Journal of Chemistry and Chemical Engineering

Journal of Chemistry and Chemical Engineering Volume 4, Number 11, November 2010 (Serial Number 36)

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Journal of Chemistry and Chemical Engineering Volume 4, Number 11, November 2010 (Serial Number 36)

Contents Analytical Chemistry 1 7

16

Contents of Selenium (IV) in Rice Samples after Oxidizing Acid Digestion Auyporn Vongkul1 and Saravut Dejmanee Intervarietal Variation in Proximate Composition and Antioxidant Potential of Dry Peas (Pisum sativum L) Majeed Mudasir, Chatha Shahzad-Ali-Shahid, Hussain Abdullah-Ijaz, ASI Muhammad-Rafique, Mahboob Shahid and Yousaf Muhammad Magnetoelectrochemical Chirality in Ag Electrodeposition Iwao Mogi and Kazuo Watanabe

Biochemistry 23 27

Production of Amylase Enzyme through Solid State Fermentation by Aspergillus niger Sharifah Soplah Syed Abdullah, Zuriani Randeran and Mohd Azizan Mohd Noor Production and Recovery of Rhamnolipids Using Sugar Cane Molasses as Carbon Source Ana Carmen Santos, Márcio Silva Bezerra, Heloize dos Santos Pereira, Everaldo Silvino dos Santos and Gorete Ribeiro de Macedo

Chemistry Engineering 34

44

Modeling and Controlling of an Active Power Filter Using Photovoltaic System with Reduced Energy Storage Capacitor Tsair-Fwu Lee, Horng-Yuan Wu, Chieh Lee and Yu Lee Waste Tire Rubber Particles using to Improve the Properties of Local Asphalt Concrete Abdulali Bashir Ahmed Ben Saleh

Inorganic Chemistry 49

Synthesis and Characterization of Oxy-Vanadium (IV) Complex of 4-(2, 4-dihydroxybenzaldimine) Antipyrine Ramadan M. El-mehdawi, Abdussallam N. Eldewik, Khaled M. Kreddan, Fathhia A. Treish, Mofida M. Ben Younes, Abtisam A. Aboushagour and Zinab A. Elkamoshi

Organic Chemistry 54

60

2-Nitrobenzofuran as Dienophile in Polar Diels-Alder Reaction: A Simple Dibenzofurans Synthesis Claudia Daniela Della Rosa, Juan Pablo Sanchez, María Nélida Kneeteman and Pedro Máximo Emilio Mancini The Synthesis of Pyridine 2,6-dicarboxylic Acid Using Microwave Irradiation Guofu Zhang, Qing Zhang, Hairui Zhang, Lin Bai, Helin Ye and Ling ling Liu

November 2010, Volume 4, No.11 (Serial No.36) Journal of Chemistry and Chemical Engineering, ISSN 1934-7375, USA

Contents of Selenium (IV) in Rice Samples after Oxidizing Acid Digestion Auyporn Vongkul1 and Saravut Dejmanee2 1. Faculty of Science and Technology, Rajamangala University of Technology Srivijaya, Thailand 2. Department of Chemistry, School of Science, Walailak University, Thailand Received: August 16, 2010 / Accepted: September 01, 2010 / Published: November 30, 2010. Abstract: Se (IV) contents in rice samples after oxidizing acid digestion were investigated by flow injection-hydride generation atomic absorption spectrometric method (FI-HG-AAS). The optimization was performed with varying the formation of gaseous hydrides and the specific pieces of instrumentation. The results of FI-HG-AAS showed good linear calibration curve at the working concentration between 0.10 and 2.00 µg/L (R2>0.999). For 7 replicate determinations of 0.75 µg/L selenium standard solution spiked in deionized water, the precision and accuracy were 2.33% and 86.63%, respectively. The detection limit (3σ/S, n=7) was 0.01 µg/L. Oxidizing acid digestions of rice samples were carried out at the ratio of 5:3 between nitric acid and perchloric acid. This method was succeeded to determine Se (IV) in acid digestion of rice samples without pre-reduction step with hydrochloric acid and heating.

Key words: Selenium, FI-HG-AAS, rice.

1. Introduction Selenium (Se) element is classified as an essential micronutrient for humans at trace level, however Se at high level is toxic to human beings. The dual Se activities as an essential or toxic element are in very narrow range between deficiency and toxic effects, which is required a minimum level at only 0.10 mg/L and a maximum at over 4.00 mg/L [1]. Meanwhile, the recommended dietary allowance-the average daily dietary intake level that is sufficient to meet the nutrient requirements of nearly all (97-98%) individuals in each life-stage and gender group-of selenium is 55 µg/day [2]. In many countries, most (> 80%) dietary selenium come from grain food, such as bread and cereals [3]. Rice is a principal food of people in Thailand that plays important role in human nutrition. In previous study, selenium was found in rice sample in Asia at 13.00 µg/L (Japan) [3], 14.00-226.00 µg/L Corresponding author: Saravut Dejmanee, Ph.D., research fields: analytic chemistry and environment chemistry. E-mail:[email protected].

(China) [4], 50.00 µg/L (Korea) [5] and 29.00-65.00 µg/L (Thailand) [6]. Selenium compounds in plants are predominantly found as selenomethionine and many other selenocompounds. Normally in plants, they were identified in many forms as selenocystien, selenocystathionine, selenohomocysteine, selenate: Se (VI) and selenite: Se (IV) [7]. Various analytical methods used in determination of total Se contents in biological materials assume that the complete destruction of the organic constituents was performed by digestion procedures. However, acidic resistance of organoselenium compounds, such as selenomethionine, selenocysteine and trimethylselenonium ion, demand agents with high oxidation potential for complete destruction. Furthermore, HNO3 alone or in a mixture with H2SO4 is insufficient to decompose materials with high fat content. And, selenium readily forms volatile species and can be lost during an uncontrolled decomposition, for example by charring. In HNO3/HClO4 or HNO3/HClO3/HClO4 enable

2

Contents of Selenium (IV) in Rice Samples after Oxidizing Acid Digestion

complete destruction of biological materials at temperatures < 250 °C without losing Se. However HClO4 must be handled with greatest care and following strict regulations; special equipment is necessary, such as special hoods and scrupulous precautions have to be taken [8, 9]. Se in food samples have mostly been determined at trace level using atomic absorption spectrometric techniques. Electrothermal atomic absorption spectrometry (ETAAS) and hydride generation-atomic absorption spectrometry (HG-AAS) are suitable and widely used techniques for the determination of this element at trace level in food samples because of its selectivity and high sensitivity [10-12]. While HG-AAS can be widely used to determine the speciation of selenium [13, 14], which are suitable for only quantitation of Se (IV) species in this work. The analyte is removed from sample matrix [15] with depending on chemical reaction. Flow injection (FI) coupled to HGAAS has several advantages over batch system. The FI-HG system is the use of low sample volumes, to make repeated determinations, high sampling frequency, low reagent consumer and the automation. The great advantage of FI-HG-AAS is the most sensitive, selective, precise and fast method [11, 15, 16]. In this work, the primary study was determined only Se (IV) species after oxidizing acid digestion (without pre-reduction step of Se (VI) to Se (IV). It is well known that only Se (IV) yields volatile hydrides [16]) that content in rice samples by using the FI-HGAAS as an analytical technique. The FI-HGAAS procedure was optimized for determination of Se (IV) species, varying the formation of gaseous hydrides and the instrumental parameters.

2. Experiment 2.1 Apparatus Rice samples were digested in Pyrex glassware using a fume hoods. The analytical instrumentation used for the determination of selenium by FI-HG-AAS was an

Analyst 800 instrument (Perkin–Elmer, Milan, Italy), controlled by a PC with WinLab 32 software. Selenium electrode discharge acts as radiation source (Perkin– Elmer, Milan, Italy). Hydrides were generated using a FIMS-100 apparatus (conventional 500 μL loop and plastic liquid separator, Perkin–Elmer, Milan, Italy). 2.2 Reagents Analytical grade nitric acid (Merck, 65% w/w) and perchloric acid (Merck, 70-72% w/w) were used in the digestion procedure. In the FI-HG-AAS analysis, 10% Hydrochloric acid (Merck) and freshly prepared solution of sodium tetrahydroborate (III) (Merck) were used as a carrier and reductant, respectively. The Se (IV) working solutions were prepared diluting a commercial standard Se (IV) solution for atomic spectroscopy (Merck, 1000 ppm) with 10% HCl, and then kept in polyethylene bottles. Argon HP grade was prepared from TIG, Thailand.

3. Procedure for Measurement 3.1 Sample Preparation The 10.00 mL (at the ratio 5 to 3) of nitric acid and perchloric acid, as oxidizing acid, were added to 1.00 g of dried rice sample that contained in the open flask and then heated on hot plate inside fumehood. Firstly, the flask was gently heated at low temperature while brown NO2 fumes evolved. Then, the heating was increased gradually until white fumes of perchloric acid appeared which completed digestion procedure with unless color solution of sample. Finally, the solution was made up volume to 25 mL with de-ionized water at ambient temperature for determination of Se (IV) by using FI-HG-AAS under optimum conditions without pre-reduction step with HCl and heating. 3.2 FI-HG-AAS Measurements The gaseous hydride was formed after mixing the reaction of the standard solution or the sample solution with NaBH4 in HCl acid solution by using the FIMS-100 apparatus. The optimal parameters were

3


investigated the affect for formation of gaseous hydrides and the specific pieces of instrumentation which were studied the operating conditions. And the analytical performances of the method were determined from optimal parameters. The selenium standard solutions were prepared with 10% HCl and then analyzed by FI-HG-AAS. In both atomic absorption spectrometer and FIAS 100 equipment, the systems were optimized to achieve maximum signal intensity and stability. Atomic absorption signals were measured on the basis of peak height. The final optimal instrumentation conditions are summarized in Table 1.

4. Results and Discussion 4.1 Optimization of the Formation of Gaseous Hydrides In order to obtain a high performance of analytical factors, the effect of different parameters affecting the selenium determination was optimized. There are a number of parameters that affect the formation of gaseous hydrides. This experiment focuses on the following parameters: the concentrations of acid media (carrier) and concentration of sodium borohydride (reductant). 4.1.1 Effect of the Concentrations of Acid Media (Carrier)

values, above 10%, show slightly decreasing of the signal due to the dilution effect of evolved hydrogen [16]. At the concentration of 6.00% and 8.00% HCl were given a similar sensitivity. Thus, the 6.00% HCl was Table 1 The optimal instrumentation parameters for determination of Se (IV) by FI-HG-AAS. Atomic absorption spectrometer setting: Conditions Studied range Wavelength (nm) 190.0–204.0 Slit width (nm) 0.7 Lamp current (mA) 200-310 Cell temperature (oC) 500–1,000 FIAS 100 flow injection program: Time (sec) Steps Studied Optimum range value Prefill 5-20 15 Fill 5-20 10 Inject 5-20 15

Optimum value 196.0 0.7 290.0 750.0 Pump speed(rpm)* 100 120 80

* controlled flow rate of carrier and reductant.

According to the Perkin-Elmer Hydride System Operation Manual, the HCl concentration should be equal to 10.00 % (v/v). The influence of hydrochloric acid concentration was studied. The concentration of HCl concentration, as a carrier for Se (ΙV) analysis (H2Se production), was set in the range of 2.00-10.00% (v/v) using a constant NaBH4 concentration of 0.20% (w/v). The results are shown in Fig. 1. And, the linear regression equations and coefficients of correlation of calibration lines for variation of HCl concentrations were shown in Table 2. Fig. 1 and Table 2 showed that the HCl concentrations had an effect on sensitivity (slope of calibration) of Se (IV) within this range. The variation of the sensitivity of Se (IV) was related to the HCl concentrations. However, the HCl concentrations upper

Fig. 1 Calibration curves of Se(IV) standard solutions by using variable concentrations of carrier (HCl) with 0.20%(w/v) reductant (NaBH4). Table 2 Linear regression equations and coefficients of correlation for variation of hydrochloric acid. Concentrations of HCl (%v/v) 2.00 4.00 6.00 8.00 10.00 12.00

Linear regression equations

R2

A = 0.0358 C + 0.0044 A = 0.0364 C + 0.0047 A = 0.0413 C + 0.0026 A = 0.0384 C + 0.0072 A = 0.0347 C + 0.0060 A = 0.0349 C + 0.0038

0.999 0.999 0.999 0.998 0.999 0.999

A is AAS peak height and C is concentration in ppb (µg/L).

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chosen as the concentration of carrier, due to show the maximum sensitivity. 4.1.2 Effect of the Concentrations of NaBH4 (Reductant) According to the Perkin–Elmer Hydride System Operation Manual, the NaBH4 concentration should be approximately 0.20% (w/v) stabilized with 0.05% NaOH. The NaBH4 concentrations were varied between 0.05 and 0.40% (w/v) using a constant HCl concentration of 6.00 % (w/v). The results are shown in Fig. 2. The linear regression equations and coefficients of correlation of calibration lines for variation of NaBH4 concentrations were shown in Table 3. In Fig. 2 and Table 3 show the NaBH4 concentration has an effect on sensitivity of Se (IV) within this range. The sensitivity rises with increasing of NaBH4 concentration, reaches a maximum between 0.20-0.30% and then drops slowly, presumably because of the generation of hydrogen exceeds considerably the rate of hydride formation [16]. The 0.20 and 0.30% NaBH4 were given a similar

Fig. 2 Calibration curves of Se(IV) standard solutions by using variable concentrations of reductant (NaBH4) with 6.00%(v/v) carrier reagent (HCl). Table 3 Linear regression equations and coefficients of correlation for variation of NaBH4. Concentrations of HCl (%v/v) 0.05% 0.10% 0.20% 0.30% 0.40%

Linear regression equations

R2

A = 0.0235 C + 0.0005 A = 0.0323 C + 0.0039 A = 0.0402 C + 0.0035 A = 0.0417 C + 0.0052 A = 0.0408 C + 0.0070

1.000 0.999 0.999 0.999 0.999

sensitivity. Thus the 0.30% NaBH4 was selected for further analysis, given the maximum sensitivity. Under the above optimum concentrations of carrier and reductant, possibility of hydride generation of Se(IV) from standard solutions and sample solutions were further examined. 4.2 Analytical Performances of the Method The linear calibration and peak profiles of Se (IV) are shown in Figs. 3 and 4, respectively. The 7 replicate determinations of 0.75 µg/L selenium standard solution,

Fig. 3 Calibration curve of Se(IV) standard solution.

the precision and accuracy were 2.67 and 2.33%, respectively, expressed as RSD and relative difference. The detection limit was estimated using the expression 3σ/S, where σ indicated the standard deviation of the response and S was the sensitivity obtained from the slope of the analytical calibration curve. The detection limit for the standard reference solution was found at 0.01 µg/L Se (IV) (n = 7).

4.3 Application for Determination of the Se(IV) Contents in Rice Samples In chosen analytical conditions, Se (IV) contents in oxidized acid digestion of rice samples without pre-reduction is successful determination. The results and some peak profile of samples are shown in Table 4 and Fig. 4, respectively.


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Fig. 4 Peak profile of Se(IV) standard solution and some rice samples. Table 4 Contents of Se(IV) after oxidizing acid digestion without pre-reduction. Se(IV) contents in digestion rice sample (µg/kg) R1 (Thai jasmine rice) 33.25 R2 (Thai jasmine rice) 27.26 R3 (Thai jasmine rice) 14.36 R4 (Sung-Yod) 18.44 R5 (Thai jasmine rice) 12.40 R6 (Leb Nok) 21.53 R7 ((Pathum Thani ) 21.42 R8 (Chai Nat) 13.27 R1 and R2: unpolished rice samples while R3-R8: polished rice samples. Samples

In this study, we successfully found Se (IV) in oxidizing acid digestion (5:3 HNO3-HClO4) of rice samples without pre-reduction step, in contrast with previous studies when Se(IV) was digested with HNO3-HClO4 (1:4 ratio) and no HCl was used, no peak was observed in the DPP polarogram, since it was oxidized to Se(VI), which is not electro active [17] and hydride active. It indicates that Se (IV) in oxidizing

The data in Table 4 show that Se (IV) contents in selected typical rice samples were 12.40 to 33.25 µg/kg. The higher Se concentration in unpolished rice (R1 and R2) is in agreement with what found in studies performed on Chinese cultivations, in which the selenium content in the rice bran was 2.58 times higher than in the milled rice [18] and the white rice hull appears significantly richer in total selenium than its flour, and this implies that the removal of the outer layer (rice hull) involves a loss of this important antioxidant element [19].

5. Conclusions The FI-HG-AAS method for Se (IV) determination after oxidizing acid digestion without pre-reduction is successful. But accurate determination method of selenium compound content in rice samples or other food stuff samples such as digestion and pre-reduction procedure will be studied in the further work.

acid digestion of rice samples which was oxidized to Se

Acknowledgment

(IV) or not. The rice samples from the process of

The authors would like to acknowledge the Thai Government Science and Technology Scholarship, Rajamangala University of Technology Srivijaya and Walailak University for supporting the experiments and the studies. The authors thank Miss Dongkamol

oxidizing acid digestion procedure still have Se(IV) which can not confirmed its incomplete process; oxidation to Se(IV). It should be investigated in further study.

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Songkonaka (M.Sc. Student), Mrs. Sudkamon Lasopha (Ph.D. candidate student) and Mrs. Sirirat Phaisansuthichol (Ph.D. candidate student) in Analytical group Walailak University, for supporting data, and collecting rice samples.

[11]

References

[12]

[1]

H.W. Lakin, Trace Elements in the Environment; Advances in Chemistry 123, American Chemical Society, Washington, 1973. [2] R. Abdulah, K. Miyazaki, M. Nakazawa, H. Koyama, Low Contribution of rice and vegetables to the daily intake of selenium in Japan, Int. J. Food Sci. Nuti. 56 (2005) 463-471. [3] V. Alexiu, L. Vladescu, Optimization of a chemical modifier in the determination of selenium by graphite furnace atomic absorption spectrometry and its application to wheat and wheat flour analysis, Anal. Sci. 21 (2005) 137-141. [4] L. Chen, F. Yang, J. Xu, Y. Hu, Q. Hu, Y. Zhang, G. Pan, Determination of selenium concentration of rice in China and effect of fertilization of selenite and selenate on selenium content of rice, J. Agric. Food Chem. 50 (2002) 5128-5130. [5] Y. Choi, J. Kim, H.S. Lee, C. Kim, I.K. Hwang, H.K. Park, C.H. Oh, Selenium content in representative Korean foods, J. Food Comp. Anal. 22 (2009) 117-122. [6] P.P. Sirichakwal, P. Puwastien, J. Polngam, R. Kongkachuichai, Selenium content of Thai foods, J. Food Comp. Anal. 18 (2005) 47-59. [7] P.D. Whanger, Selenocompounds in plants and animals and their biological significance, J. Am. Coll. Nutr. 21 (2002) 223-232. [8] M. Verlinden, On the acid decomposition of human blood and plasma for the determination of selenium, Talanta 29 (1982) 875-882. [9] V. Galgan, Determination of selenium in biological material by flow injection hydride generation atomic absorption spectroscopy, Doctoral thesis, Swedish University of Agricultural Sciences, 2007. [10] C.R. Rosa, M. Moraes, J.A.G. Neto, J.A. Nobrega, A.R.A. Nogueira, Effect of modifiers on thermal behavior of Se in

[13]

[14]

[15]

[16]

[17]

[18]

[19]

acid digestates and slurries of vegetables by graphite furnace atomic absorption spectrometry, Food Chem. 79 (2002) 517-523. M. Korenovska, Optimization of selenium determination in vegetables, fruits, and dairy products by flow injection hydride generation atomic absorption spectrometry, Chem. Pap. 57 (2003) 155-157. J.P. Diaz-Alarcón, M. Navarro-Alarcón, H.L.G. Serrana, C. Asensio-Drima, M.C. López-Martın, Determination and chemical speciation of selenium in farmlands from southeastern spain: relation to levels found in sugar cane, J. Agric. Food Chem. 44 (1996) 2423-2427. X. Dong, Y. Nakaguchi, K. Hiraki, Quantitative analysis of human hair for selenium(IV), selenium(VI) and total selenium by hydride-generation atomic absorption spectrometry, Anal. Sci. 13 (1997) 195-198. Y. Zhang, J.N. Moore, W.T. Frankenberger, Speciation of soluble selenium in agricultural drainage waters and aqueous soil-sediment extracts using hydride generation atomic absorption spectrometry, Environ. Sci. Technol. 33 (1999) 1652-1656. J. Salar-Amoli, T. Ali-Esfahani, J. Hassan, Effect of sample treatment on determination of arsenic (III) and arsenic (V) in aqueous and tissue samples by hydride generation atomic absorption spectrometry, J. Chemistry and Chemical Engineering 3 (2009) 49-53. J. Stripeikis, P. Costa, M. Tudino, O. Troccoli, Flow injection-hydride generation atomic absorption spectrometric determination of Se(VI) and Se(IV): utility of a conventionally heated water bath for the on-line reduction of Se(VI), Anal Chim Acta. 408 (2000) 191-197. G. Somer, A.N. Unlu, The effect of acid digestion on the recoveries of trace elements: recommended policies for the elimination of losses, Turk J. Chem. 30 (2006) 745-753. J.G. Zheng, J.Y. Wang, J.H. Jiang, A.H. Lin, Studies on six mineral element contents of rice bran and milled rice from three colour rice cultivars, CAN143:438932, AN 2005: 373400. M. Panigati, L. Falciola, P. Mussini, G. Beretta, R. Facino, Determination of selenium in italian rices by differential pulse cathodic stripping voltammetry, Food Chem. 105 (2007) 1091-1098.


Intervarietal Variation in Proximate Composition and Antioxidant Potential of Dry Peas (Pisum sativum L) Majeed Mudasir1, Chatha Shahzad-Ali-Shahid2, Hussain Abdullah-Ijaz1, ASI Muhammad-Rafique3, Mahboob Shahid4 and Yousaf Muhammad1 1. Department of Chemistry, GC University, Faisalabad 38000, Pakistan 2. Department of Industrial Chemistry, GC University, Faisalabad 38000, Pakistan 3. Nuclear Institute of Agriculture and Biology, Jhang Road, Faisalabad 38000, Pakistan 4. Department of Zoology, GC University, Faisalabad 38000, Pakistan

Received: July 12, 2010 / Accepted: August 04, 2010 / Published: November 30, 2010. Abstract: The present research work was carried out to investigate the proximate composition and antioxidant capacities of newly invented varieties of dry peas. The proximate composition in terms of moisture (7.00-8.50%), ash (3.85-4.97%), fiber (3.8-12.3%) and protein (17.3-18.8%) contents varied significantly with respect to various cultivars. Total phenolic contents (TPC), total flavonoids contents (TFC), percentage inhibition of peroxidation of linoleic acid system, reducing power and DPPH free radical scavenging capacity of different dry peas extracts were found in range of 0.651-0.684 g/100g of GAE, 0.021-0.041 g/100g of CE, 0.713-0.895, 40.051-84.608%, 18.097-24.591 mg/mL, respectively. The amount of TFC, and percent inhibition of per oxidation in linoleic system of different dry peas extracts varied significantly among various cultivars and solvents. From the results of present investigations it is reasonable to say that 80% methanolic extracts of dry peas have exhibited varying degree of antioxidant activity. Key words: Dry peas, proximate analysis, antioxidant activity, source of antioxidant.

