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  • 1 İnönü University, 44280, Malatya, Turkey
  • 2 Anadolu University, 26470, Eskişehir, Turkey
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A new molecularly imprinted polymer (MIP) was prepared by using catechin (C) as the template molecule. The polymer was characterized by swelling tests, scanning electron microscopy (SEM), Brunauer–Emmett–Teller (BET), and Fourier transform infrared (FTIR) spectroscopy. The MIP with high recovery was selected as a solid-phase extraction (SPE) sorbent in this work. The standard solutions were directly applied onto the SPE cartridges following loading, washing, and elution procedures. A solution of the collected fractions was analyzed by high-performance liquid chromatography–diode-array detection (DAD) and fluorescence detector. The optimization of the method and validation was achieved on a C18 column (5 μm, 250 × 4.6 mm) with methanol–water (35:65, v/v) mixture adjusted pH 2.5 as the mobile phase at a flow rate of 1 mL min−1 at room temperature. The selectivity coefficient (k) of imprinted p(HEMA–MAH) cryogel was 5.1-fold that of non-imprinted cryogel. It showed good selectivity and affinity for C molecule. A comparison was made between the results obtained with the MIP cartridges and a traditional C18 reversed-phase cartridge. It was observed that 2.3 times higher recovery of C can be obtained on catechin-MIP cryogel. The results of the presented work showed that the prepared MIP can be used as SPE sorbent for extracting of C from red wines.

Abstract

A new molecularly imprinted polymer (MIP) was prepared by using catechin (C) as the template molecule. The polymer was characterized by swelling tests, scanning electron microscopy (SEM), Brunauer–Emmett–Teller (BET), and Fourier transform infrared (FTIR) spectroscopy. The MIP with high recovery was selected as a solid-phase extraction (SPE) sorbent in this work. The standard solutions were directly applied onto the SPE cartridges following loading, washing, and elution procedures. A solution of the collected fractions was analyzed by high-performance liquid chromatography–diode-array detection (DAD) and fluorescence detector. The optimization of the method and validation was achieved on a C18 column (5 μm, 250 × 4.6 mm) with methanol–water (35:65, v/v) mixture adjusted pH 2.5 as the mobile phase at a flow rate of 1 mL min−1 at room temperature. The selectivity coefficient (k) of imprinted p(HEMA–MAH) cryogel was 5.1-fold that of non-imprinted cryogel. It showed good selectivity and affinity for C molecule. A comparison was made between the results obtained with the MIP cartridges and a traditional C18 reversed-phase cartridge. It was observed that 2.3 times higher recovery of C can be obtained on catechin-MIP cryogel. The results of the presented work showed that the prepared MIP can be used as SPE sorbent for extracting of C from red wines.

Introduction

Molecular imprinting has become an interesting method to produce recognition sites in a macromolecular matrix for the preparation of selective sorbents over the past decades [1]. Molecularly imprinted polymers (MIPs) were synthetically developed by interactive complexation of a functional monomer with a template, followed by polymerization with a cross-linker. The template was removed from the polymer matrix to form cavities sculpted around the template molecules. After the removal of template, the resting polymer is more selective. The selectivity of the polymer depends on various factors. These factors are the size and shape of the cavity and rebinding interactions. MIPs are easy to prepare, stable, and capable of molecular recognition [2, 3].

Cryogels are considered as one of the new types of polymeric hydrogels. They are a very good alternative to chromatographic supports because they have many advantages such as large pores, short diffusion path, low pressure drop, very short residence time for both adsorption and elution, and high chemical and physical stability [4]. Cryogels are also cheap materials, and therefore, they can be used as disposables, thereby avoiding cross-contamination between batches [5].

