Energy Fuels 2010, 24, 6131–6141 Published on Web 11/04/2010
: DOI:10.1021/ef1009082
Simple and Efficient Method for the Analysis of Transesterification Reaction Mixtures for Biodiesel Production by Reversed-Phase High Performance Liquid Chromatography Stephen N. Csernica and James T. Hsu* Department of Chemical Engineering, Lehigh University, 111 Research Drive, Bethlehem, Pennsylvania 18015, United States Received July 15, 2010. Revised Manuscript Received September 27, 2010
Isocratic reversed-phase high performance liquid chromatography (HPLC) methods have been developed for the analysis of biodiesel methyl esters and intermediate species associated with transesterification reactions. While most HPLC methods for biodiesel analysis employ gradient elution and ultraviolet (UV) detection, these methods employ isocratic elution with refractive index detection (RID). For the separation of methyl esters, monoglycerides, and free fatty acids, a mobile phase of 85% acetonitrileþ15% deionized water is used. Diglycerides can be separated using 100% methanol, and triglycerides can be quickly analyzed using a mixture of 60% 2-propanolþ40% methanol. While previous HPLC methods for methyl ester analysis have been shown to successfully separate components found in biodiesel mixtures, the use of UV detection is inadequate for the detection of species with saturated carbon chains. Many of the common feedstocks used for biodiesel synthesis contain a significant percentage of species with saturated carbon chains, for example, soybean, sunflower, palm, and palm kernel oils. The use of RID, however, allows for both saturated and unsaturated methyl esters to be detected. Additionally, the use of HPLC for the analysis of biodiesel methyl esters and the intermediate species associated with transesterification reactions, as opposed to the more commonly employed gas chromatography (GC) method, has the advantage that samples can be directly analyzed without any sort of sample derivatization, as is required when GC is used.
The intermediate steps, consisting of the three consecutive reactions previously mentioned, proceed as follows:
Introduction The demand for alternative and sustainable forms of energy has increased significantly over the past decade. Biodiesel has emerged as a serious potential to replace petroleum-derived diesel fuel due to its reduced emissions of carbon monoxide, sulfur dioxide, unburned hydrocarbons, soot, and particulate matter.1 The term biodiesel refers to the alkyl esters of fatty acids produced from vegetable oils, animal fats, and recycled greases when used as fuel for diesel engines.2,3 Biodiesel is often blended with petroleum-derived diesel fuel but can be used neat. Biodiesel is most commonly produced by the transesterification of acylglycerols with an alcohol, usually methanol, in the presence of an alkaline catalyst.2,3 Feedstocks containing high free fatty acid content, such as waste greases, typically require a two-step acid-alkali process to convert them to biodiesel.2,3 More recently, noncatalytic processes utilizing supercritical alcohols have been employed.4 Under supercritical conditions, both free fatty acids and acylglyercols are converted to biodiesel. When vegetable oils are used as the feedstock, three consecutive reactions give rise to the formation of the fatty acid esters. The overall transesterification reaction proceeds as follows:
Triglyceride þ R0 OHTR1 COOR0 þ Diglyceride Diglyceride þ R0 OHTR2 COOR0 þ Monoglyceride Monoglyceride þ R0 OHTR3 COOR0 þ Glycerol On the basis of the above reactions, it is clear that one mole of triglyceride will yield three moles of fatty acid esters and one mole of glycerol as a byproduct. Additionally, triglycerides from oils can be hydrolyzed with enzymes to yield free fatty acids. The pure fatty acids can then be used as a feedstock for biodiesel production. When free fatty acids react with alcohols to form fatty acid alkyl esters, the reaction is called esterification. The esterification reaction for a given free fatty acid with an alcohol is as follows: RCOOH þ R0 OHTRCOOR0 þ H2 O Esterification reactions yield one mole of fatty acid ester and one mole of water as a byproduct.5 Regardless of the feedstock used or the transesterification/ esterification process employed, the final product must meet the requirements of biodiesel fuel standards, which have recently been established in both the United States (ASTM D6751) and Europe (EN14214).2,3,6 Both standards specify the tolerable limits of free and bound glycerol (i.e., mono-, diand triglycerides) allowed in the final biodiesel product. The limits set by each standard can be found in Table 1. Residual
Triglyceride þ 3R0 OHT3R0 COOR þ Glycerol *To whom all correspondence should be addressed. Phone: (610) 7584257. Fax: (610) 758-5057. E-mail:
[email protected]. (1) Fukuda, H.; Kondo, A.; Noda, H. J. Biosci. Bioeng. 2001, 92, 405–416. (2) Fogila, T. A.; Jones, K. C.; Nunez, A.; Phillips, J. G.; Mittelbach, M. Chromatographia 2004, 60, 305–311. (3) Foglia, T. A.; Jones, K. C.; Phillips, J. G. Chromatographia 2005, 62, 115–119. (4) Saka, S.; Kusdiana, D. Fuel 2001, 80, 225–231. r 2010 American Chemical Society
(5) Tesser, R.; Casale L.; Verde, D.; Di Serio, M.; Santacesaria, E. Chem. Eng. J. 2010, 157, 539-550. (6) McCurry, J. D.; Wang, C. X. Analysis of Glycerin and Glycerides in Biodiesel (B100) Using ASTM D6584 and EN14105. Agilent Technologies, 2007. (www.agilent.com/chem).
