Renewables Saleh Al Arni1 Alex F. Drake2 Research Article Marco Del Borghi1 Attilio Converti1 Study of Aromatic Compounds Derived from Sugarcane Bagasse: II. Effect of Concentration 1 2 Department of Chemical and Process Engineering, University of Genoa, Via Opera Pia 15, I – 16145, Genoa, Italy. Pharmacy, King’s College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, UK. 523 The results of a set of experiments that were designed to integrate current knowledge on the major constituents of sugarcane bagasse lignin, specifically p-coumaric, ferulic, syringic acids, and vanillin are presented. These aromatic compounds have well-known antioxidant power and they are very important for the food, pharmaceutical, and nutraceutical industries. Following a study on the effect of pH upon the spectra of these monomers and the determination of their pKa values, their self-association properties in ethanol as a solvent, particularly at concentrations < 10–4 M were investigated using UV spectrophotometry and their Kass values were determined. The results collected allowed to better understand the chemistry of sugarcane lignin and provide a contribution to the possible exploitation of this abundant straw material for the recovery of fine chemicals. Keywords: Concentration, Ferulic acid, Lignin, p-Coumaric acid, Renewables, Self-Association, Sugarcane bagasse, Syringic acid, Vanillin Received: November 19, 2009; revised: December 22, 2009; accepted: January 5, 2010 DOI: 10.1002/ceat.200900552 1 Introduction Lignin is a lignocellulosic biomass fraction that combines with cellulose and hemicellulose. Being a straw organic material abundant in nature, it could be used as a cheap source of natural chemical compounds, because of its low cost and reduced impact on the environment. Natural lignin composition is poorly known, because it is not yet possible to definitively determine the complete structure of lignin. Since there is no compound that can be used as a standard for lignin structure, their measurements are very dependent on the methodology employed for experiments. Can we say that lignin is a heterogeneous polymer of phenolic compounds [1] made up of three principal monomers derived from the hydroxycinnamyl alcohol, such as p-coumaryl alcohol (C9H10O2), coniferyl alcohol (C10H12O3), and sinapyl alcohol (C11H14O4), whose chemical structure depends on various environmental factors. Lignin has a complex polymeric, disordered, and random structure, and consists mainly of ether linked aromatic rings, which add elasticity to the cellulose and hemicellulose matrices [2]. Some aromatic carboxylic acids, – Correspondence: Prof. A. Converti (converti@unige.it), Department of Chemical and Process Engineering, University of Genoa, Via Opera Pia 15, I – 16145, Genoa, Italy. Chem. Eng. Technol. 2010, 33, No. 3, 523–531 mainly p-coumaric acid and ferulic acid, are present in most lignins in the form of esters [3]. The hydrocarbon compounds extracted from lignin as well as its structural monomers such as p-coumaric, ferulic and syringic acids, vanillin, and others are labeled in the aromatic ring [4]. Recently, new methods have been explored using different wavelengths and absorptivities on specific biomass samples. The aromatic compounds under investigation, but vanillin, do in fact contain a carboxylic acid group whose carbonyl normally absorbs at about 290 nm, although its precise location depends on the remaining structure of their molecules [5]. The carbonyl group acts as a chromophore primarily on account of the excitation of non-bonding (O) lone pair electrons to an anti-bonding (C=O) orbital, which gives rise to a n → p* transition. Typical absorption energies are about 4 eV at k = 290 nm. The magnitude of the molar extinction coefficient (e) reflects both the size of the chromophore