MIPs were used as solid-phase extraction (SPE) sorbents in many applications. Molinelli et al. prepared MIPs selective for quercetin and applied as the material for SPE in off-line separations. The MIP achieved a capacity of 0.4 g quercetin per gram polymer and a recovery rate of 98.2%. The MIP showed excellent selectivity toward quercetin and was therefore suitable for the application in SPE [6]. Blahova et al. prepared MIP using acrylamide and ethylene glycol dimethacrylate (EGDMA) as functional and crosslinking monomers, respectively. Catechin was chosen as the template molecule and solvent acetonitrile as porogen. The polymer was investigated as SPE sorbent for the cleanup of organic extracts of green tea [7]. A synthetic polymer selective for epicatechin was prepared using the molecular imprinting technique and was used as an SPE sorbent. The capacity of 38 μg epicatechin per 100 mg polymer was achieved with recovery rates of 95.04%. The MIP has shown a good selectivity toward epicatechin, enabling an effective sample pretreatment of tea extracts and simplifying subsequent chromatographic analysis [8]. Song et al. prepared MIPs through thermal polymerization by using quercetin as the template molecule, acrylamide (AA) as the functional monomer, and EGDMA as the cross-linker in the porogen of tetrahydrofuran (THF). Besides quercetin, two structurally similar compounds of rutin and catechol were employed for molecular recognition specificity tests of MIPs. It was noticed that the MIPs showed the highest selective rebinding to quercetin. Accordingly, the MIPs were used as an SPE sorbent for the extraction and enrichment of quercetin in Cacumen Platycladi samples, followed by high-performance liquid chromatography–ultraviolet (HPLC–UV) analysis [9]. In another work, the caffeine and some catechin compounds were extracted from green tea by using two MIPs as sorbent materials in multi-SPE process. A comparison was made between the results obtained with the caffeine and catechin MIP cartridges, blank polymer, and a traditional C18 reversed-phase cartridge. It was thereupon found that the recovery of caffeine by the caffeine-MIP-based sorbent used in this work was almost two and four times greater than that by a commercially available C18 material [10]. Tian et al. prepared a quercitrin MIP using quercitrin as template, acrylamide as functional monomer, and EGDMA as cross-linker in the porogen of tetrahydrofuran. Crude extract of flavones from Chamaecyparis obtusa was purified by using this polymer as the separation medium [11]. Lopez et al. have successfully developed MIP for solid extraction and preconcentration of catechins by a thermal polymerization method. Quercetin was used as template, 4-vinylpyridine as functional monomer, and EGDMA as cross-linker in this method. The quercetin MIPs were evaluated according to their selective recognition properties for quercetin, structurally related compounds (catechin, epigallocatechin gallate, and epicatechin), and an unrelated compound of similar molecular size (α-tocopherol) [12].

The purposes of this study were as follows: (1) to synthesize and characterize catechin-imprinted cryogels, because cryogels are the new type of polymeric materials and have many advantages such as highly porous structure, nonhindered diffusion of solutes, very low flow resistance, very short residence time for both adsorption and elution; (2) to use as the alternative solid-phase extraction sorbent to extract catechin directly from red wine with higher affinity prior to chromatographic analysis; (3) to optimize and validate method for the analysis of catechin compounds in red wines by HPLC; and (4) to compare between the results obtained with MIP cartridges and a traditional C18 reversed-phase cartridge for showing selectivity of them.

The collected samples were analyzed with HPLC photodiode array and fluorescence detection because catechin and epicatechin are highly fluorescent.

Experimental

Reagents and Chemicals

The standard chemicals of (+)-catechin (C, ≥98%), (−)-epicatechin (EC, ≥90%), (−)-epigallocatechin gallate (EGCG, ≥95%), (−)-epigallocatechin (EGC, ≥95%), (−)-gallocatechin (GC, ≥98%), (−)-epicatechin gallate (ECG, ≥98%), (−)-gallocatechin gallate (GCG, ≥98%), l-histidine methylester, methacryloyl chloride, N,N′-methylenebisacrylamide, and ammonium persulfate (APS) were purchased from Sigma Chemical Co. (USA). Hydroxyethyl methacrylate (HEMA) obtained from Fluka A.G. (Buchs, Switzerland), distilled under reduced pressure in the presence of hydroquinone inhibitor, and stored at 4 °C until use. N,N,N′,N′-tetramethylene diamine (TEMED) was obtained from Fluka A.G. (Buchs, Switzerland). HPLC-grade acetonitrile, methanol, and infrared (IR) grade KBr were purchased from Merck (Darmstdt, Germany). All solvents and reagents were of HPLC or analytical grade. Water was purified (18 MΩ cm−1 quality) from New Human Power I (Korea).

Strata-X (each 6 mL, 500 mg phase) was from Phenomenex. Catechin-imprinted cryogels (300 mg phase) were prepared.

Apparatus

High-performance liquid chromatography analysis was performed on two different HP 1100, Hewlett-Packard systems. The systems are equipped with a diode-array UV detector and a fluorescence detector, respectively. They consist of quartenary pump, a degasser, a Rheodyne injecton valve furnished with 20 μL loop, and a column oven.