1. Introduction In recent years, more attention has been given to discover substances having potential to prevent the process of lipid peroxidation. Some synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoulene (BHT), propyle gallate (PG) and tertiary butyl hydroquinone (TBHQ) often used in lipids and related food products to retard the oxidative deterioration have been shown to cause liver disorder and nutritional problems, especially, if ingested in excess amount [1-2]. In various countries restrictions are imposed on the use of such synthetic phenolic compounds due to their perceived carcinogenic potential [3-4]. There is an increasing interest for

Corresponding author: Chatha Shahzad-Ali-Shahid, M. Phil., research field: analytical chemistry. E-mail:chatha222@ gmail.com.

replacing synthetic antioxidant by those of safer and natural origin in the food industry and preventive medicine. Therefore research on natural antioxidant has gained momentum as they are considered to pose no health risk to consumer [5-6]. Plants, vegetables and fruits are rich source of natural phytochemicals with antioxidants properties. The best known plant based antioxidant compounds are tocopherols, carotenoids, vitamin C and different phenolics [7]. Soft fruits being rich in polyphenolics, flavonoids, anthocyanins, catechins and vitamin C are also an excellent source of numerous natural antioxidants. Total phenolics, flavonoids and vitamin C predominately found in pear and apple are claimed to inhibit cancer and tumor formation due to their oxy-radical scavenging ability [8]. Legume seeds occupy an important place in the human diet all over the world as they are a good source of protein,

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Intervaratial Variation in Proximate Composition and Antioxidant Potential of Dry Peas (Pisum sativum L)

polysaccharides and micronutrients (vitamins, trace minerals). Legumes are also a recognized as a rich source of a variety of non-nutrient bioactive compounds of medicinal interest [9]. Leafy green vegetables and beans are reported to contain phytochemicals like carotenoids, vitamin C and phenolics, having chemo-preventive and antioxidant properties [10]. Pea (Pisum sativum L.) is a crop of moderate and temperate regions. It has been recognized as one of the major food, grown in the different parts of world and consumed by humans principally as green immature seeds. They are good source of macronutrients like proteins, polysaccharides, lipids and micronutrients like vitamins, trace minerals and other bioactive and polyphenolic compounds whose concentrations vary depending from variety to variety [11]. Dry peas are also reported to contain starch with a low glycemic index [12]. Among micronutrients, peas have high contents of ascorbic acid, β-carotene, thiamine, riboflavin and some other antioxidants compound some of which are, so far, poorly characterized [13]. In view of beneficial uses of natural antioxidants in human health; still great demand exists to exploit additional plants based food as a source of natural antioxidants. Although significant amount of work has been carried out on the nutritional aspects of pea, relatively little is reported regarding the antioxidant potential of pea in Pakistan. Therefore, the present research work was designed to investigate the proximate composition and antioxidant activity of some newly invented varieties of dry peas using different antioxidant assays.

2. Experiment 2.1 Collection of Samples Samples of different cultivars; PF-400 (Approved), DP-05-06 (Line), DP-NO.267 (Approved), DP-05-03 (Line) of dried green peas (Pisum sativum L.) were collected from Department of Pulses, Ayub Agriculture Research Institute, Jhang road, Faisalabad, Pakistan. The collected samples were authenticated by

Dr. Muhammad Naeem, Assistant professor of Botany, G.C. University Faisalabad. 2.2 Pretreatment and Storage of Samples Samples of green peas were washed with tap water to remove dust particles, remaining water was removed using paper towel. Samples of green peas were air dried till constant weight was achieved. Dried samples of green peas were ground to semi powder using grinder (LG BL 999SP) and stored in air tight polythene bags. All the reagents and chemicals used in research work were purchased from E. Merck or Sigma-Aldrich. 2.3 Proximate Composition of Dry Peas The proximate composition of four different cultivars of dry peas regarding, moisture, ash, crude fiber and crude protein contents was determined according to the following standard methods [14]. 2.4 Preparation of Antioxidant Extracts The antioxidant fractions of powdered dry pea’s samples were extracted using two solvent systems; 100% methanol, (pure methanol) and 80 % methanol (methanol: water, 80: 20 v/v). In detail, 20 g of ground sample was taken in a conical flask followed by the addition of 200 mL solvent and extraction was executed on magnetic stirrer (Utech Products Inc. Albany NY 12203 USA) for 3 hours. The filtrate was separated from the solid residue by filtering through Whatman No. 1 filter paper. The solvent was removed under vacuum at 45 ℃, using a vacuum rotary evaporator (Eyela, N-N Series, Rikakikai Co. Ltd). Viscous extracts were dried on water bath and stored at -4 ℃ till further use. 2.5 Evaluation of Antioxidant Potential 2.5.1 Determination of Total Phenolics The total phenolic contents were determined following the Folin-Ciocalteu reagent method [15]. Briefly, 50 mg of dry mass of extracts was mixed with 0.5 mL of Folin-Ciocalteu reagent and 7.5 mL deionized water.


The mixture was kept at room temperature for 10 minutes, and then 1.5 mL of 20% sodium carbonate (w/v) was added. The mixture was heated on water bath at 40 ℃ for 20 minutes and then cooled in an ice bath. Finally absorbance at 755 nm was measured with spectrophotometer (Hitachi U-2001 model 121-0032 Japan). Amounts of total phenolic were calculated using a calibration curve for Gallic acid (10-100 ppm). The results were expressed as Gallic acid equivalents (GAE) per hundred grams of dry matter. All the experiments were carried out in three replicates and analyzed in triplicate. 2.5.2 Determination of Total Flavonoids (TF) Total flavonoids were determined by following the procedure of Dewanto et al. [16]. One milliliter of aqueous extract containing 0.01g/mL of dry matter was placed in a 10 mL volumetric flask, then 5mL of distilled water added followed by 0.3 mL of 5% NaNO2. After 5 min, 0.6 mL of 10% AICl3 was added. After another 5 min 2 mL of 1M NaOH was added and volume made up with distilled water. The solution was mixed and absorbance measured at 510 nm. TF amounts expressed as catechin equivalents per hundred grams of dry matter for triplicate samples analyzed in triplicate. 2.5.3 Determination of Reducing Power Reducing power of extract was determined according to the procedure of Yen et al. [17]. Equivalent volume of green peas extracts containing 8, 10, 12 mg of dry matter was mixed with 5.0 mL of sodium phosphate buffer (0.2 M, pH= 6.6) and 5.0 mL of 1.0 % potassium ferricyanide, the mixture was incubated at 50 ℃ for 20 minutes. Then 5.0 mL of 10% trichloroacetic acid was added and centrifuged at 980 rpm for 10 minutes at 5 ℃ in a refrigerated centrifuge (CHM-17, Kokusan Denki, and Tokyo, Japan). The upper layer of solution (5.0 mL) was diluted with 5.0 mL of distilled water and 1.0 mL of 0.1 % ferric chloride solution. Finally absorbance at 700 nm was measured with spectrophotometer (Hitachi U-2001 model 121-0032 Japan). Each triplicate sample was

9

analyzed in triplicate. 2.5.4 Determination of %age Inhibition of Peroxidation of Linoleic Acid Antioxidant potential of extracts was determined in terms of measurements of inhibition of peroxidation linoleic acid system following a previously reported method [18]. Extracts of each treatment were added to a solution mixture of linoleic acid (0.13 mL), 99.8% ethanol (10 mL) and 10 mL of 0.2 M. Sodium phosphate buffer having ( p H = 7) and mixture was diluted to 25 mL with distilled water. The degree of oxidation was measured following ammonium thiocyanate method [17]. With 10 mL of 75% ethanol, 0.2 mL of aqueous solution of 30% ammonium thiocyanate, 0.2 mL of sample solution and 0.2 mL of ferrous chloride (FeCI2) solution (20 mm in 3.5% HCI) being added sequentially. After 3 minutes of stirring, the absorbance values of mixtures measured with spectrophotometer (Hitachi U-2001 model 121-0032 Japan) at 500 nm were taken as peroxide contents. A control was performed with linoleic acid but without extracts. Synthetic antioxidants, butylated hydroxytoluene (BHT) (200 ppm) were used as positive control. The system was incubated at 40 ℃ and maximum peroxidation level was observed after 360 hours (15 days) in the control (blank). Percent inhibition of linoleic acid was calculated by the following formula: 100 – [(Increase in Asample at 360 h / Increase in Ablank at 360 h) × 100] Where Asample is absorbance of sample and Ablank is absorbance of blank. 2.5.5 DPPH Radical Scavenging Assay The antioxidant potential of the dry peas extracts was assessed by measuring their ability to scavenge 2, 2-dipheny1-1-picrylhydrazyl (DPPH) free radical. The DPPH assay was performed as described by Bozin et al. [19]. The samples (from 0.2 to 500 ug/mL) were mixed with 1 mL of 90 uM DPPH solution and filled up with 95% Methanol, to a final volume of 4 mL. The absorbance of the resulting sample solutions and the blank were recorded after 1 h at room temperature.

10


Sample with BHT was used as a positive control. For each sample, three replicated measurements were recorded. The disappearance of DPPH was studied at 515 nm using a spectrophotometer (Hitachi U-2001 model 121-0032 Japan). Scavenging of free radical DPPH in percent (%) was calculated in the following way. RS% = 100 - (Ablank - Asample) / Ablank Where, Ablank is the absorbance of the control reaction mixture excluding the test compounds, and Asample is the absorbance of the test compounds. IC50 values, which represented the concentration of extracts that caused 50% neutralization of DPPH radicals, were calculated from the plot of scavenging percentage against concentration.

3. Statistical Analysis The data are reported as mean ± SD (standard deviation) of triplicate sample analyzed in triplicate. The data obtained was statistically analyzed by ANOVA using Minitab Software.

4. Results and Discussion 4.1 Yield of Extracts The data regarding the percentage yield of extracts from different cultivars is presented in Table 1. It was observed that yield of extracts from different cultivars of dry pea's varied widely and found in the range of 4.34-5.29 and 7.03-8.45% with pure and 80% methanol solvent, respectively. Overall, the maximum yield was obtained from the cultivar DP-NO.267 Table 1 Yield of extracts (g/100g of dry weight) of different cultivars of (Pisum sativum L). b

Solvent system 100% Methanol 80% Methanol a PF-400 4.76 ± 0.19 b 8.13 ± 0.25b b a DP-05-06 5.29 ± 0.26 c 7.21 ± 0.28a b a DP-NO.267 4.99 ± 0.14 b 8.45 ± 0.42b c a DP-05-03 4.34 ± 0.17 a 7.03 ± 0.21b a Values are mean ± SD of three separate experiments. a Mean followed by different subscript letters in same column represent significant difference. b Mean followed by different superscript letters in same row represent significant difference. Cultivars a

(Approved) with 80% methanol and minimum from the cultivar DP-05-03 (Line) with pure methanol. The 80% methanol was found more efficient extracting solvent as compared to pure methanol. The efficient extraction in 80% methanol might be attributed to the polarity of solvent as the most of the plant based antioxidants are polar in nature. Therefore, the 80% methanol is generally employed for the extraction of antioxidants components from plant materials due to their polarity and good solubility with many antioxidant components [20, 21]. The statistical analysis showed that percentage yield of extracts was found to vary significantly (p < 0.05) among cultivars of dry peas and solvent systems. These differences in yield of extracts may be attributed to genetic modifications of cultivars and polarity of solvents. The findings of present work regarding the yield of extracts (7.03 - 8.45%) were found in close agreement with those reported for corncob extracts (19.5%) with 80% methanol as extracting solvent [22]. Some scientific reports also revealed that 80% methanol is more efficient extracting solvent for plant based antioxidants and related phytochemicals [23]. 4.2 Proximate Composition of Dry Peas The data regarding the percentage composition of different constituents (moisture, ash, neutral fiber and protein contents) from different cultivars of dry pea is presented in Table 2. These cultivars’ were found to contain 7.00 - 8.50% moisture contents, 3.85-4.97% ash contents, 3.812.3% fiber contents and 17.3-18.8% protein contents. The statistical analysis showed that the percentages of these constituents were found to vary significantly (p < 0.05) among cultivars of dry peas. These differences are seemed to be associated with genetic variations of different cultivars. The findings of our work regarding the proximate composition were found in comparable levels of moisture (6.21-8.98%), Ash (3.6%), fiber (8.0%) and protein contents (14.5- 28.5%) reported by Reichert and Kenzie [24] for some species of pea.


Table 2

11

Proximate composition of different cultivars of Pisum sativum La.

Constit-uent (%) Moisture contents

Cultivars PF-400

DP-05-06 a

7.00 ± 0.21

Ash contents

4.03 ± 0.12

Fiber contents

12.3 ± 0.4d

Protein contents

17.7 ± 0.5

a

ab

DP-NO.267

8.00 ± 0.32

b

3.85 ± 0.15

a

3.8 ± 0.1a 17.7 ± 0.9

8.40 ± 0.42

8.50 ± 0.25b

4.09 ± 0.20

a

4.97 ± 0.24b

8.5 ± 0.4b ab

DP-05-03

b

18.8 ± 0.7

10.2 ± 0.3c b

17.3 ± 0.5a

Values are mean ± SD of three separate experiments. 0.8

4.3 Total Phenolic Contents (TPC)

extracts from different cultivars of dry peas were found in the range of 0.651- 0.676 and 0.661 - 0.684 g /100 g of dry weight as GAE with pure and 80% methanol solvent systems, respectively. Overall, the highest TPC were found to be extracted from the cultivar DP-05-06 (Line) with 80% methanol, and that of minimum TPC from DP-NO.267 (Approved) cultivar with pure methanol. In the present investigation 80% methanol was found more efficient extracting solvent for plant based phenolic compounds as compared to 100% methanol. The statistical analysis revealed that TPC of all the investigated cultivars found in comparable amounts with non-significant (p > 0.05) differences among cultivars of dry peas and solvent systems. The efficient extraction in 80% methanol might be attributed to the polarity of solvent as the most of the plant based antioxidants are polar in nature. The present findings regarding TPC of various cultivars of dry peas using pure and 80% methanol solvent systems were found in close agreement with those (0.33-2.47 g/100 g) reported for the antioxidant activity of phenolic fractions of pea in 80% methanol (solvent: water, 80:20 v/v) [26].

0.6 TPC(g/100) GAE

Total phenolic contents (TPC) of dry peas extracts were determined by using Folin-Ciocalteu method due to its high sensitivity, lower interference and quickness to quantify the phenolics as compared to other competitive test [25]. The data regarding the TPC (g/100 g of dry weight as GAE) of extracts from different cultivars is presented in Fig. 1. It was observed that TPC of

0.7 0.5 0.4 0.3

M1 M2

0.2 0.1 0

PF-400 DP-05-06 DP- NO.267 DP-05- 03 Cultivers of Pisum sativum L Fig. 1 Total phenolic contents (g/100g DW GAE) of different extracts. M1: 100% Methanol (Pure methanol), M2: 80% Methanol (methanol: water, 80:20, v/v).

The scientific investigations reported in the literature revealed that the phenolic compounds contribute directly to overall antioxidant activities of plant extracts [27]. Although exact reaction of the reagent with reducing species is not known but it is considered that a complex is formed between phosphomolybdic tungstate and reducing species, phenolic ion, changing color from yellow to blue where absorbance at 755nm is measured [28]. 4.4 Total Flavonoid Contents (TFC) The data regarding the TFC (g/100 g of dry weight as CE) of extracts from different cultivars is presented in Fig. 2. It was observed that TFC of extracts from different cultivars of dry pea's varied widely and found in the range of 0.021- 0.027 and 0.031- 0.041 g /100 g of dry weight as CE with pure and 80% methanol solvent systems, respectively. Overall, the highest TFC was obtained from the cultivar DP-05-06 (Line) with 80%


0.045 0.04 0.035 0.03 0.025 0.02 0.015 0.01 0.005 0

M1 M2 BHT

M1 M2 100 90 80 70 60 50 40 30 20 10 0 PF-400

%age

TFC (g/ 100g) CE

12

Pf-400 DP-05-06 DP.NO-267 DP-05-03 Cultivars of Pisum sativum L Fig. 2 Total flavonoid contents (g/100g DW CE) of different extracts. M1: 100% Methanol (Pure methanol), M2: 80% Methanol (methanol: water, 80:20, v/v).

DP-05-06 DP-NO.267 DP-05-03 Cultivars of Pisum sativum L Fig. 3 %age Inhibition of peroxidation in Linoleic acid system of different extracts. M1: 100% Methanol (Pure methanol), M2: 80% Methanol (methanol: water, 80:20, v/v).

methanol, and that of minimum TFC from DP-NO.267 (Approved) cultivar with pure methanol. The 80% methanol was found more efficient extracting solvent for plant based flavonoid compounds as compared to

inhibition was achieved from the cultivar DP-05-03 (Line) using 80% methanol and that of minimum from the cultivar DP-05-06 (Line) extracted with pure methanol. The 80% methanol was found more efficient extracting solvent as compared to 100% methanol. The statistical analysis showed that variation in the percentage inhibition of peroxidation in linoleic acid system was found to be significant (p < 0.05) with respect to cultivars and solvent systems. The present findings regarding % inhibition of various cultivars of dry peas using pure and 80% methanol solvent systems were found to be comparable with synthetic antioxidant, BHT (89.49%). Our results were in good agreement with the finding of Kaur and Kapoor [29] who reported the percent inhibition of methanolic extracts of pea pod to be 84%. The antioxidant activity has also been assessed as ability to prevent the oxidation. Therefore, inhibition of linoleic acid oxidation was also used to assess the antioxidant activity of dry peas extracts. Therefore, the 80% methanol is generally employed for the extraction of antioxidants components from plant materials due to their polarity and good solubility with many antioxidative components [20-21].

pure methanol. The statistical analysis revealed that TFC of all the investigated cultivars found in varied amounts with significant (p < 0.05) differences among cultivars of dry peas and solvent systems. Our findings regarding TFC of different extracts of dry pea cultivars were found to representing the similar fashion of antioxidant activity as reported by Sultana et al. [22] for the bark extracts of various plants. 4.5 Peroxidation in Linoleic Acid System Antioxidant activity of different dry peas extracts was determined by inhibition of peroxidation in linoleic acid system by using thiocyanate method [17]. The data regarding the percentage inhibition of extracts from different cultivars is presented in Fig. 3. It was observed that % inhibition of extracts from different cultivars of dry pea's varied widely and found in the range of 40.051-61.380 % and 73.988-84.608% with pure and 80% methanol solvent systems, respectively. Linoleic acid is a polyunsaturated fatty acid, upon oxidation peroxides are formed which oxidize Fe2+ to Fe3+, the later forms complex with SCN-, concentration of which is determined spectrophotometrically by measuring absorbance at 500 nm). Overall, the highest %

4.6 DPPH Radical Scavenging Activity The data regarding the 50% inhibition (IC50 mg/mL) of extracts from different cultivars is presented in Fig. 4.


30

(IC50 mg/ml)

25 20

M1 M2 BHT

15 10 5 0

PF-400

DP-05-06 DP-NO-267 DP-05-03 Cultivares of Pisum sativum L Fig. 4 DPPH free radical scavenging activity (IC50 µg/mL) of different extracts. M1: 100% Methanol (Pure methanol), M2: 80% Methanol (methanol: water, 80:20, v/v).

Lower the IC50 value of extract, more effective it will be for the inhibition of DPPH. It was observed that IC50 value of extracts from different cultivars of dry pea's varied widely and found in the range of 18.09723.571% mg/mL and 19.541-24.591% mg/mL with pure and 80% methanol solvent systems, respectively. Overall, the highest scavenging activity was obtained from the cultivar DP-05-06 (Line) because its IC50 µg/mL value was low. And lowest scavenging activity was obtained from the cultivar DP-NO.267 (Approved) because its IC50 mg/mL value was very high. Pure methanol as extracting solvent was found more efficient as compared to 80% methanol. The statistical analysis showed that scavenging activity of all invested cultivars of peas was found to be significantly (p < 0.05) different among cultivars and solvent systems. BHT being pure synthetic antioxidant always shows better radical scavenging activity with less IC50 value (12.92 µg/ mL) as compared to crude solvent extracts [22]. DPPH is a very stable organic free radical with deep violet color which give absorption maxima at 515-528 nm, upon receiving proton from any hydrogen donor species mainly, phenolics loses this absorption, resulting in a visually noticeable color change from deep violet to yellow. As the concentration of phenolic compounds or degree of hydroxylation of the phenolic compounds increases DPPH scavenging activity increases, hence antioxidant activity increases. Because

13

this radical is very sensitive to active ingredients at low concentrations and can accommodate a large number of samples in a very short time, it has been extensively used for measuring radical scavenging activity of different plant extracts. In the DPPH assay, the ability of the examined extracts to act as donor of hydrogen atoms or electrons in transformation of DPPH into its reduced from DPPH-H was investigated. The free radical scavenging capacity of examined extracts increased in a concentration dependent manner. Our results are compareable with the findings of Sultana et al. [22] for the DPPH free radicals scavenging activity of barks extracts from four different trees (Azadirachta indica, Terminalia arjuna, Acacia nilotica, and Eugenia jambolana Lam.) to be 49- 87%. 4.7 Determination of Reducing Power The data regarding the reducing power of extracts from different cultivars of dry pea extracts is presented in Fig 5. The extracts exhibited a close dependent reducing power with in a concentration range 8-10 mg of extract /mL. It was observed that reducing power of extracts

Fig. 5 Reducing power, extracts from different cultivars of Pisum sativum L. PF-400 (M1): 100% Methanol (Pure methanol) PF-400 (M2): 80% Methanol (methanol: water, 80:20, v /v) DP-05-06 (M1):100% Methanol (Pure methanol) DP-05-06 (M2):80% Methanol (methanol: water, 80:20, v /v) DP-NO.267 (M1):100% Methanol (Pure methanol) DP-NO.267 (M2):80% Methanol (methanol:water, 80:20, v /v) DP-05-03 (M1):100% Methanol (Pure methanol) DP-05-03 (M2):80% Methanol (methanol: water, 80:20, v /v) BHT:Synthetic antioxidant (Positive control)

14


from different cultivars of dry peas varied to some extent and found in the range of (0.722-0.895) and (0.713-0.859 mg/mL) with pure and 80% methanol solvent systems, respectively. The highest reducing power was obtained from the cultivar PF-400 (Line) pure methanol and that of lowest from the cultivar DP-05-03 (Line) with 80% methanol. In this investigation the pure methanol proved to be the more efficient extracting solvent as compared to 80% methanol. Literature reports demonstrated that the measurement of reducing power is a significant

extraction, isolation and purification of bioactive constituents that might be utilized for the formulation of functional and medicinal foods.

Acknowledgments We would like to extend our special gratitude to Dr. Noor Ahmad, Director, Division of Pulses, Ayub Agriculture Research Institute, Faisalabad, Pakistan for providing the seed samples of newly invented varieties of dry peas.

References

reflection of antioxidant activity of the extracts. In this method Fe (III) is reduced to Fe (II), resulting change in color from yellow to bluish green and the intensity of color depends on the reducing power of the compound present in the reaction media. The scientists have revealed in their investigations that greater the intensity of the colors will reflect the greater reducing power and consequently, greater the antioxidant activity [30]. The data regarding the reducing power of pea’s extracts is not available in the literature, however, our findings showed the similar trend of antioxidant activity in terms of reducing power of peas extracts as reported by Anwar et al. [31] for seeds extracts from four cultivars of mungbean.