and the probability that the light of a given wavelength will be absorbed when it strikes the chromophore. p-Coumaric, ferulic, and syringic acids all contain a core styrene chromophore. This chromophore has many transitions which are responsible for the UV absorption spectrum; in isolation, this group is expected to have a UV maximum at ∼290 nm [5]. However, various substituents such as -CO2H, -OH, and -OCH3 will induce progressive shifts to longer wavelengths. For this reason, the absorptivity of lignin compounds typically extracted from sugarcane bagasse has been determined at k between 200 and 450 nm. © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.cet-journal.com 524 S. Al Arni et al. Since lignin has received less attention for production of chemicals compared to polysaccharides and has not yet been used in any large-scale industrial application for the recovery of aromatic compounds, there is a great deal in the use of the lignin fraction of sugarcane bagasse as source of antioxidants of particular concern to the food, pharmaceutical, and nutraceutical industries. Sugarcane bagasse was already taken into consideration in a previous study as a source of lignin compounds and its alkaline solubilization was investigated [6]. Huge amounts of this agricultural residue from sugar extraction from sugarcane accumulate in the mills, thus creating environmental problems. Although utilized in the sugar factories as fuel for the boilers [7], recently, there has been an increasing trend towards alternative utilization. It is in fact a large and inexpensive raw material that can be used in several biotechnological processes: as carbohydrate source for the productions of xylitol [8, 9], ethanol [10], and several different enzymes [11–13], or even as a support for cell immobilization [14–17]. Following a previous study on the effect of pH upon the spectra of the main constituents of its lignin fraction, e.g., p-coumaric acid, ferulic acid, syringic acid, and vanillin, and the determination of their pKa values [18], their self-association properties to form dimers were investigated by optical spectroscopy. range 180–450 nm. The baseline spectra of the solvent used to make up the samples were obtained on the same day as the sample spectra and subtracted from them. After obtaining the spectra, data were transferred to the standard analysis software, Grams 32 Al (Galactic Industries Inc., Salem, NH), for baseline correction and other studies. Molar extinction coefficients (e, expressed as M–1cm–1) were determined for wavelengths (k, expressed in nm) over the whole spectral range. This was done through the Beer-Lambert law (A = ecl), by dividing the absorbance (A) by the concentration (c) and the cell path length. Relevant data obtained from spectra at different concentrations at constant wavelength were plotted to obtain self-association curves. The MathCad 8 Professional program was used to analyze the maximum values (emax) of e versus concentration to obtain the limiting monomer and dimer spectra, as well as the association constant (Kass) values. 2 Experimental m+m>d 2.1 Reagents Setting the total concentration (Ct) equal to the sum of the molar concentrations of the dimer (Cd) and the monomer (Cm): p-Coumaric acid, ferulic acid, syringic acid, and vanillin used as standard reagents were pure chemicals purchased from Sigma-Aldrich (Steinheim, Germany). 