Cryogel samples were examined using a JEOL-JEM 1220EX (Tokyo, Japan) scanning electron microscope (SEM). Quantachrome Nova 2200e Brunauer–Emmett–Teller (BET) apparatus was used for specific surface area. Fourier transform infrared (FTIR) spectra of the imprinted p(HEMA–MAH) beads were obtained by using a Shimadzu FTIR 8000 Series (Japan) spectrophotometer.

A rotary evaporator was used from Heidolph for the evaporation of the extracts. The SPE study was performed on Vac-Elut manifold system, having a 12-position capacity, obtained from Machery Nagel.

Water was purified (18 MΩ cm−1 quality) from New Human Power I (Korea).

Standard Solutions

All the standards were dissolved in methanol to a concentration of 1 mg mL−1 and were stored in darkness at −20 °C until analyzed. Five standard mixtures, containing all the catechins at different concentration levels between 0.25 and 20 mg L−1 were prepared prior to use.

Sample Preparation

Wine samples were purchased from local stores in Elazığ and Malatya. Buzbağ Öküzgözü 2011 from Elazığ (Öküzgözü grapes), Güzay Öküzgözü 2007 from Malatya (Karaoğlan and Öküzgözü grapes), Buzbağ Klasik 2011 Öküzgözü-Boğazkere from Elazığ (Öküzgözü and Boğazkere grapes), and Güzay Öküzgözü Ahenk 2009 from Malatya (Arapgir Öküzgözü grapes) were analyzed. Samples were opened, protected against sunlight, and stored at 4 °C. Analyses were carried out within a few days.

Preparation of a Synthetic Wine Sample

The synthetic wine solution (1 L) was prepared from 120 mL ethanol, 2.5 g of l-(l)-tartaric acid and water to the mark, and adjusted to pH 3.2 with NaOH [13]. In order to compensate for the wine matrix effect, the calibration curves were constructed in a polyphenol-free synthetic wine matrix. Five milliliters of synthetic wine samples was spiked with analytes at different concentration levels.

Synthesis of N-Methacryloylhistidine Monomer

Details of the preparation and characterization of N-methacryloylhistidine (MAH) were reported elsewhere [14].

Preorganization of Catechin-MAH Complex

Catechin-MAH precomplexes were synthesized at different ratios for determining the most appropriate ratio. In this process, the amount of catechin is constant and the amount of MAH was increased. For synthesis of the first catechin-MAH (MAH-1) precomplex, 7.5 mg catechin and 10 mg MAH were used; for the second catechin-MAH (MAH-2) precomplex, 7.5 mg catechin and 20 mg MAH were used; and for the third catechin-MAH (MAH-3) precomplex, 7.5 mg catechin and 30 mg MAH, respectively, were used. They dissolved in 100 μL acetone and 1 mL deionized water, and these solutions were stirred for 2 h in magnetic stirrer. Following this process, they were dried at room temperature for 1 day.

Preparation of Catechin-p(HEMA–MAH) Cryogel Columns

A typical preparation procedure is as follows: 1 mL of 0.36 M N,N′-methylenebisacrylamide was used as a cross-linker. A total of 260 μL of HEMA monomer and 740 μL deionized water were added to this solution for monomer–cross-linker mixture. After 60 μL acetone and 3 mL deionized water were added to 6 mg each catechin-MAH precomplexes, both solutions were mixed. This mixture was stirred in a magnetic stirrer for 10 min, followed by immersion in an ultrasonic bath for 5 min. The cryogel was produced by free radical polymerization initiated by by 4 mg APS. After adding APS, the solution was cooled at −4 °C for 20 min. Five microliters of TEMED was added to this mixture. Then, the reaction mixture was poured into plastic syringe (5 mL; i.d., 0.8 cm) with closed outlet at the bottom. Firstly, the polymerization solution in the syringe was frozen at −12 °C for 2 h and then −20 °C for 16 h. After this time, cryogel matrix was thawed at room temperature for 4 h. MAH-1, MAH-2, and MAH-3 cryogels were washed with water and was stored in acetate buffer containing 0.02% sodium azide at 4 °C until use. Nonimprinted (NIP) cryogel was also prepared in the absence of catechin using the same polymerization procedure which was given above.

