- Email: [email protected]
Purification of vanillin by a molecular imprinting polymer technique
Separation and Purification Technology 66 (2009) 450–456
Contents lists available at ScienceDirect
Separation and Purification Technology journal homepage: www.elsevier.com/locate/seppur
Purification of vanillin by a molecular imprinting polymer technique M.N. Mohamad Ibrahim ∗ , C.S. Sipaut, N.N. Mohamad Yusof School of Chemical Sciences, Universiti Sains Malaysia, 11800 Minden, Pulau Pinang, Malaysia
a r t i c l e
i n f o
Article history: Received 31 October 2008 Received in revised form 16 February 2009 Accepted 17 February 2009 Keywords: Black liquor Lignin Crystallization method Vanillin compound Molecular imprinting
a b s t r a c t The successful separation of vanillin from soda lignin, extracted from the black liquor of oil palm empty fruit bunches (EFBs) with 20% (v/v) sulfuric acid may be negated by the high impurity content in the vanillin obtained. A molecular imprinting polymers (MIPs) technique was developed for vanillin to remove the impurities attached to it. A synthesis method was used in preparing the MIPs, where impure vanillin molecules were used as the template, dimethylsulfoxide (DMSO) as the solvent, and methacrylic acid (MAA) and ethylene glycol dimethacrylate acid (EGDMA) respectively functioning as the monomer and cross-linker. The results were confirmed by gas chromatography (GC), mass spectrometry (MS), Fourier transform infrared (FTIR) spectroscopy and thermal gravimetric analysis (TGA) of the sample against a standard vanillin. © 2009 Elsevier B.V. All rights reserved.
1. Introduction A molecular imprinting polymer is an efficient technique for creating a 3D cross-linked polymer network with specific binding site (memory) for a specific template [1]. Theoretically, molecular imprinting polymers (MIPs) are capable of selectively recognizing most of the known molecules through the synthesis processes of imprinting the specific template molecule into the polymer. The concept of MIPs is based on host–guest chemistry. The hypothesis is, polymers can mimic the recognition mechanisms of biological enzymes and antibodies [2]. However, molecular imprinting polymers (MIPs) became a reality and part of the host–guest chemistry field in the 1970s when polymers displaying properties that mimic the biological enzymes and monoclonal antibody were successfully synthesized [2,3]. MIPs can be classified into two specific categories, covalent and non-covalent [1]. The publications on MIPs have increased steadily over the last few years with the non-covalently imprinted MIPs the more popular. Non-covalent MIPs rely on hydrogen bonding between the host and the guest, which depends on their molecular structures, levels of crosslinking in the polymer, bond distances, dipole moments and types of interactions between the template and polymer [4–9]. The advantages of non-covalent MIPs include simple synthesis procedure, easy binding and extraction of templates as well as wide varieties of commonly available starting materials and chemical [10–13].
∗ Corresponding author. Tel.: +60 4 6533554; fax: +60 4 6574854. E-mail address: [email protected] (M.N.M. Ibrahim). 1383-5866/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.seppur.2009.02.010
The MIPs technique was used to involve preparing polymers by arranging methacrylic acid (MAA) as functional monomers around a template compound (vanillin) and fixing them with ethylene glycol dimethacrylate acid (EGDMA) as a cross-linker and is described schematically in Fig. 1. Vanillin (4-hydroxy-3-methoxybenzaldehyde) is a unique and highly prized flavor compound used as an ingredient in food flavors, in pharmaceuticals and as fragrance in perfumes and odor-masking products. In the vanilla bean, vanillin is the major compound that characterizes the aromatic compound of vanilla. The natural source of vanillin is from the seeds of the vanilla plant, a member of the orchid family. However, this process is very expensive compared to synthetic processes. Fortunately, nature provides a second source of vanillin, available from lignin extracted from the black liquor of oil palm empty fruit bunches (EFBs) by using a crystallization process [14,15]. The characteristics of the vanillin compound obtained from lignin were compared with the characteristics of standard vanillin. It was observed that the vanillin obtained from the crystallization process contains high level of impurities. Therefore, a molecular imprinting polymers technique was applied in order to purify the vanillin. This technique was selected because it has been used successfully with biomolecule mimics, and has high selectivity for compounds comparable to vanillin. The goal of this study is to create molecularly imprinted polymers that can recognize specific molecules such as vanillin from several other compounds. In this process, monofunctional monomers containing an acidic group such as methacrylic acid are used to allow hydrogen bonding with vanillin, the target molecule during polymerization. Then, the complexes were assembled in the liquid phase and fixed by cross-linking polymerization with ethylene glycol dimethacrylate acid (EGDMA) used as the