3 Theory of Dimerization In ethanol as a solvent, the aromatic compounds under investigation self-associate to form dimers at high concentration. The dimerization equation was derived as follows. In the case of self-association equilibrium, a monomer can be said to form a dimer as described by the following equation: (1) Ct = Cm + Cd (2) the equilibrium constant of dimerization (Kass) is defined as: 2.2 Sample Preparation Kass = Cd / Cm2 Weighing of samples was carried out using an electronic microbalance, model MT5 (Mettler Toledo, Columbus, OH), and the use of an ultrasonic bath was necessary to ensure their complete dissolution. The preparation of stock solutions was the first step in conducting these experiments. For concentration studies, 1- to 3-mg samples were put into 25 mL tubes, and 96 % (v/v) ethanol was added as a solvent up to the following concentrations: 0.717 mM p-coumaric acid, 0.254 mM ferulic acid, 0.266 mM syringic acid, and 0.239 mM vanillin. From these solutions, all dilutions were derived. 2.3 Analytical Techniques For spectral measurements, rectangular cuvettes of 1.0-cm path length (l) or cylindrical cuvettes with l = 1.0, 2.0, and 5.0 cm were used in a UV-VIS-IR spectrophotometer, model 17DS (Aviv Associates, Lakewood, NJ). The instrumental setting was: spectral band width = 1.0 nm, measurement step size = 0.5 nm, scan speed = 0.2 s. Scans were typically over the www.cet-journal.com (3) Absorbance based on Beer’s law will now be [19]: Aobs = Am + Ad = (em Cm l) + (ed Cd l) (4) and the extinction coefficient: eobs = (em Cm + ed Cd) / Ct (5) Combining Eqs. (2), (3), and (5), and solving the system gives: 1 eobs Ct † ˆ Ct (   p " p2 #) 1± 1 ‡ 4Kass Ct 1± 1 ‡ 4Kass Ct em ‡ ed Kass 2Kass 2Kass (6) Applying this equation shows that the positive sign before the square root is the only acceptable solution. © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Chem. Eng. Technol. 2010, 33, No. 3, 523–531 Renewables In Eq. (6), the dependent variable, eobs, depends upon three parameters, specifically the limiting values of the extinction coefficients of both the monomer (em) and dimer (ed), as well as Kass. Using the MathCad 8.0 software, plots of the experimental data of eobs (Ct) against concentration resulted in graphs that could be simulated by Eq. (6) and fitted with the best values of ed, em, and Kass. 4 Results and Discussion 525 ∼310, 323, and 275 nm for p-CA, FA, and SA, moved at low concentrations to ∼291, 310, and 262 nm, respectively. On the contrary, no clear effect was evident for vanillin, for which a variation in the percentage of conversion of monomer to dimer was observed, when a change in the concentration took place at a given wavelength. Such shifts indicate that the BeerLambert law was not obeyed. Whereas, the extinction coefficient of p-CA remained relatively constant (≈ 22000 M–1cm–1), the extinction coefficients from high to low concentrations for FA and SA decreased from 18600 to 17000 M–1cm–1 and from 10800 to 9600 M–1cm–1, respectively. As depicted in Fig. 2, all the selected compounds were present as monomeric species at the lowest concentrations and self-associated at the highest concentrations, dimerization being the likely result of the monomer in equilibrium with its dimer. Although, at this stage, the precise molecular nature of self-associated vanillin in 96 % ethanol is not known, a proposal is presented in Fig. 2D). Figs. 3–6 show the simulated curves for p-CA, FA and SA, and vanillin, respectively, referred to wavelengths corresponding either to maximum or to minimum absorption, specifically k = 210, 227, 291, and 310 nm for p-CA; k = 216, 232, and 323 nm for FA; k = 215, 262, and 275 nm for SA; k = 232, 280, 310, and 358 nm for vanillin. The equilibrium constants, Kass, as well as the other parameters that appear in Eq. (6) were calculated for all the compounds with the help of the MatCad software package that allowed the simulation of the dimerization curves from the experimental data. Their values are listed in Tab. 1 together with those of the extinction coefficients of both the monomeric and dimeric species. As far as the dimerization constants are concerned, although they were somehow influenced by the wavelength, p-CA, FA, and