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UV detector set at 205 nm. This method provides good separation of many components found in biodiesel mixtures. The components were found to be retained with respect to increasing equivalent carbon number (ECN), defined as CN - 2DB, where CN is the total number of carbon atoms in the acyl chain(s) of a given acylglycerol and DB is the total number of double bonds in the chain(s).8 In most cases, the ECN is normally assumed to be representative of free fatty acids, mono-, di-, or triglycerides. In this study, however, ECN values were also assigned to fatty acid methyl esters. For clarification purposes, the ECN values of the free fatty acids, fatty acid methyl esters, monoglycerides, diglycerides, and triglycerides will be differentiated by ECNFFA, ECNFAME, ECNMG, ECNDG, and ECNTG, respectively. Despite the different variable names, however, the calculation of ECNi will be identical, that is ECNi =CN - 2DB. Using similar mobile phases, Di Nicola et al. optimized a binary gradient method for analyzing biodiesel mixtures.7,8 Five different parameters were optimized including total flow rate, gradient start time, gradient end time, compositions of mobile phases in the gradient, and the mixing proportion of the two components in the nonaqueous mobile phase. It was determined that the best gradient profile consisted of acetonitrilemethanol (4:1, v/v) at 0 to 2.2 min and then from 2.2 to 25.5 min a linear gradient up to 34% acetonitrile-methanol (4:1, v/v) þ 66% n-hexane-isopropanol (8:5, v/v), followed by isocratic elution to 30 min. The optimal flow rate was determined to be 1.3 mL/min. Detection was achieved using a UV detector set at 210 nm. While each of the above methods employing gradient elution has been successful in their ability to separate the components of biodiesel mixtures, they have all focused on UV detection. A major drawback to the use of UV detection for biodiesel mixtures is that some acylglycerols and methyl esters do not absorb in the UV region at wavelengths above 220 nm.8,9 Additionally, many species in biodiesel mixtures contain saturated carbon chains. These species provide very weak UV detection. A list of common oils used as biodiesel feedstocks and their fatty acid composition can be found in Table 2. Soybean oil is commonly used as a feedstock in the United States, this oil can contain up to 16% by mass species that contain saturated carbon chains. Furthermore, palm oil is commonly used as a feedstock in tropical regions and can contain greater than 50% by mass species with saturated carbon chains. To improve the sensitivity of detection of the saturated species, fatty acid derivatives can be formed.10 While useful, this procedure still requires the samples to be derivatized prior to analysis. Another possibility to overcome the absorbance problems associated with the UV detection of saturated species is to use refractive index detectors. These detectors, however, cannot be used with gradient elution, and therefore, isocratic elution must be used.11 The analysis of biodiesel mixtures using an RID has been shown effective using size-exclusion chromatography.12 When size-exclusion chromatography is used, however, all similar species have the same retention time. For example, all fatty acid methyl esters would have the same retention time; likewise, all triglycerides would have the same
Table 1. Free and Total Glycerol Specifications for Biodiesel component free glycerol monoglycerides diglycerides triglycerides total glycerol related compounds
EN14214 limit ASTM D6751 limit (% m/m) (% m/m) 0.02 max 0.80 max 0.20 max 0.20 max 0.25 max
0.020 max NA NA NA 0.240 max
glycerol, both free and bound, has been shown to aversely affect engine performance and emission properties of the fuel.6 All things considered, a reliable and sensitive analytical method is needed to quantify the tri-, di-, and monoglycerides as well as free glycerol in a biodiesel sample. High temperature capillary gas chromatography (GC) is widely used and forms the basis for the ASTM D6584 and EN14105 protocols for determining total glycerol related compounds in biodiesel.2,6 These chemical test methods outline procedures for sample preparation, instrument configuration, operating conditions, and reporting. The best advantage of the standardized GC methods is the use