Comparison of Chromatographic Methods for the Determination of Bound Glycerol in Biodiesel

2004, 60, 305311

T. A. Foglia1,&, K. C. Jones1, A. Nun ez, J. G. Phillips1, M. Mittelbach2

1

U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, 600 East Mermaid Lane, Wyndmoor, PA 19038, USA; E-Mail: tfoglia@errc.ars.usda.gov 2 Institute for Chemistry, University of Graz, Heinrichstrasse 28 8010, Graz, Austria

Received: 5 January 2004 / Revised: 1 April 2004 / Accepted: 18 May 2004 Online publication: 1 July 2004

Abstract
An important fuel criterion for biodiesel is bound glycerol, which is a function of the residual amount of triglycerides and partial glycerides in the biodiesel. Either high-temperature gas chromatography or high performance liquid chromatography can be used for determining these minor but important components in biodiesel. In this paper we have conducted a statistical study on the accuracy of the two methods for ascertaining the bound glycerol in biodiesel fuels obtained from different feedstocks. Analysis of variance showed that with one exception, namely diacylglycerols in some soy oil based biodiesel, there was no statistical difference in bound glycerol for the biodiesel samples analyzed or a difference between methods. Operationally, the high performance liquid chromatographic method is superior to the high temperature gas chromatographic method in that it requires no sample derivatization, has shorter analysis times, and is directly applicable to most biodiesel fuels.

Keywords
Gas chromatography APCI mass spectrometry Column liquid chromatography evaporative light-scattering detection Biodiesel Glycerol

Introduction
The term biodiesel is used to dene the simple alkyl esters of fatty acids from vegetable oils, animal fats, and recycled greases when used as fuels for diesel engines, either neat or blended with petroleum diesel [1, 2]. Biodiesel typically is produced from the neat oil or fat by transesterication with an alcohol, usually methanol, in the presence of an alkaline catalyst [35]. Alkali metal

hydroxides are the most common catalysts used but other metal catalysts [6, 7] as well as biocatalysts [8, 9] have been used to catalyze the transesterication reaction. When greases, which have a high free fatty acid content, are used as feedstocks a two-step acid-alkali process is used [10] to convert them to biodiesel. Regardless of the feedstock used or the transesterication process employed, however, it is imperative that the degree of purity and the type of contaminants

present in biodiesel be known prior to its use. To this end, biodiesel fuel standards have been established both in Europe (Germany DIN 51606) and the United States (ASTM D6751-02). Both standards specify the tolerable limits of free and total glycerol, i. e., the sum of free glycerol and glycerol bound as mono-, di-, and triacylglycerols, allowed in the nal product, since total residual glycerol adversely aects the performance and emission properties of the fuel. To this end, methods have been developed to monitor the residual glyceride composition of the transesterication reaction and to determine total glycerol in the nal fuel. Initial methods for the analysis of biodiesel employed thin-layer chromatography with ame-ionization detection (TLC/FID) [11], which also was used to study the variables aecting the yields of alkyl esters from transesteried vegetable oils [12]. This technique, although quantitative, is time consuming, dicult to conduct, and labor-intensive. Transesterication reaction products subsequently were analyzed by high temperature capillary gas chromatography (HTGC), which detected and quantitated esters, triglycerides, diglycerides, and monoglycerides in one run [13]. Since the standards require a determination of total glycerol in the biodiesel, the HTGC approach was augmented to include the determination of both free and bound glycerol [14]. The latter protocol for determining total glycerol in biodiesel, which is commonly referred to as the Plank Method, forms the basis for the

Original DOI: 10.1365/s10337-004-0372-z 0009-5893/04/09

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ASTM protocol for determining total glycerol in biodiesel. Analysis of biodiesel by HTGC [13, 14] requires that the hydroxyl groups of the partial glycerides and free glycerol be derivatized by silylation before analysis, which makes the method operator-dependent and of limited use for monitoring the transesterication process. A method, however, that can be adapted for the continuous analysis of the transesterication process is near-infrared spectroscopy in combination with a ber optic probe [15]. Other shortcomings of the HTGC method are: the use of internal glyceride standards; speciation of alkyl esters and residual partial glycerides; a need for standards for each feedstock used to produce biodiesel; and a general limitation to fatty acid methyl esters (FAME) as biodiesel. As an alternative to HTGC, high performance liquid chromatographic (HPLC) methods have been developed for analyzing reaction mixtures obtained from transesteried fats and oils [1619]. Advantages of the methods are that derivatization of samples is not required, analysis times are less than 30 min and all neutral lipid classes, including alkyl esters, free fatty acids, triglycerides, 1,2- and 1,3diglycerides and 1(2)-monoglycerides, are readily quantitated. To date, however, HPLC methods have been unsuccessful at including the analysis of free glycerol and bound glycerol into a single protocol determination as does HTGC. In this paper we report the HTGC and HPLC quantitation of the lipid classes in biodiesel fuels prepared from dierent feedstocks and a statistical analyses of the accuracy, precision (repeatability), recoveries, and sensitivity of the two methods. The analysis also includes a statistical comparison of results between the two methods. The operational advantages and disadvantages of both analytical methods also are presented.