Surface Morphology

The surface morphologies of the cryogels were examined by SEM. Cryogel samples were dried at room temperature and then coated with gold (100 Å).

Surface Area Measurements

The specific surface area of all cryogels was measured in a BET apparatus using multipoint analysis.

Swelling Test

Swelling ratio of the cryogel was determined in distilled water. The experiment was conducted as follows. Initially, cryogel sample was washed until washing solution was clear. Then, it was dried until reaching to constant mass; the mass of dried sample was weighed (±0.0001 g). This dried sample was placed into a 50-mL vial containing distilled water for 4 h. The bead sample was taken out from the water, wiped using a filter paper, and weighed. The weight ratio of dry and wet samples was recorded.

The swelling ratio was calculated by using the following equation (eq. (1)):

Swelling ratio%=WsW0/W0×100
where W0 and Ws are the weights of cryogel (g) before and after swelling, respectively.

FTIR Studies

The dry beads (about 0.1 g) were thoroughly mixed with 0.1 g KBr and pressed into a pellet. The FTIR spectrum was then recorded in the range of 4.000 cm−1 to 400 cm−1.

Solid-Phase Extraction

The MAH-1, MAH-2, MAH-3, and a traditional C18 reversed-phase cartridges were previously conditioned with 5 mL methanol, followed by 5 mL water. The pH of calibration control was adjusted to pH 2 with 3 M HCl. Five milliliters of this sample was introduced in the column. Finally, the compounds were eluted with 5 mL methanol which acidified the pH 2.0 with 3 M HCl. This eluent was evaporated to dryness on a rotary evaporator, the residue being dissolved in 5 mL of mobile phase. Twenty microliters of this solution was injected into the HPLC system. Recoveries were studied using an artificial wine sample containing the seven standards at 5 mg mL−1.

HPLC Analysis

The separation was carried out using Eclipse XDB C18 column (250 mm × 4.6 mm, 5 μm) with a guard cartridge of the same material at room temperature.

The optimized mobile phase was methanol–water (35:65, v/v) mixture adjusted pH 2.5 with formic acid. Analysis was run at a flow rate of 1 mL min−1 with an 8 min run time at room temperature. The elution conditions were isocratic. The mobile phase was vacuum-filtered through a 0.45-μm nylon filter and degassed on-line by micro vacuum degasser. The chromatograms were monitored by UV detection at a wavelength of 280 nm. The fluorescence detector was set at λem 315 nm and λex 280 nm. The injection volume was 20 μL.

Catechins were identified by comparing retention times with those of pure standards and by spiking the samples with standard solutions.

Validation Procedure

System suitability test parameters were checked to ensure that the system was working correctly during the analysis [15], using 20 μg mL−1 catechins solution. Parameters which are typically used in suitability evaluations are capacity factor (k′), selectivity factor (α), resolution (R), number of theoretical plates (N), and tailing factor (T). For an optimum separation, capacity factor should be in the range of 0.5 < k′ < 10. A value of 1.5 for resolution implies a complete separation of two compounds. The number of theoretical plates must be higher than 2000. The calculated tailing factors of polyphenols were obtained in the acceptable range of 0.5 ≤ T ≤ 2.

A full validation of assay consisting of selectivity, linearity, lower limit of detection and quantitation (LOD and LOQ), and intra-day and inter-day accuracy and precision of the method was performed according to the International Conference on Harmonization (ICH) description [16].

Selectivity of the Imprinted Cryogel

Here, three parameters are used for evaluation. The distribution coefficient (Kd) for catechin and epicatechin was calculated by the following equation (eq. (2)):

Kd=C0Cf/CfxV/m
in which C0 and Cf are the initial and the final concentration of catechins (mg L−1), respectively; V is the volume of the solution (mL); and m is the weight of the cryogel used in the column (g). Kd was used to control the molecular selectivity of polymers.

The selectivity coefficient (k) displays the differences of two substances adsorbed by one absorbent and is described as the ratio of the Kd values of the two competitive solutes. It was calculated by the following equation (eq. (3)):

k=Kd1/Kd2
in which Kd1 (template) is the distribution coefficient of catechin and Kd2 (competing) is the distribution coefficient of epicatechin.

To generate a simpler comparison, the relative selectivity coefficient (k′) is suggestive, which is defined as the ratio of the k values of the two competetive solutes (eq. (4)).

k=kMIP/kNIP
where kMIP is selectivity coefficient for MIP cryogel and kNIP is that for NIP cryogel.