M.N.M. Ibrahim et al. / Separation and Purification Technology 66 (2009) 450–456
451
Fig. 1. Schematic illustration of the vanillin imprinting performed in this study. Blue colors represent cross-linked polymer network. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
crosslinking monomer. Subsequently, the target molecule (vanillin) was removed through extraction, utilizing the appropriate solvents (i.e. xylene) thus leaving behind vacant sites exhibiting a high affinity to the template [16–18]. The selectivity response of the imprinted polymers to vanillin compound is characterized by gas chromatography (GC) of the sample vis-à-vis standard vanillin. 2. Experimental 2.1. Materials Sulfuric acid, hydrochloric acid, nitrobenzene solution, nhexane (analytical grade) and xylene were purchased from QRëC. Sodium hydroxide, methacrylic acid, 2,2 -azobisisobutyronitrile (AIBN) and ethylene glycol dimethacrylate acid (EGDMA) were purchased from R&M Chemicals. Vanillin, chloroform and acetone were obtained from Fluka and Fisher Chemicals. 2.2. Vanillin production Soda lignin was extracted from the soda black liquor, derived from oil palm empty fruit bunches by using 20% sulfuric acid. The degradation of soda lignin using alkaline nitrobenzene oxidation was carried out at 165 ◦ C for 3 h in a 7 mL 2 M NaOH and 0.4 mL nitrobenzene solution. After 3 h, the mixture was cooled and then extracted with chloroform (5× 20 mL) to remove excess nitrobenzene. The oxidation mixture was acidified with concentrated HCl to pH 3–4 and extracted again with chloroform (15 mL). The solvent from the chloroform solution was removed by using a rotary evaporator at 40 ◦ C under reduced pressure to obtain the residue. A crystallization process was used to separate the vanillin component from the hydrolysis–oxidation of lignin. Based on the
solubility of vanillin in acetone, 15 mL of acetone was added to the residue. The mixture was slowly heated to 60 ◦ C for 15 min until vanillin precipitated. 2.3. Preparation of control polymer (CP) and imprinted polymers by crosslinking A typical procedure for preparing an imprinted polymer P1 involves the mixture of 5 mL of dimethyl sulfoxide, 2 mmol of standard vanillin with methacrylic acid (5 mmol), ethylene glycol dimethacrylate acid (EGDMA) (5 mmol) and 2,2 azobisisobutyronitrile (24 mg). The mixture was de-gassed in a test tube with nitrogen and placed in a water-bath at 80 ◦ C for 6 h. The bulk polymer was then taken from the reaction and freeze-dried. The dried polymer was then grounded and tested. Polymer P2 was prepared in a similar manner but the standard vanillin sample was replaced with vanillin obtained from the crystallization process. A non-imprinted blank polymer was prepared following the same method as used to prepare P1. However, no template component was added to this polymerization mixture, which served as the control. 2.4. Gel content determination Gel content is a method to determine the percentage of crosslinking level of the polymer. This method is used with standard procedures by refluxing the samples in boiling xylene for 24 h [19]. The crosslinked matrix samples (P1 and P2) were placed in 100 mesh cages, labeled and weighed. After refluxing in xylene, the residual solvent was removed in a preheated vacuum oven with a cold trap at a temperature of 140 ◦ C for 4 h at <0.1 bar. The cages were removed, allowed to cool and reweighed. Five samples were used to determine the insoluble fraction (gel content) and having
452
M.N.M. Ibrahim et al. / Separation and Purification Technology 66 (2009) 450–456
Table 1 The percentage of crosslinking level. Percentage of crosslinking level (%) Control (without template) P1 Total of standard vanillin P2 Total of sample X
95.83 92.17 3.67 92.33 3.49
an experimental error within 3% of the mean. The gel content was calculated as: Gel content (%) =
1−
W − W 3 4 W2 − W1
× 100%
(1)
where W1 = the weight of the mesh cage, W2 = the weight of the mesh cage and sample, W3 = the weight of the stapled cage and sample, and W4 = the weight of the stapled cage and sample after extraction and drying. 2.5. Gas chromatography analysis All gas chromatography data were collected by using an SRI 8610 C Gas chromatograph systems equipped with a flame ionization detector (FID) and helium as the carrier gas. The injector temperature was 280 ◦ C. Samples (0.5–1.0 L) were injected by using the splitless mode with a purge time of 1.0 min. Fused-silica capillary columns DB-5 (30 mm × 0.25 mm i.d., 0.32 m film thickness) was used in the process to qualitatively and quantitatively determine the residual products. 3. Results and discussion The identification of an unknown compound obtained from a crystallization process requires multiple forms of identification. In the case of vanillin extracted from lignin, there are four independent forms present for identification. 3.1. Mass spectral identification
Fig. 2. Comparison of mass spectrum between standard vanillin and the sample.