5. Conclusions From these investigations it can be concluded that the variations in the amount of TFC, and percent inhibition of peroxidation in linoleic acid system were found significant but non significant in the amount of TPC, DPPH radical scavenging capacity and reducing power among various cultivars of dry peas and extracting solvents. 80% methanol was found most efficient solvent for the extraction of antioxidant fractions from dry peas. By comparing the results of all the performed assays it is reasonable to say that 80% methanolic extracts of dry peas have exhibited varying degree of antioxidant activity. People should develop the habit to take dry pea in different food recipes. However, further research is recommended for the

[1]

G.O. Adegoke, V. Kumar, A.A. Gopala, G. Varadarj, K. Sambaiah, B.R. Lokesh, Antioxidants and lipid oxidation in foods, Journal of Food Science and Technology 35 (1998) 283-289. [2] H.Y. Chung, Development of natural antioxidants stable at frying temperature, Hanguk Sikpum Youguage Hkhoechi, 10 (1997) 564-573. [3] I. Razali, H. Norhaya, A.S. Norasaimah, Determination of antioxidants in palm oil products by HPLC, Elaeis 9 (199) 25-32. [4] S. Iqbal, S. Haleem, M. Akhtar, M. Zia-ul-Haq, A. Akbar, Efficiency of pomegranate peel extracts in stabilization of sunflower oil under accelerated conditions, Journal of Food Chemistry 41 (2008) 194-200. [5] N.K. Andrikopoulos, H. Brueschweilier, H. Felber, C. Taeschler, HPLC analysis of phenolic antioxidant, Tocopherols and Triglycerides 68 (1991) 359-364. [6] A. Moure, D. Franco, J. Sineiro, H. Domínguez, M.J. Nunez, J.M. Lema, Antioxidant activity of extracts from Gevuina avellana and Rosa rubiginosa defatted seeds, Journal of Food Res Chemistry 34 (2001) 103-109. [7] S.A.S. Chatha, F. Anwar, M.R. Asi, B. Sultana, Antioxidant potential of extracts from different agro wastes: stabilization of corn oil, Grasasyaceites 59 (2008) 205-217. [8] G. Black, D. Vila, Chromatographic analysis of some polyphenols of Iberian Thymus, Journal of Ethnopharmacol 24 (1992) 147-157. [9] J.W. Anderson, B.M. Smith, C.S. Washnock, Cardiovascular and renal benefits of dry bean and soybean intake, Journal of Clinics and Nutrition 70 (2002) 464-474. [10] T. Yoshida, T. Hatano, H. Ito, Chemistry and function of vegetable polyphenols with high molecular weights, Bio. Factors 13 (2000) 121-125. [11] M. Duenas, T. Hernandez, I. Estrella, Assessment of in vitro antioxidant capacity of the seed coat and the cotyledon of legumes in relation to their phenolic contents,

Intervaratial Variation in Proximate Composition and Antioxidant Potential of Dry Peas (Pisum sativum L) Journal of Food Chemistry 98 (2006) 95-103. [12] P.K. Foster, J.B. Miller, International tables of glycemic index, Am. J. Clin. Nat. 62 (1995) 871-893. [13] R. Alonso, E. Orue, F. Marzo, Effects of extraction and conventional processing methods on protein and antinutritional factor contents in pea seeds, Journal of Food Chemistry 63 (1998) 505-512. [14] Official Methods of Association of Official Analytical Chemists, 14th eds., AOAC, Arlington, VA Method, 28 (1984) 110. [15] A. Chaovanalikit, R.E. Wrolstad, Total anthocyanins and total phenolics of fresh and processed cherries and antioxidants properties, Journal of Food Science 69 (2004) 67-72. [16] V. Dewanto, X. Wu, K. Adom, R. Hailiu, Enzymes and proteins containing manganese and overview, Met. Ions Biology 37 (2002) 209-278. [17] G. Yen, E.D. Duh, C.E. Lister, Antioxidants of anthraquinones and anthrone, Journal of Food Chemistry 70 (2000) 307-315. [18] S. Iqbal, M.I. Bhanger, F. Anwar, Antioxidant properties and compounds of some commercially available varieties of rice in Pakistan, Journal of Food Chemistry 93 (2006) 317-328. [19] B. Bozin, N. Simin, G. Anackov, Characterization of the volatile composition of essential oil of some Lamiaceae species and the antimicrobial and antioxidant activities of the entire oils, Journal of Agriculture and Food Chemistry 54 (2006) 1822-1828. [20] P. Siddhuraju, K. Becker, Antioxidant properties of various solvent extracts of total phenolic constituents from three different agro climatic origins of drum stick tree (Moringa oleifera lam) leaves, Journal of Agricultural and Food Chemistry 51 (2003) 2144-2155. [21] K. Zhou, L. Yu, Total phenolic contents and antioxidant properties of commonly consumed vegetables grown in

15

Colorado, LWT 39 (2005) 155-1162. [22] B. Sultana, F. Anwar, R. Przybylski, Antioxidant activity of phenolic and components present in barks of Azadirachta indica, Terminalia arjuna, Acacia nilotica and Eugenia jambolana Lam trees, Food Chemistry 104 (2007) 1106-1114. [23] B. Hsu, I.M. Coupar, K. Nia, Antioxidant activity of hot water extracts from the fruits of the Doum plam, hyphened the baica, Journal of Food Chemistry 98 (2006) 317-328. [24] R.D. Reichert, S.L.M. Kenzie, Composition of pea (Pisum sativum L.) varying widely in protein content, Journal of [25]

[26]

[27]

[28]

[29]

[30]

[31]

Agricultural and Food Chemistry 30 (1982) 312-317. P.G. Waterman, S. Mole, Analysis of phenolic plants metabolites, Oxford, Blackwell Scientific Publication, 1994, pp. 116-133. R. Amarowicz, M. Karamac, S. Weidner, Antioxidant activity of phenolic fraction of pea (Pisum sativum L.) Czech, Food Science 19 (2001) 139-142. J.M. Awiki, L.W. Rooney, X. Wu, R.L. Prior, L. Cisenneros-Zevallos, Screening methods to measure antioxidant activity of sorghum (Sorghum bicolor) and Sorghum products, Journal of Food Science and Technology 51 (2003) 6657-6662. D. Huang, O. Boul, R. Prior, The chemistry behind antioxidant capacity assays, Journal of Agricultural and Food Chemistry 53 (2005) 1841-56. C. Kaur, C. Kapoor, Anti-oxidant activity and total phenolic content of some Asian vegetables, Int. Journal of Food Science and Technology 37 (2002) 153-161. Y. Zou, Y. Lu, D. Wei, Antioxidant activity of a flavonoid rich extract of Hypericum perforatum L. in vitro, Journal of Agricultural and Food Chemistry 52 (2004) 5032-5039. F. Anwar, S. Latif, B. Sultana, M. Ashraf, Chemical composition and antioxidant activity of seeds of different cultivars of mungbean, Journal of Food Science 72 (2007) 503-510.


Magnetoelectrochemical Chirality in Ag Electrodeposition Iwao Mogi and Kazuo Watanabe Institute for Materials Research, Tohoku University Katahira, Aoba-ku, Sendai 980-8577, Japan

Received: July 26, 2010 / Accepted: August 10, 2010 / Published: November 30, 2010. Abstract: Chiral behaviors of magnetoelectrodeposited Ag film electrodes were investigated for the electrochemical reactions of glucose and cysteine. The Ag films were electrodeposited under a magnetic field of 2 T perpendicular to the electrode surface at various potentials of –0.06~–0.4 V (vs Ag | Ag+). The current-time curves during the electrodeposition and the surface morphology of the films implied that the film growth was considerably affected by the micro-MHD (magnetohydrodynamic) effect. Such Ag films were employed as an electrode, and voltammograms of glucose and cysteine were examined. Chiral behavior was clearly observed as current difference between the enantiomers on the film electrodes prepared at electrodeposition potentials less than –0.1 V. Such electrochemical chirality disappeared after the oxidation and reduction of the surface monolayer, and this fact indicates that the chiral sites must be just on the film surface. Key words: Magnetoelectrodeposition, chirality, micro-MHD effect, silver film, glucose.

1. Introduction For the past two decades, the development of a liquid-He-free and cryocooled superconducting magnet promoted applications of high magnetic fields into various scientific fields [1], and magnetoelectrochemistry (electrochemistry under magnetic fields) is never exceptional. Magnetoelectrolysis allows control of convection of electrolytic solution, morphology and texture of electrodeposits, kinetics of electrode reactions, etc. [2, 3]. One of the most intriguing advantages of magnetoelectrolysis is to induce chirality in the electrodeposits [4, 5]. This is called as “magnetoelectro-chemical chirality”. When a magnetic field is imposed to an electrochemical cell, the Lorentz force acting on the faradaic current causes convection of the electrolytic solution (MHD effect) [2]. The Lorentz force leads to spiral structures in the two-dimensional growth of

Corresponding author: Iwao Mogi, Ph.D., assistant professor, research fields: magneto-science and electrochemistry. E-mail: [email protected].

magnetoelectrodeposited metals [6-9] and conducting polymers [10], and the three-dimensional helical structures in silicate membrane growth [11]. If the chiral structures can be formed in nanometer scales on the magnetoelectrodeposited (MED) films, such film surfaces would serve as an enantioselective catalyst. Aogaki [12] proposed the micro-MHD effect in electrodeposition under the magnetic fields parallel to the faradaic current and perpendicular to the electrode surfaces. In electrodeposition processes, non-equilibrium fluctuation produces a lot of humps on the deposit surfaces. The faradaic current around such humps is not parallel to the magnetic field, so that the Lorentz force could cause vortex-like convection in the local areas around the humps. Such local vortices could be expected to produce chiral surfaces on the MED films [13]. We performed the magnetoelectrodeposition of Ag films and employed them as an electrode. Such Ag film electrodes exhibited chiral recognition of the enantiomers of glucose and amino acids (cysteine and histidine) in the voltammograms of their oxidation

Magnetoelectrochemical Chirality in Ag Electrodeposition

17

reactions [5, 14]. Generally the surface morphology and electrochemical properties of electrodeposited films depends on the deposition potential. It is thus interesting to examine the potential dependence on the magnetoelectrochemical chirality of the Ag films. In this paper, we report the chiral electrode behavior and surface morphology of the MED Ag films prepared at various potentials. Here we also show the surface oxidation effects on the chiral electrode behavior and discuss a possible chiral site on the MED films.

2. Experiment All chemicals were reagent grade and were used as received. For electrochemical experiments, a conventional system with the following three electrodes was employed: a polycrystalline platinum disc working electrode with a diameter of 3 mm, a platinum plate counter electrode, a Ag|Ag+ (50 mM) reference electrode for the Ag electrodeposition or a Ag |AgCl| NaCl (3 M) reference electrode for the voltammetric measurements. A potentiostat (Princeton Applied Research model 263A) was used for all the electrochemical experiments. Fig. 1 shows a schematic of the magnetoelectrodeposition. The electrochemical cell was placed at the bore center in a cryocooled superconducting magnet (Sumitomo Heavy Industries Ltd.), which can produce magnetic fields of up to 5 T. An applied magnetic field B was perpendicular to the working electrode surface, and it was parallel (+B) or antiparallel (-B) to the faradaic current. The films prepared at +2 T or -2 T, for examples, are called the +2T-film or the -2T-film, respectively. The temperature within the magnet bore was controlled at 25 ºC by circulating thermo-regulated water. Ag electrodeposition was conducted in a 50 mM AgNO3 aqueous solution containing 0.1 M NaClO4 as a supporting electrolyte. Fig. 2 shows a linear sweep voltammogram of Ag+, and it shows that the reduction of Ag+ occurs at potentials less than -0.05 V. The current increase at potentials less than -0.4 V is ascribed

Fig. 1 Schematic of magnetoelectrodeposition in a superconducting magnet. Magnetic fields B are applied to parallel (+) or antiparallel (–) to faradaic currents, and they are perpendicular to the electrode surface.

Fig. 2 Voltammograms of 50 mM AgNO3 in a 0.1 M NaClO4 aqueous solution. The potential sweep rate was 10 mVxs–1.

to the hydrogen evolution reaction. The Ag films were formed on the Pt working electrode by the potentiostatic electrodeposition at various potentials of –0.06~–0.4 V by a passing charge of 0.2 C cm-2. The magnetoelectrodeposited (MED) films were used as an electrode, and their chiral properties were examined by measuring cyclic voltammograms (CVs) of the enantiomers of glucose and cysteine in a 0.1 M NaOH aqueous solution. In the case to examine the surface oxidation effect on the chiral properties of the MED film electrodes, the MED films underwent

18


pretreatment of a CV measurement (pre-CV) in a 0.1 M NaOH aqueous solution before the measurements of glucose or cysteine, and the surface oxidation levels were controlled by the potential sweep range.

3. Results and Discussion The magnetoelectrodeposition was conducted at the various potentials under a magnetic field of 2 T. Fig. 3 shows current-time curves during the Ag electrodeposition at –0.3 V in 0 and +2 T. It shows that the magnetic field enhances the electrodeposition currents, even though the applied field is parallel to the faradaic current. The MHD effect is the minimum in such a configuration, but it never disappears because the current is not parallel to the magnetic field at the electrode edge and the fluctuated deposit surface. The latter is the micro-MHD effect [12], as mentioned above. The current increase under the magnetic field was observed at all the deposition potentials from –0.06 V to –0.4 V. Fig. 4 shows the microphotographs of the surfaces of

electrodes, which were electrodeposited at potentials of -0.06, -0.2, -0.3 and -0.4 V in the absence of magnetic field. The figure shows that oxidation current increases with increasing the overpotential at the electrodeposition. The peak current on the film electrode prepared at

Fig. 3 Current-time curves during the Ag electrodeposition at –0.3 V (vs Ag | Ag+) in 0 T (solid line) and +2 T (dotted line). The electrodeposition was conducted up to a passing charge of 0.2 C cm-2 in a 50 mM AgNO3 aqueous solution containing 0.1 M NaClO4.

the Ag 0T-films (left) and +2T-films (right) electrodeposited at –0.06 V ((a), (b)), –0.2 V ((c), (d)) and –0.3 V ((e), (f)). Both films at –0.06 V consist of several crystals and a powder-like background. The crystals have relatively large sizes (~ 5 mm) with well-defined crystallographic forms. With increasing overpotential, the crystal size becomes small ((c)~(f); 1~2 mm), and the number of crystal increases considerably. Comparing the 0T- and 2T-films at–0.2 V ((c) and (d)), the number of crystal in the +2T-film is less than that in the 0T-film. This tendency is noticeable at –0.3 V ((e) and (f)). While the 0T-film is aggregates of the crystals, the crystals are island-like in the +2T-film. This morphological change could arise from the micro-MHD effect. A similar morphological change due to the micro-MHD effects was reported in the MED Ni-Al2O3 composite films [15] and magnetocorrosion of Zn single crystals [16]. Fig. 5 shows voltammograms of 20 mM L-glucose in a 0.1 M NaOH aqueous solution on the Ag 0T-film

Fig. 4 Microphotographs of the surfaces of the Ag films electrodeposited at (a) 0 T, –0.06 V; (b) +2 T, –0.06 V; (c) 0 T, –0.2 V; (d) +2 T, –0.2 V; (e) 0 T, –0.3 V; (f) +2 T, –0.3 V.


Fig. 5 Voltammograms of 20 mM L-glucose in a 0.1 M NaOH aqueous solution on the Ag 0T-film electrodes. The Ag films were electrodeposited at –0.06 V (solid line), –0.2 V (dashed line), –0.3 V (dotted line) and –0.4 V (dash-dotted line). The potential sweep rate is 10 mVxs–1.

–0.3 V is approximately 10 times larger than that at –0.06 V. Viewing these voltammograms together with the surface morphologies of the 0T-films (Figs. 4(a),

19

4(c) and 4(e)) indicates that the oxidation current of glucose increases with increasing number of crystal. This implies that the electrode reaction of glucose occurs on the individual crystals. Fig. 6 shows the chiral electrode behaviors of the +2T- and –2T-films for the enantiomers of glucose, and the dependence of the chiral behavior on the electrodeposition potentials. The voltammograms of glucose were measured on the +2T-film electrode, which were electrodeposited at (a) –0.06 V, (b) –0.2 V and (c) –0.3 V, and on the –2T-film electrode prepared at (d) –0.2 V. On the 0T-film electrode, both voltammograms of enantiomers were coincident each other [5]. On the +2T-film electrode at –0.2 V, the oxidation current of L-glucose is larger than that of D-glucose (Fig. 6(b)). On the contrary, the result is opposite for the–2T-film electrode. The oxidation current of D-glucose is larger than that of L-glucose (Fig. 6(d)). These results indicate that the magnetoelectrodeposition induces chirality

Fig. 6 Voltammograms of 20 mM L- and D-glucoses in a 0.1 M NaOH aqueous solution on the Ag film electrodes. The Ag films were prepared at (a) +2T, –0.06 V; (b) +2T, –0.2 V; (c) +2T, –0.3 V; (d) –2T, –0.2 V. The potential sweep rate is 10 mVxs–1.


20

in the Ag electrodeposited films and that the films possess the ability to recognize the molecular chirality of glucose. The fact that the chirality of the MED Ag film depends on the polarity of the magnetic field indicates that the origin of the chirality is the Lorentz force and that the micro-MHD effect induces the chiral structure on the electrodeposit surfaces. The +2T-film electrode prepared at–0.06 V shows small oxidation currents of glucose, and the voltammograms of both enantiomers are coincident each other (Fig. 6(a)). The chiral behavior of the voltammograms appeared in the films prepared at the potentials less than –0.1 V, and the current difference between the enantiomers increases with increasing overpotential (Figs. 6(b) and 6(c)). Viewing these voltammograms

together

with

the

surface

morphologies in Figs. 4(b), 4(d) and 4(f), the chiral behavior of the +2T-film becomes clearer with increasing number of crystals. These results imply that the chiral sites could be on the individual crystals. A similar result was observed in the electrochemical reaction of an amino acid of cysteine. Fig. 7 shows the CVs of 20 mM D- and L-cysteines in a 0.1 M NaOH aqueous solution on the Ag +2T-film electrode prepared at (a) –0.06 V and (b) –0.2 V. The oxidation reaction of cysteine occurs around a current peak at 0.15 V. While the +2T-film electrode prepared at –0.06 V shows no chiral behavior, the electrode prepared at –0.2 V exhibits clear current difference between the enantiomers. The crystal structure of silver is face-centered cubic (fcc), and generally such a highly symmetrical crystal has no chirality. However, Attard et al. [17] pointed out that there exist chiral kinks in the {643} surface of the fcc crystals such as Pt, Ag and Cu, and demonstrated that such a surface exhibits chiral electrode behaviors for the oxidation of D- and L-glucoses. Similarly, they proposed that corner kinks of crystals could be a chiral site [18]. These ideas might be crucial for considering the chirality of the MED Ag films. If the chiral sites exist on the surface of the individual

Fig. 7 Cyclic voltammograms of 20 mM L-and D-cysteines in a 0.1 M NaOH aqueous solution on the Ag +2T-film electrodes, which were prepared at (a) –0.06 V and (b) –0.2 V. The potential sweep rate is 10 mVxs–1.

crystals in the MED Ag films, the chiral behavior of the film electrodes would be sensitive to the surface oxidation. To examine this, we pretreated the MED Ag films as follows. The MED film electrodes underwent a CV measurement (pre-CV) in a 0.1 M NaOH aqueous solution without glucose and cysteine. The potential sweep range of the pre-CV was –0.30~0.23 V or –0.30 ~ 0.30 V. While the latter potential range includes the oxidation and reduction between Ag and Ag2O on the surface monolayer of the Ag crystals [19], the former range does not include them. Fig. 8 shows the voltammograms of the enantiomers of glucose after the pre-CV measurement with the potential ranges of (a) –0.30 ~ 0.23 V and (b) –0.30 ~ 0.30 V. In the case of Fig. 8(a), the current of the preCV is slight and the pre-CV curve does not have the


21

Fig. 8 Voltammograms of 20 mM L- and D-glucoses in a 0.1 M NaOH aqueous solution on the Ag +2T-film electrode. The pre-CVs were measured at (a) –0.30 ~ 0.23 V or (b) –0.30 ~ 0.30 V in a 0.1 M NaOH aqueous solution before the measurement of glucose. The potential sweep rate is 10 mVxs–1.

Fig. 9 Cyclic voltammograms of 20 mM L- and D-cysteines in a 0.1 M NaOH aqueous solution on the Ag +2T-film electrode. The pre-CVs were measured at (a) –0.30~ 0.23 V or (b) –0.30~ 0.30 V in a 0.1 M NaOH aqueous solution before the measurement of cysteine. The potential sweep rate is 10 mVxs–1.

oxidation peak of the surface monolayer. The oxidation currents of the enantiomers are clearly different in the voltammograms of glucose, and thus the chiral behavior of the MED film survives after this pre-CV treatment. On the contrary, the pre-CV curve clearly exhibits an oxidation peak at 0.26 V and a reduction peak at 0.16 V in Fig. 8 (b). These peaks correspond to the oxidation (Ag to Ag2O) and the reduction (Ag2O to Ag) of the surface monolayer of the Ag electrode [19]. After this pre-CV treatment, the voltammograms of the enantiomers of glucose are coincident each other and the chiral behavior disappears. A similar surface oxidation effect was observed for cysteine. Fig. 9 shows the CVs of the enantiomers of

cysteine after the pre-CV measurement with the potential ranges of (a)–0.30~0.23 V and (b) –0.30~0.30 V. While the CVs exhibit chiral behavior for the enantiomers after the pre-CV treatment without the oxidation of the surface monolayer, as seen in Fig. 9(a), the chiral behavior disappears after the pre-CV treatment with the oxidation and reduction of the surface monolayer in Fig. 9(b). These results in Figs. 8 and 9 mean that the chiral sites are wiped out by the oxidation and reduction of the surface monolayer of the MED Ag film, and this fact indicates that the chiral sites must be just on the surface of the MED films. As suggested by Attard et al. [17, 18], a possible chiral site is considered to be a kink, or a corner kink on the


22

surface of the Ag individual crystals. Visualization of such chiral sites is an intriguing issue and in progress by means of atomic force microscopy.

[6]

[7]

4. Conclusions We have demonstrated that the MED Ag film electrodes exhibit chiral behaviors for the oxidation of the enantiomers of glucose and cysteine, and such magnetoelectrochemical chirality depends on the electrodeposition potential. Considering the voltammetric behavior in connection with the surface morphology indicates that the electrochemical reactions of glucose and cysteine occur on the individual crystals with sizes of 1~2 mm on the film surfaces. The chiral sites were wiped out by the oxidation and reduction of the surface monolayer of the MED Ag films. These results imply that the chiral sites must be just on the surface of the individual crystals, and thus possible chiral sites are considered to be kinks or corner kinks on the crystal surfaces. The magnetoelectrochemical chirality would be useful for the preparation of chiral surfaces without any chiral chemical reagents.

Acknowledgment The magnetoelectrochemical experiments were performed in the High Field Laboratory for Superconducting Materials, IMR Tohoku University. The author wishes to thank Prof. R. Aogaki of Polytechnic University in Kanagawa, Japan, for illuminating discussion.

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

References [1] [2] [3] [4]

[5]

M. Yamaguchi, T. Tanimoto, Magneto-Science, Kodansha-Springer, Tokyo, 2006. T.Z. Fahidy, Magnetoelectrolysis, J. Appl. Electrochem. 13 (1983) 553. R.A. Tacken, L.J.J. Janssen, Applications of magnetoelectrolysis, J. Appl. Electrochem. 25 (1995) 1. I. Mogi, K. Watanabe, Chirality of magnetoelectropolymerized polyaniline electrodes, Jpn. J. Appl. Phys. 44 ( 2005) L199. I. Mogi, K. Watanabe, Chiral electrode behavior of magneto-electrodeposited silver films, ISIJ Int. 47 (2007) 585.

[17]

[18]

[19]

I. Mogi, S. Okubo, Y. Nakagawa, Dense radial growth of silver metal leaves in a high magnetic field, J. Phys. Soc Jpn. 60 (1991) 3200. I. Mogi, M. Kamiko, S. Okubo, Magnetic field effects on fractal morphology in electrochemical deposition, Physica B 211 (1995) 319. J.M.D. Coey, G. Hinds, M.E.G. Lyons, Magnetic-field effects on fractal electrodeposits, Europhys. Lett. 47 (1999) 267. V. Heresanu, S. Bodea, R. Ballou, P. Molho, Electrochemical growth of metal arborescences under magnetic field, Proc. Int. Symp, Magneto-Science, Yokohama, 2005, p.1. I. Mogi, M. Kamiko, Pattern formation in magnetoelectropolymerization of pyrrole, Electrochemistry 64 (1996) 842. W. Duan, S. Kitamura, I. Uechi, A. Katsuki, Y. Tanimoto, Three-dimensional morphological chirality induction using high magnetic fields in membrane tube prepared by a silicate garden reaction, J. Phys. Chem. B 109 (2005) 013445. R. Aogaki, Micro-MHD effect on electrodeposition in the vertical magnetic field, Magnetohydrodynamics 39 (2003) 453. R. Aogaki, R. Morimoto, A. Sugiyama, M. Asanuma, Origin of chirality in magnetoelectrodeposition-control of micro-MHD flows by rotation, in Proc. 6th Int. Conf. Electromagnetic Processing of Materials, Dresden, 2009, p. 439. I. Mogi, Chiral electrode behaviors of magnetoelectrodeposited Ag films for amino acids, ECS Trans. 13 (2008) 45. T. Yamada, S. Asai, Distribution control of dispersed particles in a film fabricated by composite plating method using a high magnetic field, J. Jpn. Ins. Met. 69 (2005) 257. K. Shinohara, K. Hashimoto, R. Aogaki, Magnetic field effect on copper corrosion in nitric acid, Chem. Lett. (2002) 778. G.A. Attard, C. Harris, E. Herrero, J. Feliu, The influence of anions and kink structure on the enantioselective electrooxidation of glucose, Faraday Disccus. 121 (2002) 253. G.A. Attard, A. Ahmadi, D.J. Jenkins, O.A. Hazzazi, P.B. Wells, K.G. Griffin, P. Johnston, J.E. Gillies, The characterization of supported platinum nanoparticles on carbon used for enantioselective hydrogeneration: a combined electrochemical-STM approach, Chem. Phys Chem. 4 (2003) 123. L.C. Nagle, A.J. Ahern, L.D. Burke, Some unusual features of the electrochemistry of silver in aqueous base, J. Solid State Electrochem. 6 (2002) 320.


