and sorption surface increased. The tendency to increasing the sorption capacities with decreasing the size of the sorbent particles was observed at all concentrations. The sorption capacities increased significantly when dp reached 60 μm, since the sorption proceeded practically in the full volume of the particle. Further grinding did not significantly affect the capacities values. Thus, the l value was chosen to be equal 30 μm. The kinetic curves had the extended linear initial parts and indicated the intradiffusional mass transfer of L-lysine into the sorbents (Fig. 11). The Boyd and the “core-shell” models were used for the kinetic data processing (Table 6). As the particle size of sorbents was reduced, the kinetic parameters were improved. This fact proves the correct application of the used sorption kinetics models. At the same time, on the smallest fraction of the sorbent particles, the kinetic parameters calculated using both models had close values. This confirmed the fact that the sorption could proceed in a limited «superficial» layer. In addition, the best kinetic sorption properties were obtained on the lysMIP-6. This could be due to the distribution of the sorbate in imprint sites, which were easily accessible in a narrow surface layer of sorbent particles. 3.6. Enantioselectivity of sorbents toward L-lysine in sorption dynamics The preparative isolation of enantiomers from multicomponent biological media is one of the most urgent problems of biotechnology. Therefore, it is important to evaluate the capability of the synthesized MIPs in the realization of the molecular recognition of L-lysine in the racemic mixture. When desorption of L-lysine and D,L-lysine was occurred from the NIP, the unimodal concentration profiles were obtained (Fig. 12a). The presence of imprint-sites in the MIPs matrices led to some division of D and L forms of amino acid on lysMIP-3 (Fig. 12b) and to full division on lysMIP-6 (Fig. 12c). 4. Conclusions In this study, lysMIPs based on the MMA-EGDMA copolymer molecularly imprinted with L-lysine were synthesized using the imprinting technique in bulk copolymerization. In order to select the most suitable matrix for molecular imprinting, the influence of the relative content of crosslinking agent on the structure of the copolymer was studied. There were shown the fundamental differences in the formation of the polymer network, which arise at varying content of EGDMA in the reaction mixture. It was demonstrated that introduction of > 12 mol% of EGDMA promotes abnormal growth of the sorbents swelling coefficients due to structural segregation of the polymer network (formation of strongly crosslinked macrospheres and transport channels). It is obvious that growth of

Fig. 11. The effect of particle size on the sorption kinetics of L-lysine on the NIP (a), on the lysMIP-3 (b), and on the lysMIP-6 (c). The sorption was carried out from 0.2 M sodium acetic buffer at pH 8.3.

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Fig. 12. Dynamic desorption of L-lysine and D,L-lysine on the NIP (a), on lysMIP3 (b) and lysMIP-6 (c). The sorption was carried out from 0.2 M sodium acetic buffer at pH 8.3, the initial concentration was 1 mg/mL; flow rate 0.5 mL/min. Desorption was carried out with 0.4 M sodium acetic buffer at pH 12.7; D × H = 10 mm × 50 mm; flow rate 0.15 mL/min.