The mass spectra (Fig. 2) can be used to identify vanillin. The library spectrum (a) is a fairly good match with sample (c) with the purity score of 96–97%. The purity measures the similarity between the mass spectrum and the library reference mass spectrum. To confirm these results, a standard vanillin was diluted with n-hexane and injected as well. The resulting mass spectrum (b) also has a 97% purity score (Fig. 2). The base, which peaks at m/z 151 and 152, shows the major ions of vanillin (Fig. 2). The mass spectrum of both samples has a much more intense molecular ion than that of aromatic compounds, since fragmentation requires the cleavage of an alkyl bond. An aromatic ring in a vanillin molecule stabilizes the molecular ion peak, which often is sufficiently large enough that accurate intensity measurements can be made on the M + 1 and M + 2 peaks. Therefore, there was no significant difference in the molar mass of sample (c) with the standard vanillin based on the molar mass analyses. The sample obtained from the crystallization process had the same molecular formula as that of standard vanillin.
obtained by using the MIPs technique. The chromatogram (Fig. 3) for standard vanillin, P1 and P2 all contain peaks at retention times, Rt of 4.416, 4.433 and 4.450 min, respectively. The GC results are consistent with the hypothesis that vanillin from the MIPs can be considered pure since the peaks of the samples were similar.
3.2. Gas chromatography analysis There were no significant differences in gel content between the two samples (Table 1). As both samples were subjected to higher amounts of cross-linking agent, the probability of the formation of polymerization networks around the samples increased. All the imprinted and non-imprinted polymers prepared were tested by gas chromatography to determine the purity of the sample
Fig. 3. Comparison between chromatogram of P1 and P2 with chromatogram of standard vanillin.
M.N.M. Ibrahim et al. / Separation and Purification Technology 66 (2009) 450–456
453
Fig. 4. FTIR spectra of polymer control and polymer P1.
3.3. Fourier transform infrared (FTIR) analyses The polymers P1 and P2 obtained from the polymerization process were characterized using FTIR analyses to determine the functional group presence in the polymer. Fig. 4 shows the FTIR spectra of polymer control (without vanillin) and polymer P1. All two spectra are characterized by the presence of a broad band of OH stretching in 3400 cm−1 region and one band of CH stretching at 2900 cm−1 . The band of OH stretching which appear at the lower frequency indicates strong hydrogen bonding. The band at 1494, 1439 and 1402 cm−1 are assigned to C–H deformation in the –CH3 of a methoxyl group. Similarly, these bands are also present in polymer P1.
Fig. 5 shows the comparison of FTIR spectra of polymer control, polymers P1 and P2. Spectral differences in polymers P1 and P2 are observed in the fingerprint region (1800–800 cm−1 ). Bands 2140, 1487, 1021, 875 and 823 cm−1 are not present in polymer P2. The band at 1487 cm−1 is assigned to CH deformation in –CH2 groups. Roczniak et al. identified the region between 1500 and 1400 cm−1 as characteristics of the deformation vibration of –CH– bands in a –CH2 group and observed some differences from the spectra of polymers [20]. These differences can provide information as to the structure of a methylene bridge. In polymer P1, the appearance of bands at 1487 cm−1 indicates the presence of a methylene bridge. Polymer P2 shows a band at 1390 cm−1 , which is attributed to an in-plane
Fig. 5. The comparison of FTIR spectra.
454
M.N.M. Ibrahim et al. / Separation and Purification Technology 66 (2009) 450–456
Fig. 6. TGA thermogram of sample obtained from crystallization process.
Fig. 7. TGA thermogram of standard vanillin.
M.N.M. Ibrahim et al. / Separation and Purification Technology 66 (2009) 450–456
Fig. 8. TGA thermogram of polymer P2.
Fig. 9. TGA thermogram of polymer control.
455
456
M.N.M. Ibrahim et al. / Separation and Purification Technology 66 (2009) 450–456
deformation of phenolic OH. However, the differences show a reaction between polymer P2 with the sample in the purification process. It can be concluded that the sample had been successfully purified using MIP process. 3.4. Thermal gravimetric analysis (TGA) Figs. 6 and 7 shows the TGA thermograms of sample obtained from crystallization and standard vanillin. The study was carried out up to 900 ◦ C in N2 atmosphere. In the case of the sample obtained from crystallization, two thermal events were observed, one in the range 34.60–268.13 ◦ C and the other in the range of 268.13–894.73 ◦ C. As shown in Fig. 6, the decomposition temperature at 70.4% weight loss appeared at the range of 34.60–268.13 ◦ C. When the temperature was raised up to 300 ◦ C, the weight loss accounted was at 60%. In the case of standard vanillin, only oneweight loss event was observed. It started at 31.00 ◦ C and ended at 894.10 ◦ C, and the standard vanillin lost 47.31 × 10−3 % in this event. From both of the thermograms, the ranges of decomposition