Production of Amylase Enzyme through Solid State Fermentation by Aspergillus niger Sharifah Soplah Syed Abdullah, Zuriani Randeran and Mohd Azizan Mohd Noor Department of BioEngineering Technology, University Kuala Lumpur-MICET, Alor Gajah 78000, Melaka ,Malaysia Received: May 26, 2010 / Accepted: June 08, 2010 / Published: November 30, 2010. Abstract: Traditionally, coconut dregs will be used as animal feed after the extraction of coconut oil and coconut milk from the copra. This study was carried out to discover the commercial value of coconut dregs as a solid substrate in the production of amylase through solid state fermentation (SSF) since this agro-waste is fairly rich in nutrients, providing the necessary nutrients supplementation for better microbial activity to produce enzymes. In this study, amylase is to be produced from coconut dregs by Aspergillus niger through solid state fermentation (SSF). Three parameters were covered, which are incubation time, initial moisture content of substrate and inoculum sizes. SSF was carried out by using incubator at 37 oC to test for enzyme activity at these following parameters: incubation time: 24, 48, 72, 96 and 120 hours; substrate moisture content: 64, 66, 68, 70 and 72% (w/w); inoculum sizes: 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mL spore suspension (5.5 × 106 spores/mL). Enzyme activities were measured through the estimation of liberated reducing sugars after the assay of amylase enzyme by using DNS (3, 5 dinitrosalicylic acid) method. Highest enzyme activities were obtained at these following parameters: incubation time: 72 hours (31.76 U/gds); initial moisture content of substrate: 66% (26.66 U / gds) and inoculum sizes: 2.0 mL (30.56 U/gds). Key words: Amylase, solid state fermentation, Aspergillus niger.

1. Introduction Amylases are enzymes that break down starch or glycogen by hydrolyzing starch molecules into vary shorter chains of polymer that consist of glucose structure. They are produced by a variety of living things ranging from microorganisms to plants and humans. Bacteria and fungi, which are common classes of microorganism that can produce amylase, excrete this enzyme outside their cells to carry out extracellular digestion [1]. After the insoluble starch has been broken down, the soluble end product such as glucose and maltose are absorbed into the cells. Solid state fermentation (SSF) provides an alternative to submerged fermentation (SmF) culturing for better control of water availability in the fermenting substrate. In SSF, Corresponding author: Syed Abdullah Sharifah Soplah, Ph.D., research fields: fermentation technology, enzyme technology, bioprocess engineering. E-mail: sharifahsoplah@micet. unikl.edu.my.

microbial growth and product synthesis occur at or near the surface of solid substrate particles which are low in moisture content [1]. Thus, it is always possible to optimize the water content and water activity (aw) of substrate to prevent an adverse effect on microbial activity since water has profound impact on the physico-chemical properties of solids and this, in turn will affect the overall process productivity. The use of agricultural wastes as the main substrate in solid state fermentation (SSF) to produce starch degrading amylase has gained researchers’ attention in carrying out studies to discover some potent agro-wastes and to study the optimum conditions in which they are capable in producing high yield of starch degrading amylase. Some of the agro-wastes that have been used for the cultivation of microorganisms included sugar cane bagasse, rice bran, banana waste, tea waste, cassava waste and oil cakes. The optimum conditions in which SSF is essential to be carried out involves

24

Production of Amylase Enzyme through Solid State Fermentation by Aspergillus niger

some important variables which are, the initial moisture content of substrate, inoculum size, incubation time and temperature, medium pH and nutrients supplementation. Thus, the objectives of this research was firstly to study the suitable conditions required for amylase production from coconut dregs (CD) by Aspergillus niger through SSF, and secondly, to discover the commercial value of coconut dregs as a solid substrate in the production of amylase through SSF.

2. Experiment 2.1 Organism and Culture Maintenance Aspergillus niger strain ATCC 1004 was collected from the culture collection of Bioprocess Laboratory, Department of BioEngineering Technology, Universiti Kuala Lumpur, Malaysia. It was maintained on potato dextrose agar slants and stored at 4 °C in a refrigerator. All the culture media, unless otherwise stated, were sterilized at 15-lb/in. [2] pressure (121 °C) for 15 min. 2.2 Substrate and Fermentation Coconut dregs (CD) were obtained from a stall in Pasar Tampin, Negeri Sembilan, Malaysia, whose owner used a press machine to obtain the coconut milk from the shredded coconut. The CD was then dried in the drying oven for 24 hours at 80 oC. Five grams of dried CD was weighed and put into a 250 mL conical flask before adding 2 mL of salt solution (containing w/v, 0.2% KH2PO4 , 0.5% (NH4)2NO3, 0.1% NaCl and 0.1% MgSO4.7H2O). Sterile distilled water was added to adjust the required moisture level. The contents of the flask were mixed thoroughly and were autoclaved at 121 oC for 15 minutes. The fermentation was initiated by the inoculation of 5.5 × 106 spores/mL per flask. SSF was carried out at 37 oC with substrate initial moisture content of 66% (w/w) by using 2 mL spore suspension for 24, 48, 72, 96 and 120 hours. This initial test was carried out to find the optimum hours required for production of maximum yield of the enzyme. The test was further proceeding by adjusting the initial moisture content of substrate. The moisture content

that was tested were 64, 66, 68, 70 and 72% (w/w). After the optimum incubation time and substrate initial moisture content had been identified, the test was once again been carried out to test for optimum inoculum size that is required to produce the highest production of amylase. The inoculum sizes that were tested were 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mL. 2.3 Analysis The enzyme produced through the fermentation of CD was extracted by adding 150 mL of distilled water into the fermented matter and shaken at 180 rpm for one hour. The fermented matter were then filtered and the filtrate obtained was assigned as the crude enzyme extract. Enzyme activities were measured through the estimation of liberated reducing sugars after the assay of amylase enzyme by using DNS (3, 5 dinitrosalicylic acid) method. The absorbance of the mixture was read at 575 nm by using Spectroquant 400. One unit (IU) of amylase was defined as the amount of enzyme releasing one μmol glucose equivalent per minute under the assay conditions.

3. Results and Discussion 3.1 Effect of Incubation Time SSF was first carried out by using 2 mL spore suspension (5.5 × 106 spores / mL) onto 5 g of CD with initial moisture content adjusted to 66% (w/w) at 37 oC. The enzyme activities were measured through the liberated reducing sugars estimated by DNS method and the unknown concentration of glucose was determined from the equation obtained from glucose standard curve. Fig. 1 presents the effect of incubation time on enzyme production. As incubation time increased from 24 hours, the concentration of liberated reducing sugar also increased which indicates an increase in enzyme activities. Enzyme activity is at maximum, of 31.76 U/gds at 72 hours of incubation. Enzyme activity was then decreased when the incubation time was prolonged to 96 hours and 120 hours. This might be mainly

25


appearance of spores in flasks with 68% moisture. Besides having less significant cells growth, the substrate with 68% moisture tend to be more sticky and wetter which may not be suitable for crucial growth of cells.

30 25 20 15

3.3 Effect of Inoculum Size

5 0 0

20

40

60

80

100

120

140

Incubation time (hours)

Fig. 1 Effect of incubation time on enzyme activity.

due to the denaturation of enzyme which is caused by the interaction with other components in the medium. The maximum enzyme activity could also be due to the fact that the microorganism was on its exponential phase during the third day of fermentation and resulting in maximum production of amylase. As incubation period was extended beyond 72 hours, this may cause depletion in nutrients availability and thus, the microorganism reached its stationary phase and could also have started producing secondary metabolites which resulted in lower yield of enzyme [3]. 3.2 Effect of Initial Moisture Content of Substrate To find the optimum moisture content of substrate that is crucial for mycelium growth and high enzyme activity, SSF was carried out for 72 hours at temperature 37 oC by using 2 mL spore suspension (5.5 × 106 spores/mL). Two flasks were incubated for each percentage of moisture content to find the average value of liberated reducing sugar obtained from the assay of amylase by using DNS method. Fig. 2 shows how initial moisture content of substrate affecting enzyme activity. For initial moisture content of substrate, this experiment recorded the highest activity of enzyme when the moisture content of substrate was adjusted to 66% w/w (26.66 U/gds). By observation, conical flasks containing substrate with 66% moisture possessed cells growth which was more significant than that possessed by substrate of 68% moisture. This was indicated by clear black spores that can be seen as compared to white

To test for the optimum inoculum size required for maximum enzyme activity, SSF was carried out with initial substrate moisture content adjusted to 66% at temperature 37 oC. Two flasks containing substrate with specified amount (mililiter) of inoculum were incubated. Fig. 3 presents the effect of inoculum size on enzyme activity. From the results obtained, it was shown that inoculum size of 2.0 mL produced the highest enzyme activity with an activity 30.56 U/gds. At inoculum size of 0.5 mL and 1.0 mL, it was seen that there exist only a slight difference in enzyme activity. This might be due to the fact that smaller number of cells present per mL as compared to other inoculum sizes required longer 30 Enzym e activity (U/gds)

10

25 20 15 10 5 0 62

64

66

68

70

72

74

Substrate moisture content (%)

Fig. 2 Effect of substrate moisture content on enzyme activity. 35 Enzyme activity (U/gds)

E n z ym e activity (U /g d s)

35

30 25 20 15 10 5 0 0

0.5

1

1.5

2

2.5

3

Inoculum size (mL)

Fig. 3 Effect of inoculum size on enzyme activity.

3.5

26


time to multiply to sufficient number in order to utilize the substrate in order to produce amylase enzyme [4]. There was a gradual increase in enzyme activity when the inoculum size was increased to 2.0 mL spore. However, the activity of enzyme decreased as the inoculum size was increased to 2.5 mL and 3.0 mL. An increase in the number of spores in inoculum will provide competition among cells to utilize the available nutrients for microbial activity. The limitation of nutrients reduced the activity of microbe and hence, reducing the synthesis of enzyme.

were proved experimentally to produce maximum enzyme activity, proving coconut dregs a promising substrate for its production.

References [1]

[2]

[3]

4. Conclusions These studies showed that coconut dregs could be a good substrate for α-amylase synthesis by fungal culture of A. niger. Evidently the substrate provided necessary nutrients for the microorganism to grow and synthesize the enzyme. Incubation period for 72 hours at 66% moisture content and 2 mL of inoculum size

[4]

A. Pandey, P. Selvakumar, C.R. Soccol, P. Nigam, Solid state fermentation for the production of industrial enzymes, Current Science 77 (1999) 149-162. S. Ramachandran, A.K. Patel, K.M. Nampoothiri, F. Francis, V. Nagy, G. Szakas, A. Pandey, Coconut oil cake -a potential raw material for the production of α-amylase, Bioresource Technology 93 (2004a) 169-174. S. Ramachandran, A.K. Patel, K.M. Nampoothiri, S. Chandran, G. Szakacs, C.R. Soccol, A. Pandey, Alpha amylase from a fungal culture grown on oil cakes and its properties, Brazilian Archives of Biology and Technology 47 (2004b) 309-317. E.A. Abu, S.A. Ado, D.B. James, Raw starch degrading amylase production by mixed culture of Aspergillus niger and Saccharomyces cerevisae grown on sorghum pomace, African Journal of Biotechnology 4 (2005) 785-790.


Production and Recovery of Rhamnolipids Using Sugar Cane Molasses as Carbon Source Ana Carmen Santos, Márcio Silva Bezerra, Heloize dos Santos Pereira, Everaldo Silvino dos Santos and Gorete Ribeiro de Macedo Federal University of Rio Grande do Norte, Center of Technology, Chemical Engineering Department, Biochemical Engineering Laboratory, Campus Universitário, Lagoa Nova, 3000, CEP:59072-970 Natal, Rio Grande do Norte, Brazil

Received: July 10, 2010 / Accepted: July 17, 2010 / Published: November 30, 2010. Abstract: Biosurfactants were synthesized by Pseudomonas aeruginosa (P.A.), using sugar cane molasses as carbon source. Assays were conducted in a shaker with agitation speed of 200 rpm, temperature of 38 °C and aeration ratio (Vm/Vf) of 0.4 and 0.6. A concentration of 3.0% was used for the carbon and energy source (molasses) and of 0.3% for the nitrogen source (NaNO3). Samples were removed at regular times until 96 hours of cultivation. The reduction in surface tension was measured using the ring method; cell concentration was obtained by the dry mass and substrate consumption by the DNS method. The metabolite produced was extracted and quantified by the thioglycolic method. The results showed a maximum surface tension reduction of 46.57% after 60 h, 3.63 g/L of biomass after 8 h (µXmáx =0.15 h-1), 79.60% of substrate consumption (µs= 0.67 h-1) and 4.47 g/L of rhamnolipid (µp=0.029 h-1). Key word: Biosurfactants, molasses, rhamnolipids, Pseudomonas aeruginosa.

Nomenclature µx - specific growth rate (h-1) µs - substrate consumption (h-1) µp - product formation (h-1) YX/S - conversion factors the substrate-cell (gram cells/gram substrate) YP/S - conversion factors substrate-product (gram product/gram substrate) Vm - volume of the medium (mL) Vf - volume of the flask (mL)

1. Introduction The properties of biosurfactants allow their use in a large number of industrial processes, including the food, pharmaceutical, textile, paper, polymer, cosmetic and petrochemical industries [1], efficiently substituteing chemically synthesized surfactants [2, 3]. Natural surfactants or biosurfactants have several Corresponding author: Ana Carmen Santos, Master, research field: biochemical engineering. E-mail:[email protected]

advantages over surfactants of synthetic origin, such as, low toxicity, high biodegradability, high foaming power, elevated selectivity, greater tolerance to high temperatures, pH and salinity, and the metabolite can be synthesized from renewable sources [4]. However, the high cost of biosurfactant synthesis has also posed a problem. The choice of low-cost renewable source material may reduce the cost of biosurfactant production by up to 50% [5, 6]. Several renewable substrates obtained from agri-industrial residue have been applied successfully in experimental biosurfactant production [5]. In general, these substrates contain high levels of carbohydrates or lipids that supply the need for a carbon source for production of this bioproduct [7]. Olive oil mill wastewaters as well as those containing animal fat, frying oil, soapstock, molasses, milk serum and other starch-rich residues [2, 8, 9] have been used as a substrate for biosurfactant production. Manioc residue [7] and cashew apple residue [10] have also

28

Production and Recovery of Rhamnolipids Using Sugar Cane Molasses as Carbon Source

been used, in addition to other residues such as bran and wheat and rice stalk, soybean, corn and rice hull; sugar cane and cassava bagasse, coffee pulp; fruit processing industry residues such as apple and grape pulp, pineapple and carrot wastes, banana wastes; coconut, soy, peanut, canola and cottonseed oil wastes [11-13]. The main classes of biosurfactants are the glycolipids, lipopeptides, lipoproteins, phospholipids, fatty acids, polymer surfactants and particulate surfactants [14]. Some authors prefer to group biosurfactants according to their molecular weight [15, 16]. Low molecular weight biosurfactants are generally glycolipids, the main example being the rhamnolipids. This class of biosurfactants is produced by several species of Pseudomonas sp., especially by P. aeruginosa [17, 18]. Rhamnolipids (Fig. 1) have a structure containing one or two rhamnose molecules, linked to one or two hydroxydodecanoic acid molecules [2]. Several factors influence the amount of rhamnolipid produced, including the type of microorganism

Co. (Germany). Sugar cane molasses (Estivas company, Brazil). PCA and Agar-Agar (Himedia Laboratories Pvt Ltd, India), 3,5 dinitro-salicilic acid P.A (VETEC Química Fina, Brazil), Sulfuric acid, 95-98% purity (CRQ, Cromato Produtos Químicos Ltda, Brazil), Petroleum Ether (Labsynth Produtos para Laboratório Ltda, Brazil), Thioglycolic acid (CRQ, Cromato Produtos Químicos Ltda, Brazil). All chemicals were of analytical grade and were used without further purification. 2.2 Microorganism We used Pseudomonas aeruginosa (AP029G-VII-A) isolated from oil wells and belonging to the culture collection of the Department of Antibiotics at the Universidade Federal de Pernambuco (UFPE). The culture was kept in solid medium at an inclined plane with PCA and 25% agar-agar. Cell renewal was performed using cell transfer in the medium and incubation at 38 ºC for 48 h. The culture was stored in a refrigerator at 5 ºC [21]. 2.3 Fermentation

bacterial or yeast, the carbon source used and the process strategy [19, 20]. Accordingly, the aim of this study was to synthesize biosurfactants using sugar cane molasses as carbon source under pre-established conditions of agitation, temperature and aeration ratio, as well as recover the metabolite produced.

2. Materials and Methods 2.1 Reagents Rhamnose (batch 075k0660) was acquired from Sigma

Fig. 1 Molecular structure of a rhamnolipid with two rhamnose molecules.

2.3.1 Inoculum The culture medium used in 1 liter was composed of: 1.0 g of NaNO3, 0.25 g of MgSO4.7H2O, 1 mL of stock salt solution (0.01g/100mL of EDTA, 0.3g/100mL of MnSO4.H2O, 0.01g/100mL of FeSO4.7H2O, 0.01g/ 100mL of CaCl2, 0.01g/100mL of CoCl2.6H2O and 0.01g/100mL of ZnSO4.7H2O) in pH 6.8-7.0 [21]. Phophorous source in the medium was that contained in the molasses. Erlenmeyer flasks (500 mL) containing 150 mL of culture medium were sealed with gauzecovered cotton wool plugs and sterilized by autoclaving at 121 ºC for 20 min. After the flasks were sterilized and cooled at ambient temperature, they were taken to the laminar flow chamber where loops of cells from the maintenance medium were aseptically transferred to the flasks containing the culture medium. The flasks were placed in a rotating incubator at 38 ºC for 10 h, under agitation of 200 rpm. This culture was used as inoculum in biosurfactant production assays [9].


2.3.2 Cultivation The cultivations were performed in a shaker, where in each assay 11 Erlemeyers (250 mL) were used with two different aeration ratios, 0.4 and 0.6 (volume of the medium over volume of the flask-Vm/Vf), fixed medium composition, molasses concentration of 3% and 0.3% of NaNO3. The flasks were then submitted to agitation of 100 rpm and temperature of 38 °C. In each cultivation 7.0% (v/v) of previously prepared inoculum was added. Samples were removed every 4 h in the first 12 h of cultivation and then every 12 h until 96 h of cultivation. 2.4 Analyses 2.4.1 Biomass 2.0 mL of fermented medium was placed in previously tared Eppendorff tubes and centrifuged for 15 min at 13.200 rpm. After the supernatant was removed, centrifugation was repeated twice, using distilled water to wash the biomass obtained. The tubes were placed in an oven at 80 °C for 24 h and then weighed. The samples were taken in duplicate and the mean value obtained was considered. 2.4.2 Surface Tension Surface tension was measured for the cell-free broth using a Du Noüy tensiometer and the ring method. The tension was applied as an indirect measure of biosurfactant production. 2.4.3 Determination of Substrate Concentration The substrate was quantified using the modified DNS method [22]. In this case it was carried out a previous hydrolysis to convert sucrose into glucose and fructose that were used in the standard curve. 2.4.4 Biosurfactant Extraction 25 mL of cell-free fermented broth was acidified at pH 2.0 with H2SO4 (2N) and refrigerated for 15 h. The medium was then centrifuged at 3,500 rpm for 20 minutes. 20 mL of the centrifuged broth was placed in a separation funnel where 20 mL of petroleum ether was added to extract the synthesized rhamnolipid. The funnel was vigorously agitated and after a 30 minute

29

interval for phase separation, the stage was repeated three times. In each period, the ether petroleum containing the rhamnolipid was stored in a beaker for subsequent biosurfactant quantification. 2.4.5 Rhamnolipid Quantification The biosurfactant produced was quantified by the thioglycolic method, expressed in rhamnose, according to Ref. [23]. The method consists of adding 4.5 mL of diluted sulfuric acid (6:1) to the cell-free broth, agitating vigorously for 1 minute in vortex. The mixture was then heated at 100 °C for 10 minutes and cooled at ambient temperature. Next, 0.1 mL of a recently prepared 3% thioglycolic acid solution was added to the mixture and agitated again in vortex for 1 minute. The mixture was kept in the dark for 3 h. Absorbance was measured at 400 and 430 nm in a Thermospectronic Genesys 10 UV Spectrophotometer. Rhamnose concentration was calculated by using the difference between the two wavelengths on a calibration curve, previously determined with commercial rhamnose. The difference was used because the absorbance value obtained on the reading at 430 nm indicates the interference of other sugars. The value obtained was multiplied by 3.4, according to Ref. [24].

3. Results and Discussion The use of sugar cane molasses, a complex substrate, as carbon source for biosurfactant synthesis, requires a balanced composition of nutrients for metabolite production to occur [24]. The obstacles lie in the natural variations of sugar cane composition and in the molasses production process. The main reasons for the generalized use of molasses as substrate is its reduced price compared to other carbon forms, in addition to its containing other compounds such as proteins and vitamins (B and C) that are important for fermentation [25]. Sugar cane molasses has high density and viscosity, requiring dilution to prevent the carbon source from acting as a microbial growth inhibitor [24]. The

30


molasses concentration used in the assays was fixed at 3.0% (m/v) and despite containing several nitrogenated compounds, phosphorous and traces of a number of salts, which help in synthesizing the desired metabolite, 0.3% (m/v) of NaNO3, a synthetic source of nitrogen, was added to obtain maximum rhamnolipid production. The aim of the assays was to study the kinetic parameters of cell growth, substrate growth and evaluate the synthesis of the biosurfactant produced by direct measurement, quantification using the thioglycolic method and an indirect measure using surface tension. Table 1 shows the operational conditions of these assays. Substrate consumption after the 96 h of cultivation was reduced from 21.94 to 5.21 g/L (76.24%) for assay 1 and from 20.64 to 4.20 g/L (79.60%) for assay 2, and is a source that is very assimilable by the microorganism. It was observed that the rate of substrate consumption (µs) was 59.7% higher when the smaller aeration ratio was used (Table 3). Raza [24], using molasses as carbon source and a P. aeruginosa mutant, obtained a 77% reduction in substrate consumption. Thus, the results obtained in the present study are compatible with the work of these authors. It

can be observed that the substrate conversion factor in rhamnolipids (YP/S) was 0.18 for assay 1 and 0.22 for assay 2. Santa Anna et al. [26] obtained a substrateproduct conversion factor of 0.13 (g of rhamnolipid/g of glycerol). The experiments were conducted in duplicate, varying only the influence of the aeration ratio for rhamnolipid synthesis. According to Kronemberger et al. [27], most biosurfactant producing microorganisms need aerobic conditions for efficient production, mainly when rhamnolipid biosurfactants are involved. Figs. 2 and 3 show the kinetic curves of these assays and Tables 2 and 3 present the parameters obtained. According to Figs. 2 and 3, the synthesized metabolite is not associated to an increase in biomass and is produced during the stationary phase of cell growth, characterizing secondary metabolism behavior, confirming previously published studies, such as those by Costa et al. [28], Santa Anna et al. [26] and Patel and Desai [29]. However, considering surface tension reduction as an indirect measure of biosurfactant production, it was found that after 12 h of cultivation, the surface tension of the broth decreased from 42.58

Table 1 Operational conditions established for the assays performed. Assays

Conditions Concentration of NaNO3 (%)

Concentration of molasses (%)

Agitation (rpm)

Aeration ratio

1

0.3

3.0

200

0.4

2

0.3

3.0

200

0.6

Fig. 2 Substrate consumption, cell growth, surface tension reduction and rhamnolipid concentration curves for conditions: 0.3%NaNO3, 3% molasses, agitation 200 rpm and aeration ratio 0.4.