SA showed average Kass values of ∼2 · 105, ∼1 · 106, and ∼4 ·105, respectively. On the other hand, that of vanillin (Kass ∼ 3 · 104) was 1–2 orders of magnitude lower, as the likely result of the presence of an aldehyde rather than a carboxylic acid group. Tab. 2 shows the limiting values of the extinction coefficients of both monomeric and dimeric vanillin estimated at different wavelengths. Using these values, a plot of percentage of self-association versus concentration has been drawn up for vanillin Previous work on the effect of pH on titration curves of aromatic compounds derived from sugarcane bagasse confirmed the suitability of the titration method to determine the values of pKa of the selected compounds [18, 20]. In this way, the pKa values of p-coumaric (p-CA), ferulic (FA) and syringic acids (SA), and vanillin in two ionization states were determined and the effect of pH on their molecular structures associated with the two equilibrium states was shown. Here, the effect of varying concentrations of these aromatic compounds on their respective spectral and self-association behavior was investigated. The absorbance spectra obtained at a series of concentrations were investigated in 96 % ethanol as solvent, using 1.0-, 2.0-, and 5.0-cm path length cuvettes. The self-association properties of the selected aromatic compounds were investigated by UV spectrophotometry, particularly at concentrations < 10–4 M. Several isosbestic points were observed where the spectra do not depend on the concentration of the species. In fact, when an isosbestic point is present at a given wavelength, the two chemical species have the same molar absorptivity (e) and then, the system either possesses more than two states, or more than one complex state has formed. According to Pouët et al. [21], the presence of isosbestic points also means a quality and quantity conservation in the global composition of samples, with given relations between the concentration of absorbing compounds or mixtures of compounds. Once the spectra of the self-associated species had been defined, the molecular structures associated with the two equilibrium states and the association constants were determined. Fig. 1 shows the extinction coefficient spectra of p-CA (graph A), FA (graph B), SA (graph C), and vanillin (graph D) versus wavelength at different concentrations using only 1.0-cm path Table 1. Values of the dimerization constant (Kass) at different wavelengths for p-coumaric, ferulic length cuvettes. The isosbestic and syringic acids, and vanillin, as well as of the molar extinction coefficients (e) of both the monomeric and dimeric species. points were observed in the spectra of these compounds at the followp-Coumaric acid Ferulic acid Syringic acid Vanillin ing wavelengths: a) p-CA: 224, 245, and 290 nm; b) FA: 209, 231, 255, Kass (ethanol) 1.25 · 105 (210 nm) 1.0 · 106 (216 nm) 3.3 · 105 (215 nm) 2.3 · 104 (232 nm) and 291 nm; c) SA: 233 and 1.20 · 105 (227 nm) 1.0 · 106 (232 nm) 5.0 · 105 (262 nm) 3.0 · 104 (280 nm) 5 265 nm; d) vanillin: 335 nm. The e 3.50 · 10 (291 nm) 1.0 · 106 (323 nm) 2.5 · 105 (275 nm) 1.6 · 104 (310 nm) 1.85 · 105 (310 nm) 3.7 · 104 (358 nm) spectra of p-CA either at low or high concentrations confirm the e292 = 13450 e261 = 9460 e280 = 10075 Monomer e286 = 22080 two-state model of monomer in e219 = 15404 e231 = 12250 e210 = 27800 e358 = 1860 equilibrium with dimer. Dimer e311 = 22370 e323 = 18619 e273 = 10723 e310 = 10570 As shown in the same figure, the e226 = 11540 e233 = 11970 e215 = 27160 e232 = 15470 absorbance maxima, which oce210 = 10490 e215 = 13662 curred at high concentrations at Chem. Eng. Technol. 