for the sample obtained from the crystallization process were similar with the standard vanillin. However, most probably the sample which was obtained from the crystallization process still contained impurities. Fig. 6 shows the sample obtained from the crystallization process has two thermal events compared to the standard vanillin thermogram which showed only one thermal event. Thus, the sample obtained from crystallization process was synthesized with the polymer template for the purification process. In this process, the polymer templates were reacting with the sample and cross-linked polymers were formed around the sample. Then, a sample is removed by refluxing using xylene as a solvent. After that, the polymers freed from the sample were analyzed by TGA analysis to identify the behavior of the polymer. The TGA thermogram for the polymer is shown in Fig. 8. For comparison, the thermogram for polymer control is also shown in Fig. 9. As shown in Fig. 8, the decomposition temperature at 80.78% weight loss appeared at 119.95 ◦ C. While at temperature of 890.32 ◦ C, the weight loss decreased significantly to −8.43%. As a result, the polymer template reacted with the sample, showing that the sample obtained after the purification process is highly pure. These results suggest that MIPs prepared in situ were successfully applied in the purification process of vanillin obtained from soda lignin. 4. Conclusion In this study, we successfully separated the vanillin component was from EFB lignin by using a crystallization process. The result of the molar mass obtained confirmed that the obtained sample had characteristics similar to those of standard vanillin. The MIPs
technique was then used to remove any impurities in the sample. The molecularly imprinted polymer prepared in situ with vanillin as a template, was successfully used to purify sample obtained from the crystallization process. These results were confirmed by gas chromatography, FTIR and TGA analysis of the sample against the standard vanillin, which showed that the characteristics of the sample from the crystallization process were similar to those of standard vanillin. This research project has two benefits in that it aims to overcome the problem relating to the disposal of pulping waste in the form of toxic black liquor, as well as providing a novel method for recycling unwanted materials into valuable products. Acknowledgments The authors would like to express their appreciation to Universiti Sains Malaysia and the Malaysian Ministry of Science, Technology and Innovation for financial support of this project through a research grant (305/PKIMIA/613306). References [1] K. Makoto, T. Toshifumi, M. Takashi, A. Hiroyuki, Molecular Imprinting: From Fundamentals to Applications, Wiley-VCH Verlag Gmbh & Co., KGaA, Weinheim, 2003. [2] T. Takeuchi, J. Haginaka, Separation and sensing based on molecular recognition using molecularly imprinted polymers, J. Chromatogr. B 728 (1999) 1–20. [3] L. Ye, R. Weiss, K. Mosbach, Macromolecules 23 (2000) 8239. [4] L.I. Andersson, Molecular imprinting: developments and applications in the analytical chemistry field, J. Chromatogr. B 745 (2000) 3–13. [5] B. Sellergren (Ed.), Molecularly Imprinted Polymers: Man-made Mimics of Antibodies and their Application in Analytical Chemistry (Techniques and Instrumentation in Analytical Chemistry), vol. 23, Elsevier, Amsterdam, 2001. [6] A. Lars, M. Akiyoshi, J.O. Daniel, M. Klaus, J. Chromatogr. 22 (1990) 323–331. [7] K. Takaomi, Y.W. Hong, F. Nobuyuki, Chem. Lett. (1995) 927–928. [8] A. Lars, J.O. Daniel, M. Klaus, J. Chromatogr. 513 (1990) 167–179. [9] M. Jun, A.N. Ian, T. Toshifumi, M. Klaus, K. Isao, Anal. Chem. Acta 335 (1996) 71–77. [10] L. Jia-Ping, H. Xi-Wen, J. Yue, C. Feng, Anal. Bioanal. Chem. 375 (2003) 264–269. [11] M.M. Wayne, P.C.L. Edward, S. Borje, Anal. Commun. 36 (1999) 217–220. [12] G. Vlatakio, L.I. Andersson, R. Muller, K. Mosbach, Nature 361 (1993) 645–647. [13] M.C. Blanco-Lopez, M.J. Lobo-Castanon, A.J. Miranda-Ordieres, P. Tunon-Blanco, Trend Anal. Chem. 23 (2004) 36–48. [14] J.L.R. Kirkaldy, J.B. Susanto, Possible utilization of by-products from palm oil industry, Planter 72 (838) (1996) 27–39. [15] K.C. Khoo, T.W. Lee, Pulp and paper from oil palm, Appita 44 (6) (1991) 385–388. [16] G. Wulff, Molecular imprinting in cross-linked materials with the aid of molecular templates-a way towards artificial antibodies, Angew. Chem. Int. Ed. 34 (1995) 1812–1832. [17] W.L. Xu, Y.Q. Wang, Y.B. Huang, P. Lu, Studies on the selection of solvents for recrystallization purification of phytosterol, J. Yangzhou Univ. 5 (2002) 58. [18] T. Takeuchi, D. Fukuma, J. Matsui, Combinatorial molecular imprinting: an approach to synthetic polymer receptors, Anal. Chem. 71 (1999) 288–290. [19] ASTM D2765-95. Standard Test Method for Determination of Gel Content and Swell Ratio of Crosslinked Ethylene Plastic, ASTM, Philadelphia, 1995. [20] C. Rocznaik, T. Biernacka, M. Skarzynski, Anal. Chem., Some properties and chemical structure of phenolic resins and their derivatives 28 (1983) 531.