31

Fig. 3 Substrate consumption, cell growth, surface tension reduction and rhamnolipid concentration curves for conditions: 0 .3%NaNO3, 3% molasses, agitation 200 rpm and aeration ratio 0.6. Table 2 Results obtained for substrate consumption, surface tension reduction, biomass concentration and rhamnolipid concentration. Assay

Conditions Substrate consumption (%)

Tension reduction (%)

Biomass (g/L)

Concentration of rhamnolipid (g/L)

1

76.24%

45.12%

3.18

3.35

2

79.60%

46.56%

3.63

4.47

mN/m to 23.48 mN/m in assay 1 and from 45.13 mN/m to 26.00 mN/m in assay 2, giving the false impression that biosurfactant synthesis is occurring in primary metabolism. In the present work the specific growth rate (µx) was not influenced by the aeration ratio. Table 2 shows the results obtained for substrate consumption after fermentation, surface tension reduction after 60 h of cultivation, biomass concentration and rhamnolipid concentration. According to Desai and Banat [2], surface tension during rhamnolipid production by P. aeruginosa can reach up to 29 mN/m. Thus, the results obtained in the present study indicate that the microorganism produces a biosurfactant with promising properties for application in the petroleum industry, given that surface tension was below 29 mN/m. The kinetic parameter values obtained for specific growth rate (µx), substrate consumption (µs) and product formation (µp), as well as the substrate-cell (YX/S) and

Table 3 Kinetic parameter results for the assays performed. Assay 1 2

µx (h-1) 0.14 0.15

Parameters kinetics µs (h-1) µp (h-1) YX/S 1.07 0.020 0.11 0.67 0.029 0.12

substrate-product (YP/S) presented in Table 3.

conversion

YP/S 0.18 0.22

factors

are

It can be seen that the increase in aeration ratio (Vm/Vf), from 0.4 to 0.6, had a slight influence on the increase in biosurfactant production, obtaining 3.35 (µp = 0.20 h-1) and 4.5 g/L (µp = 0.29 h-1) for assay 1 and 2, respectively, establishing an increase of 34.3%. Rhaman et al. [23], using soybean oil as carbon source, obtained 4.31 g/L of rhamnolipid with Pseudomona aeruginosa as microorganism. This slight increase in aeration ratio also caused a greater reduction in surface tension (45.13% for assay 1 and 46.56% for assay 2), similar to the value of

32


48.2% obtained by Santa Anna et al. [26], using glycerol as substrate and P. aeruginosa PA1.

[9]

4. Conclusions Based on the results obtained in this study, the aeration ratio, the process variable under study, was not significant in relation to the biosurfactant produced. The use of molasses as carbon source seems to be a potential substrate for rhamnolipid production, reaching nearly 4.5 g/L of biosurfactant under the study conditions, attaining a tension of 23.48 mN/m, corresponding to a reduction of 46.56% compared to the medium, when the higher aeration ratio was used.

[10]

Acknowledgments

[13]

The authors thank the National Council for Scientific and Technological Development (CNPq) and the Federal Agency for Support and Evaluation of Postgraduate Education (CAPES) for financial support.

References [1]

[2]

[3]

[4]

[5]

[6] [7]

[8]

D. Kitamoto, H. Isoda, T. Nakahara, Functions and Potential applicartions of glycolips biosurfactants from energy-saving materials to gene delivery carries, Journal of Bioscience and Bioengeneering 94 (2002) 187-201. J.D. Desai, I.M. Banat, Microbial production of surfactants and their commercial potencial, Microbiology and Molecular Biology Reviews 61 (1997) 47-64. M. Nitschke, G.M. Pastore, Biosurfactants: proprierties and applications, Revista Química Nova 25 (2002) 772-776. N. Kosaric, Biosurfactants and their application for soil bioremediation, Food Technology Biotechnology 39 (2001) 295-304. R.S. Makkar, S.S. Cameotra, Sintesis of biosurfactants in extreme conditions, Applied Microbiology and Biotechnology 50 (1999) 520-529. K.K. Gautam, V.K. Tyagi, Microbial surfactants: a review, Journal of Oleo Science 55 (2006) 155-166. M. Nitschke, C. Ferraz, G.M. Pastore, Selection of microorganism for biosurfactant producting using agroindustrial wastes, Brazilian Journal of Microbiology 35 (2004) 81-85. S. Maneerat, Production of biosurfactants using substrates from renewable-resources, Songklanakarin J. Sci. Technol. 27 (2005) 675-683.

[11]

[12]

[14] [15]

[16]

[17]

[18]

[19]

[20]

[21]

M.S. Bezerra, Investigation of important parameters for Scale-up of biosufactants using molasses as substrate. 101f, Master dissertation, Federal University of Rio Grande do Norte, Chemical Engineering Departament, Natal/RN, 2006. M.V.P. Rocha, S.C.L. Benedicto, G.A. Pinto, M.S. Bezerra, G.R. Macedo, L.R.B. Gonçalves, M.C.M. Sousa, Production of biosurfactant by Pseudomonas aeruginosa grown on cashew apple juice, Applied Biochemistry and Biotechnology 136 (2007) 185-194. R.S. Makkar, S.S. Cameotra, An update on use unconventional substrates for biosurfactants production and their applications, Applied Microbiology and Biotechnology 58 (2002) 428-434. S. Mukherjee, P. Das, R. Sem, Towards commercial production of microbial surfactants, Trends in Biotechnology 24 (2006) 509-515. J.M. Luna, L. Sarubbo, G.M.C. Takaki, A new biosurfactant produced by Candida glabrata UCP 1002: characteristics of stability and application in oil recovery, Brazilian Archives of Biology and Technology 52 (2009) 785-793. N. Kosaric, Biosurfactants in Industry, Pure & Appl. Chem. 64 (1992) 1731-1737. E.Z. Ron, E. Rosenberg, Biosurfactants and oil bioremediation, Current Opinion in Biotechnology 13 (2002) 249-252. H.B.Y. Bach, D. Gutnick, An exocellular protein from the oildegrading microbe Acinetobacter venetianus RAG-1 enhances the emulsifying activity of the polymeric bioemulsifier Emulsan, Applied and Environmental Microbiology 69 (2003) 2608-2615. G. Georgiou, S.C. Lin, M.M Sharma, Surface-active compounds from microorganism, Biotechnology 10 (1992) 60-65. A.B.J. Matsuura, Production and characterization of biosurfactants aiming industrial applications and bioremediation processes. 98f. PhD thesis, Faculty of Food Engineering. State University of Campinas, CampinasBrazil/SP, 2004. S. Lang, D. Wullbrandt, Rhamnose lipids-biosynthesis, microbial production and application potencial, Appl. Microbiol. Biotechnol. 51 (1999) 22-32. E. Déziel, F. Lépine, D. Dennie, D. Boismenu, O.A. Mamer, R. Villemur, Liquid chromatography/mass spectrometry analysis of mixtures of rhamnolipids produced by Pseudomonas aeruginosa strain 57RP grown on mannitol or naphthalene, Biochimica et Biophysica Acta 1440 (1999) 244-252. A.K.C. Lobato, Study of biosurfactants production by microorganisms isolated from oil wells.. 148f. Masters


[22]

[23]

[24]

[25]

dissertation, Federal University of Rio Grande do Norte, Chemical Engineering Departament, Natal/RN, 2003. J.B. Summers, The estimation of sugar in diabetic urine, using dinitrosalicylic acid, J. Biol. Chem. 62 (1924) 287-290. K.S.M. Rahman et al., Rhaminolipid biosurfactant production by strains of Pseudomonas aeruginosa using low-cost raw materials, American Chemical Society and American Institute of Chemical Engineers 18 (2002) 1277-1281. Z.A. Raza, M.S. Khan, Z.M. Khalid, Physicochemical and surface-active properties of biosurtactant produced using molasses by a Pseudomonas aeruginosas mutant, Journal of Environmental Science and Health Part A 42 (2007) 73-80. R.S. Makkar, S.C. Cameotra, Utilization of molasses for biosurfactant production by two Bacillus strains at thermophilic conditions, J. Am. Oil. Chem. Soc. 74 (1997)

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887-889. [26] L.M. Santa Anna, G.V. Sebastian, E.P. Menezes, T.L.M. Alves, A.S. Santos, J.N. Pereira, D.M. Freire, Production of biosurfactants from Pseudomonas aeruginosa PA1 isolated in oil environments, Brazilian Journal of Chemical Engineering 19 (2002) 159-166. [27] F.A. Kronemberger, L.M.M. Santa Anna, R.R. Menezes, A.C.L.B. Fernandes, C.P. Borges, D.M.G. Freire, Oxygen control on biosurfactants production in bioreactor, paper presented at XVI Sinaferm, 07-July to 01-August, Curitiba-PR, 2007. [28] S. Costa, M. Nitschke, R. Haddad, M.N. Eberlin, J. Contiero, Production of Pseudomonas aeruginosa LBI Rhamnolipids following growth of brazilian native oils, Process Biochemstry 41 (2006) 483-488. [29] R.M. Patel, A.J. Desai, Biosurfactant production by Pseudomonas aeruginosa GS3 from molasses, Letters in Applied Microbiology 25 (1997) 91-94.


Modeling and Controlling of an Active Power Filter Using Photovoltaic System with Reduced Energy Storage Capacitor Tsair-Fwu Lee1, Horng-Yuan Wu2, Chieh Lee3 and Yu Lee4 1. Department of Electronics Engineering, National Kaohsiung University of Applied Sciences, Kaohsiung 807, Taiwan 2. Electrical Engineering, National Kaohsiung University of Applied Sciences, Kaohsiung 807, Taiwan 3. Chemical and Materials Engineering, National Kaohsiung University of Applied Sciences, Kaohsiung 807, Taiwan 4. Department of Chemical and Materials Engineering, Tunghai University, Taichung City 40704, Taiwan Received: August 03, 2010 / Accepted: August 25, 2010 / Published: November 30, 2010. Abstract: A novel three-phase active power filter (APF) circuit with photovoltaic (PV) system to improve the quality of service and to reduce the capacity of energy storage capacitor is presented. The energy balance concept and sampling technique were used to simplify the calculation algorithm for the required utility source current and to control the voltage of the energy storage capacitor. The feasibility was verified by using the Pspice simulations and experiments. When the APF mode was used during non-operational period, not only the utilization rate, power factor and power quality could be improved, but also the capacity of energy storage capacitor could sparing. As the results, the advantages of the APF circuit are simplicity of control circuits, low cost, and good transient response. Key words: Active power filter, sampling, energy-storage capacitor, harmonic current, energy balance.

1. Introduction Power electronics circuits are widely used in many different types of industrial equipment, such as frequency changers, motor drive systems, etc. This equipment presents nonlinear impedance to the utility, generating large harmonic currents with well known negative effects, such as a low power factor, low efficiency and destruction of other equipment (e.g. the power capacitor can be damaged by the resonant over voltage etc. [1-2]). Also, some precision instruments and communication equipment will be interfered with the electromagnetic interference (EMI). Therefore, utility power quality has become an important issue recently. Many research papers and methods [3-5] Corresponding author: Tsair-Fwu Lee, Ph.D., research field: innovative algorithm applications. E-mail: [email protected]. tw.

have been proposed to solve these problems. Conventionally, a passive L-C filter was used to suppress the harmonics. Capacitors were used to compensate the lag power factor. However, they have many disadvantages, such as large size, resonance, and fixed compensation characteristics [6-7]. Therefore, conventional passive power filter can not provide a complete solution. Many specialists approach the solution from the viewpoint of preventing the generation of harmonics, such as high power factor switching power supply, frequency changer and uninterruptible power supply (UPS) etc. [8-12]. However, the present harmonic pollution sources still need to be improved. Some APF methods have been proposed to compensate for the present harmonic loads. They are in the type of parallel with the non-linear load to provide the

Modeling and Controlling of an Active Power Filter Using Photovoltaic System with Reduced Energy Storage Capacitor

reactive power and to compensate for the harmonic current, in order to achieve the goal of high quality of service of utility sources [13-19]. Due to the limitations of sunshine, the average operating time of a PV system is only 6-8 hours. Hence, the equipment of a PV system operates at a low utilization rate. If the system is transferred to the APF operation mode during non-operational moments, so that the utilization rate, power factor and power quality can be improved [20]. This paper proposes a new three-phase active power filter (APF) technique, based on the concept of energy balance. At the end of each period, we use a sampling technique to detect the energy deviation of the energy-storage capacitor and to calculate the peak value of utility command current at the end of each period. By employing a set of a three-phase sinusoidal reference voltage in phase with the utility source to multiply with this peak value, we can obtain the required utility source current. The difference between the instantaneous load current and this current is the command current for the APF. This command current includes reactive fundamental power and harmonics. The current is provided by a bilateral converter using a hysteresis current control technique [21-25]. Because the instantaneous fundamental reactive power of a balanced three-phase system is zero, the instantaneous fundamental reactive power transfers only among phases. In theory, the required capacity of the energy-storage capacitor is zero. But, harmonics power transfers between the load and the energy-storage capacitor (charging and discharging), hence we still

35

the energy balance concept, is proposed. As there is no delay element (such as LPF or PI control) used in this control circuit, and the transient response is fast and good.

2. Principles of Operation 2.1 System Description The basic circuit configuration of a utility interactive PV system is shown in Fig. 1. The battery bank of a DC bus is replaced by a capacity due to less power storage in the DC bus. The system configuration is the same as APF except for the DC converter stage. DC converter stage will be disabled and the system will be transferred to APF mode during non-operation duration. The Fig. 1(a) shows the utility interaction PV system operates on generation mode, and the Fig. 1(b) shows the system operates on APF mode. The fundamental building block of the threephase APF system is shown in Fig. 2. Under normal

Fig. 1

The block diagram of PV power supply system.

need to use a small capacitance for the energy-storage, and the average capacitor voltage can be maintained at a constant value. However, due to the losses in the converter such as switching loss, capacitor leakage current etc., the utility must provide not only the real power needed by the load but also these overhead required by converter to maintain the capacitor voltage at a prescribed value. In this paper, a new DC bus voltage controller, using

Fig. 2 The block diagram of the three-phase APF system.

36


circumstances, the utility can be assumed as a sinusoidal voltage source.

vs,k (t) = Vsm Ree

j (ωt −k

2π ) 3

(1)

k = -1, 0 and 1 If a nonlinear load is applied, then the load current will consist of the fundamental component and all the higher order harmonics. It can be represented as ∞

i l , k (t ) = ∑ I l , n Re e

j ( n (ωt − k

2π ) +θ n ) 3

,

Where k = -1, 0 and 1. Therefore, the three-load power can be expresses as 1

∑vs,k (t ) × il ,k (t )

k =−1

j ( 2ωt −k 3 1 1 = Vsm I l ,1 cosθ1 + ∑Vsm I l ,1 Re e 2 2 k =−1

+ Vsm

1

∞

∑ ∑ I l ,n

2π j (ωt −k ) 3 Re e

4π +θ1 ) 3

2.2 Calculation of Fundamental Component is,k(t) For a three-phase APF in a steady state, the energy transformations are shown in Fig. 3. The power consumption of the load is PL =

(2)

n =1

ptotal ,l (t ) =

Hence, the APF needs to calculate the current is,k(t) accurately and instantaneously as described in the following.

I

In Eq. (3), the first term is the real power supplied by the utility source, the second term is the reactive power, and the third term is the harmonic power. The last two terms are supplied by the APF. For a balanced three phase system, the second item is zero, i.e.

ptotal,l (t ) = Ps + ptotal,c (t )

(4)

3V I 3 Ps = VsmI l,1 cosθ1 = sm m 2 2

(5)

∞

∑Il,n Ree

j(ωt −k

2π 2π ) j(n(ωt −k )+θn ) 3 Re e 3

k =−1 n=2

(6)

wherek = -1,0, and 1 As the APF provides the harmonic power Ptotal,c, the current supplied by the utility will be

is ,k (t ) = I m Re e

j ( ωt − k

2π ) 3

sm

(7)

m

=

V

DC

R

2

⋅

L

2 Ps = I l ,1 cos θ1 3Vsm

(8)

The current is,k (t) is in phase with the utility voltage and is pure sinusoidal. Therefore, the APF must provide the compensation current.

ic,k (t) = il,k (t) − is,k (t)

m

(11)

2 3 V sm

(12)

In theory, the energy-storage capacitor does not need to provide real power to the load. Therefore, at the end of one complete utility cycle, the terminal voltage of storage energy capacitor of converter will keep no change. However, owing to the switching loss and conductive loss of the power converter, the utility must provide not only the real power needed by the load (i.e. the in phase pure sinusoidal current is, k (t)), but also the additional power required by the converter to maintain the capacitor voltage at the prescribed value. The change of energy-storage capacitor in one period will be 1 ΔEc = Cc (Vref 2 - Vc 2 ) (13) 2

Where Im =

I

2

where

where k = -1, 0, and 1

1

3V

Ps =

k =−1 n=2

ptotal,c (t) =Vsm ∑

(10)

This power will be provided by the utility source. Because the reactive power and harmonic power are supplied by APF, the utility only needs to supply the pure sinusoidal current and in phase with the utility voltage (i.e. pf=1.0). The power supplied by the utility is

(3)

2π j ( n (ωt −k )+θ n ) 3 Re e ,

VDC 2 RL

(9)

Fig. 3

The energy transfers in APF.


Where, Vref: the reference voltage of the energy-storage capacitor in steady state; Vc: the voltage of the energy-storage capacitor at the end of each utility period. If the energy loss of the capacitor can be compensated to the set value desired at the end of the next utility cycle by the extra utility current ΔImsinωt, then in this cycle the energy compensated by the utility will be ΔEc =

3V ΔI 3 T (Vsm sin ωt × ΔI m sinωt)dt = sm m × T (14) ∫ 0 T 2

Based on the concept of energy balance, the compensation energy from the utility must equal to the energy loss of the energy-storage capacitor, i.e. ΔEs =ΔEc. Therefore, in the next period the variation of the utility current is ΔI m =

Where

Cc (Vref 2 - Vc 2 ) 3VsmT

= K (Vref 2 - Vc 2 )

(15)

37

by K2 (K2 =K12×K) to get ΔIm. This current ΔIm adds to the previous command current IUP1 to obtain the steady-state utility command current of this coming period, IUP. At the same time, one more ΔIm adds to the steady-state utility command current IUP, to get Im (Im=IUP+ΔIm), and it will include the component to compensate the energy loss of the previous period. At the end of this coming period, the terminal voltage of energy-storage capacitor will return to Vref. These values are sampled at the end of each period by the sample and hold the circuit to keep the value until the end of next period. Hence, during each period, the change of energy-storage capacitor voltage does not affect the compensation characteristics of the APF. That means the capacitor can endure a much larger voltage ripple. As the results, we can reduce the capacity of this capacitor. 2.3 The Current Tracking Circuit

Cc K = 3 V sm T

(16)

Keep in mind, this energy-loss is caused by the increment of load. If the utility makes only this variation of current, ΔIm, it will just keep the capacitor

The block diagram of current tracking and control circuit are shown in Fig. 5. The voltage of phase A, v A (t ), is connected to a zero cross detecting circuit (ZCD) to get a synchronous reference square wave. It

voltage from not dropping again, and be unable to pull the capacitor voltage back to the reference level. For the capacitor voltage to return to its reference value Vref at the end of the next cycle, the utility must provide 2ΔIm current variation in the next cycle (one ΔIm is for the load increment and other ΔIm is for the compensation of energy loss in the previous cycle).

Fig. 4 The control block diagram of energy-storage capacitor voltage.

Fig. 4 shows the control block diagram of energy-storage capacitor voltage. The input is the terminal voltage of the capacitor. It follows an attenuation circuit (K1) to adjust this voltage to an appropriate level (Vc/K1). Then, it is connected to an isolation amplifier to isolate the main power circuit from the control circuit. The value of Vc2 is calculated by a square circuit. The other input is the reference voltage (Vref/K1), it also follows a square circuit to calculate Vref 2. The difference between (Vc/K1)2 and the set value (Vref /K1)2 is multiplied

Fig. 5 The block diagram of current tracking and control circuit.

38


will be used as the input of the phase lock loop (PLL). Using the PLL technique, we can get 256 times of the frequency of the utility source and the most significant bit of the frequency divider (Q7) which is synchronous with the utility source. The output of frequency divider of PLL (Q0~Q7) is used as the phase inputs of the three-phase sinusoidal reference voltage generator. The value of Im is used as the reference voltage of the multiplier type digital to analog converter (DAC) to generate the utility command current is*. We subtract the utility command current is* from the load current iL to get the compensation command current of the APF, ic*. This is used as the input signal for the three sets of hysteresis-type current tracking circuits which control the power switching devices (Insulated Gate Bipolar Transistor, IGBT) to generate a precise compensation current for each phase. The compensation current for each phase can compensate the utility current to be purely sinusoidal with unit power factor. The three-phase sinusoidal reference waveform is generated by a look-up table (which was stored in an EPROM), as shown in Fig. 6. The three-phase sinusoidal waveform data are pre-programmed in EPROM and read out sequentially according to the phase input Q0~ Q7. Then we use a multiplier type digital to analog converter (DAC) to convert these binary data to a set of three-phase sinusoidal reference voltage data. The reference voltage of DAC is directly fed from the command current Im of the capacitor voltage

control circuit. We can omit two sets of multipliers and directly generate the three-phase sinusoidal command current is* by using a multiplier type DAC. This is the reason for using the multiplier type DAC. 2.4 Improvement of the Transient Response In a balanced three-phase system, the summation of three-phase instantaneous reactive power is zero, which means the reactive power transfers only among phases. In the steady state, the energy-storage capacitor is not the same as the single-phase APF capacitor that must be provide large reactive power (reactive power transfers between load and converter). Therefore, the energy-storage capacitor of three-phase APF can take a small size capacitor to operate. However, in practical experimental circuits, we use a 22 µf capacitor for compensating the unbalance of line voltage, the unbalance of the load impedance and also the harmonic power. The test results show that under the condition of 2 A load current, it has good compensation characteristics. But, in the transient state, due to the command current of APF is calculated according to the utility current in a previous period, the extra current in current period will be supplied from the energy-storage capacitor. Using a smaller size of energy-storage capacitor, the loss of energy will make the terminal voltage of the energy-storage capacitor discharge rapidly. The capacitor voltage may drop to below the peak valve of the rectified utility ( VC −limit), as shown in Fig. 7. For example, when the load changes at t1 (increasing load current), the capacitor voltage will discharge down to under the value of VC −limit . However, because the anti-paralleled diode of the power switching device (IGBT or power MOSFET), the capacitor voltage can not drop down to under the value of VC −limit = 2V L− L . The voltage will keep at 2V L − L until the start of next period. Since the value of ΔVC , that is used for calculating the command current

Fig. 6 The block diagram for generating three phase sinusoidal reference waveform.

of the next period, is not in linear region (which shall be ΔVC ), the variation of the utility current ΔI m , that


Fig. 7 The voltage of energy-storage capacitor under the transient state (load change at t1).

is calculated with Eq. (15), is not correct. Hence it can not recharge the capacitor back to Vref at the end of the new period. Therefore, in the transient state, the control circuit must be modified. For example, in the case of the increase of load current is as shown in Fig. 8, the load current changes at t1, and the charging current is(t) is smaller than the load current. The voltage VC drops down. At t2, when the VC is equal to Vlow , we begin to modify the utility command current in advance. Under this new loading state, the changes of the utility command current are analyzed as follows: The energy loss of the capacitor until t2 is 1 ΔEc' = Cc (Vref 2 - Vlow2 ) 2

(17)

If we wish to recover the capacitor voltage to Vref during the remaining time in this period, the utility command current must be increased by an amount of 2

ΔI m =

2

C c (Vref - Vc )T (t 2 − t1 )3V sm (T − t 2 ))

Where K' =

= K ' (Vref 2 - Vc 2 )

KT 2 (t 2 − t1 )(T − t 2 )

(18)

(19)

Since the modified value of K’ changes according to t1 and t2, it is difficult to implement in the hardware. If Im is updated immediately, using the K’ in Eq. (18), it will be often too large and will push the control system into saturation. Furthermore, the capacitor voltage cannot drop down to under the value of VC−lim, hence we cannot accurately calculate the utility increment current directly from the capacitor voltage. In practice, when the capacitor voltage drops down

39

Fig. 8 The voltage of energy-storage capacitor under the transient state (using advanced sampling technique).

to below Vlow , the controller sends out the sampling signal for updating the utility command current, using original K in order to increase the utility current. Although, this current is smaller than the real need for the new load current, it can decrease the drop-down speed of the capacitor voltage. If the new Im is updated at the rate of Δt, this is equivalent to increasing the utility command current by ΔIm at every update time. Therefore, after n sampling pulses, it is equivalent to increasing the utility command current by n × Δ I m. Eventually, the utility command current will be able to provide the energy needed for the new loading current. The capacitor voltage will be charged up from VC −limit and return to the linear region again. Then the amount ΔIm decays progressively and the capacitor voltage will approach to V ref . After the capacitor voltage rises over Vlow, the system will be stayed in the linear region and the APF can work in steady-state during the next new period. 2.5 Determination of Energy-storage Capacitor When the voltage or the load of a three-phase system is in an unbalanced condition, the instantaneous summation of the line frequency volt-ampere supplied from the energy-storage capacitor is not equal to zero. Assuming that under the steady state condition, the volt-ampere which needs to be compensated is equal to SUB, then the maximum energy supplied by the capacitor will be equal to T Δ E UB , max = S UB × (20) 2

40


If this energy output make the capacitor voltage drop down to Vlow , (i.e. VC −limit + 0.2(Vref − VC −limit ) ), then

1 ΔEUB,max = CC,UB (Vref 2 -Vlow2 ) (21) 2 SUB ⋅ T CC,UB = (22) (Vref 2 - Vlow2 ) For example, if V L − L =220 V, S=10 kVA, SUB

=10% ×S=1.0 kVA, VC −limit =311 V, V ref =1.5 VC −limit , and Vlow =1.1VC−limit, then CC,UB=165 μf, i.e., 6.25 μf/amp. On the other hand, the power absorbed by the nonlinear load can be represented as [17]

pL (t) = With

1

1

k=−1

k=−1

∑ vs,k (t) ⋅ ∑ il,k (t) = PL + ~p(t)

3V ⋅ I cos φ1 PL = sm L1 2

(23)

(24)

consumed by the load.

