Simultaneous Determination of Fatty Acid Methyl Esters Contents in the Biodiesel by HPLC-DAD Method

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1 2016 International Conference on Applied Mechanics, Mechanical and Materials Engineering (AMMME 2016) ISBN: Simultaneous Determination of Fatty Acid Methyl Esters Contents in the Biodiesel by HPLC-DAD Method Yan-gang HAN 1,2,*, Yan LU 1, Shang-fu LIAO 1, Hong-bo CHEN 2, De-qing YU 1, Ming YANG 1 and Zheng-jie MO 1 1 Zhejiang Fangyuan Test Group Co., Ltd., Xiasha Road 300#, Hangzhou , China 2 Zhejiang Institute of Quality Inspection Science, Xiasha Road 300#, Hangzhou , China *Corresponding author Keywords: Fatty acid methyl ester, Biodiesel, HPLC analysis, DAD. Abstract. This paper presents a reliable method for simultaneous analysis of ten kinds of fatty acid methyl esters in biodiesel with high performance liquid chromatography equipped with diode array detector. The separation column was a Pursuit XRs C18 (250 mm 4.6 mm) and the mobile phase was a mixture of methanol and acetonitrile (80:20, V/V). The analytical performance parameters such as linearity, accuracy, precision, limit of detection and limit of quantification were discussed. The calibration curves of ten components show good linearity with the correlation coefficient greater than The proposed method is time and cost-effective, which could be used for the determination of the FAMEs in the commercial biodiesel product. Introduction With the increasing depletion of traditional petroleum resources and the improvement consciousness of environmental protection, biodiesel has become the focus of development in a steadily growing number of countries around the world because of its potential advantages in energy conservation and air pollution reduction [1-5]. According to its chemical nature, biodiesel is mainly composed of several kinds of fatty acid methyl esters (FAMEs) such as myristic acid methyl ester, palmitic acid methyl ester, stearic acid methyl ester, behenic acid methyl ester and so on [6]. It is stipulated in Chinese National Standard GB/T <Biodiesel Blend Stock (BD100) for Diesel Engine Fuels> that the total content of esters should be no less than 96.5%, and the testing method cites that in Standard NB/SH/T 0831<Test method for determination of fatty acid methyl esters and linolenic acid methyl ester contents in biodiesel by gas chromatography>. However, the composition and content of biodiesel produced from diverse raw materials are different, which directly affect the applications of biodiesel and its subsequent products [7,8]. So it is very important to accurately determine the composition and content of fatty acid methyl esters in biodiesel, which is critical to the quality control of biodiesel and its subsequent products [9]. Gas chromatography (GC) has the advantage of high sensitivity, but has some limitations in the determination of samples with large molecular weight, high boiling point or low volatility [10]. In this paper, ten kinds of fatty acid methyl esters in biodiesel were determined simultaneously using high performance liquid chromatography (HPLC) with diode array detector (DAD). Experimental Instrumentation The HPLC system (model Agilent 1200) equipped with a silica-based column model Pursuit XRs C18 (250 mm 4.6 mm) was used. The HPLC system has a quaternionic pump, an autosampler, and a DAD (model Agilent G1315C). The DAD could supply the UV spectrum of nm for every component in the HPLC separation.

2 Reagents and Solutions Methanol and acetonitrile were purchased from Tedia High Purity Solvents Co., Ltd. All solvents were of HPLC-grade and were used as obtained, without further purification. Ultrapure water was obtained from a Milli-Q Direct Water Purification System. FAME Standards The standards of ten kinds of FAMEs, including myristic acid methyl ester (C14:0), myristoleic acid methyl ester (C14:1), palmitic acid methyl ester (C16:0), stearic acid methyl ester (C18:0), oleic acid methyl ester (C18:1), linoleic acid methyl ester (C18:2), linolenic acid methyl ester (C18:3), arachidic acid methyl ester (C20:0), behenic acid methyl ester (C22:0) and erucic acid methyl ester (C22:1), were purchased from ANPEL Laboratory Technologies (Shanghai) Incorporated. These FAME standard solutions were prepared at the predetermined concentrations (100 µg/ml for every component) by dilution with methanol for HPLC analysis. Heptadecanoic acid methyl ester (C17:0) with the concentration of 50 µg/ml was used as the internal standard. HPLC Conditions The column temperature was kept constant at 35 o C. The mobile phase was a mixture of methanol and acetonitrile (80:20, V/V) at a flow rate of 1 ml/min. The injection volume of the samples was set at 10 µl. The detection wavelength was set at 210 nm. FAME standards and biodiesel samples were analyzed in duplicate to confirm the observations. Peaks of the samples were identified by comparing their UV spectra with those of FAME standards. Results and Discussion Chromatograms of Standard FAMEs The chromatograms of standard FAMEs under optimized conditions are shown in Figure 1. A good separation with distinct peak shapes was achieved within 35 min. Linearity Figure 1. HPLC chromatograms showing separation of standard FAMES. To determine linearity, FAME standard solutions were diluted at concentrations of 50, 100, 200, 500 and 1000 µg/ml. Every calibration solution was injected into HPLC in triplicate. The calibration curve was prepared by plotting the peak area against the concentration of the compound. Linearity was evaluated by linear regression analysis. Excellent linearity was observed in Table 1. The regression coefficients of the calibration curves obtained for all the FAMEs were more than 0.99, thereby confirming the linearity of the developed method.