of flame ionization detectors (FID). The response of these detectors is linear and proportional to the number of carbon atoms in the molecule.7 Despite being the current industry standard, however, biodiesel analysis by GC requires samples to be derivatized before analysis to improve the constituents’ volatility as well as reduce their reactivity to therefore make the GC method feasible.6-8 Complicating the derivatization procedure is the derivatizing agent, N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA). According to the material safety data sheet (MSDS) for MSTFA, this compound can cause serious health issues, is extremely flammable, and reacts violently with water. In addition to the derivatization of the nonvolatile species, oven temperatures as high as 350 °C are specified to assist these species in reaching the gas phase. Oven temperatures this high can quickly reduce the life of a given gas chromatography column.9 Furthermore, both standardized methods ASTM D6584 and EN14105 were only developed for biodiesel produced from vegetable oils such as rapeseed, soybean, sunflower, and palm. When biodiesel is produced from less common sources, such as lauric acid oils like coconut and palm kernel oil, these methods are not suitable.6 High performance liquid chromatography (HPLC) makes feasible the direct analysis of all biodiesel components without derivatization.2,3,7-9 Numerous detection methods have been suggested ranging from ultraviolet (UV) or fluorescence detection, density detection, flame ionization detection (FID), refractive index detection (RID), evaporative light scattering detection (ELSD), and mass spectrometric detection.2,3,8 Many HPLC methods for biodiesel analysis have focused on gradient elution profiles.7-9 One of the most common and variable HPLC techniques is based on a linear ternary gradient consisting of aqueous-organic and nonaqueous mobile phase steps: 70% acetonitrile þ 30% water at 0 min, 100% acetonitrile at 10 min, 50% acetonitrile þ 50% isopropylhexane (5:4, v/v) at 20 min followed by 5 min of isocratic elution of the latter mixture.8 Detection was achieved using a
(10) Lin, J. T.; MeKeon, T. A.; Stafford, A. E. J. Chromatogr., A 1995, 699, 85–91. (11) Snyder, L. R.; Kirkland, J. J.; Dolan, J. W. Introduction to Modern Liquid Chromatography, 3rd ed.; John Wiley & Sons, Inc.: Hoboken, NJ, 2010. (12) Arzamendi, G.; Argui~ narena, E.; Campo, I.; Gandı´ a, L. M. Chem. Eng. J. 2006, 122, 31–40.
(7) Santori, G.; Arteconi, A.; Di Nicola, G.; Moglie, M.; Stryjek, R. Energy Fuels 2009, 23, 3783–3789. (8) Holcapek, M.; Jandera, P.; Fischer, J.; Prokes, B. J. Chromatogr., A 1999, 858, 13–31. (9) Di Nicola, G.; Pacetti, M.; Polonara, F.; Santori, G.; Stryjek, R. J. Chromatogr., A 2008, 1190, 120–126.
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Table 2. Typical Fatty Acid Composition of Common Oils Used as Biodiesel Feedstocksa fatty acid composition (mass %) oil canola17 palm18 rapeseed19 sunflower17 soybean18 a
C14:0 1 >1
C16:0
C18:0
C18:1
C18:2
C18:3
6 48 3 6 10
4 1 4 5
61 37 24 17 23
22 9 15 72 52
7 >1 8
C20:0
C20:1
C22:1
C24:1
13
35
1
8
Compositions may not sum to 100% due to rounding or the presence of trace amounts of other species.
retention time (different from the methyl esters). When analyzing a transesterification reaction mixture using this procedure, up to five peaks may be present, each one corresponding to methyl esters, monoglycerides, diglycerides, triglycerides, and glycerol, respectively. This method is adequate for validating biodiesel samples against the requirements set by the ASTM D6751 or EN14214 standards; however, it provides no information about individual chemical species present in a given sample. In the present work, simple reversed-phase HPLC methods have been developed utilizing isocratic elution and refractive index detection that is capable of separating and detecting species in biodiesel mixtures containing both saturated and unsaturated carbon chains. The first method presented has been shown to successfully separate and detect free fatty acids, monoglycerides, and most importantly, fatty acid methyl esters. Two other methods have been developed for a more rapid quantitation of di- and triglyceride compounds. Together these methods allow for a complete analysis of transesterification reaction samples for biodiesel production.
