Synthesis of molecular imprinting polymers for extraction of gallic acid from urine
© The Author(s) 2018
Received: 13 December 2017
Accepted: 13 February 2018
Published: 21 February 2018
The molecularly imprinted polymers for gallic acid were synthesized by precipitation polymerization. During the process of synthesis a non-covalent approach was used for the interaction of template and monomer. In the polymerization process, gallic acid was used as a template, acrylic acid as a functional monomer, ethylene glycol dimethacrylate as a cross-linker and 2,2′-azobisisobutyronitrile as an initiator and acetonitrile as a solvent. The synthesized imprinted and non-imprinted polymer particles were characterized by using Fourier-transform infrared spectroscopy and scanning electron microscopy. The rebinding efficiency of synthesized polymer particles was evaluated by batch binding assay. The highly selective imprinted polymer for gallic acid was MIPI1 with a composition (molar ratio) of 1:4:20, template: monomer: cross-linker, respectively. The MIPI1 showed highest binding efficiency (79.50%) as compared to other imprinted and non-imprinted polymers. The highly selective imprinted polymers have successfully extracted about 80% of gallic acid from spiked urine sample.
Gallic acid (GA) is a polyphenolic naturally occurring compound in fruits such as blueberries, strawberries, apples, and bananas or other variety of plants and herbs such as oak bark, tea leaves and witch hazel. Gallic acid is diversely used in various applications because of various pharmacological properties like antitumor and anti-inflammatory . Gallic acid is main member of the polyphenolic family that provides vital antioxidant properties . The extensive usage of gallic acid made an emphases on the researchers to design and develop new materials and/or approach for monitoring GA from different real samples. Molecular imprinting technology is a promising approach for the monitoring of gallic acid in real samples.
Molecularly imprinted polymers are the cross-linked polymeric materials and are able to resist chemical and physical stresses such as organic solvents, heat, acid, bases and others . The concept of polymer that can selectively recognize desired molecules have captured many attentions from scientific community over recent years. These recognition systems in polymers are analogue of the biological recognition systems in the body such as enzymes, DNA, antibodies and aptamers. The imprinted polymers produced from the polymerization process have cavities that can complement to the shape of the desired molecules. The developments in molecular imprinting polymers as chromatography stationary phases especially in high performance liquid chromatography have been driven by the advantage of physicochemical stability and high selectivity in the polymers .
The three binding approaches have been used in the synthesis of MIPs such as, covalent method, non-covalent method and semi-covalent method. The most widely used is the non-covalent approach. In non-covalent imprinting method, templates bond to monomers with a non-covalent intermolecular bonding which can be destroyed and created easily. Weak metal coordination, electrostatic interactions, hydrogen bonds and hydrophobic interactions are included in non-covalent forces used by both molecules of chemically and geometrically complement to each other . Simple diffusion can be used to remove templates once polymerized with a polar or acidic solvent and is enough to destroy the non-covalent interaction between template and polymer . While covalent imprinting is less economical and usually troublesome, in this research, all polymers were synthesized by non-covalent approach by precipitation polymerization method.
Materials and methods
Materials and reagents
Gallic acid (GA) and 2,2′-azobis(isobutyronitrile) (AIBN) were obtained from R & M Marketing company located in Essex, United Kingdom, acetonitrile was obtained from Avantor Performance Materials Incorporated located in Phillipsburg, New Jersey, acrylic acid (AA), ethylene glycol dimethacrylate (EGDMA) and syringic acid were obtained from Sigma-aldrich Corporated located in St. Louis, Missouri, acetone was obtained from HmbG Chemicals company located in Hamburg, Germany and methanol and acetic acid was obtained from R & M Marketing company located in Essex, United Kingdom.
Fourier-transform infrared spectroscopy (FTIR) (Nicholet iS10), scanning electron microscopy, (SEM) (JEOL JSM 6930 LA), sonic bath (Branson 2510), shaker (Multi Shaker NB-101MT), high-performance liquid chromatography (HPLC) (Model Shimadzu LC-20), and water bath (Model Memmert W350T).
Preparation of imprinted and non-imprinted polymer beads
Composition of polymers for synthesis
Template, GA (mM)
Monomer, AA (mM)
Cross-linker, EGDMA (mM)
Initiator, AIBN (mg)
Extraction of template from the polymer matrix
Extraction of template from the imprinted polymer beads was carried out by washing with mixture of methanol and acetic acid (9:1, v/v). The extraction of template was monitored by using HPLC. This process was repeated until template was not detected by HPLC.
Characterization of polymer beads
Morphology of polymers surface was observed with SEM coated with gold under reduced pressure. Polymer samples were dried in a vacuum oven at 60 °C for 6 h until constant weight is achieved before analysis. Then, polymer samples were analysed at 32 scans by FTIR.
Competitive binding capacity
The competitive binding test was performed by using gallic acid along with syringic acid. In this study, 500 mg of both MIPI1 and NIPN1 polymer beads were immersed in two different flasks containing 30 mL of feed solution (10 ppm of both GA and syringic acid). The reaction flasks were agitated on shaker followed by the same procedure of batch binding. The collected samples were analysed by HPLC. The extraction percentage (%) of MIPI1 polymer beads and NIPN1 polymer beads was calculated by using Eq. 1.
Spiking of human urine and extraction efficiency
Firstly, urine was collected from a drug free human. Prior to spiking, urine was filtered and kept in a refrigerator. The spiked human urine was prepared by adding a 30 mL of 10 ppm of GA solution to a 30 mL of human urine. After that 500 mg of MIP I1 and NIPN1 were added into the two different flasks containing spiked human urine. The extraction efficiency was obtained by following the batch binding process and calculated by using Eq. 1. The analysis was performed by using HPLC.
Results and discussion
Synthesis of imprinted polymer beads and non-imprinted polymer beads
The polymeric microspheres with adequate control of product morphology can be achieved by using precipitation polymerisation . It produces microspheres with smooth and clean surfaces and gives suitable particle sizes. This method is simple and have many advantages because stabilisers are abandoned during the polymerisation process as compared to suspension polymerisation. On the other hand, non-covalent approach has been adopted during polymerization process. In this study five different polymers varying in chemical composition have been synthesized by precipitation polymerization using non-covalent approach.
Morphology of polymer beads
A quantitative energy dispersive X-ray (EDS) analysis was performed to establish amount of main chemical elements such as carbon (C) and oxygen (O) present in the imprinted polymer. It was (Fig. 1b) found that the significant amount of carbon (67.92%) and oxygen (32.08%) were present in the sample.
Competitive binding capacity
Specific parameters of polymers for the competitive uptake from feed solution
The extensive use of GA for many pharmacological purposes is the main reason to synthesize selective imprinted polymers for the extraction of GA from urine. The MIPI1 was used for the extraction of GA from urine and it was found that about 80% of GA was successfully extracted from the spiked urine sample.
The imprinted polymers for GA were prepared by precipitation polymerisation method using non-covalent approach. The selected MIPI1 have successfully extracted 80% of GA from the spiked urine sample. This could open diverse applications of imprinted polymers for the extraction of compounds from various biological samples.
All authors have equally contributed to the paper and have given approval to the final version of the paper. All authors read and approved the final manuscript.
Authors are thankful to Faculty of Resource Science and Technology, UNIMAS, Sarawak for providing necessary research facilities.
The authors declared that they have no competing interests.
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