2010, 33, No. 3, 523–531 © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.cet-journal.com 526 S. Al Arni et al. 25000 -1 10000 5000 A 0 200 1 225 15000 -1 15000 Extinction coefficient, ε (M .cm ) Conc.1.42E4 Conc.9.45E5 Conc.6.30E5 Conc.4.20E5 Conc.2.80E5 Conc.1.87E5 Conc.1.24E5 Conc.8.30E6 20000 Extinction coefficient (ε) Conc1.69E4M Conc1.13E4M Conc7.53E5M Conc5.02E5M Conc3.35E5M 20000 2 10000 5000 3 250 275 B 0 300 325 200 350 250 300 350 Wavelength, λ (nm) Wavelengths, λ (nm) 30000 16000 Conc1.18E04 Conc7.89E05 Conc5.26E05 Conc3.26E05 -1 15000 10000 5000 0 200 12000 -1 20000 Conc2.39E04 Conc1.59E04 Conc1.06E04 Conc7.08E05 Conc4.72E05 Conc3.15E05 Conc2.10E05 14000 Extinction coeffecient, ε (M .cm ) -1 -1 Extinction coeffecient, ε (M .cm ) 25000 C 10000 8000 6000 4000 2000 D 0 220 240 260 280 300 320 200 340 250 300 350 400 Wavelength, λ (nm) Wavelenghth, λ (nm) Figure 1. Molar extinction coefficients in 96 % ethanol solution versus wavelength obtained at different concentrations using 1.0-cm path length cuvettes: A) p-coumaric acid; B) ferulic acid; C) syringic acid; D) vanillin. Table 2. Limiting values of extinction coefficients of both monomeric (em) and dimeric (ed) vanillin obtained at different wavelengths. k [nm] em [M–1cm–1] ed [M–1cm–1] 232 12000 16100 280 8500 11000 310 9500 11000 358 2700 10 as a function of its concentration in 96 % ethanol at different wavelengths (Fig. 7). At about 280 nm, the extent of vanillin self-association turned out to be greater in comparison with either lower or higher wavelengths at all the concentrations tested in this study. Under these conditions, its dimerization constant was www.cet-journal.com ∼3 · 104, while the maximum values of the molar extinction coefficients were ∼11 000 and ∼8500 M–1cm–1 for the dimeric form (high concentrations) and the monomeric form (low concentrations), respectively. 5 Conclusion The self-association properties of the main aromatic compounds present in sugarcane bagasse lignin, specifically p-coumaric acid (p-CA), ferulic acid (FA), syringic acid (SA), and vanillin, have been investigated in ethanol at different concentrations, in particular < 10–4 M. The UV spectra of both monomeric and self-associated species allowed determination of their association constants (Kass), which turned out to be so low that special 5.0-cm path length cuvettes were required for analysis. © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Chem. Eng. Technol. 2010, 33, No. 3, 523–531 Renewables O 527 OH H O O Kass H HO O O O H H O O HO HO Monomer Dimer Low concentration High concentration A) p-Coumaric acid O OH O H H3 C O O Kass H O HO CH3 O O O H H O O HO HO O H 3C O Monomer Dimer H 3C Low concentration High concentration B) Ferulic acid O O O H H3C CH3 OH O O HO H O Kass O CH3 O H3C O O O O H H3C H H3C O O HO HO O O H3C H3C Monomer Dimer Low concentration High concentration C) Syringic acid H H O O O O H3CO H3CO + OCH3 O OCH3 O O H O H D) Vanillin Figure 2. Molecular structure in 96 % ethanol solution at high and low concentrations of A) p-coumaric acid; B) ferulic acid; C) syringic acid; D) vanillin. Chem. Eng. Technol. 2010, 33, No. 3, 523–531 © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.cet-journal.com 528 S. Al Arni et al. B Mol. Extin.Coeff.at 227 nm (M-1 cm-1) Mol. Extin.Coeff.at 210 nm (M-1 cm-1) A 4 2 .10 4 1.8 .10 4 Ecalc( Ct ) 1.6 .10 Eobs j 4 1.4 .10 4 1.2 .10 4 1 .10 0 5 4 1.5 .10 4 1.4 .10 4 Ecalc( Ct ) 1.3 .10 Eobs j 4 1.2 .10 4 1.1 .10 4 1 .10 5 4 4 4 4 .10 8 .10 1.2 .10 1.6 .10 2 .10 Ct , Ctot j Concentration (M) 4 .10 0 D Mol. Extin.Coeff.at 310 nm (M-1 cm-1) Mol. Extin.Coeff.at 291 nm (M-1 cm-1) C 4 2.3 .10 4 2.2 .10 4 Ecalc( Ct ) 2.1 .10 j 4 2 .10 4 1.9 .10 4 1.8 .10 0 4 .10 5 4 4 4 8 .10 1.2 .10 1.6 .10 2 .10 Ct , Ctot j Concentration (M) Simulated Experimental Simulated Experimental Eobs 5 5 5 4 4 4 8 .10 1.2 .10 1.6 .10 2 .10 Ct , Ctot j Concentration (M) 4 2.3 .10 4 2.14.10 4 Ecalc( Ct ) 1.98.10 Eobs j 4 1.82.10 4 1.66.10 4 1.5 .10 