region at wavelengths higher than 220 nm; therefore, a wavelength of 205 nm was chosen for detection. Hydrolysis of Oil Triglycerides. Soybean oil triglycerides and water were mixed in a jacketed vessel by constant agitation with a magnetic stirrer. The temperature was maintained at 37 °C using a constant temperature water bath equipped with a pump. The initial water to soybean oil triglycerides molar ratio was 6:1. The hydrolysis reaction was initiated by addition of Candida rugosa lipase (100 mg per mL DI H2O) and allowed to continue for 24 h. To ensure complete hydrolysis, equal amounts of water and lipase as added initially were added again after 12 h. The free fatty acids were collected by centrifugation at 10 000 rpm using a Beckman Coulter Avanti J-E model centrifuge.13 This procedure was repeated using palm oil triglycerides. Due to the high saturated fatty acid content of palm oil, the temperature was maintained at 45 °C to ensure the reaction mixture remained in the liquid phase. Esterification of Free Fatty Acids. The free fatty acids derived from the hydrolysis of soybean and palm oils were used as feedstocks to produce biodiesel methyl esters by esterification with a homogeneous catalyst. The reaction was conducted in a jacketed vessel maintained at 60 °C by a constant temperature water bath equipped with a pump. The initial methanol to free fatty acid molar ratio was 6:1. The reaction was initiated by the addition of 3% (by mass) sulfuric acid. The reaction was allowed to continue for 24 h to ensure complete conversion. The fatty acid methyl esters were collected by centrifugation at 10 000 rpm. The upper phase, consisting of the methyl esters, was placed in a rotary evaporator to ensure any residual methanol was removed. This procedure was conducted twice, once with the fatty acids from soybean oil and once with the fatty acids from palm oil. Sample Preparation. Standard solutions (20 mg/mL) of myristic, palmitic, stearic, oleic, linoleic, and linolenic acids, as well as methyl palmitate, methyl stearate, methyl linoleate, the monoglyceride mixture, and the soybean oil triglycerides were prepared in 2-propanol. Further dilutions were carried out to prepare 2.0, 1.0, 0.5, and 0.25 mg/mL solutions. Ten microliters of the latter solutions were injected into the HPLC for analysis. Each sample was analyzed in triplicate. Sample solutions of the free fatty acids derived from the enzymatic hydrolysis of soybean oil, the enzymatic hydrolysis of palm oil, the biodiesel methyl esters produced from the esterification of each of the free fatty acid sources, and the commercial B100 were prepared in the same fashion.
Experimental Section Materials. Refined soybean oil was purchased from SigmaAldrich and used as the triglyceride standard. Free fatty acid standards used in this study were of high purity (g98%) and obtained from the following suppliers: myristic acid (C14:0), palmitic acid (C16:0), and stearic acid (C18:0), Sigma-Aldrich; oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18:3), Fluka Analytical. Methyl palmitate, methyl stearate, and methyl linoleate were used as fatty acid methyl ester standards and were purchased from Sigma-Aldrich. Methyl myristate, methyl oleate, and methyl linolenate were synthesized from myristic, oleic, and linolenic acids, respectively. HPLC grade chemical solvents were obtained from the following suppliers: acetonitrile, 2-propanol, and methanol, Fischer Scientific. Free fatty acids from soybean oil triglycerides as well as palm oil triglycerides were obtained from Candida rugosa lipase-catalyzed hydrolysis. Soybean and palm oil methyl esters were prepared by homogeneous acid-catalyzed transesterification with sulfuric acid (H2SO4). Commerical B100 biodiesel was purchased from a local BP distributor (Hamburg, PA). Methods. HPLC Instrumentation. HPLC analyses were performed with an Agilent Technologies 1200 Series HPLC equipped with a vacuum degasser, quaternary pump, autosampler, temperature controlled column compartment, UV detector, and refractive index detector. Separations were obtained with a ZORBAX Eclipse XDB-C18 column (150 4.6 mm, I.D., 5 μm). Method 1 consisted of isocratic elution with 85% acetonitrileþ15% deionized water at a flow rate of 1.0 mL/min. Method 2 consisted of isocratic elution with 60% 2-propanol þ 40% methanol at a flow rate of 1.0 mL/min, and finally, method 3 consisted of isocratic elution with 100% methanol also at a flow rate of 1.0 mL/min. The column temperature was maintained at 35 °C for all methods. For comparison, detection was carried out using both UV and RID. As mentioned, acylgylcerols and methyl esters do not adsorb in the UV
Results and Discussion Free Fatty Acids. As mentioned, pure standards were first analyzed to determine retention times. Samples of myristic, palmitic, stearic, oleic, linoleic, and linolenic acids were prepared and analyzed individually. Properties of these fatty acids and their retention times can be found in Table 3. Under the conditions of method 1, it was determined that each component had a different retention time. Furthermore, the (13) Su, C. H.; Fu, C. C.; Gomes, J.; Chu, I. M.; Wu, W. T. AIChE J. 2008, 54, 327–336.