∑ P3n cos(3nωt − ϕ 3n )

n =1

(25)

will be expressed as

∞

~ p L (t ) = ∑ P6n cos(6nωt − ϕ 6n ) n=1

(26)

In this equation, the six-order harmonic will be the largest, and this six-order harmonic energy supplied by the capacitor will be

T 1 P6,L ⋅ = CC,HR (Vref 2 − Vlow2 ) 12 2 CC , HR =

m

n =

Where

′ + ΔI ΔI

m

ΔI

m

′ + ...... + Δ I

m

′ = ΔI

m

″

″ m

(30)

′

ΔI m′ = K (Vref 2 − Vc,limit 2 )

(31)

Δt =

T − t2 n

(32)

3. Simulation and Experimental Results

∞

For the six-pulse rectified load, the above equation

i.e.

ΔI

Hence Δt will be determined as.

PL is the DC component relating to the real power

~ p L (t ) =

of this control system to be Δ I m ' ' , at this time t2, the capacitor voltage drops down to the value of VC−limit. Therefore, the control system will modify the utility command current, using the advanced sampling technique. Since the capacitor voltage already drops down to the value of VC −limit, the updated amount, ΔI m ' , for each sampling is constant. In the time duration of T-t2, the total advanced modified amount of the utility command current will be

T ⋅ P6, L 6(Vref 2 − Vlow 2 )

(27)

(28)

PL , 2 then C C , HR = 132 μf (6 μf/amp). If we consider the both cases simultaneously, then the capacitor value is suggested as.

As in the previous case, if we assume P6,L =

CC = CC,UB2 + CC,HR2

(29)

2.6 Determination of the Advanced Sampling Time Interval Δt

Assuming the maximum allowed transient current

Fig. 9 shows the simulation results, using Pspice simulation software, assuming V AC =220 V, L=1 mh, C C =22 μf, I L,DC =10 A. To verify this control algorithm, we have made a small scale three-phase APF. The three-phase voltage reduces to 55 volts, with DC load R L =40 ohm, L=1 mh, and the energy-storage capacitor C C =22 μf. The capacitor reference voltage is set at 120 V. The steady-state test results for I DC=1.0 p.u. are shown in Fig. 10. The waveform of ch1 is the energy-storage capacitor voltage, ch2 is the utility current, ch3 is the converter output compensation current, and ch4 is the load current. The test results of transient response, using the advanced sampling technique, are shown in Fig. 11. The load current varies between I DC =0.5 per unit (p.u.) and I DC =1.0 p.u.. When the load current increases from I DC =0.5 p.u. to I DC =1.0 p.u., the capacitor voltage drops down to Vlow and the control circuit generates the sampling clock immediately to update the utility command current. From the test results, we can see that the transient time is only one period. At the next new period, the


Fig. 9 The simulation results. Trace 1 is the capacitor voltage, trace 2 is line current, trace 3 is the compensation current and trace 4 is the load current.

(a)

41

(a)

(b) Fig. 11 The transient response test results for load current varies between I DC =0.5 p.u. and I DC =1.0 p.u. (a) R load. (b) R-C load

(b) Fig. 10 The steady-state test results for load current varies between I DC =0.5 p.u. and I DC =1.0 p.u.. (a) R load. (b) R-C load.

Fig. 12 The sampling clock of the proposed advanced sampling technique.

system is already in steady-state. On the other hand, when the load current decreases from IDC=1.0 p.u. to IDC=0.5 p.u., the capacitor voltage is also kept in a

reasonable range. Fig. 12 shows the sampling clock of the proposed advanced sampling technique. From the test results, we can see that even we use a small size

42


capacitor (11 μf/amp) which may caused a large ripple, the compensation characteristics are still good enough.

4. Conclusions Due to the nonlinear load characteristics of much electronic equipment, the utility power system is polluted by harmonics and the power factor is decreased. In this paper, a new APF technique is proposed, using the sampling technique to simplify the calculation of the real fundamental component of the load current. In addition, the energy-balance concept in the energy-storage capacitor is used to simplify the design of the conventional APF capacitor voltage control circuit. The merits of this APF are: (1) Simplifying the calculation of the required utility source current is(t), (2) A large ripple voltage can be tolerated in the energy-storage capacitor. Therefore, a smaller energy-storage capacitor is needed. (3) Only a proportional control method is applied in the controller design of the capacitor voltage. Hence the transient response is fast and good. The feasibility of the above scheme is verified by Pspice simulation and experimental results. The results demonstrate that the harmonics are suppressed and nearly unity power factor is obtained.

References [1]

[2]

[3]

[4]

R. Walling, N. Miller, G. Consulting, N. Schenectady, Distributed generation islanding-implications on power system dynamic performance, in: Power Engineering Society Summer Meeting, 2002 IEEE, Chicago, IL, USA, 2002. M. Shwehdi, M. Sultan, Power factor correction capacitors; essentials and cautions, in: Power Engineering Society Summer Meeting, IEEE Seattle, WA, 2000, pp. 1317-1322. L. Tolbert, F. Peng, Multilevel converters as a Utility Interface for Renewable Energy Systems, 2000, pp. 1271-1274. W. Radasky, C. Baum, M. Wik, Introduction to the special issue on high-power electromagnetics (HPEM) and intentional electromagnetic interference (IEMI), IEEE Transactions on Electromagnetic Compatibility, vol. 46, 2004.

[5]

[6]

[7]

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

H. Jou, J. Wu, Y. Chang, Y. Feng, A novel active power filter for harmonic suppression, IEEE Transactions on Power Delivery 20 (2005) 1507-1513. F.M.P. Pamplona, B.A. Souza, Harmonic passive filter planning in radial distribution systems using genetic algorithms, in: Transmission and Distribution Conference and Exposition, Latin America, 2004, pp. 126-131. G. Spiazzi, E. da Silver Martins, J.A. Pomilio, A simple line-frequency commutation cell improving power factor andvoltage regulation of rectifiers with passive LC filter, in: Power Electronics Specialists Conference, PESC, 2001 IEEE 32nd Annual Vancouver, BC, Canada, 2001. K. Chatterjee, G. Venkataramanan, M. Cabrera, D. Loftus, Unity power factor single phase AC line current conditioner, Industry Applications Conference, in: Conference Record of the 2000 IEEE, Rome , Italy, 2000. H.L. Do, B.H. Kwon, Single-stage line-coupled half-bridge ballast with unity power factor and ripple-free input current using a coupled inductor, IEEE Transactions on Industrial Electronics 50 (2003) 1259-1266. K.W. Siu, Y.S. Lee, C.K. Tse, Analysis and experimental evaluation of single-switch fast-response switching regulators with unity power factor, IEEE Transactions on Industry Applications 33 (1997) 1260-1266. M. Van der Berg, J.A. Ferreira, W. Hofsajer, A unity power factor low EMI battery charger for telecommunication applications, in: Telecommunications Energy Conference, INTELEC’95., 17th International, The Hague , Netherlands, 1995, pp. 458-465. C. Zhang, Q. Chen, Y. Zhao, D. Li, Y. Xiong, A Novel Active Power Filter for High-Voltage Power Distribution Systems Application, IEEE Transactions on Power Delivery 22 (2007) 911-918. G.W. Chang, C.M. Yeh, Optimization-based strategy for shunt active power filter control under non-ideal supply voltages, IEE Proceedings-Electric Power Applications 152 (2005) 182-190. W.U. Jin-Chang, H.L. Jou, Novel Circuit Topology for Three-Phase Active Power Filter, IEEE Transactions on Power Delivery 22 (2007) 444-449. H.L. Jou, J.C. Wu, Y.J. Chang, Y.T. Feng, A novel active power filter for harmonic suppression, IEEE Transactions on Power Delivery 20 (2005) 1507-1513. H.H. Kuo, S.N. Yeh, J.C. Hwang, Novel analytical model for design and implementation of three-phase active power filter controller, IEE Proceedings-Electric Power Applications 148 (2001) 369-383. B. Singh, B.N. Singh, A. Chandra, K. Al-Haddad, A. Pandey, D.P. Kothari, A review of three-phase improved power quality AC-DC converters, IEEE Transactions on Industrial Electronics 51 (2004) 641-660.

Modeling and Controlling of an Active Power Filter Using Photovoltaic System with Reduced Energy Storage Capacitor [18] K.P. Sozanski, Shunt active power filter with improved dynamic performance, in: Power Electronics and Motion Control Conference, EPE-PEMC 2008, 13th, Poznan, 2008, pp. 1995-1999. [19] X. Wei, K. Dai, X. Fang, P. Geng, F. Luo, Y. Kang, Parallel control of three-phase three-wire shunt active power filters, Power Electronics and Motion Control Conference, IPEMC 2006, CES/IEEE, 5th International, Shanghai, 2006. [20] Y.C. Kuo, T.J. Liang, J.F. Chen, A high-efficiency single-phase three-wire photovoltaic energy conversion system, IEEE Transactions on Industrial Electronics 50 (2003) 116-122. [21] Z. Chunyu, L. Yabin, P. Yonglong, A direct phase control scheme for unity power factor three-phase buck type rectifier based on SVPWM, Machine Learning and Cybernetics, International Conference in Dalian, China, 2006. [22] H. Do, R. Akkaya, A Simple Control Scheme for Single-

43

Phase Shunt Active Power Filter with Fuzzy Logic Based DC Bus Voltage Controller, in: Proceedings of the International Multi Conference of Engineers and Computer Scientists, IMECS, Hong Kong, 2009. [23] C.M. Liaw, T.H. Chen, T.C. Wang, G.J. Cho, C.M. Lee, C.T. Wang, Design and implementation of a single phase current-forced switching mode bilateral converter, in: IEE Proceedings B [see also IEE Proceedings-Electric Power Applications] Electric Power Applications 138 (1991) 129-136. [24] L. Bowtell, A. Ahfock, Comparison between Unipolar and Bipolar Single Phase Grid Connected Inverters for PV Applications, 2007, pp. 1-5. [25] T.F. Lee, Y.C. Hsiao, H.Y. Wu, T.L. Huang, F.M. Fang, M.Y. Cho, Optimization of reactive power compensation and voltage regulation using artificial immune algorithm for radial transmission networks, Engineering Intelligent Systems 15 (2007) 107-113.


Waste Tire Rubber Particles using to Improve the Properties of Local Asphalt Concrete Abdulali Bashir Ahmed Ben Saleh Department of Chemistry, Misurata University, Misurata, Libya Received: August 10, 2010/Accepted: September 03, 2010/Published: December 30, 2010. Abstract: The research considered urgent ecological reasons linked to environment such as worn tires, the waste tire rubber’s powder was collected from the tire cars repair shops (passed from the sieve No 18 μm), and used to improve the asphalt concrete properties. Raw materials used were prepared and tested. Varies of asphalt concrete mixtures were prepared with different ratios of bitumen (5, 5.5, 6, 6.5, 7% % of concrete weight). The Marshall mix design method was used to determine optimum conditions for bitumen in asphalt concrete with specific weight, stability and flow Test, the optimum amount of bitumen was 6.1 % of whole asphalt concrete. The different percentages of waste tire rubber powder (0.0, 0.05, 0.10, 0.15% of bitumen weight) were added in optimum bitumen of asphalt concretes, then specific weight and Marshall test were evaluated. These asphalt-rubber mixtures were found to act quite differently from traditional, unmodified asphalt mixtures. However, these results indicate that improved pavement performance can be achieved with asphalt-rubber binder. Key words: Asphalt concrete, additives to asphalt concrete, waste rubber tiers, modified asphalt.

1. Introduction Experimenting with particle rubber for asphalt modification began in the 1920’s. However, development of rubber modified asphalt binders, as they are now most often used in many countries started in the 1960s [1]. There two processes were used to development the asphalt concretes, first one call dry process which was developed in the late 1960’s in Sweden under the trade name Rubit [2]. It differs from the second one wet process in that the crumb rubber is used as a part of the aggregate and is directly mixed with the aggregate. This process also requires more liquid asphalt than a conventional hot-mix [3]. In the wet process, when asphalt cement and scrap rubber are mixed together, there is an interaction between rubber and bitumen, which results in swelling and softening of the crumbs. This in turn increases the viscosity of the modified binder. The wet process requires the use of at least 20% more liquid asphalt than is used in a conventional hot-mix pavement [4]. In Corresponding author: Abdulali Bashir Ahmed Ben Saleh, Ph.D., research field: polymer. E-mail:abdu702002 @yahoo.com.

1992 and in 1994, Liang and Woodhams [5, 6] described a process for devulcanization of scrap tire rubber in asphalt with the aid of aromatic oils and high shear and subsequently further stabilizing the devulcanized or disintegrated rubber particles by reacting the product with liquid polybutadiene and sulphur. Another paper by the same authors describes the work done on of the asphalt rubber mixes development [7]. Many papers had been presented or published [8-10] on the utilization of waste rubber in asphalt modification, some of them introduced in an attempt to increase the high temperature stiffness and low-temperature flexibility of asphalt pavements, these two properties (high temperature stiffness and low-temperature flexibility) are mostly responsible for decreasing a pavement’s susceptibility to environmental challenges and increasing its lifetime. In this paper, we aimed to improve local asphalt concrete’s mechanical properties by using directly additives of particles of waste tire rubber

2. Materials Asphalt, the asphalt used from the Ashgal Amah

45

Waste Tire Rubber Particles using to Improve the Properties of Local Asphalt Concrete

company with gravity weight 1.025 g/cm3. All aggregates, coarse, fine and filler were supplier from Ashgal Amah company, the requires tests and require aggregate gradation were carried out according to Libyan specification. Additives, the waste rubber powder was collected from rubber tier repair shops and passed from the sieve No 18 μm. 2.1 Design of Mixture’s Asphalt Concrete The four aggregates and the asphalt selected for Marshall mix design [11]. The gradations of aggregates compared with Libyan specifications [12], and the percent of each aggregate to be used are determined so resulting blend meets specified limits. The aggregate blend is determined by trial and error method, the aggregate percent are determined to be 10% of aggregate No 1.5, 37%of aggregate No. 1, 50% of fine aggregate, and 3% of filler aggregate. The percents of asphalt were 5, 5.5, 6, 6.5, and 7 of whole weight of mixture.

0.050, 0.100, 0.150% of bitumen weight) to the optimum amount of bitumen. 2.3 Bulk Density of the Compacted Specimen The bulk density of the sample is usually determined by weighting the sample in air and in water. It may be necessary to coat samples with paraffin before determining density. The specimen is then marked and stored for stability and flow measurements [11]. 2.4 Stability-Flow Test In conducting the stability test, the specimen is immersed in a bath of water at a temperature of 60 ºC for a bout 30 minutes. It is then placed in the Marshall stability testing machine and loaded at a constant rate of deformation of 5 mm per minute until failure. The total maximum in Kgf (that causes failure of the specimen) is taken as Marshall Stability. The total amount of deformation is units of 0.25 mm that occurs at maximum load is recorded as Flow Value (!1).

2.2 Preparation of Test Specimens

3. Result and Discussion

The aggregate, is dried at 105-110 ºC and sufficient amount is weighed (about 1200 g) to give a height of 64 mm and diameter of 102mm when compacted in the mould. The required quantity of bitumen (5, 5.5, 6, 6.5, 7% of specimen whole weight) is weighed out and heated to a temperature reduce the viscosity of bitumen. The aggregates are heated to a temperature about of 175 ºC the compaction mould assembly and rammer are cleaned and kept pre-heated to a temperature of 100 ºC to 145 ºC. The required amount of first trial of bitumen is added to the heated aggregate and thoroughly mixed until all the aggregate is coated. The mix is placed in a mould and compacted with number of blows specified (75 blows each face). The sample is taken out of the mould after few minutes using sample extractor and transferred to a smooth flat surface and allowed to cool to room temperature [11]. The modified asphalt concretes were prepared by directly addition of varies ratio of waste tire rubber (0.025,

Five unmodified asphalt concretes with deferent ratio of bitumen were prepared and tested. Following results and analysis is performed on the data obtained from the experiments: The bulk specific gravity Gb cm of the unmodified asphalt concretes specimens is determined for all specimens, and given by Eq. 1. (1)

Gbcm (g/cm3)

Where, Wa = weight of sample in air (g) Ww = weight of sample in water (g) Fig. 1 shows the relationship between density and 2.26 2.24 2.22 2.20 2.18 4.5

5

5.5

6

6.5

7

7.5

bitumen content (%)

Fig. 1 Relationship between density and bitumen content.

46


bitumen content in unmodified asphalt concretes. The density is gradually increase with bitumen content is increased, which may be related to the bitumen filled the concrete air voids. The percent voids in mineral aggregate (VMA) is the percentage of void spaces between the granular particles in the compacted paving mixture, including the air voids and the volume occupied by the effective asphalt content (2) Where, VMA = percent voids in mineral aggregates. Gbcm = bulk specific gravity of compacted specimen Gbam = bulk specific gravity of aggregate. Pta = aggregate percent by weight of total paving mixture. Fig. 2 shows the relationship between the percent voids in mineral aggregate (VMA) and its asphalt content. Percent air voids in compacted mixture (Pav) is the ratio (expressed as a percentage) between the volume of the air voids between the coated particles and the total volume of the mixture.

then the stability decrement gradually as shown in Fig. 5. For this design all volumes were in the required range (ASTM D1559). After all the data was collected, optimum bitumen content is selected as the average binder content for maximum density, maximum stability and specified percent air voids in the total mix [11]. The optimum amount of bitumen was 6.1 of total pavement weight. The modified of asphalt concrete is prepared according to bitumen optimum amount by adding different ratios of waste particles rubber to ratio specified. The bulk specific gravity Gbcm of the modified specimens is determined using equation (1). Fig. 6 describes the relation ship between specific gravity and bitumen content. It can be easy note that specific gravity is decreasing with the bitumen content is increased, that may be related to the rubber’s light weight compare to other concrete component.

(3)

Fig. 2 The percent voids in mineral aggregate (VMA) against bitumen concentration in asphalt concrete.

Fig. 3 Percent air voids in compacted mixture against bitumen content. Flow (mm)

Where, Pav = percent air voids in compacted mixture Gmp = maximum specific gravity of the compacted paving mixture Gbcm = bulk specific gravity of the compacted mixtures. Fig. 3 describes the relation ship between Percent air voids in compacted mixture (Pav) and bitumen content. After the samples were weighted in air and water and all calculations mad, the samples were then tested for stability and flow. Flow value is determined from Marshall test’s machine. Fig. 4 shows the relationship between flow value to specimens and its asphalt content. The flow values is increment with the rubber particles concentrations were increased. Marshall’s stability is evaluated from Marshall test machine. The stability values is increased with increasing of asphalt content until a proximately of 6%

6 4 2 0 4.5

5

5.5

6

6.5

7

7.5

bitumen content%

Fig. 4 Relationship between flow value to specimens and its asphalt content.

Marsh all stabi li ty (kgf)


addition about 0.05-0.1% of bitumen but the increasing of the rubber impact leads to decrease in Marshall’s stability.

1,500 1,300 1,100 900 700 4.5

4. Conclusion 5

5.5

6

6.5

7

7.5

bitumen content%

Fig. 5 Marshall stability values against bitumen loading.

Gbcm g/cm3

47

2.30 2.25 2.20 2.15 2.10 2.05 0

0.05

0.1

0.15

0.2

Directly addition of waste tire rubber particles process employed to incorporate particles rubber with asphalt seems to be an effective method for the preparation of rubber modified asphalt concrete with improved mechanical properties such as Marshall stability. Small amount of rubber seems to be more active to improve the stability, whereas the increasing of rubber content may leads to decrease Marshall stability.

Rubber content %

Fig. 6 Relationship between specific gravity and bitumen content of modified asphalt concrete.

Acknowledgments Thanks are due to Abdulhameed M. Abufalga, Abdu-Allah A. Alogab, Mohmmed S. Zogainin, Mohmmed D. Gazaly students from the Misurata higher institute for trainers preparation, for their assistance in the preparation of specimens, and grateful thank to Mr. abdu-Alsalam Abu-Setah with his staff in Amashroaat engineering consulting company for their assistance in specimens preparation and testing.

Mashall stability (kgf)

Fig. 7 The correlation between flow and rubber content.

References [1]

1,400.00 1,300.00

[2]

1,200.00 1,100.00

[3] 0

0.05

0.1

0.15

0.2

rubber content %

Fig. 8 Correlation between Marshall’s stability and loading of rubber particles.

The correlation between flow and rubber content as shown in Fig. 7 is indicates that the 0.1 ratio of rubber is beast ratio because it is give value of 3.53 mm which in the standard range 2-4 mm. Fig. 8 shows the relationship between Marshall’s stability and loading of rubber particles. There are some improvements in the Marshall stability with

[4] [5] [6]

[7] [8]

L. Zanzotto, G. Kennepohl, Presented at the 75th Annual Meeting of the Transportation Board, Washington, D.C., 1996. A. Bjorklund, in Proceedings of the VI1 World Road Conference, Vienna, 1979. H.B. Takallon, R.G. Hicks, TRR 1171, TRB, National Research Council, Washington, D.C., 1988. M. Heitzman, Washington, D.C. Preprint 20549, 7151 Annual Meeting, January 12-16, 1992. Z.Z. Liang, R.T. Woodham, GB Patent Application, Filed on December 29, 1992. Z.Z. Liang, R.I. Woodhams, World Patent Application 94/14896, Priority date: December 1994, p.29, 1992 - GB 92 27035.4. A. Coomarasamy, S.A.M. Hesp, Paving in Cold Areas, Niigata. Japan (Paper to be presented), October 1996. R. Kim Jong, Characteristics of crumb rubber modified (CRM) asphalt concrete, KSCE Journal of Civil Engineering Volume 5, Number 2 / June, 2001.

48 [9]


M. Tuncan, A. Tuncan, A. Cetin, The use of waste materials in asphalt concrete mixtures, Waste Management & Research 21 (2003) 83-92. [10] M.A. Mull, K. Stuart, A. Yehia, Fracture resistance characterization of chemically modified crumb rubber asphalt pavement, Journal of Materials Science 37 (2002) 557-566.

[11] F.L. Roberts, P.S. Kandhal, E. Ray Browm, Dah-Yinn Lee, T.W. Kennedy, Hot mix Asphalt, Materials, Mixture design, and construction, first edition, NAPA Education Foundation, Lanham, Maryland, 1991. [12] Libyan specification of general constructions and ways pavement, forth issue, 1976.


Synthesis and Characterization of Oxy-Vanadium (IV) Complex of 4-(2, 4-dihydroxybenzaldimine) Antipyrine Ramadan M. El-mehdawi1, Abdussallam N. Eldewik2, Khaled M. Kreddan3, Fathhia A. Treish1, Mufida M. Ben Younes1, Abtisam A. Aboushagour1 and Zinab A. Elkamoshi1 1. Chemistry Department, Faculty of Science, Al-Fateh University, Tripoli, Libya 2. Chemistry Department, Faculty of Science, University of Aljabal Algharbi, Zawia, Libya 3. Libyan Petroleum Institute, Tripoli, Libya Received: June 13, 2010 / Accepted: July 29, 2010 / Published: November 30, 2010. Abstract: The reaction of VCl3 with 4-(2, 4-dihydroxybenzaldimine) antipyrine (LH) in a 1:1 molar ratio results in formation of the general complex of [VOL(Cl)OH]. The nature of bonding and stereochemistry of the complex was deduced from the elemental analysis, IR, UV-Vis, 1H and 13C NMR spectroscopy. This complex has a coordination number five (square pyramidal) as known for many vanadyl complexes. The formation of the vanadyl ion (VO2+) and the mode of bonding of the Schiff base through the oxygen of the pyrazol ring and the nitrogen of the azomethine group to the vanadyl ion were established by IR technique. Key words: Schiff base, Oxy-Vanadium(IV) complex, 1H and 13C NMR.