0 Simulated Experimental 4 .10 5 5 4 4 4 8 .10 1.2 .10 1.6 .10 2 .10 Ct , Ctot j Concentration (M) Simulated Experimental Figure 3. Molar extinction coefficients of p-coumaric acid in 96 % ethanol solution versus concentration. k (nm): (A) 210, (B) 227, (C) 291, (D) 310. (×) Experimental values; (line) simulated curves. The isosbestic points were observed in the spectra of these compounds at the following wavelengths: a) p-CA: 224, 245, and 290 nm; b) FA: 209, 231, 255, and 291 nm; c) SA: 233 and 265 nm; d) vanillin: 335 nm. p-CA exhibited Kass ∼ 2 · 105 and a maximum molar extinction coefficient (emax = 22000 M–1cm–1) at ∼291 nm at low concentrations (monomer), which shifted to ∼310 nm at high concentrations (dimer). However, emax appeared to be almost independent of the concentration. FA exhibited Kass ∼ 1 · 106, emax ≈ 18600 M–1cm–1 at k ∼ 323 nm as a dimer and emax ∼ 17000 M–1cm–1 at 318 nm as a monomer. SA behaved similarly to FA, showing a value of Kass ∼ 4 · 105, emax ∼ 1800 M–1cm–1 at k ∼ 275 nm as a dimer and www.cet-journal.com emax ∼ 9600 M–1cm–1 at k ∼ 262 nm as a monomer. The dimerization constant of vanillin (Kass ∼ 3 · 104) was 1–2 orders of magnitude lower than those of the other compounds, and the maximum molar extinction coefficients of its dimeric and monomeric forms were emax ≈ 11 000 M–1cm–1 and 8500 M–1cm–1, respectively, both at k ∼ 280 nm. These results allowed us to increase our understanding of the chemistry of sugarcane lignin and provide a contribution to the possible exploitation of this abundant straw material for the recovery of fine chemical products. © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Chem. Eng. Technol. 2010, 33, No. 3, 523–531 Renewables A A 4 2.9 .10 4 1.44 .10 Ecalc Eobs 4 ( Ct ) 1.38 .10 j 1.32 .10 4 4 1.26 .10 4 1.2 .10 1 .10 5 3.2 .10 5 5 4 7.4 .10 1.16 .10 1.58 .10 Ct , Ctot j Concentraion (M) 4 2 .10 4 Mol. Extin.Coeff.at 215 nm (M-1 cm-1) Mol. Extin.Coeff.at 216nm (M-1 cm-1) 4 1.5 .10 4 2.82 .10 4 Ecalc ( Ct ) 2.74 .10 Eobs j 4 2.66 .10 4 2.58 .10 4 2.5 .10 0 4 .10 Simulated Experimental Eobs ( Ct ) 1.18 .10 4 j .10 4 1.12 1.06 .10 4 4 1 .10 1 .10 5 3.2 .10 5 5 7.4 .10 1.16 Ct , Ctot 4 .10 1.58 .10 4 2 .10 4 Eobs j 7600 5 4 .10 0 4 1.48 .10 4 1.34 .10 3.2 .10 5 5 4 7.4 .10 1.16 .10 1.58 .10 Ct , Ctot j Concentraion (M) 4 2 .10 4 1.02 Ecalc Eobs ( Ct ) j .10 4 9400 8600 7800 7000 0 Simulated Experimental Figure 4. Molar extinction coefficients of ferulic acid in 96 % ethanol solution versus concentration. k (nm): (A) 216, (B) 232, (C) 323. (×) Experimental values; (line) simulated curves. C 4 1.1 .10 (M-1 cm-1) 4 ( Ct ) 1.62 .10 5 5 4 4 4 8 .10 1.2 .10 1.6 .10 2 .10 Ct , Ctot j Concentration (M) Simulated Experimental Mol. Extin.Coeff.at 275nm Mol. Extin.Coeff.at 323 nm (M-1 cm-1) 4 8200 7000 4 1.76 .10 4 1.2 .10 1 .10 2 .10 8800 Ecalc ( Ct ) C 4 1.9 .10 j 4 9400 Simulated Experimental Eobs 4 1.6 .10 4 1 .10 j Concentration (M) Ecalc 5 8 .10 1.2 .10 Ct , Ctot j Concentration (M) B 4 Mol. Extin.Coeff.at 262 nm (M-1 cm-1) Mol. Extin.Coeff.at 232 nm (M-1 cm-1) Ecalc .10 5 Simulated Experimental B 4 1.3 .10 1.24 529 4 .10 5 5 8 .10 1.2 .10 Ct , Ctot j Concentration (M) 4 1.6 .10 4 2 .10 4 Simulated Experimental Figure 5. Molar extinction coefficients of syringic acid in 96 % ethanol solution versus concentration. k (nm): (A) 215, (B) 262, (C) 275. (×) Experimental values; (line) simulated curves. Acknowledgements References The authors thank the Italian Ministry of Education, University and Research (MIUR), and the University of Genoa for the PhD fellowship of Dr. S. Al Arni. [1] E. N. Aquino-Bolaños, E. Mercado-Silva, Postharvest Biol. Technol. 2004, 33 (3), 275. DOI: 10.1016/j.postharvbio. 