Contents lists available at ScienceDirect
Separation and Purification Technology journal homepage: www.elsevier.com/locate/seppur
Purification of vanillin by a molecular imprinting polymer technique M.N. Mohamad Ibrahim ∗ , C.S. Sipaut, N.N. Mohamad Yusof School of Chemical Sciences, Universiti Sains Malaysia, 11800 Minden, Pulau Pinang, Malaysia
a r t i c l e
i n f o
Article history: Received 31 October 2008 Received in revised form 16 February 2009 Accepted 17 February 2009 Keywords: Black liquor Lignin Crystallization method Vanillin compound Molecular imprinting
a b s t r a c t The successful separation of vanillin from soda lignin, extracted from the black liquor of oil palm empty fruit bunches (EFBs) with 20% (v/v) sulfuric acid may be negated by the high impurity content in the vanillin obtained. A molecular imprinting polymers (MIPs) technique was developed for vanillin to remove the impurities attached to it. A synthesis method was used in preparing the MIPs, where impure vanillin molecules were used as the template, dimethylsulfoxide (DMSO) as the solvent, and methacrylic acid (MAA) and ethylene glycol dimethacrylate acid (EGDMA) respectively functioning as the monomer and cross-linker. The results were confirmed by gas chromatography (GC), mass spectrometry (MS), Fourier transform infrared (FTIR) spectroscopy and thermal gravimetric analysis (TGA) of the sample against a standard vanillin. © 2009 Elsevier B.V. All rights reserved.
1. Introduction A molecular imprinting polymer is an efficient technique for creating a 3D cross-linked polymer network with specific binding site (memory) for a specific template [1]. Theoretically, molecular imprinting polymers (MIPs) are capable of selectively recognizing most of the known molecules through the synthesis processes of imprinting the specific template molecule into the polymer. The concept of MIPs is based on host–guest chemistry. The hypothesis is, polymers can mimic the recognition mechanisms of biological enzymes and antibodies [2]. However, molecular imprinting polymers (MIPs) became a reality and part of the host–guest chemistry field in the 1970s when polymers displaying properties that mimic the biological enzymes and monoclonal antibody were successfully synthesized [2,3]. MIPs can be classified into two specific categories, covalent and non-covalent [1]. The publications on MIPs have increased steadily over the last few years with the non-covalently imprinted MIPs the more popular. Non-covalent MIPs rely on hydrogen bonding between the host and the guest, which depends on their molecular structures, levels of crosslinking in the polymer, bond distances, dipole moments and types of interactions between the template and polymer [4–9]. The advantages of non-covalent MIPs include simple synthesis procedure, easy binding and extraction of templates as well as wide varieties of commonly available starting materials and chemical [10–13].
∗ Corresponding author. Tel.: +60 4 6533554; fax: +60 4 6574854. E-mail address: [email protected] (M.N.M. Ibrahim). 1383-5866/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.seppur.2009.02.010
The MIPs technique was used to involve preparing polymers by arranging methacrylic acid (MAA) as functional monomers around a template compound (vanillin) and fixing them with ethylene glycol dimethacrylate acid (EGDMA) as a cross-linker and is described schematically in Fig. 1. Vanillin (4-hydroxy-3-methoxybenzaldehyde) is a unique and highly prized flavor compound used as an ingredient in food flavors, in pharmaceuticals and as fragrance in perfumes and odor-masking products. In the vanilla bean, vanillin is the major compound that characterizes the aromatic compound of vanilla. The natural source of vanillin is from the seeds of the vanilla plant, a member of the orchid family. However, this process is very expensive compared to synthetic processes. Fortunately, nature provides a second source of vanillin, available from lignin extracted from the black liquor of oil palm empty fruit bunches (EFBs) by using a crystallization process [14,15]. The characteristics of the vanillin compound obtained from lignin were compared with the characteristics of standard vanillin. It was observed that the vanillin obtained from the crystallization process contains high level of impurities. Therefore, a molecular imprinting polymers technique was applied in order to purify the vanillin. This technique was selected because it has been used successfully with biomolecule mimics, and has high selectivity for compounds comparable to vanillin. The goal of this study is to create molecularly imprinted polymers that can recognize specific molecules such as vanillin from several other compounds. In this process, monofunctional monomers containing an acidic group such as methacrylic acid are used to allow hydrogen bonding with vanillin, the target molecule during polymerization. Then, the complexes were assembled in the liquid phase and fixed by cross-linking polymerization with ethylene glycol dimethacrylate acid (EGDMA) used as the