Results and Discussion

Characterization Studies

The surface structure of the catechin-p(HEMA–MAH) cryogels was visualized by SEM, which are presented at different magnification scales. SEM photograph of 1:2 catechin-MAH precomplex containing p(HEMA–MAH) cryogel is shown in Figure 1. The specific surface area of the 1:1, 1:2, and 1:3 catechin-MAH precomplex containing p(HEMA–MAH) cryogels was found to be 3135.6 m2 g−1, 1808.8 m2 g−1, and 1409.3 m2 g−1, respectively.

Figure 1.
Figure 1.

SEM photographs of 1:2 catechin-MAH precomplex containing p(HEMA–MAH) cryogel

Citation: Acta Chromatographica Acta Chromatographica 30, 1; 10.1556/1326.2016.00193

The equilibrium swelling ratios for the first, second, and third catechin–MAH precomplex containing p(HEMA–MAH) cryogels were found to be 217.2%, 144.4%, and 114.2%, respectively.

As shown in Figure 2, FTIR spectra of 1:2 catechin–MAH precomplex have the characteristic stretching vibration band of hydrogen bonded alcohol, O–H, around 3350–3400 cm−1, N–H stretching vibration band around 3200 cm−1, C–H stretching vibration band around 2900 cm−1, C=O–NH stretching vibration band around 1635 cm−1, N–H bending around 1538 cm−1, asymetric C–O–C streching band of catechin molecule at 1145 cm−1, C–O streching band of catechin molecule around 1340 cm−1, and C–H stretching band of the catechin molecule at 780 cm−1. It was observed that the peak intensity increased depending on the increase of the MAH monomer in the complex.

Figure 2.
Figure 2.

FTIR spectra of 1:2 catechin–MAH precomplex

Citation: Acta Chromatographica Acta Chromatographica 30, 1; 10.1556/1326.2016.00193

The FTIR band characterizing catechin-imprinted p(HEMA–MAH) cryogel structure is assigned in Figure 3 as follows: a strong and broad band at 3200–3450 cm−1 for the stretching vibration mode of O–H groups and a strong band at 1727 cm−1 for the stretching vibration mode of C=O groups. The appearance of strong bands at around frequencies of 1642 cm−1 (C=O–NH) and 1538 cm−1 (N–H bending, amide II) indicates the incorporation of MAH monomer into the polymer structure.

Figure 3.
Figure 3.

FTIR spectra of 1:2 catechin-imprinted p(HEMA–MAH) cryogel

Citation: Acta Chromatographica Acta Chromatographica 30, 1; 10.1556/1326.2016.00193

Method Optimization

The first step of the study was the optimization of the chromatographic conditions. Methanol was used as the organic solvent because polyphenols are highly soluble in this solvent. Thus, experiments were carried out to separate catechins mixture from each other using different ratio of methanol–water mobile phases with a flow rate of 1 mL min−1. pK values of polyphenols are around 9; consequently, pH control was essential to avoid ionization of hydroxyl groups [17]. The retention behavior of catechins was studied in the presence of formic acid. Easily ionizing ability of phenolic hydroxyl groups makes the tailing phenomenon at the end of the standard peaks. By adding formic acid, all the standard peaks were separated successfully [18].

The effect of methanol–water ratio on retention times of catechin species was investigated. The ratio of mobile phase solution was chosen methanol–water (35:65, v/v) mixture adjusted to pH 2.5 with formic acid for optimum separation (Figure 4).

Figure 4.
Figure 4.

Effect of mobile phase ratio on separation of the mixture

Citation: Acta Chromatographica Acta Chromatographica 30, 1; 10.1556/1326.2016.00193

Under these conditions, the retention times of gallocatechin (GC), (−)-epigallocatechin (EGC), (+)-catechin (C), (−)-epigallocatechin gallate (EGCG), (−)-gallocatechin gallate (GCG), (−)-epicatechin (EC), and (−)-epicatechin gallate (ECG) were 2.85, 3.50, 3.86, 4.31, 5.32, 5.75, and 7.94 min, respectively (Figure 5).

Figure 5.
Figure 5.