1. Introduction The metal ions of the early transition elements with high oxidation states usually found in complex oxy cations of the type MOn+. The total coordination number of vanadium is always five or six when VO2+ ion occur in coordination with some ligands in both solution or solid state. Vanadyl complexes of bidentate and tetra dentate ligands such as, acetylacetonato, benzoato, oxalate, salen and salaphen are well known [1, 2]. These complexes have five coordinate structure ranged from distorted trigonal bipyramidal to square pyramidal [3]. The vanadyl ion and its complexes are effective not only in treating and relieving both types of diabetes mellitus, but also in preventing the onset of diabetes mellitus [4]. Bis (maltelato) oxo vanadium (IV) complex has an anti diabetic therapeutic potential which exceeded vanadyl sulphate in glucose-lowering Corresponding author: Abdussallam N. Eldewik, Prof./Ph.D., research field: solid state chemistry. E-mail: [email protected].

ability [5]. In addition, some insulin mimetic vanadyl complexes are currently prepared [6, 7]. Vanadyl complexes are very effective in catalytic processes especially in epoxidation of olefins [8]. The aim of this work is to synthesize a vanadyl complex that may have both catalytic and biological activity.

2. Experiment All chemicals were AR grade and used without further purification. All manipulations carried out under atmospheric pressure. The elemental analysis (C, H, N) was performed on Vario-EL (III) elemental analyzer. FTIR spectra were recorded at room temperature with a Bruker IFS-25 DPUS/IR spectrometer over the wave number range 400-4000 cm-1 with a resolution of about 4 cm-1. The electronic absorption spectra were carried out by using Perkin-Elmer lambda 413 1 13 spectrophotometer. H and C NMR experiments were performed on a Bruker Aviance 5mm QNP probe with resonance frequency of 75.47 MHz for 13C nuclei and

50

Synthesis and Characterization of Oxy-Vanadium (IV) Complex of 4-(2, 4-dihydroxybenzaldimine) Antipyrine

300.13 MHz for 1H nuclei. The 1H and 13C chemical shifts were internally referenced to tetramethyl silane (TMS) in DMSO solvent. 2.1 Preparation of 4-(2, 4-dihydroxybenzaldimine) Antipyrine EtOH (LH) The Schiff base (LH) was prepared by heating 2.0 g (14.4 mmol) of 2, 4-dihydroxy benzaldehyde with 3.0 g (14.8 mmol) of 4-amino antipyrine in 30 mL ethanol under reflux for 2 h. A yellow solid was formed after addition of H2O to the filtered solution and the Schiff base formed was recrystallized by using ethanol-ether mixture to give a bright yellow solid which was characterized by elemental analysis, Anal. Calc., for C20H23N3O4: C, 65.04; H, 6.23; N, 11.38; found: C, 64.98; H, 6.45; N, 11.15, IR and 1H and 13C NMR. 2.2 Preparation of [VOL(Cl)OH] 4-(2,4-dihydroxybenzaldimine) Antipyrine)

H3C 12 N 2

11

10

6 9 8

N 1

CH3 13 3 4

5

7

N 14

O 22

21

15

20

1

19

17

HO 23 Scheme 1 Structure of Schiff base ligand (L).

18

OH 24

(LH:

To a solution of 0.1 g (0.63 mmol) VCl3 in 10 mL ethanol, Schiff base (LH) 0.2 g (0.6 mmol ) in≈20 mL ethanol was added and the mixture were refluxed for 2 h. A green coloured product was filtered off, washed with acetone and air dried. The vanadyl complex was characterized by elemental analysis. Anal. Calc., for C18H18N3O5VCl: C, 48.8; H, 4.07; N, 9.49; found: C, 49.1; H, 4.11; N, 9.53. IR, UV-Vis and 1H and 13C NMR.

Scheme 2 Proposed structure of metal complex (•V, •O, •Cl, •H, •N and •C).

coordinated to the vanadium metal ion. In addition, from the IR spectrum we can see that the ν(C=O) bands at 1603 cm-1 was shifted to lower wavenumbers with about 12 cm-1 relative to the corresponding free ligand (Free ligand at 1615 cm-1) indicating that the ligand coordinated through the carbonyl oxygen of the pyrazole ring. Furthermore, the stretching frequency of the azomethine group (HC=N) which observed at 1580 cm-1 was shifted to lower frequency with about 5 cm-1

3. Results and Discussion

relative to the corresponding ligand suggesting the

Tridentate Schiff base ligand (L) was formed by the reaction of 2, 4-dihydroxy benzaldehyde with 4-amino antipyrine. Vanadyl complex was synthesized from the free ligand and vanadium trichloride. The free ligand and its vanadyl complex (Scheme 2) were characterized by UV-Vis, IR,1H and 13C NMR specroscopy.

involvement of the nitrogen atom in the coordination. The medium to strong band at 988 cm-1 was assigned to the oxyvanadium double bond which indicates that the vanadyl complex is monmeric in nature. 3.2 Ultraviolet- Visible Spectroscopy (UV-Vis) The

UV-Vis

spectra

of

VO2+

complex

of

3.1 Infrared Spectroscopy (IR)

oxyvanadium (IV) compounds is not as simple as it

The IR spectroscopic data as iullstrated in Table 1 of the complex exhibited sharp moderate to strong band at 3264 cm-1. This band may be assigned to the free (OH)

would be for ordinary vanadium complexes due to the strong VO π-bonding. The absorption around 28409 cm-1 and 24390 cm-1 may be assigned to the spin allowed


51

Table 1 Infrared and UV-Vis spectroscopic data for the ligand and metal complex. Compound

νC-H (cm-1)

νO-H (cm-1)

νC=O (cm-1)

νVO (cm-1)

νHC=N (cm-1)

Metal complex [VOLCl(OH)]

2916.0 3059.0

3148.0 3461.0

1603.0

988.0

1580.0

Free Ligand (LH)

2936.0 3065.0

3264.0 3465.0

1615.0

-

1585.0

Phenyl group UV-Vis (cm-1) (λ, cm-1)(Abs) 39062, 1.6 (LMTC) 864.0 28409, 1.5 773.0 24390, 0.5 830.0 767.0 697.0

LMTC=Ligand to metal charge transfer.

* (b)

* (a)

14

13

12

11

10

9

8

7

6

5

4

3

2

1

0

-1

ppm

Fig. 1

1

H NMR spectrum of (a) Ligand and (b) metal complex. (*) DMSO peak.

b2 (dxy)→2e (dxz, dyz) and 2b2 (dxy)→2b1 (dx2-y2) transitions respectively. The third band corresponds to

2

the 2b2 (dxy)→2a1 (dz2) not observed because it is obscured by the tail end of a strong charge transfer band at 39062 cm-1 [9]. 3.3 1H and 13C NMR Spectroscopy 1

H NMR spectra of the free ligand show peaks at 9.56 ppm and 13.3 ppm assignable to the azomethine proton HC═N and 2-hydroxy proton of the Schiff base respectively. The aromatic protons appears at 6.26 to 7.51 ppm and methyl groups attached to carbon atoms number 12 and 13 (Scheme 1) appears at 2.5 ppm and 2.4 ppm respectively as shown in Fig. 1a.

The 1H NMR spectra of the complex shows single resonances at 9.56, 10.5 and 13.40 ppm assignable to the azomethine ( HC═N ), the OH group coordinated to VO2+ and OH of the 2-hydroxy linked to aromatic group of the schiff base respectively. The later resonance indicates that the two hydroxy groups of the aromic ring of the Schiff base neither deprotonated nor coordinated. The aromatic protons of the complex appears at 6.3 to 7.9 ppm (Fig. 1b). 13 C NMR spectral data of the free ligand and complex are shwon in Fig. 2. The assignments of the resonances of the carbon atoms of the complex seems to be straight forward, the aromatic carbons appears in the range 102-135 ppm, the resonances of the azomethine

52


(c)

*

(b) *

21

8, 10 (a)

* 5 3 4 15

160

19

150

17 9 7,11

140

130

12

6 16 20 18

120

110

100

90

80

70

60

50

40

30

13

20

10

0

ppm

Fig. 2 (a) 13C NMR spectrum of ligand (number of carbon atoms in Scheme 1), (b) its DEPT spectrum and (c) metal complex, (*) DMSO peak.

carbon and carbon of carbonyl appears at 161.73 and 158.08 ppm respectively as shown in Fig. 2. Clearly, Distortion-less Enhancement by Polarlization Transfer, DEPT, technique is a very useful adjunct to 13 C NMR spectroscopy. Whereas the peaks in a typical 13

C NMR spectrum appear as singlets, the result of the DEPT experiment (Fig. 2b) can tull us whether a given peak arises from a carbon on a methyl group, a methylene group, or a methine group. By comparing the results of the DEPT spectrum with the original 13C NMR spectrum, we can also identify the peaks which arise from quaternary carbons. Quaternary carbons, which bear no hydrogens, appear in the 13C NMR spectrum but are missing in the DEPT spectrum. The appearance of pronounced signal to niose ratio in the 13 C NMR spectrum of metal complex (Fig. 2c) is attributed to the presence of paramagnetic V4+ ion close to the carbon environment.

to the vanadyl ion through the carbonyl oxygen of the pyrazole ring and the nitrogen of the azomethine group. The 1H NMR spectrum indicates that the hydroxyl groups of the aromatic ring of the Schiff base neither deprotonated nor coordinated. All of these results with the support of elemental analysis indicate that the complex has a square pyramidal structure.

Acknowledgment The authors acknowledge the help from the advanced laboratory of chemical analysis for their C, H, N, IR and UV-Vis and the Libyan Petroleum Institute for their 1H and 13C NMR analyses.

References [1]

[2]

4. Conclusions Finally, from the above results we conclude that the IR spectrum indicates that the ligand was coordinated

[3]

J. Selbin, G. Maus, D.L. Johnson, Spectral studies of β-ketoenolate complexes of oxovanadium(IV), J. Inorg. Nucl. Chem. 29 (1967) 1735-1744. X. Wang, X.M. Zhang, H.X. Liu, Synthesis, properties and structure of the vanadium(IV) Schiff base complex [VO(salphen)]·MeCN, Transition Metal Chemistry 19 (1994) 611-613. M. Pasquali, F. Marchetti, C. Floriani, S. Merlino, J. C. S. Dalton (1977) 139.

Synthesis and Characterization of Oxy-Vanadium (IV) Complex of 4-(2, 4-dihydroxybenzaldimine) Antipyrine [4]

[5] [6] [7]

H. Sakurai, A New Concept: The use of Vanadium complexes in the treatment of diabetes mellitus, Chem. Rec. 2 (2002) 237-248. R.K.Y. Ho, S.E. Livingstone, Australian J. of Chemistry 18 (1956) 659. A. Katoh, M. Yamaguchi, R. Saito, Y. Adachi, H. Sakurai, Chem. Lett. 33 (2004) 1274-1275. Y.H. Xing, K. Aoki, F.Y. Bai, A new insulin-like vanadyl

[8] [9]

53

complex: synthesis and structure of V(IV)O(H2O)2 (2,6-PyridineDicarboxylate)E2H2O, Journal of Coordination Chemistry 57 (2004) 157-165. G.F. Miralamov, Ch.I. Mamedov, Petrol. Chem. 46 (2006) 25-27. A.B.P. Lever, Inorganic Electronic Spectroscopy, Elsevier, 1984.


2-Nitrobenzofuran as Dienophile in Polar Diels-Alder Reaction: A Simple Dibenzofurans Synthesis Claudia Daniela Della Rosa, Juan Pablo Sanchez, María Nélida Kneeteman and Pedro Máximo Emilio Mancini Area Organic Chemistry, Chemistry Department, Chemical Engeeniering School, Litoral National University, Santa Fe, Argentine Received: September 04, 2010 / Accepted: September 20, 2010 / Published: November 30, 2010. Abstract: 2-nitrobenzofuran is studied as dienophile in polar thermal Diels-Alder reactions with normal electron demand using several structurally different dienes. A very strong electron-acceptor group, such as nitro group, enhances the dienophilic character of these heterocyclic compounds and owing to the fact that this substituent is easily extruded under thermal conditions this reaction sequence becomes a simple method of organic compound’s families with heteroatom rings preparation. Part of this work is specifically concerned with theoretical studies. The global and local electrophilicity and nucleophilicity indexes were calculated for the dienophile and dienes used in this work. A concise synthesis of dibenzofurans has been development via cycloaddition reactions. Key words: 2-Nitrofurane, dienophile, Diels-Ader, dibenzofurans.

1. Introduction Due to our interest in the cycloaddition chemistry of substituted aromatic heterocycles with electron withdrawing groups, we have reported that 2-nitrofuran, 2-nitrothiophene, 2-nitro-N-tosylpirrole and 2-nitroselenophene react with strong and poor dienes under different conditions [1]. Additionally, we have performed studies on the dienophilic character of other aromatic systems such as nitronaphthalenes in Diels-Alder (D-A) reactions [2]. While the use of these substrates as dienes in thermal and high-pressure D-A reactions has been widely analyzed, the employment of such compounds as dienophile has received relatively little attention in the literature. In the latter direction we have shown the dienophilic behaviour of naphthalenes properly mono and disubstituted with electron withdrawing groups nitro, cyano, acetyl, and chloro [3]. In general, when these dienophiles were exposed to different dienes, it yielded the correponding initial cycloadducts products which normally suffer the Corresponding author: Pedro Máximo Emilo Mancini, professor, research field: organic reactions. E-mail: pmancini@ fiq.unl.edu.ar.

thermal extrusion of the nitro group. Unexpected results were observed when using nitronaphthalenes with less reactive dienes. Interestingly, when 1- and 2-nitronaphthalenes reacted with isoprene, they produced the corresponding N-naphthylpyrroles. From the results obtained in all these reactions, we can conclude that there are two competitive reactions that might take place on mononitronaphthalenes: the addition to the nitro group (hetero D-A process) and the normal D-A reaction on the C1-C2 bond, depending on the strength of the diene partner. This behaviour can be extended to the nitrothiophenes probable due its high aromaticity in relation to the others aromatic heterocyclopentadienes [4]. Dibenzofurans are important heteroatomic compounds which display a wide variety of biological activities. The dibenzofuran containing pytoalexins show manifold biological activities, eliciting a strong interest from chemist and biologists [5]. Considerable effort has been devoted to the development of eficient methods for the building of this heteroatomic ring system. More of the general procedures involved several steps and the overall yield usually are not very good [6].

2-Nitrobenzofuran as Dienophile in Polar Diels-Alder Reaction: A simple Dibenzofurans Synthesis

Benzofurans have been shown in a few cases, to react as dienophiles in D-A reaction. The benzo[b]furan itself can act an electron-rich dienophile in an inverse electron demand process. In other way, several years ago was informed the reaction between 3-formylbenzofuran and isoprene as an example of a D-A cycloaddition [7]. Recently is has been reported a study of acyl substituted benzofurans as efficient dienophiles in a normal electro demand [4+2] cycloaddition using hight pressure reaction conditions [8]. On the other hand, theoretical studies were performed to estimate reactivity. The global electrophilicity index (ω), introduced by Parr et al. [9] is a useful descriptor of reactivity that allows a quantitative classification of the global electrophilicity character of a molecule within a unique scale [10]. This index is defined as,

ω=

μ2 2η

(1)

where µ is the electronic chemical potential and η the chemical hardness. Useful information about polarity of D-A processes may be obtained from the difference in the global electrophilicity power of the reactants. This difference has been proposed as a measure of the polar character of the reaction. On the other hand, local reactivity indexes are associated with site selectivity in a chemical reaction. These descriptors should reflect the sites in a molecule where the reactivity pattern stated by the global quantities should take place. For instance, an important local reactivity parameter was introduced by Parr et al. And it was defined as the Fukui function [11]. Eq. 2 provides a simple and direct formalism to obtain the Fukui function from an approach based on a relationship with the FMOs [12]. The condensed Fukui function for electrophilic (nucleophilic) attack involves the HOMO (LUMO) FMO coefficients (c) and the atomic overlap matrix elements (S).

f kα = ∑ | cμα |2 + ∑ cμα cvα S μv μ∈k

v≠ μ

(2)

This scheme has been corroborated for several reactions that are well documented [13].

55

Eq. 3 has been introduced to analyze at which atomic site of a molecule the maximum electrophilicity power will be developed [14]. (3) ωk = ωf k+ Furthermore, the first approaches toward a quantitative description of nucleophilicity, in the form of a regional reactivity index, have also been reported. Eq. 4 has been developed by Domingo et al.[10] with the purpose of identifying the most nucleophilic site of a molecule and assessing the activation/deactivation caused by different substituents on the electrophilic aromatic substitution reactions of aromatic compounds, N k = Nf k− (4) N = ( ε HOMO,Nu − ε HOMO,TCE ) (5) where εHOMO,TCE is the HOMO energy of tetracyanoethylene (TCE) (taken as a reference molecule because it exhibits the lowest HOMO energy in a large series of molecules previously considered in the framework of polar D-A cycloadditions). N is the global nucleophilicity index and Nk is its local counterpart [15, 16]. This nucleophilicity index has been useful to explain the nucleophilic reactivity of some molecules towards electrophiles in cycloaddition as well as substitution reactions [17]. In this work the polarity of the normal electron demand D-A process propose has been studied by means of global electrophilicity index difference between reactants and the regioselectivity of the normal electron demand D-A reaction shown in Schemes 2, 3 and 4 using the local electrophilicity index for dienophiles (electrophiles in the reaction) and the local nucleophilicity index for dienes (nucleophiles in the reaction).

2. Computational Details Studies show that the B3LYP method [18], using 6-311+G(d,p) basis set, is adequate to model D-A reactions concerning medium-sized molecules. Hence, the gas-phase equilibrium geometries of all species described here were obtained by full optimization at the B3LYP/6-311+G(d,p) level using the GAUSSIAN03W program [19]. All the stationary points found were

56


characterized as true minima by frequency calculations. The chemical hardness, the electronic chemical potential and the global electrophilicity index have been calculated. Regional Fukui functions for the +

−

dienophiles ( f k ) and for the diene ( f k ) were obtained from single-point calculations on the optimized structures at the ground state (Eq. 2). A program that reads the FMO coefficients and the overlap matrix from the Gaussian output files and performs the required calculation was developed and tested. Once the Fukui functions were computed, the local electrophilicity and nucleophilicity values were calculated (Eqs. 3 and 4). Herein we report a simple, economical and efficient one-step procedure to synthesize the dibenzofuran ring system in good to excellent yield through the D-A reaction of 2-benzofuran and different dienes. A very strong electron-acceptor group, such as nitro group, push the dienophilic character of these heterocyclic compounds and owing to this substituent is easily extruded under thermal conditions make this reaction sequence a simple method of organic compound’s families with heteroatom rings preparation. Part of this work is specifically concerned with theoretically studies using DFT methods.

it showed its dienophilic character taking part in DA cycloaddition reactions. The thermal reactions of 2-nitrobenzofuran 1 with isoprene 2 in a sealed ampoule at 150 ºC or 200 ºC for 72 h using benzene as solvent afforded the mixture of isomeric cycloadducts 5a and 5b with reasonable yield (Scheme 2, Table 1). On the other hand, reactions of 1 with 1-N-acetylN-propyl-1,3-butadiene 3 afforded dibenzofuran 6 with loss of N-acetyl-N-propyl and nitro groups (Scheme 3, Table 2). In the same way, in the reactions with Rawal’s diene cycloadduct 7 was obtained in good yield and complete regioselectivity (Scheme 4, Table 3). This product resulted from the expected aromatization of the nitro-adduct promoted by the loss of the nitro and diethylamine groups, respectively.

Scheme 1 Dienophile and dienes.

3. Results and Discussion To explore the normal electron demand D-A dienophilicity of nitrobenzofurans (1), we choose isoprene (2), 1-N-acetyl-N-propyl-1,3-butadiene (3), and 1-diethylamino-3-tert-butyldimethylsilyloxy-1,3butadiene (4) as diene (Scheme 1). The selection the dienes taken into account the type of substitution present in their structure and the relative nucleophilicity. The nitrobenzofuran (1) was prepared by nitration of benzo[b]furan and subsequent chromategraphic purification. All reactions were performed in sealed ampoule using benzene or toluene as solvent and different temperatures and time of reaction. When 2-nitrobenzofuran was reacted with the abovementioned dienes under different reaction conditions,

Scheme 2 D-A reaction 2-nitrobenzofuran with isoprene. Table 1 Diels-Alder reactions of 2-nitrobenzofuran with isoprene. T (ºC) Time D:Da 12:1 200 72 h 12:1 150 72 h a Diene/Dienophile ratio, b Based on consumed dienophile.

Scheme 3 dienamide.

Product 5a, 5b 5a, 5b

Yieldb 70% 70%

D-A reaction2-nitrobenzofurane with a

2-Nitrobenzofuran as Dienophile in Polar Diels-Alder Reaction: A simple Dibenzofurans Synthesis Table 2 Diels-Alder reactions of 2-nitrobenzofuran with 1-N-acetyl-N-propyl-1,3-butadiene. D:Da T (ºC) Time 3:1 140 72 h 3:1 120 72 h a Diene/Dienophile ratio, b Based on consumed dienophile.

Product 6 6

Yieldb 65% 70%

57

Table 4 Global electrophilicity index for dienes and the dienophile. Compound 1 2 3 4

ω(eV) 3.3275 1.2746 1.2202 0.7945

Table 5 Local indexes for dienes and the dienophile. Compound 1

Scheme 4 D-A Reaction 2-nitrobenzofurane with Rawal´s diene. Table 3 Diels-Alder reactions of 2-nitrobenzofuran with Rawal’s diene. a

D:D T (ºC) Time 2:1 Reflux Tol. 48 h 2:1 Reflux Tol. 72 h a Diene/Dienophile ratio b Based on consumed dienophile

Product 7 7

2 3 4

b

Yield 75% 85%

By analogy, the reactions of nitrobenzofurans with dienes 2, 3 and 4 could be considered a domino process that is initialized by a polar DA reaction; the latter concerted elimination of nitrous acid from the [2+4] cycloadduct yields the corresponding dibenzofurans. According to the global electrophilicity index ω shown in Table 4, the dienes will act as nucleophiles and the dienophiles as electrophiles. To study the regioselectivity, we used the local electrophilicity and nucleophilicity indexes for dienophiles and dienes respectively (Table 5). The sites of study were C2 and C3 in 1 and C1 and C4 in dienes 2, 3, 4. The more favoured aducts are the ones where the most electrophilic and nucleophilic sites interact first. In the reactions, in which it is possible discussed the regioselectivity the experimental data agree with the computational results. However, the influence of the methyl group when isoprene is the diene is less than the groups present in Rawal’s diene.

4. Conclusions The ease of thermal extrusion of nitrous acid accompanying the D-A reaction followed by the further

Site 2 3 1 4 1 4 1 4

ωk(eV) 0.1571 0.5359 -

Nk 1.1182958 0.7518751 0.5727111 0.2524620 0.404231 1.199553

aromatization makes this reaction sequence a simple, economical and efficient one-step procedure to synthesize the dibenzofuran ring systems in good to excellent yields through a polar Diels-Alder reaction of 2-nitrobenzofuran and diverse dienes. This route should be applicable for the preparation of many biologically interesting molecules. DFT calculations of the electrophilicity and nucleophilicity indexes agree with the experimental results and they are a good reactivity and regioselectivity predictors in these type of reactions.

5. Experimental Section 5.1 General Aspects 1

H and 13C NMR espectra were recorded in CDCl3 on 300 and 75 MHz FT-spectrometers, respectively, using TMS as the internal standard; GC-MS analyses were performed in an instrument equipped with a PE-5-type column. IR spectra were recorded from NaCl cells. Melting points were observed on a Winkle-Zeiss Gottingen microhot stage and were uncorrected. The silica gel and neutral alumina used for chromatography were 70-230 mesh. The synthesis of 2-nitrobenzofuran was performed starting from benzofuran adapting a procedure proposed by A. Katritzky et al. [20].