EGDMA leads to disappearance of exchange groups which are predominantly contained in microspheres. Besides, this process can contribute to the loss of specific capacity due to irreversible crosslinking of pre-polymerization complexes (“template-functional monomer”) in dense microspheres. In this study, there was also shown how minor changes in the physicochemical conditions of synthesis and sorption media can fundamentally affect the specific affinity of lysMIPs toward L-lysine. Effects of the ionic strength and pH on sorption of the amino acid on the lysMIPs and the corresponding NIP showed that the increase in the positive entropy change predominated in the template – imprint sites binding. At the same time, the affinity of the lysMIPs toward L-lysine became higher in the sorption conditions, which were close to those of the polymerization (formation of recognition points in the imprint sites). The better agreement of the sorption of L-lysine on the lysMIP-6 with the Langmuir isotherms evidenced about the preferable binding with imprint sites. Simultaneously, the kinetic parameters of sorption testified to the availability of imprint sites in the surface sorbing layer. This contributed to the rapid kinetics and the separation of the enantiomers of the amino acid in the dynamic sorption. Study of the sorption on the synthesized sorbents has also demonstrated how the effects of ionic strength and pH on specific binding of template molecule can be minimized. This result can be useful in performing sorption processes aimed at the preparative obtaining of biologically active substances directly from native solutions and culture fluids (which usually contain large amounts of mineral salts). References [1] C. Alexander, H.S. Andersson, L.I. Andersson, R.J. Ansell, N. Kirsch, I.A. Nicholls, J. O'Mahony, M.J. Whitcombe, J. Mol. Recognit. 19 (2006) 106–180. [2] D.A. Spivak, K.I. Shea, J. Mol. Recognit. 25 (2012) 320–382. [3] D.A. Spivak, Science and technology, in: M. Yan, O. Ramström (Eds.), Molecularly Imprinted Materials, Marcel Dekker, New York, NY, USA, 2005, p. 413. [4] H.S. Anderson, I.A. Nicholls, A historical perspective of the development of molecular imprinting, in: B. Sellergreen (Ed.), Molecular Imprinted Polymers: Man-Made Mimics of Antibodies and their Application in Analytical Chemistry, Elsevier, Mainz; Amsterdam, 2001, p. 582. [5] L. Ye, K. Mosbach, Chem. Mater. 20 (2008) 859–868. [6] O. Brügemann, K. Haupt, Y. Lei, E. Yilmaz, K. Mosbach, J. Chromatogr. A 889 (2000) 15–24. [7] C.J. Allender, Adv. Drug Deliv. Rev. 57 (2005) 1731–1732. [8] M. Kempe, K. Mosbach, J. Chromatogr. A 694 (1995) a–13. [9] G. Wulff, H.G. Poll, M. Minarik, 9 (1986) a–405. [10] B. Shellergren, B. Ekberg, K. Mosbach, J. Chromatogr. 347 (1985) 1–10. [11] M. Kempe, K. Mosbach, J. Chromatogr. A 691 (1995) 317–323. [12] H. Kim, G. Guiochon, J. Chromatogr. A 1097 (2005) 84–97. [13] A.P. Leshchinskaya, N.M. Ezhova, O.A. Pisarev, React. Funct. Polym. 102 (2016) 101–109. [14] J.A. Garćia-Galzón, M.E. Díaz-Garćia, Sens. Actuat. B 123 (2007) 1180–1194. [15] A.P. Leshchinskaya, A.R. Groshikova, I.V. Polyakova, O.A. Pisarev, E.F. Panarin, Mol. Imprint. 1 (2013) 17–26. [16] O.A. Pisarev, I.V. Polyakova, Trends Chromatogr. 7 (2013) 85–106. [17] E. Verheyen, J.P. Schillemans, M. van Wijk, M.-A. Demeniex, W.E. Hennink, C.F. van Nostrum, Biomaterials 32 (2011) 3008–3020. [18] M. Lehmann, M. Dettling, H. Brunner, E.M. Günter, J. Chromatogr. B 808 (2004) 43–50. [19] Y. Lu, Ch. Li, H. Zhang, X. Liu, Anal. Chim. Acta 489 (2003) 33–43. [20] R. Panahi, E. Vasheghani-Farahani, S.A. Shojaosadati, Biochem. Eng. J. 35 (2007) 352–356. [21] G.V. Samsonov, O.A. Pisarev, Isolat. Purif. 2 (1996) 93–102. [22] I.V. Polyakova, V.M. Kolikov, O.A. Pisarev, J. Chromatogr. A 1006 (2003) 251–260. [23] N.M. Ezhova, I.V. Polyakova, O.A. Pisarev, Appl. Biochem. Microbiol. (Russ) 45 (2009) 221–225. [24] G.V. Samsonov, O.A. Pisarev, A.T. Melenevskiy, Pure Appl. Chem. 65 (1993) 2287–2290. [25] V.S. Soldatov, Z.I. Sosinovich, T.A. Korshunova, T.V. Mironova, React. Funct.

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