3 Table 1. Regression parameters of the calibration curves (n=3). Component Linear equation Linear range (µg/ml) Correlation coefficient (R 2 ) C14:0 y= x C14:1 y= x C16:0 y= x C18:0 y= x C18:1 y= x C18:2 y= x C18:3 y= x C20:0 y= x C22:0 y= x C22:1 y= x Limits of Detection and Quantification Limits of detection (LOD) and quantification (LOQ) were calculated as follows: LOD=3 S/N LOQ=10 S/N where: S/N is the signal to noise ratio. The results for LOD and LOQ are listed in Table 2, which clearly indicate that the method has good sensitivity. Table 2. LOD and LOQ results of ten kinds of FAMEs. Component LOD (µg/ml) LOQ (µg/ml) C14: C14: C16: C18: C18: C18: C18: C20: C22: C22: Precision and Accuracy The recoveries were determined by the method of standard addition. Two known concentrations of each of the ten standards were spiked into the sample. Six replicate analyses were run for all the ten standards at defined concentrations. The precision was expressed in terms of RSD. The accuracy was expressed as the percentage of the analyte concentration measured in each sample relative to the known amount of the analyte spiked to the sample. The precision and accuracy data are listed in Table 3. The proposed method was found to be precise and accurate.

4 Table 3. Precision and accuracy data of ten kinds of FAMEs (n=6). Component Spiked level (µg/ml) Recovery (%) RSD (%) C14: C14: C16: C18: C18: C18: C18: C20: C22: C22: Method Application The proposed method was used to evaluate the contents of ten kinds of FAMEs in the commercial biodiesel obtained from Zhejiang Eastriver Energy S&T Co., Ltd.. The biodiesel was pretreated by water extraction and filtration with 0.45 µm organic membrane, and injected into HPLC for analysis. The results are presented in Table 4. Table 4. FAMEs contents in the commercial biodiesel. Component Content (%) C14: C14: C16: C18: C18: C18: Summary Ten kinds of FAMEs in biodiesel could be analyzed simultaneously by HPLC-DAD method. The analytical procedure has a 35 min chromatographic run time, which allows the analysis of a large number of samples in a short period of time. The calibration curves were linear over the concentration range. The methodology was also accurate and precise as observed from the recovery and RSD values, which made it reliable and practical for quantification of various FAMEs in biodiesel. References [1] A. Demirbas, I. Demirbas, Importance of rural bioenergy for developing countries, Energy Conversion and Management 48 (2007) [2] M. Balat, Production of biodiesel from vegetable oils: a survey, Energy Sources Part A 29 (2007) [3] Z.L. Zhang, J.B. Ji, Research progress of feedstocks and deep-processing technologies for biodiesel, Chemical Industry and Engineering Progress 33 (2014) [4] L.C. Meher, S.D. Vidya, S.N. Naik, Technical aspects of biodiesel production by transesterification a review, Renewable & Sustainable Energy Reviews 10 (2006) [5] A. Demirbas, New biorenewable fuels from vegetable oils, Energy Sources Part A 32 (2010)

5 [6] Y. Li, J. Zeng, W. Du, D.H. Liu, Progress and prospect of biodiesel industry in China, Chinese Journal of Biotechnology 31 (2015) [7] S.N. Shi, L.Y. Jin, R.M. Pei, C.Y. Liu, C.Z. Qiao, Analysis of fatty acid methyl ester in the biodiesel, Chemical Research 27 (2016) [8] X.C. Wang, J.H. Fang, B.S. Chen, E.Y. Chen, L. Chen, J. Wang, Effect of unsaturated fatty acid methyl esters on oxidation and lubrication performance of diesel engine oil, China Oils and Fats 40 (2015) [9] M.M. Wu, W.B. Zhang, Analysis of fatty acid methyl ester of three kinds of biodiesel by GC/MS, Journal of Fuzhou University (Natural Science Edition) 5 (2014) [10] M.M. Fan, W.T. Mao, J. Yang, P.B. Zhang, Qualitative and quantitative analysis on fatty acid methyl ester (biodiesel), Chemical Industry and Engineering Progress 31 (2012)

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