Chromatogram of 20 mg mL−1 reference standards of catechins monitored by UV detection at a wavelength of 280 nm and by fluorescence detection at λem 315 nm and λex 280 nm wavelengths. 1. GC, 2. EGC, 3. C, 4. EGCG, 5. GCG, 6. EC, 7. ECG

Citation: Acta Chromatographica Acta Chromatographica 30, 1; 10.1556/1326.2016.00193

Method Validation

System Suitability

The important parameter t0 was 2.11 ± 0.01 min in the analysis. This was the time of KBr peak. The capacity factor (k′) values were in the range of 0.5 < k′ < 10 except GC. The resolution value for separation of GCG and EC was 1.10. The resolution values of other compounds were higher than 1.5. The therotical plate numbers of all compounds were higher than 2000, and the calculated tailing factors of them were obtained in the acceptable range of 0.5 ≤ T ≤ 2.

Establishment of the Calibration Curves Using Photodiode Array Detection

Quantifications of catechins were based on the calibration curves constructed under optimum conditions as the peak areas of analyzed subtance. Linearity of the method was determined by performing injections at six different concentration levels in the linear range over six different days. Retention time (RT), linear range, R2, LOD, and LOQ values were listed in Table 1.

Table 1.

Calibration graphs using photodiode array detection

CompoundsRange (mg L−1)Calibration curve (y = mx + b)R2%CV (slope)LOQ (mg L−1)LOD (mg L−1)
(+)-Catechin (C)1–20y = 11.372x + 1.3670.99992.110.750.25
(−)-Epicatechin (EC)1–20y = 13.10x − 3.70910.99990.111.000.50
Gallocatechin (GC)0.25–4.0y = 45.993x − 4.5060.99732.340.250.10
(−)-Gallocatechin gallate (GCG)0.25–4.0y = 532.58x − 48.4420.99382.220.250.05
(−)-Epigallocatechin (EGC)1.50–4.0y = 54.868x − 5.61680.99831.961.500.50
(−)-Epicatechin gallate (ECG)0.50–4.0y = 401.93x − 10.9280.99901.670.500.10
(−)-Epigallocatechin gallate (EGCG)0.20–4.0y = 365.97x − 12.1730.99570.760.200.05

Establishment of the Calibration Curves Using Fluorescence Detection

Catechin and epicatechin are highly fluorescent. Consequently, the sensitivity of the procedure could be improved using a fluorescence detector. In this case, the chromatographic conditions were same. Calibration graphs were performed also by plotting concentration (mg L−1) against peak area. Parameters of retention time, linear range, R2, LOD and LOQ were presented in Table 2.

Table 2.

Calibration graphs using fluorescence detection

CompoundsRange (mg L−1)Calibration curve (y = mx + b)R2%CV (slope)LOQ (mg L−1)LOD (mg L−1)
(+)-Catechin (C)1–20y = 73.96x − 6.17330.99990.240.080.04
(−)-Epicatechin (EC)1–20y = 62.863x + 6.1450.99921.800.100.05

Solid-Phase Extraction Efficiency

MAH-1, MAH-2, and MAH-3 cartridges were used. The different cartridges were previously conditioned, followed by 5 mL water. Five milliliters of 5 mg L−1, 10 mg L−1, and 15 mg L−1 catechin standards were introduced in the column. Recoveries were calculated as peak area ratios of reference standard/analyte (spiked synthetic wine samples). Recovery of catechin from MAH-1, MAH-2, and MAH-3 cartridges at three replicates with relative standard deviation was 55.44 ± 2.36, 87.82 ± 4.09, and 38.48 ± 1.48, respectively. Higher recoveries from analyte were obtained with MAH-2 cartridges. Therefore, next studies continued with MAH-2 cartridge.

Recovery of catechin from non-imprinted p(HEMA–MAH) cryogels was found <2%.

Elution Solvents

Methanol and acetonitrile with addition of formic acid were tested as elution solvents in 300 mg of polymer placed into polypropylene cartridge. Each effluent was collected and analyzed by HPLC (Table 3).

Table 3.