2004.03.009 [2] M. Paster, Industrial bioproduct: Today and tomorrow, U. S. Department of Energy, Office of Energy Efficiency and Renewable Energy, Office of the Biomass Program, Washington, D.C. 2003, online: http://www.bioproducts-bioenergy.gov/ Chem. Eng. Technol. 2010, 33, No. 3, 523–531 © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.cet-journal.com 530 S. Al Arni et al. B A 4 1.1 .10 Ecalc ( Ct ) Eobs j 1.4 .10 4 1.3 .10 4 1.2 .10 4 1.1 .10 4 1 .10 4 Mol. Extin.Coeff.at 280 nm (M-1 cm-1) Mol. Extin.Coeff.at 232 nm (M-1 cm-1) 4 1.5 .10 0 4 .10 5 8980 6960 Ecalc ( Ct ) Eobs j 4940 2920 900 5 4 4 8 .10 1.2 .10 1.6 .10 2 .10 Ct , Ctot j Concentraion (M) 0 4 .10 Simulated Experimental C D Mol. Extin.Coeff.at 358 nm (M-1 cm-1) Mol. Extin.Coeff.at 310 nm (M-1 cm-1) 1800 4 1.06 .10 4 Ecalc ( Ct ) 1.02 .10 j 5 4 4 4 8 .10 1.2 .10 1.6 .10 2 .10 Ct , Ctot j Concentraion (M) Simulated Experimental 4 1.1 .10 Eobs 5 9800 9400 9000 0 4 .10 1560 Ecalc ( Ct ) 1320 Eobs j 1080 840 600 5 5 4 4 8 .10 1.2 .10 1.6 .10 2 .10 Ct , Ctot j Concentraion (M) 0 4 .10 5 5 4 4 . 4 8 .10 1.2 .10 1.6 .10 2 10 Ct , Ctot j Concentraion (M) Simulated Experimental Simulated Experimental Figure 6. Fitting of the self-association spectral data of vanillin to a two-component dimer model. k (nm): (A) 232, (B) 280, (C) 310, (D) 358. (×) Experimental values; (line) simulated curves. 100 90 80 Percentage 70 60 Vanillin 50 percent232 percent280 percent310 percent358 40 30 20 10 0 0,00004 0,00008 0,00012 0,00016 0,00020 0,00024 Concentration (M) Figure 7. Percentage of conversion of monomeric to dimeric vanillin as a function of the concentration in 96 % ethanol. k (nm): (×) 232, (䊊) 280, (~) 310, (q) 358. www.cet-journal.com © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Chem. Eng. Technol. 2010, 33, No. 3, 523–531 Renewables [3] [4] [5] [6] [7] [8] [9] [10] [11] pdfs/bioproductsopportunitiesreprotfinal.pdf (accessed June, 2005). R. D. Hartley, J. Sci. Food Agric. 1972, 23 (11), 1347. DOI: 10.1002/jsfa.2740231110 R. Ji et al., Chemosphere 2005, 60 (9), 1169. DOI: 10.1016/ j.chemosphere.2005.02.014 P. W. Atkins, Physical Chemistry, 5th ed., Oxford University Press, Oxford 1994. S. Al Arni, M. Zilli, A. Converti, Cienc. Tecnol. Aliment. 2007, 5 (4), 271. J. Zandersons et al., Biomass Bioenerg. 1999, 17 (3), 209. DOI: 10.1016/S0961-9534 (99)00042-2 W. Carvalho, S. S. Silva, A. Converti, M. Vitolo, Biotechnol. Bioeng. 2002, 79 (2), 165. DOI: 10.1002/bit.10319 A. A. Cunha et al., Appl. Biochem. Biotechnol. 2009, 157 (3), 527. DOI: 10.1007/s12010-008-8301-5 B. F. Sarrouh, S. S. Silva, D. T. Santos, A. Converti, Chem. Eng. Technol. 2007, 30 (2), 270. DOI: 10.1002/ceat.200600271 S. Mathew, T. E. Abraham, Enzyme Microb. Technol. 2005, 36 (4), 565. DOI: 10.1016/j.enzmictec.2004.12.003 Chem. Eng. Technol. 2010, 33, No. 3, 523–531 531 [12] J. C. Meza et al., Process Biochem. 2005, 40 (10), 3365. DOI: 10.1016/j.procbio.2005.03.004 [13] M. Mazutti, J. P. Bender, H. Treichel, M. Di Luccio, Enzyme Microb. Technol. 2006, 39 (1), 56. DOI: 10.1002/jctb.2273 [14] A. Pandey, C. R. Soccol, P. Nigam, V. T. Soccol, Bioresour. Technol. 2000, 74 (1), 69. DOI: 10.1016/S0960-8524(99) 00142-X [15] L. Sene, A. Converti, M. G. A. Felipe, M. Zilli, Bioresour. Technol. 2002, 83 (2), 153. DOI: 10.1016/S0960-8524(01) 00192-4 [16] M. Zilli et al., Biodegradation 2004, 15 (2), 87. DOI: 10.1023/ B:BIOD.0000015613.91044.a4 [17] D. T. Santos et al., J. Food Eng. 2008, 86 (4), 542. DOI: 10.1016/j.jfoodeng.2007.11.004 [18] S. Al Arni, A. F. Drake, M. Del Borghi, A. Converti, Chem. Eng. Technol., under revision. [19] S. B. Brown, An Introduction to Spectroscopy for Biochemists, Academic Press, London 1980. [20] S. Al Arni, Ph.D. Thesis, University of Genoa, Italy 2008. [21] M.-F. Pouët, E. Baures, S. Vaillant, O. Thomas, Appl. Spectrosc. 2004, 58 (4), 486. DOI: 10.1366/000370204773580365 © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.cet-journal.com