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Figure 1. HPLC chromatogram of FFA standards with (a) RID and (b) UV. Ln = linolenic acid; M = myristic acid; L = linoleic acid; P = palmitic acid; O=oleic acid; S=stearic acid. Table 3. Retention Times and Various Properties of Free Fatty Acids fatty acid
abbreviation
retention time (min)
acyl chain type
ECNFFA
molecular mass (g/mol)
linolenic acid myristic acid linoleic acid palmitic acid oleic acid stearic acid
Ln M L P O S
5.260 6.166 7.506 10.939 11.755 20.321
C18:3 C14:0 C18:2 C16:0 C18:1 C18:0
12 14 14 16 16 18
278.4 228.4 280.4 256.4 282.3 284.4
ECNFFA. In Figure 1, however, it can be seen that each species has a different retention time. In reversed-phase HPLC, fatty
retention times of the fatty acids increased with increasing ECNFFA.7,14,15 From Table 3, it can be seen that myrisitc and linoleic acids, as well as palmitic and oleic acids, have the same
(15) Christie, W. W. Analysis of Fatty Acids by High-Performance Liquid Chromatography. Scottish Corp Research Institute: Invergowrie, Dundee (DD2 5DA), Scotland, 2007.
(14) Holcapek, M.; Jandera, P.; Fischer, J. Crit. Rev. Anal. Chem. 2001, 31, 53–56.
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Figure 2. HPLC chromatograms of FFA from the (a) hydrolysis of soybean oil triglycerides with RID and (b) hydrolysis of palm oil triglycerides with RID. Ln=linolenic acid; L=linoleic acid; P=palmitic acid; O=oleic acid; S=stearic acid.
acids are separated by both chain length and degree of unsaturation; hence, these two parameters make up the mathematical definition of the ECNFFA. The first double bond acts to reduce the effective chain length by a little less than two carbon units so that a C18:1 fatty acid (oleic acid) elutes just after a C16:0 fatty acid (palmitic acid). Second and further double bonds have smaller effects on retention time so that a C18:3 fatty acid (linolenic acid) elutes just before a C14:0 fatty acid (myristic acid).15 Physically, the double bond of an unsaturated fatty acid
creates a rigid bend in the acyl chain.16 This bend may result in steric exclusion from the column stationary phase and account for the reduced retention time of these species. It should be noted that the UV detector was only capable of detecting the species with double bonds, i.e., linolenic, linoleic, and oleic acids, as seen in Figure 1b, while the RID was capable of detecting both unsaturated and saturated components. It should also be noted that the two detectors were connected in series, with the UV detector positioned
(16) Voet, D; Voet, J. G. Biochemistry, 3rd ed.; John Wiley & Sons, Inc.: Hoboken, NJ, 2004. (17) Jaeger, W. K.; Siegel, R. Special Report 1081; Oregon State University, Corvallis, OR, 2008.
(18) Lotero, E.; Liu, Y.; Lopez, D. E.; Suwannakarn, K.; Bruce, D. A.; Goodwin, J. G., Jr. Ind. Eng. Chem. Res. 2005, 44, 5353–5363. (19) Ehrensing, D. T. EM 8955-E; Oregon State University, Corvallis, OR, 2008.