M.N.M. Ibrahim et al. / Separation and Purification Technology 66 (2009) 450–456
451
Fig. 1. Schematic illustration of the vanillin imprinting performed in this study. Blue colors represent cross-linked polymer network. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
crosslinking monomer. Subsequently, the target molecule (vanillin) was removed through extraction, utilizing the appropriate solvents (i.e. xylene) thus leaving behind vacant sites exhibiting a high affinity to the template [16–18]. The selectivity response of the imprinted polymers to vanillin compound is characterized by gas chromatography (GC) of the sample vis-à-vis standard vanillin. 2. Experimental 2.1. Materials Sulfuric acid, hydrochloric acid, nitrobenzene solution, nhexane (analytical grade) and xylene were purchased from QRëC. Sodium hydroxide, methacrylic acid, 2,2 -azobisisobutyronitrile (AIBN) and ethylene glycol dimethacrylate acid (EGDMA) were purchased from R&M Chemicals. Vanillin, chloroform and acetone were obtained from Fluka and Fisher Chemicals. 2.2. Vanillin production Soda lignin was extracted from the soda black liquor, derived from oil palm empty fruit bunches by using 20% sulfuric acid. The degradation of soda lignin using alkaline nitrobenzene oxidation was carried out at 165 ◦ C for 3 h in a 7 mL 2 M NaOH and 0.4 mL nitrobenzene solution. After 3 h, the mixture was cooled and then extracted with chloroform (5× 20 mL) to remove excess nitrobenzene. The oxidation mixture was acidified with concentrated HCl to pH 3–4 and extracted again with chloroform (15 mL). The solvent from the chloroform solution was removed by using a rotary evaporator at 40 ◦ C under reduced pressure to obtain the residue. A crystallization process was used to separate the vanillin component from the hydrolysis–oxidation of lignin. Based on the
solubility of vanillin in acetone, 15 mL of acetone was added to the residue. The mixture was slowly heated to 60 ◦ C for 15 min until vanillin precipitated. 2.3. Preparation of control polymer (CP) and imprinted polymers by crosslinking A typical procedure for preparing an imprinted polymer P1 involves the mixture of 5 mL of dimethyl sulfoxide, 2 mmol of standard vanillin with methacrylic acid (5 mmol), ethylene glycol dimethacrylate acid (EGDMA) (5 mmol) and 2,2 azobisisobutyronitrile (24 mg). The mixture was de-gassed in a test tube with nitrogen and placed in a water-bath at 80 ◦ C for 6 h. The bulk polymer was then taken from the reaction and freeze-dried. The dried polymer was then grounded and tested. Polymer P2 was prepared in a similar manner but the standard vanillin sample was replaced with vanillin obtained from the crystallization process. A non-imprinted blank polymer was prepared following the same method as used to prepare P1. However, no template component was added to this polymerization mixture, which served as the control. 2.4. Gel content determination Gel content is a method to determine the percentage of crosslinking level of the polymer. This method is used with standard procedures by refluxing the samples in boiling xylene for 24 h [19]. The crosslinked matrix samples (P1 and P2) were placed in 100 mesh cages, labeled and weighed. After refluxing in xylene, the residual solvent was removed in a preheated vacuum oven with a cold trap at a temperature of 140 ◦ C for 4 h at <0.1 bar. The cages were removed, allowed to cool and reweighed. Five samples were used to determine the insoluble fraction (gel content) and having
452
M.N.M. Ibrahim et al. / Separation and Purification Technology 66 (2009) 450–456
Table 1 The percentage of crosslinking level. Percentage of crosslinking level (%) Control (without template) P1 Total of standard vanillin P2 Total of sample X
95.83 92.17 3.67 92.33 3.49
an experimental error within 3% of the mean. The gel content was calculated as: Gel content (%) =
1−
W − W 3 4 W2 − W1
× 100%
(1)
where W1 = the weight of the mesh cage, W2 = the weight of the mesh cage and sample, W3 = the weight of the stapled cage and sample, and W4 = the weight of the stapled cage and sample after extraction and drying. 2.5. Gas chromatography analysis All gas chromatography data were collected by using an SRI 8610 C Gas chromatograph systems equipped with a flame ionization detector (FID) and helium as the carrier gas. The injector temperature was 280 ◦ C. Samples (0.5–1.0 L) were injected by using the splitless mode with a purge time of 1.0 min. Fused-silica capillary columns DB-5 (30 mm × 0.25 mm i.d., 0.32 m film thickness) was used in the process to qualitatively and quantitatively determine the residual products. 3. Results and discussion The identification of an unknown compound obtained from a crystallization process requires multiple forms of identification. In the case of vanillin extracted from lignin, there are four independent forms present for identification. 3.1. Mass spectral identification
Fig. 2. Comparison of mass spectrum between standard vanillin and the sample.