58


The following reagents were prepared by literature methods: 1-N-acetyl-N-propyl-1,3-butadiene (3) [21] and 1-diethylamino-3-tert-butyldimethylsilyloxy-1,3butadiene (4) [22]. Other reagents were obtained from commercial sources used as received or purified as required by standard methods. 5.2 General Procedure for the Thermal Reactions of 2-Nitrobenzofuran The temperature, the length of the reaction, and the diene/dienophile ratio were dependent on the starting material and are indicated in Tables 1, 2 and 3. An ampule containing a solution of 1.0 mmol of the dienophile and the required amount of diene in 0.5 mL of dry benzene was cooled in liquid nitrogen, sealed, and then heated in an oil bath. After the reaction time was completed, it was cooled once more in liquid nitrogen and opened. The solution was evaporated and the residue purified by column chromatography on silica gel or alumina using hexane/ethyl acetate mixtures as eluent. 3-methyldibenzofuran (5a): 1H NMR (CDCl3) δ: 1.56 (s, 3H), 6.91 (dd, J= 7.6 Hz J= 2.1Hz, 1H), 7.58 (d,

(7): δ: 0.20 (s, 6H), 1.05 (s, 9H), 6.90 (dd, 1H, J= 8.0 Hz, J=1.9 Hz), 6.93 (d, 1H; J=3.8Hz), 7.78-7.80 (m, 2H); 7.98 (d, J=7.7Hz, 1H); 8,21(dd, J= 7.6, J=1.2, 1H), 8.43 (dd, J=7.8, J= 1.3, 1H); 13C NMR (CDCl3) δ:-4.4, 18.7, 25.7, 109.3, 111.6, 112.0, 114.1, 120.5; 122.2; 125.2; 126.6; 138.1; 145.5; 152.2; 154.0. HRMS m/z 298.4582 (Calcd C18H22O2Si 298.4574).

Acknowledgments We are indebted to the Universidad Nacional del Litoral (UNL), Santa Fe, República Argentina. This work received finatial support from the Science and Technology Secretariat, UNL, CAI+D Program (Projects 2009, 12/H618 y 12/H619)

References [1]

[2]

J= 2.0 Hz, 1H), 7.78-7.80 (m, 2H); 8.20 (dd, J= 8.4 Hz J= 1.1 Hz, 1H), 8.42 (d, J= 7.9 Hz, 1H), 8.56 (dd, J= 8.2 Hz, J=1.5 Hz, 1H), 13C NMR (CDCl3) δ: 23.6; 111.6; 111.8; 113.5; 121.2; 124.8; 125.9, 132.0; 132.8; 136.6,

[3]

146.5; 156.2. HRMS m/z 182.2226 (Calcd C13H10O 182.2218). 2-methyldibenzofuran (5b): 1H NMR (CDCl3) δ: 1.56 (s, 3H); 6.95(dd, J=7.7 Hz, 1.2 Hz, 1H ), 7.71 (d, J= 2.1Hz, 1H); 7.78-7.80 (m, 2H ); 8.18 (d, J= 7.8 Hz, 1H ), 8.20 (dd, J= 7.6 Hz, J= 1.2 Hz, 1H), 8.45 (dd, J=7.8 Hz, J= 1.3 Hz, 1H)), 13C NMR (CDCl3) δ: 23.5; 111.7; 114.5; 120.8; 122.5; 123.6; 124.0; 126.3; 132.7; 135.6; 146.5; 155.2. HRMS m/z 182.2224 (Calcd C13H10O 182.2218). 5.3 Dibenzofuran (6): Spectral Data Identical with Those of Commercial Material Terc–butyl (dibenzofuran-2-yloxy) dimethylsilane

[4]

[5]

[6]

(a) C. Della Rosa, M.N. Kneeteman, P.M.E. Mancini, Comparison of the reactivity between 2- and 3-nitropyrroles in cycloaddition reactions. A simple indole synthesis, Tetrahedron Lett. 48 (2007) 1535-1538. (b) C. Della Rosa, M.N. Kneeteman, P.M.E. Mancini, 2-Nitrofurans as dienophile in Diels-Alder reactions, Tetrahedron Lett. 48 (2007) 7075-7078. E. Paredes, B. Biolatto, M. Kneeteman, P.M.E. Mancini, Tetrahedron Lett. 43 (2002) 4601-4603. (b) E. Paredes, B. Biolatto, M. Kneeteman, P.M.E. Mancini, Nitronaphthalenes as Diels-Alder dienophiles, Tetrahedron Lett. 41 (2000) 8079-8082. E. Paredes, R. Brasca, M. Kneeteman, P.M.E. Mancini, A novel application of the Diels-Alder reaction: nitronaphthalenes as normal demand dienophiles, Tetrahedron 63 (2007) 3790-3799. C. Della Rosa, E. Paredes, M.N. Kneeteman, P.M.E. Mancini, Behaviour of thiophenes substituted with electron-withdrawing groups in cycloaddition reactions, Lett. Org. Chem. 1 (2004) 369-371. Z. Liu, R. Larock, Synthesis of carbazoles and dibenzofurans via cross-coupling of o-iodoanilines and o-iodophenols with silylaryl triflates, Organic Lett. 6 (2004) 3739-3741. (a) T. Kokubun, Harborne, J. Eagles, P. Waterman, Four dibenzofurans phytoalexins fron the sapwood of Mosfilus, Phytochemistry 39 (1995) 1039-1042; (b) R. Miller, R. Kleiman, R. Powell, Germination and Growth Inhibitors of Alfalfa, J. Natural Products 51 (1988) 328-330; (c) M. Hussain, N.T. Hung, P. Langer, Efficient synthesis of functionalized dibenzofurans by domino “twofold Heck/6π electrocyclization” reactions of 2,3-di- and


[7]

[8]

[9] [10]

[11]

[12]

[13]

[14]

2,3,5-tribromobenzofurans, Tetrahedron Lett. 50 (2009) 3929-3932. (a) E. Wenkert, P.D.M. Moller, S. Piettre, Five-membered aromatic heterocyclic as dienophiles in Diels-Alder reactions. Furan, pyrrole and indole, J. Am. Chem. Soc. 110 (1988) 7188-7194; (b) E. Wenkert, S. Piettre, Reaction of alpha- and beta-acylated furans with conjgated dienes, J. Org. Chem. 53 (1988) 5850-5853. N. Chopin, H Gérard, I. Chataigner, S.R. Piettre, Benzofurans as efficient dienophiles in normal electron demand [4 +2] cycloadditions, J. Org. Chem. 74 (2009) 1237-1246. R. G. Parr, L. Von Szentpaly, S. Liu, Electrophilic index, J. Am. Chem. Soc. 121 (1999) 1922-1924. (a) L.R. Domingo, M.J. Aurell, R. Contreras, Quantitative characterization of the global electrophilicity power of common diene/dienophile pairs in Diels-Alder reactions, Tetrahedron 58 (2002) 4417; (b) P. Pérez, L.R. Domingo, M.J. Aurell, R. Contreras, Quantitative characterization of the global electrophilicity pattern of some reagents involved in 1,3-dipolar cycloaddition reactions, Tetrahedron 59 (2003) 3117-3125. R.G. Parr, W. Yang, Density functional approach to the frontier-electron theory of chemical reactivity, J. Am. Chem. Soc. 106 (1984) 4049-4050. (a) P. Fuentealba, P. Pérez, R. Contreras, On the Condensed Fukui function, J. Chem. Phys. 113 (2000) 2544-2551; (b) R.R. Contreras, P. Fuentealba, M. Galván, P. Pérez, A direct evaluation of regional Fukui function in molecules, Chem. Phys. Lett. 304 (1999) 405-413. P. Fuentealba, R. Contreras, Fukui function in chemistry, Reviews of Modern Quantum Chemistry, World Scientific, 2002. L. R. Domingo, M. J. Aurell, P. Pérez, R. Contreras, Quantitative characterization of the local electrophilicity of organic molecules, Understanding the regioselectivity

[15]

[16]

[17]

[18]

[19] [20]

[21]

[22]

59

on Diels-Alder Reactions, J. Phys. Chem. A 106 (2002) 6871. P. Pérez, L.R. Domingo, M. Duque-Noreña, E. Chamorro, A condensed-to-atom nucleophilicity index. An application to the director effects on the electrophilic aromatic substitution, J. Mol. Struct. (THEOCHEM), 895 (2009) 86-91. (a) L.R. Domingo, E. Chamorro, P. Pérez, Understanding the reactivity of captodative ethylenes in polar cycloaddition reactions, a theoretical study, J. Org. Chem. 73 (2008) 4615-4624; (b) P. Jaramillo, L.R. Domingo, E. Chamorro, P. Pérez, A further exploration of a nucleophilicity index based on the gas-phase ionization potentials, J. Mol. Struct. (THEOCHEM) 865 (2008) 68-72. L.R. Domingo, J.A. Saéz, R.J. Zaragozá, M. Arnó, Understanding the participation of cuadricyclanes as nucleophile in Polar [2σ + 2σ + 2π] cycloadditions toward electrophilic π molecules, J. Org. Chem. 73 (2008) 8791-8799. (a) A. D. Becke, J. Chem. Phys. 98 (1993) 5648; (b) C. Lee, W. Yang, R.G. Parr, Development of the Colle-Salvetti correlation-energy formula into a functional of the electron density, Phys. Rev. B 37 (1988) 785-789. M. J. Frisch., Gaussian 03, Revision B.04, Gaussian Inc., Pittsburgh, PA, 2003. A.R. Katritzky, A.V. Vakulenko, J. Sivapacki, B. Draghici, R. Damavarapu, Synthesis of dinitro-substituted Furans, Thiophenes, and Azoles, Synthesis 5 (2008) 699-706 W. Oppolzer, L. Bieber, E. Francotte, An improved, versatile preparation of trans-N-alkyl-1-amino-1,3-dienes, Tetrahedron Lett. (1979) 981-984. S. Kozmin, J. Janey, V. Rawal. 1-Amino-3siloxy-1,3-butadienes: highly reactive dienes for the Diels-Alder reaction, J. Org. Chem. 64 (1999) 3039-3052.


The Synthesis of Pyridine 2,6-dicarboxylic Acid Using Microwave Irradiation Guofu Zhang1, Qing Zhang1, Hairui Zhang2, Lin Bai1, Helin Ye1 and Lingling Liu1 1. School of Chemistry and Environmental Science, Lanzhou City University, Lanzhou 730070, China 2. College of Information Enginnering, Lanzhou city University, Lanzhou 730070, China

Received: July 16, 2010 / Accepted: August 03, 2010 / Published: November 30, 2010. Abstract: This research took 2,6-dimethylpyridine as raw materials under microwave irradiation to syntheses pyridine 2,6-dicarboxylic acid by adding oxidation potassium permanganate. However, there are lots of factors affecting the yield including the amount of potassium permanganate and sulfuric acid, reaction time, power, 2,6-dimethylpyridine dosage. We made the further research, using orthogonal experiment to find the optimal process conditions. Thus our research changed the synthesis process from a traditional method to a new type of microwave technology. Key words: 2,6-dimethylpyridine, pyridine 2,6-dicarboxylic acid, potassium permanganate, microwave, synthesis.

1. Introduction In this paper, the synthesis of pyridine, 2,6-dicarboxylic acid under microwave irradiation is simple , high yield, short reaction time, etc. It is a better way of the synthesis of pyridine 2,6-dicarboxylic acid. Pyridine 2,6-dicarboxylic acid, known as 2,6-pyridinedicarboxylate, (molecular formula: C7H5NO4), referred to as H2DPC, H2DPC in vivo which is used as is biologically active material. Pyridine 2, 6-dicarboxylic have many advantages, which are widely applied in the field of medicine. It can form stable complexes with gallium, indium ions. And three gallium complex (NaGaL2xH2O, NaGaL2x3H2O, NaGaL2x2H2O) significantly inhibited Gram-positive bacteria activities [1], which can be a foundation for further study in vitro, in vivo anti-cancer trials, and provides important theoretical information to development new anticancer drugs. The poly 2,6-pyridine-dicarboxylic acid/multi-walled carbon nanotubes can be used for composite modified electrode [2]. Corresponding author: Guofu Zhang, professor, research fields: spectrometry, electroplating and environment protecting. E-mail: [email protected].

In recent years, to understand the foundation of H2DPC complexes in vivo, it has attract great interests [3]. H2DPC is very interesting and flexible rigid ligands, which has been called “universal ligand” in the world [4]. It can form a stable chelate complex and coordinated manner eclectic with the transition, non-transition, lanthanide and actinide metal ions [5]. The way may be either bidentate, tridentate, or be bridged ligand, which can act as a bridge abutment with two metal ions. As a binary acid, H2DPC in coordination has three forms, namely two-anion (DPC2-), monoanion (HDPC-) and neutral molecules (H2DPC). Pyridine dicarboxylic acid has another important function of their biological activity. Because gallium has anti-cancer, anti-tumor activity, gallium pyridine acid complex may be a very promising new drug. Based on these facts, X.Y. Zhang et al. [6] synthesized six novel pyridine dicarboxylic acid (2,3-, 2,5-, 2,6-) gallium (III), Indium (III) complex, and tested the biological activity of the three types of gallium complex. As the price of pyridine 2,6-dicarboxylic acid is very high, the traditional oil bath heating method must

The Synthesis of Pyridine 2,6-dicarboxylic Acid Using Microwave Irradiation

cost 3.5 hours under the conditions, particularly time-consuming, the study prepared in the microwave conditions, to make pyridine-2, 6-dicarboxylic acid was necessary. This research work will give us an efficiency and low cost method.

2. Preparation of Pyridine 2,6-dicarboxylic Acid Compounds 2.1 Conventional Methods The earliest document on the methods to the reparation of H2DPC is in University of Wisconsin, 1935, Alvin. W. Singer and S.M. Mcelvain used KMnO4 via water as a solvent to oxidation of 6-dimethyl-pyridine. The yield of this method was only 64%. As the 2, 6-dimethyl-pyridine prices is high, the traditional oil bath heating method must cost a few hours under the conditions, particularly time-consuming therefore, in order to enhance its synthesis conversion rates, reduce return time to study the preparation of pyridine 2,6-dicarboxylic acid under microwave irradiation is necessary. H2SO4

4 KMnO4 H3C

N

Scheme 1

CH3

heat

KOOC

N

COOK

HOOC

N

COOH

synthesis steps.

The amount of KMnO4 in this method is 1.1 times than the theoretical amount, and the amount is divided into two portions, then in the beginning half of the reaction 30 min to add the first portion, and in the end add the second half. H2O dosage (mL) was 11.3 times than the amount of KMnO4, without stirring at 100 °C for 2 h, the yield reached 64%. 2.2 Improved Synthesis Methods In Guangxi Normal University, L.F. Tan et al. [7] synthesized pyridine 2,6-dicarboxylic acid with 2,6-lutidine as the raw material by KMnO4 oxidation. Meanwhile, the factors affecting productivity: KMnO4 and H2O dosage, reaction time and reaction temperature were discussed. They found the optimum conditions for the production rate through orthogonal experiment from 64% reported to 81%.

61

The method of optimal conditions is adding KMnO4 by 1.4-fold theoretic mass once, H2O dosage is 9.4 times the amount of KMnO4, without stirring, at 110 °C for 3.5 h, yield 81%.

3. Experiments 3.1 Reagents and Solvents 2,6-dimethylpyridine(AR), Potassium permanganate (AR), Sulfuric acid (AR), commercially available; Distilled water is self-made. 3.2 Laboratory Instruments Microwave oven (Galanz WP 750B), frequency 2450 MHz, power 750 W; Digital melting point apparatus (WRS-1A); Nicolet 5700 FR-IR Fourier transform infrared spectrophotometer, KBr tablet press (United States); SHANGPING FA 1004 electronic balance (division value 0.1mg, Beijing Sartorius Instrument Co.); Nuclear magnetic resonance spectroscopy with Avanci-D2X-200 type NMR determination; elemental analysis values from Vario El elemental analyzer. 3.3 Experimental Steps Add 0.50 g 2, 6-dimethyl pyridine, 2.30 g of potassium permanganate and 20.0 mL of distilled water in a 250 mL round bottom flask once. In a microwave oven, take a good casing and condenser, set the microwave radiation power of 300 W, start the microwave, radiation reaction 30 min, after reaction, when reaction is complete, filtered to remove manganese dioxide when hot. In the hot filtrate added 3.0 mL 70% sulfuric acid, cooled in ice bath 30 min, 2,6 pyridine-dicarboxylic acid precipitated needle, obtained crude product. Recrystallized twice with distilled water to obtain a more pure white needle-like product of pyridine 2,6-dicarboxylic acid, the yield was 85-90% (Ref. [7] 81%). With a digital melting point apparatus (WRS-1A), its melting point (mp) was 231.0 ~232.4 °C (mp. 227~228 °C in Ref. [7]), and it’s accord with the reference.

62


Fig. 1 IR Spectrum of 2,6 pyridine - dicarboxylic acid.

3.4 Characterization IR spectra (Fig. 1), at the 1680 cm-1 occurring the C=O characteristic absorption peaks, at 1550 cm-1 appears the skeleton of the benzene ring stretching vibration peaks. The characteristic absorption peak which appears at 3200 cm-1 can be attributed to OH absorption peak, consistent with the Ref. [8], to prove the product is pyridine 2,6-dicarboxylic acid. Element analysis of the observed: C, 50.04%; H, 4.96%; O, 38.79%; N, 8.06%. Theoretical value: C, 50.31%; H, 5.04%; O, 38.30%; N, 8.38%.

4. Results and Discussion In the experiment, pyridine 2,6-dicarboxylic acid was synthesized by using of microwave radiation method. The main factors affected the results: potassium permanganate and 2,6-dimethylpyridine material ratio, the microwave irradiation time, microwave power sulfuric acid dosage etc. And our research group used the orthogonal experiments to decide the better reaction conditions. 4.1 Radiation Power on the Yield Reaction conditions were same to the experimental procedures, microwave irradiation time fixed to 30 min

Table 1 Microwave power on the reaction. Serial No 1 2 3 4 5 6

Reaction time(min) 30 30 30 30 30 30

Radiation power (W) Yield % 225 40.0 300 67.0 375 86.3 450 83.2 600 74.4 675 57.0

and the reactant ratio fixed to 1:1.5, to change the microwave power, radiation power obtained different reaction yield, the results shown in Table 1. The results showed that: a fixed irradiation time and the ratio of reactant materials, the yield increases to 375 W maximum with the microwave radiation power. When the reaction of the radiated power over 600 W, the reaction temperature is too high for some charcoal black so that the yield reduction, therefore, the best radiation power was 350 W, pyridine 2,6-dicarboxylic yield was the highest. 4.2 Microwave Radiation Time on the Reaction Yield Fixed radiation power 375 W, reactant materials ratio of 1:1.5, to change the microwave irradiation time, obtained different reaction yields, the results shown in Table 2. The results showed that: the fixed radiation power


Table 2 Microwave radiation time on the reaction yield. Serial No Radiation power(W) 1 375 375 3 375 4 375 5 375 6 375

Reaction time(min) 15 20 25 30 35 40

Yield/% 34.5 53.6 65.1 77.4 86.2 85.3

and reactant ratio, the yield increased with time, to the maximum time 30 min, the reaction of radiation for more than 39 min, the system temperature is too high for some charcoal black thus yield decreased, therefore, the optimum irradiation time was 30 min, pyridine 2,6-dicarboxylic acid yield was the highest. Radiation time was less than 30 min, incomplete oxidation, and the reaction time more than 39 min,

Table 3 reaction. Serial No. 1 2 3 4 5 6

63

Dosage of potassium permanganate on the Mass ratio of 2,6-lutidine to potassium permanganate /g 1:1.0 1:1.1 1:1.2 1:1.3 1:1.4 1:1.5

Yeild/% 54.2 62.5 71.4 85.6 85.2 85.0

Table 4 Dosage of sulfuric acid on the reaction. Serial No. 1 2 3 4 5 6

70%dosage of sulfuric acid(mL) 2.0 2.5 3.0 3.5 4.0 4.5

Yield (%) 58.4 69.2 85.6 85.4 80.3 71.7

increased side effects. 4.3 Dosage of Potassium Permanganate on the Reaction

acid, obtained the yield under different amount, the results shown in Table 4.

Reaction conditions were same to the experimental

The results showed that: the fixed microwave irra-

procedures, fixed microwave power 375 W, microwave

diation time, microwave power and reactant materials

irradiation time was 30 min, fixed 2,6–dimethylpyridine

ratio, the yield with the increase of sulfuric acid to

mass to 0.5000 g, change the amount of potassium

reach the maximum of 3 mL, more than 4.0 mL, the

permanganate, the yield in the reaction under different

reaction yield begins to decrease, therefore, the amount

quality, the results shown in Table 3.

of sulfuric acid should be appropriate. The results sh-

The results showed that: the fixed radiation power,

owed that: 70% of the dosage of 3.0 mL sulfuric acid,

and radiation time, the yield with the increase of the

the pyridine 2,6-dicarboxylic acid yield was the highest.

amount of potassium permanganate, when 2,6-lutidine

5. Conclusions

with potassium permanganate mass ratio 1:1.5, the yield reached the maximum; when more than 1:1.5,

The right experiment conditions with a microwave

the yields no significant increase, thus the choice of

oven, 2,6-dimethylpyridine, potassium permanganate

2,6-lutidine and potassium permanganate mass ratio of

and distilled water, and sulfuric acid in our research

1:1.5, pyridine-2,6-dicarboxylic acid reached the

work are better than that of microwave radiation

maximum yield rate.

preparation of pyridine 2,6-dicarboxylic acid. Its yield can reach 85% -86%. This method is simple, high

4.4 Dosage of Sulfuric Acid on the Reaction

yield, short reaction time etc. And it is a better method

Reaction conditions were same to the experimental procedures, fixed microwave radiation power 375 W, microwave irradiation time was 30 min, the mass ratio of 2,6-lutidine and potassium permanganate was 1:1.5. Take 2,6-dimethylpyridine 0.5000 g, potassium permanganate 0.800 g, change the amount of sulfuric

of synthesis of pyridine 2,6-dicarboxylic acid. We can predict microwave chemistry in the various branches such as the chemical and pharmaceutical fields, has broad application prospects in the future [9]. For its research, there is still a wide space. We need a long way to go.


64

References [1]

[2]

[3]

[4]

X.Y. Zhang, S.J. Li, Pyridine acid gallium, indium Synthesis and biological activity, Chemical Research and Application 17 (2005) 772-776. (in Chinese) C.X. Li, Y.L. Zen, Poly 2,6-pyridine dicarboxylic acid/multi-walled carbon nanotube modified electrode and its electrocatalytic property, Analytical Chemistry 6 (2006) 45-49. (In Chinese) T.Z. Jin, J.K. Liu, H.Z. Zhang, RE amine acid complexes II Pyridine-2, 6-dicarboxylate La complex Synthesis and crystal structure, Acta of Rare Earth 9 (1991) 97. (In Chinese) D.P, Murtha, R.A. Walton, Metal carboxylates. IV. Mixed ligand com plexes of copper(II) con ta in ing pyridine-2,6-dicarboxylic acid in its monoanionic and dianionic forms, Inorganica Chimica Acta 8 (1974) 279-284.

[5]

[6]

[7]

[8] [9]

I.E. Erikson, Gren the I Puegdom enech, A kinetic investigation of canthanide(III)com plex form ation with picolinic acid, Inorganica Chimica Acta 126 (1987) 131-135. X.Y. Zhang, S.J. Li, Pyridine acid gallium, indium Synthesis and biological activity, Chemical Research and Application 17(2005) 772-776. (in Chinese) L.F. Tan, Y.M. Jiang, F.X. Wei, Research on the synthesis of Pyridine 2,6-dicarboxylic acid, Guangxi Chemical 27 (1998) 10-12. (In Chinese) M.H. Zhu, Instrumental Analysis, 3rd ed., Beijing Higher Education Press, 2002, pp. 294-303. (In Chinese) C.L. Ma, Y.H. Zhang, 2,6-pyridine dicarboxylic acid and thio 2,2-chloro-benzoic acid methyl triethyl amine synthesis of several quaternary ammonium salts and crystal structure, Applied Chemistry 20 (2003) 296-298. (In Chinese)