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Figure 3. HPLC chromatogram of FAME standards from commercial B100 biodiesel with (a) RID and (b) UV. MeLn = methyl linolenate; MeM = methyl myristate; MeL = methyl linoleate; MeP = methyl palmitate; MeO = methyl oleate; MeS = methyl stearate. Table 4. Retention Times and Various Properties of Fatty Acid Methyl Esters fatty acid methyl ester methyl linolenate methyl myristate methyl linoleate methyl palmitate methyl oleate methyl stearate
abbreviation
retention time (min)
acyl chain type
ECNFAME
molecular mass (g/mol)
MeLn MeM MeL MeP MeO MeS
12.451 16.691 19.097 30.356 32.195 60.184
C18:3 C14:0 C18:2 C16:0 C18:1 C18:0
12 14 14 16 16 18
292.46 242.40 294.47 270.45 296.49 298.50
before the RID. The retention times for the species as detected by the UV are, therefore, slightly shorter than the retention times as detected by the RID. Additionally, free fatty acids were derived from the enzymatic hydrolysis of triglycerides from soybean oil and palm
oil. The typical free fatty acid composition of soybean oil and palm oil can be seen in Table 2. The RID chromatograms of the resulting hydrolyzed free fatty acids from the two oils can be seen in Figure 2. The UV chromatograms are not shown due to the detector’s inability to detect the saturated species; 6136
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they are similar, however, to the UV chromatogram seen in Figure 1b. From Figure 2a, it is clear that five free fatty acids have been hydrolyzed from soybean oil; they are, in the order of shortest retention to longest: linolenic acid, linoleic acid, palmitic acid, oleic acid, and stearic acid. From Figure 2b, it can be seen that six free fatty acids were hydrolyzed from palm oil, in order of shortest retention to longest: linolenic acid, myristic acid, linoleic acid, palmitic acid, oleic acid, and stearic acid. Calibration curves were prepared from the free fatty acids standards and used to determine the composition of the hydrolyzed samples. The free fatty acid compositions of both the hydrolyzed soybean oil and palm oil were in agreement with the typical fatty acid compositions found in Table 2. Fatty Acid Methyl Esters. The standard compounds methyl palmitate, methyl stearate, and methyl linoleate were first analyzed to determine each components retention time. Similarly, methyl myristate, methyl oleate, and methyl linolenate were synthesized from the respective free fatty acids by acid-catalyzed esterification and analyzed. The retention times and various properties of these methyl esters can be found in Table 4. Since methyl myristate, methyl palmitate, and methyl stearate are the methyl esters of myristic, palmitic, and stearic acids, respectively, all of these esters contain saturated chains. Like saturated fatty acids, it was observed that saturated methyl esters provide weak UV absorbance and are essentially undetectable relative to the unsaturated species. Using RID, however, provides strong detection of all species, both unsaturated and saturated. Like the fatty acids, the retention times of the methyl esters increased with increasing ECNFAME. A sample of methyl ester standards from commercial B100 biodiesel was also analyzed. The RID and UV chromatograms of methyl ester standards from commercial B100 can be found in Figure 3. Calibration curves for the RID were also generated for the methyl ester compounds. Calibrations of methyl palmitate, methyl stearate, and methyl linoleate were produced from the purchased standards. Calibrations of methyl linolenate, methyl myristate, and methyl oleate were generated by esterifying linolenic, myristic, and oleic acids, respectively. Using the previously determined calibration curves for the fatty acids, the concentration of linolenic, myristic, and oleic acids were determined at various points throughout the reaction. The change in concentration of the fatty acids relative to their initial concentration is proportional to the respective methyl ester concentration as follows: mFAME ¼ ðmFFA, i -mFFA Þ :
Figure 4. Calibration curves of FAME. MeLn = methyl linolenate; MeM = methyl myristate; MeL = methyl linoleate; MeP = methyl palmitate; MeO = methyl oleate; MeS = methyl stearate. Table 5. Calibration Constants Associated with Methyl Esters component
slope
R2
MeLn MeM MeL MeP MeO MeS
8.18 106 5.56 106 8.41 106 7.08 106 6.26 106 5.61 106
0.990 0.958 0.999 0.999 0.987 0.999
mental biodiesels and the commercial B100; the results can be found in Table 6. When a fatty acid is esterified to a fatty acid methyl ester, the length of the acyl chain and number of double bonds in the chain do not change. Therefore, the value of ECNFFA will be identical to the value of the ECNFAME. When the retention times of a given free fatty acid to that of its respective fatty acid methyl ester are compared, it can be seen that the retention time of the methyl ester is always greater than that of the free fatty acid. This indicates that the molecular interactions between the ester moiety of a methyl ester and the column stationary phase are greater than the interactions between the carboxyl group of a fatty acid and the column stationary phase. In reversed-phase chromatography, hydrophobic interactions are the most important of all molecular interactions that contribute to retention time.11 The most commonly cited mechanism of reversed-phase retention is the so-called Horvath’s solvophobic-interaction model, which simply states that hydrophobic solute molecules prefer to adhere to the hydrophobic alkyl ligands.11 Therefore, the greater
MFAME MFFA
where mFAME, mFFA,i, and mFFA represent the mass of the FAME, the initial mass of FFA, and the present mass of FFA; MFAME and MFFA represent the molecular masses of the FAME and FFA, respectively. The resulting calibration curves can be seen in Figure 4, and the constants associated with the calibration can be found in Table 5. As mentioned, the fatty acids derived from the hydrolysis of soybean oil and palm oil triglycerides were used as feedstocks to produce fatty acid methyl esters. The RID chromatograms of the soybean methyl esters and palm methyl esters can be seen in Figure 5. The UV chromatograms are again not shown due to the detector’s inefficient response to the saturated species. The calibration curves from Figure 4 were used to determine the compositions of both experi6137
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Figure 5. HPLC chromatogram of FAME produced from (a) the esterification of FFA from soybean oil with RID and (b) the esterification of FFA from palm oil with RID. MeLn=methyl linolenate; MeM=methyl myristate; MeL=methyl linoleate; MeP=methyl palmitate; MeO= methyl oleate; MeS=methyl stearate.