The mass spectra (Fig. 2) can be used to identify vanillin. The library spectrum (a) is a fairly good match with sample (c) with the purity score of 96–97%. The purity measures the similarity between the mass spectrum and the library reference mass spectrum. To confirm these results, a standard vanillin was diluted with n-hexane and injected as well. The resulting mass spectrum (b) also has a 97% purity score (Fig. 2). The base, which peaks at m/z 151 and 152, shows the major ions of vanillin (Fig. 2). The mass spectrum of both samples has a much more intense molecular ion than that of aromatic compounds, since fragmentation requires the cleavage of an alkyl bond. An aromatic ring in a vanillin molecule stabilizes the molecular ion peak, which often is sufficiently large enough that accurate intensity measurements can be made on the M + 1 and M + 2 peaks. Therefore, there was no significant difference in the molar mass of sample (c) with the standard vanillin based on the molar mass analyses. The sample obtained from the crystallization process had the same molecular formula as that of standard vanillin.
obtained by using the MIPs technique. The chromatogram (Fig. 3) for standard vanillin, P1 and P2 all contain peaks at retention times, Rt of 4.416, 4.433 and 4.450 min, respectively. The GC results are consistent with the hypothesis that vanillin from the MIPs can be considered pure since the peaks of the samples were similar.
3.2. Gas chromatography analysis There were no significant differences in gel content between the two samples (Table 1). As both samples were subjected to higher amounts of cross-linking agent, the probability of the formation of polymerization networks around the samples increased. All the imprinted and non-imprinted polymers prepared were tested by gas chromatography to determine the purity of the sample
Fig. 3. Comparison between chromatogram of P1 and P2 with chromatogram of standard vanillin.
M.N.M. Ibrahim et al. / Separation and Purification Technology 66 (2009) 450–456
453
Fig. 4. FTIR spectra of polymer control and polymer P1.
3.3. Fourier transform infrared (FTIR) analyses The polymers P1 and P2 obtained from the polymerization process were characterized using FTIR analyses to determine the functional group presence in the polymer. Fig. 4 shows the FTIR spectra of polymer control (without vanillin) and polymer P1. All two spectra are characterized by the presence of a broad band of OH stretching in 3400 cm−1 region and one band of CH stretching at 2900 cm−1 . The band of OH stretching which appear at the lower frequency indicates strong hydrogen bonding. The band at 1494, 1439 and 1402 cm−1 are assigned to C–H deformation in the –CH3 of a methoxyl group. Similarly, these bands are also present in polymer P1.
Fig. 5 shows the comparison of FTIR spectra of polymer control, polymers P1 and P2. Spectral differences in polymers P1 and P2 are observed in the fingerprint region (1800–800 cm−1 ). Bands 2140, 1487, 1021, 875 and 823 cm−1 are not present in polymer P2. The band at 1487 cm−1 is assigned to CH deformation in –CH2 groups. Roczniak et al. identified the region between 1500 and 1400 cm−1 as characteristics of the deformation vibration of –CH– bands in a –CH2 group and observed some differences from the spectra of polymers [20]. These differences can provide information as to the structure of a methylene bridge. In polymer P1, the appearance of bands at 1487 cm−1 indicates the presence of a methylene bridge. Polymer P2 shows a band at 1390 cm−1 , which is attributed to an in-plane
Fig. 5. The comparison of FTIR spectra.
454
M.N.M. Ibrahim et al. / Separation and Purification Technology 66 (2009) 450–456
Fig. 6. TGA thermogram of sample obtained from crystallization process.
Fig. 7. TGA thermogram of standard vanillin.
M.N.M. Ibrahim et al. / Separation and Purification Technology 66 (2009) 450–456
Fig. 8. TGA thermogram of polymer P2.
Fig. 9. TGA thermogram of polymer control.
455
456
M.N.M. Ibrahim et al. / Separation and Purification Technology 66 (2009) 450–456
deformation of phenolic OH. However, the differences show a reaction between polymer P2 with the sample in the purification process. It can be concluded that the sample had been successfully purified using MIP process. 3.4. Thermal gravimetric analysis (TGA) Figs. 6 and 7 shows the TGA thermograms of sample obtained from crystallization and standard vanillin. The study was carried out up to 900 ◦ C in N2 atmosphere. In the case of the sample obtained from crystallization, two thermal events were observed, one in the range 34.60–268.13 ◦ C and the other in the range of 268.13–894.73 ◦ C. As shown in Fig. 6, the decomposition temperature at 70.4% weight loss appeared at the range of 34.60–268.13 ◦ C. When the temperature was raised up to 300 ◦ C, the weight loss accounted was at 60%. In the case of standard vanillin, only oneweight loss event was observed. It started at 31.00 ◦ C and ended at 894.10 ◦ C, and the standard vanillin lost 47.31 × 10−3 % in this event. From both of the thermograms, the ranges of decomposition