Comparison of Elution Solutions

CatechinsACN–HCOOHMeOH–HCOOH
Recovery (%)Recovery (%)
DADFluorescence detectorDADFluorescence detector
(+)-Catechin (C)46.59 ± 1.7440.64 ± 1.3692.86 ± 2.3991.40 ± 2.06
(−)-Epicatechin (EC)42.70 ± 1.8534.96 ± 1.1161.75 ± 1.2061.11 ± 1.61
(−)-Epigallocatechin (EGC)16.29 ± 0.9635.51 ± 2.35
(−)-Gallocatechin gallate (GCG)21.43 ± 1.2141.94 ± 1.41
Gallocatechin (GC)28.02 ± 1.6755.16 ± 2.90
(−)-Epicatechin gallate (ECG)20.27 ± 1.0962.83 ± 1.82
(−)-Epigallocatechin gallate (EGCG)15.75 ± 1.0659.84 ± 1.11

Recovery for catechins using MeOH solution was higher than using ACN solution in SPE procedure. Therefore, MeOH–HCOOH (adjusted to pH 2.5 with formic acid) was selected as elution solution (Figure 6).

Figure 6.
Figure 6.

Recovery for catechin compounds using different elution solutions

Citation: Acta Chromatographica Acta Chromatographica 30, 1; 10.1556/1326.2016.00193

Volume of Elution Solution

The extraction was achieved in 5, 6, and 7 mL analyte and elution solutions. The increase in the volume of analyte and elution solutions did not change recovery values. Therefore 5 mL of analyte and 5 mL methanol-formic acid solution were selected.

Adsorption Ability and Selectivity of the Imprinted Cryogel

The adsorption capacity of MIP was increased with increasing of catechin concentration. The maximum adsorption capacity was found to be 0.51 ± 0.03 mg g−1 dry weight of weight of cryogel.

The measured values of the three parameters (Kd, k, and k′) were shown in Table 4. The k value of imprinted p(HEMA–MAH) cryogel was 5.1-fold that of non-imprinted p(HEMA–MAH) cryogel. These findings indicated that catechin-imprinted p(HEMA–MAH) cryogel has a high selectivity for C over the interference compound EC.

Table 4.

Competetive adsorption of catechin and epicatechin on MIP and NIP (n = 3)

SamplesC0 (mg L−1)Cf (mg L−1)Kdkk
CECCECCEC
MIP40407.4 ± 0.617.2 ± 0.973.4 ± 2.722.1 ± 1.13.32 ± 0.35.1 ± 0.5
NIP404038.5 ± 1.537.7 ± 1.20.65 ± 0.11.01 ± 0.20.65 ± 0.1

Comparison of C18 and Catechin-Imprinted Cryogel Column

Catechin (15 ppm) and epicatechin (10 ppm) solutions passed through a conventional C18 column and catechin-imprinted cryogel column, respectively. Recoveries were calculated. Then, other standards of 1 ppm added to this solution. After the same process, recovery values were calculated. The best recoveries were obtained by catechin-imprinted cryogel column (Table 5).

Table 5.

Comparison of C18 and catechin-imprinted cryogel column

Recovery (%)
MIP columnC18 column
DADFluorescence detectorDADFluorescence detector
C (15 mg L−1)90.32 ± 1.9289.53 ± 1.5339.71 ± 0.5039.30 ± 0.80
C (15 mg L−1) in mixture84.99 ± 1.1185.51 ± 2.0916.90 ± 0.6917.42 ± 0.54
EC (10 mg L−1)61.75 ± 0.0261.11 ± 0.6129.05 ± 0.0929.72 ± 0.25
EC (10 mg L−1) in mixture55.25 ± 0.6654.72 ± 0.35

Analysis of Local Red Wines

Samples of red wine were diluted two times with 13% (v/v) ethanol. Then, they passed through the MIP cryogel column. After sample application, the cartridge was washed with water. Next, elution was performed by means of acidic methanol. The results are summarized in Table 6.

Table 6.

C and EC of local red wines mg L−1 obtained by HPLC method

Red wine samplesC (mg L−1)EC (mg L−1)
DADFluorescence detectorDADFluorescence detector
Buzbağ Öküzgözü13.92 ± 0.5915.94 ± 0.483.76 ± 0.153.61 ± 0.19
Buzbağ Klasik11.80 ± 0.4214.37 ± 0.763.25 ± 0.153.58 ± 0.21
Güzay Öküzgözü6.98 ± 0.848.71 ± 0.131.82 ± 0.261.51 ± 0.08
Güzay Ahenk11.56 ± 0.9212.10 ± 0.372.03 ± 0.542.48 ± 0.63

Chromatogram of the a sample was shown below (Figure 7). The amount of each active compound was appointed using calibration curve method.

Figure 7.
Figure 7.