the hydrophobicity of a given solute molecule, the longer its retention time. On the basis of the relative retention times of a fatty acid and its respective methyl ester, it can, therefore, be stated that the methyl ester is a more hydrophobic molecule. Intermediate Glycerides. In addition to the analysis of fatty acid methyl esters present in a given biodiesel sample, it is also important to determine the amount of unreacted acylglycerides. Since most biodiesel processes fail to achieve 100% conversion, there often exists trace amounts of bound glycerol in the form of mono-, di-, and triglycerides, all of which can aversely affect engine performance. The chromatogram seen in Figure 6 is an intermediate transesterification reaction sample indicating the presence of various monoglyceride species. As this is an intermediate sample, there
exists some methyl ester compounds; these species have been labeled as such. As expected, the retention times of the monoglycerides increased with increasing ECNMG. In a similar manner, as seen between a free fatty acid and its respective methyl ester, the ECNMG will be identical to the ECNFFA for any given monoglyceride and its respective free fatty acid. When the retention times of the free fatty acids and monoglycerides studied are compared, it can be seen that a given free fatty acid will always have a slightly longer retention time than its respective monoglyceride. Since the acyl chains of a given free fatty acid and its respective monoglyceride are identical, the difference in the retention times is caused by the differences in the molecular interactions, specifically the 6138
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Table 6. Methyl Ester Composition of Commercial B100 and Experimental Biodiesels from Soybean and Palm Oilsa composition (mass %) soybean methyl esters component MeLn MeM MeL MeP MeO MeS a
theoretical 0.08 0.01 0.52 0.10 0.23 0.05
18
palm methyl esters
experimental
theoretical
0.073
18
experimental
experimental
0.016 0.089 0.437 0.419 0.038
0.047 0.017 0.330 0.147 0.368 0.091
0.01 0.01 0.09 0.48 0.37 0.04
0.505 0.105 0.259 0.059
commerical B100
Compositions may not sum to 100% due to rounding or the presence of trace amounts of other species.
Figure 6. HPLC chromatogram of an intermediate transesterification reaction sample for monoglyceride analysis with RID.
hydrophobic interactions, between the respective functional groups and the column stationary phase. On the basis of the retention theory in reversed-phase chromatography, it can be stated that the hydrophobicity of a given monoglyceride is less than that of its respective fatty acid. This is most likely due to the two hydroxyl groups present on the glycerolbackbone of monoglycerides. Soybean oil triglycerides were also analyzed using the RID. Since values of ECNTG are approximately three times larger than values of ECNFFA, the retention times of triglycerides are expected to be very long. To expedite the analysis, the mobile phase was changed to a mixture of 60% 2-propanol þ 40% methanol. From the solvent polarity index (P) values found in Table 7,11 which provide a measure of overall solvent polarity, it can be seen that by changing the mobile phase from 85% acetonitrile þ 15% deionized water (P = 6.26) to 60% 2-propanol þ 40% methanol (P = 4.38) the polarity of the mobile phase decreases. A reduction in the polarity of the mobile phase facilitates the desorption of the highly hydrophobic triglyceride molecules from the column surface back into the mobile phase. This results in shorter retention times and, hence, faster analysis. The RID chromatogram of refined soybean oil using 60% 2-propanol þ 40% methanol as the mobile phase can be seen in Figure 7. From this figure, it is clear there exists two different groups of peaks. The set of smaller peaks, with retention times in the range of 2.5-3.5 min, represent diglycerides; the set of larger
Table 7. Solvent Polarity Index Values solvent
polarity index
2-propanol methanol acetonitrile water
3.9 5.1 5.8 10.2
peaks, between 4.5 and 10 min, represent triglycerides. The individual peaks within each group represent specific di- or triglycerides, each with a different ECNDG or ECNTG. On the basis of the available fatty acids present in soybean oil, there theoretically can exist triglycerides with ECNTG values ranging from 36 to 54 (even integers only). A triglyceride with ECNTG value of 36 is formed from three linolenic acid chains (ECNFFA = 12) whereas a triglyceride with an ECNTG value of 54 is composed of three stearic acid chains (ECNFFA=18). Given there are only seven peaks corresponding to triglycerides seen in Figure 7, it is highly probable that some peaks correspond to multiple triglyceride species with the same retention time. Without standard compounds of specific triglyceride species, it is difficult to determine the identity of individual peaks. To be of any practical value for studying transesterification reactions, standards of biodiesel methyl esters must also be analyzed using the mobile phase consisting of 60% 2-propanol þ40% methanol to determine if there exists any overlap between any of the groups of molecules. It was 6139
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: DOI:10.1021/ef1009082
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Figure 7. HPLC chromatogram of triglycerides from soybean oil with RID (mobile phase is 60% 2-propanol þ 40% methanol).