for the sample obtained from the crystallization process were similar with the standard vanillin. However, most probably the sample which was obtained from the crystallization process still contained impurities. Fig. 6 shows the sample obtained from the crystallization process has two thermal events compared to the standard vanillin thermogram which showed only one thermal event. Thus, the sample obtained from crystallization process was synthesized with the polymer template for the purification process. In this process, the polymer templates were reacting with the sample and cross-linked polymers were formed around the sample. Then, a sample is removed by refluxing using xylene as a solvent. After that, the polymers freed from the sample were analyzed by TGA analysis to identify the behavior of the polymer. The TGA thermogram for the polymer is shown in Fig. 8. For comparison, the thermogram for polymer control is also shown in Fig. 9. As shown in Fig. 8, the decomposition temperature at 80.78% weight loss appeared at 119.95 ◦ C. While at temperature of 890.32 ◦ C, the weight loss decreased significantly to −8.43%. As a result, the polymer template reacted with the sample, showing that the sample obtained after the purification process is highly pure. These results suggest that MIPs prepared in situ were successfully applied in the purification process of vanillin obtained from soda lignin. 4. Conclusion In this study, we successfully separated the vanillin component was from EFB lignin by using a crystallization process. The result of the molar mass obtained confirmed that the obtained sample had characteristics similar to those of standard vanillin. The MIPs
technique was then used to remove any impurities in the sample. The molecularly imprinted polymer prepared in situ with vanillin as a template, was successfully used to purify sample obtained from the crystallization process. These results were confirmed by gas chromatography, FTIR and TGA analysis of the sample against the standard vanillin, which showed that the characteristics of the sample from the crystallization process were similar to those of standard vanillin. This research project has two benefits in that it aims to overcome the problem relating to the disposal of pulping waste in the form of toxic black liquor, as well as providing a novel method for recycling unwanted materials into valuable products. Acknowledgments The authors would like to express their appreciation to Universiti Sains Malaysia and the Malaysian Ministry of Science, Technology and Innovation for financial support of this project through a research grant (305/PKIMIA/613306). References [1] K. Makoto, T. Toshifumi, M. Takashi, A. Hiroyuki, Molecular Imprinting: From Fundamentals to Applications, Wiley-VCH Verlag Gmbh & Co., KGaA, Weinheim, 2003. [2] T. Takeuchi, J. Haginaka, Separation and sensing based on molecular recognition using molecularly imprinted polymers, J. Chromatogr. B 728 (1999) 1–20. [3] L. Ye, R. Weiss, K. Mosbach, Macromolecules 23 (2000) 8239. [4] L.I. Andersson, Molecular imprinting: developments and applications in the analytical chemistry field, J. Chromatogr. B 745 (2000) 3–13. [5] B. Sellergren (Ed.), Molecularly Imprinted Polymers: Man-made Mimics of Antibodies and their Application in Analytical Chemistry (Techniques and Instrumentation in Analytical Chemistry), vol. 23, Elsevier, Amsterdam, 2001. [6] A. Lars, M. Akiyoshi, J.O. Daniel, M. Klaus, J. Chromatogr. 22 (1990) 323–331. [7] K. Takaomi, Y.W. Hong, F. Nobuyuki, Chem. Lett. (1995) 927–928. [8] A. Lars, J.O. Daniel, M. Klaus, J. Chromatogr. 513 (1990) 167–179. [9] M. Jun, A.N. Ian, T. Toshifumi, M. Klaus, K. Isao, Anal. Chem. Acta 335 (1996) 71–77. [10] L. Jia-Ping, H. Xi-Wen, J. Yue, C. Feng, Anal. Bioanal. Chem. 375 (2003) 264–269. [11] M.M. Wayne, P.C.L. Edward, S. Borje, Anal. Commun. 36 (1999) 217–220. [12] G. Vlatakio, L.I. Andersson, R. Muller, K. Mosbach, Nature 361 (1993) 645–647. [13] M.C. Blanco-Lopez, M.J. Lobo-Castanon, A.J. Miranda-Ordieres, P. Tunon-Blanco, Trend Anal. Chem. 23 (2004) 36–48. [14] J.L.R. Kirkaldy, J.B. Susanto, Possible utilization of by-products from palm oil industry, Planter 72 (838) (1996) 27–39. [15] K.C. Khoo, T.W. Lee, Pulp and paper from oil palm, Appita 44 (6) (1991) 385–388. [16] G. Wulff, Molecular imprinting in cross-linked materials with the aid of molecular templates-a way towards artificial antibodies, Angew. Chem. Int. Ed. 34 (1995) 1812–1832. [17] W.L. Xu, Y.Q. Wang, Y.B. Huang, P. Lu, Studies on the selection of solvents for recrystallization purification of phytosterol, J. Yangzhou Univ. 5 (2002) 58. [18] T. Takeuchi, D. Fukuma, J. Matsui, Combinatorial molecular imprinting: an approach to synthetic polymer receptors, Anal. Chem. 71 (1999) 288–290. [19] ASTM D2765-95. Standard Test Method for Determination of Gel Content and Swell Ratio of Crosslinked Ethylene Plastic, ASTM, Philadelphia, 1995. [20] C. Rocznaik, T. Biernacka, M. Skarzynski, Anal. Chem., Some properties and chemical structure of phenolic resins and their derivatives 28 (1983) 531.