Chromatogram of Buzbağ-Öküzgözü wine (A) using fluorescence detection at λem 315 nm and λex 280 nm wavelengths (B) using UV detection at a wavelength of 280 nm

Citation: Acta Chromatographica Acta Chromatographica 30, 1; 10.1556/1326.2016.00193

Conclusions

Cryogels are considered as one of the new types of polymeric hydrogels. The research focused on developing new imprinting cryogel which have low pressure drop and short residence time for both adsorption and elution and high chemical and physical stability for separation and determination of catechin from red wines. The prepared catechin-MIPs were characterized by BET, SEM, and FTIR and were applied to the cleanup of catechins from red wine samples successfully. The selectivity coefficient (k) of imprinted p(HEMA–MAH) cryogel was 5.1-fold that of non-imprinted cryogel. It was observed that the best recovery was obtained for C and the prepared cryogel showed good selectivity for target molecule C. A comparison was made between the results obtained with the MIP cartridges and a traditional C18 reversed-phase cartridge. It was found that the recovery of catechin by the MIP-based cryogel was 2.3 times greater than that by a commercially available C18 material. It is suggested that the developing catechin-MIP material be used as an analytical tool for the enrichment and determination of catechin from natural products, fruit juices, bevarages, tea extracts, and herbal extracts.

Acknowledgments

This research was financially supported by the Scientific and Technological Research Council of Turkey, as part of project no. 112T644.

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    Andersson, L. I.; Nicholls, I. A. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2004 , 804 , 1.

  • 2.

    Ozkara, S.; Andac, M.; Karakoc, V.; Say, R.; Denizli, A. J. Appl. Polym. Sci. 2011 , 120 , 1829.

  • 3.

    Ozcan, A. A.; Say, R.; Denizli, A.; Ersoz, A. Anal. Chem. 2006 , 78 , 7253.

  • 4.

    Bereli, N.; Andac, M.; Baydemir, G.; Say, R.; Galaev, I. Y.; Denizli, A. J. Chromatogr. A 2008 , 1190 , 18.

  • 5.

    Arvidsson, P.; Plieva, F. M.; Savina, I. N.; Lozinsky, V. I.; Fexby, S.; Bulow, L.; Galaev, I. Y.; Mattiasson, B. J. Chromatogr. A 2002 , 977 , 27.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6.

    Molinelli, A.; Weiss, R.; Mizaikoff, B. J. Agri. Food Chem. 2002 , 50 , 1804.

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    Blahova, E.; Lehotay, J.; Skacani, I. J. Liq. Chromatogr. Relat. Technol. 2004 , 27 , 2715.

  • 8.

    Ding, L.; Li, H.; Tang, F.; Yao, S. Z. Anal. Lett. 2006 , 39 , 2373.

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    Song, X. L.; Li, J. H.; Wang, J. T.; Chen, L. X. Talanta 2009 , 80 , 694.

  • 10.

    Jin, Y. Z.; Xuan, Y. H.; Jin, Y. S.; Row, K. H. J. Liq. Chromatogr. Relat. Technol. 2011 , 34 , 1604.

  • 11.

    Tian, M.; Han, D.; Row, K. H. Anal. Lett. 2011 , 44 , 737.

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    Lopez, M. D. C.; Perez, M. C. C.; Garcia, M. S. D.; Vilarino, J. M. L.; Rodriguez, M. V. G.; Losada, L. F. B. Anal. Chim. Act. 2012 , 721 , 68.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13.

    Garcia-Falcon, M. S.; Perez-Lamela, C.; Martinez-Carballo, E.; Simal-Gandara, J. Food Chem. 2007 , 105 , 248.

  • 14.

    Say, R.; Garipcan, B.; Emir, S.; Patir, S.; Denizli, A. Macromol. Mater. Eng. 2002 , 287 , 539.

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    Zakeri-Milani, P.; Barzegar-Jalali, M.; Tajerzadeh, H.; Azarmi, Y.; Valizadeh, H. J. Pharmaceut. Biomed. 2005 , 39 , 624.

  • 16.

    Text on Validation of Analytical Procedures Q2A, International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use , London , 1994.

    • Search Google Scholar
    • Export Citation
  • 17.

    Snyder, L. R.; Kirkland, J. J.; Glajch, J. L. Practical HPLC Method Development , 2nd edn. , A Wiley-Interscience Publication , Danvers , 1997 , pp. 294 300.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18.

    Porgali E. ; Buyuktuncel, E. Food Res. Int. 2012 , 45 , 145.

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