Figure 8. HPLC chromatogram of a mixture of methyl ester standards from commercial B100 and diglycerides from soybean oil with RID (mobile phase is 100% methanol).
determined that methyl esters overlap with the peaks corresponding to diglycerides in Figure 7. To resolve the overlapping peaks, the solvent was changed to 100% methanol. The polarity index for methanol is 5.1, which is between the polarity values of the previous mobile phases studied. It was found that using 100% methanol as the mobile phase the peaks corresponding to diglycerides no longer overlap with any other components. A mixture of the standard methyl esters from commercial B100 and diglycerides from soybean oil was prepared and analyzed using 100% methanol. The chromatogram seen in Figure 8 shows the resolution of the methyl esters and diglycerides. Triglycerides can also be resolved using 100% methanol; however, the time required for their analysis is greater than 3 h; it is, therefore, suggested that the mobile phase of 60% 2-propanolþ 40% methanol be used for their analysis. Free Glycerol. The final component associated with transesterification reactions for biodiesel production is glycerol.
Glycerol is produced after a monoglyceride is transesterified to a fatty acid methyl ester. As compared to the hydrophobic tri-, di-, and monoglycerides, fatty acid methyl esters, and free fatty acids, the glycerol molecule is extremely polar and hydrolphilic. This hydrophilic nature results in no retention within the C18-based column used in this study. It was observed in the laboratory, however, that, when a ZORBAX Carbohydrate Analysis column (1504.6 mm I.D.) was used with 85% acetonitrileþ15% deionized water at a flow rate of 0.5 mL/min, the free glycerol could be detected with the RID. The polar nature of the column also results in essentially no retention of the methyl esters and other hydrophobic species. Conclusion An isocratic reversed-phase HPLC method employing refractive index detection has been shown to successfully separate and detect both saturated and unsaturated fatty acids 6140
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and their respective methyl esters when 85% acetonitrileþ15% deionized water (method 1) is used as the mobile phase. The use of RID has been shown to be superior to UV detection due to its capability of detecting all species present in biodiesel from soybean and palm oils. Under the employed conditions, monoglycerides were retained between 3 and 15 min, free fatty acids between approximately 5 and 22 min, and methyl esters between approximately 12 and 60 min. The retention time of the individual monoglycerides, free fatty acids, and fatty acid methyl esters increased with increasing ECNMG, ECNFFA, and ECNFAME, respectively. The analysis of triglyceride species was facilitated using a mixture of 60% 2-propanol þ 40% methanol (method 2) as the mobile phase. The retention times of triglycerides under the conditions of method 2 were between approximately 4 and 10 min. The diglycerides were best separated using 100% methanol (method 3) as the mobile phase; retention times ranged from approximately 5 to 40 min under these conditions. While the calibration results were only reported for the methyl esters, calibrations can be made in a similar fashion for each of the other groups of species present in transesterificaiton mixtures. On the basis of the theory of
retention in reversed-phase chromatography, the relative hydrophobicity of different species can be used to help predict retention. The results obtained in this study indicate that triglycerides are the most hydrophobic, followed by diglycerides, then methyl esters, fatty acids, and monoglycerides, and finally, glycerol was found to be the least hydrophobic species studied. The biggest advantage of the use of HPLC to analyze biodiesel methyl esters over GC is the fact that HPLC allows for the direct analysis of samples without the need for derivatization. Additionally, as compared with other HPLC methods developed for the analysis of biodiesel-related compounds, the use of RID has been shown effective for detecting both unsaturated and saturated species. Furthermore, this entire process can be made more efficient by analyzing the methyl esters using an increased mobile-phase flow rate of 2.0 mL/min and, thus, cutting the analysis time significantly. Acknowledgment. This project has been partially financed by a grant from the Commonwealth of Pennsylvania Department of Community and Economic Development through the Pennsylvania Infrastructure Technology Alliance (PITA).
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