DE, USA) to remove traces of partial glycerides and free fatty acids. Standards used in this study were the highest purity available (>99%) and were obtained from the following suppliers: oleic acid, Fluka Chemical (Milwaukee, WI, USA); triolein, diolein, monoolein, tricaprin 1,2,4-butanetriol, Sigma Chemical (St. Louis, MO, USA); glycerol and methyl t-butyl ether (MTBE), J. T. Baker (Phillipsburg, NJ, USA); glacial acetic acid, Mallinckrodt (Paris, KY, USA); pyridine and bis-trimethylsilyltriuoroacetamide (BSTFA), Sigma Chemical. TLC standard mixture 18-1a (composed of 25-wt% each of methyl oleate, triolein, 1,3(1,2)-diolein, and 1(2)-monoolein) was obtained from NuChek Prep Inc. (Elysian, MN, USA). All solvents used were HPLC grade and were obtained from Burdick & Jackson (Muskegon, MI, USA).

Gas Chromatography (GC)

Standard and Sample Solutions

instrument was equipped with an on-column capillary injector and a ame ionization detector. Sample analyses were carried out on a DB-1ht fused silica capillary column (15 m 0.32 mm i.d., 0.1 lm lm thickness, J&W Scientic, Folsom, CA, USA). Acquisition and processing of data were obtained using an HP Vectra XA computer with HP-Chemstation Software. Samples (1 lL) were injected oncolumn by an HP 7673 auto sampler injector at an oven temperature of 50 C. After an isothermal period of 1 min at 50 C, the GC oven was heated at 15 C min)1 to 180 C, then at 7 C min)1 to 230 C and at 30 C min)1 to 370 C (hold for 10 min for a total run time of 31.5 min). Helium was the carrier gas at a linear velocity of 62 cm s)1 measured at 50 C. The injector and detector temperatures were 380 C and helium was the detector makeup gas (26.4 mL min)1).

Experimental
Materials
Soybean and rapeseed oil methyl esters (SME and RME, respectively) were prepared by alkali transesterication of soybean and rapeseed oils (ADM Co., Decatur, IL, USA), respectively. The distilled esters (100 mL) were passed through two successive columns of silica (35 g, , 3575 lm, Analtech, Wilmington, 60 A

Stock solutions of glycerol (0.5 mg mL)1), 1,2,4-butanetriol (1.0 mg mL)1) tricaprin (8.0 mg mL)1), mono-, di-, and triolein (each 5.0 mg mL)1) in pyridine were used to prepare a series of standard solutions that contained from .001 to .007 mg mL)1 glycerol, from 0.006 to 0.064 mg mL)1 of di- and triolein, and from 0.013 to 0.127 mg mL)1 of monoolein. Standard solutions of 1,2,4-butanetriol and tricaprin containing 0.013 mg mL)1 and 0.101 mg mL)1, respectively were used as internal standards. Selected amounts of the standard of glycerol, mono-, di-, and triolein solutions were transferred to screw-capped vials (10 mL) and 100 lL of the butanetriol and tricaprin standard solutions added to each vial. For analysis of alkyl ester (biodiesel) samples, 100 lL of the butanetriol and tricaprin standard solutions were added to 95105 mg of the esters contained in a 10 mL screw-capped test tube. BSTFA solution (100 lL) was added to the standard and sample and after 15 min at room temperature the silylated mixtures were diluted with 8 mL of n-hexane and analyzed by GC.
GC Instrumentation

High Performance Liquid Chromatography (HPLC)

Standard and Sample Solutions

Stock solutions of reference mixture 18-1a (4.00 mg mL)1, 1.00 mg of each component) and oleic acid (1.04 mg mL)1) in n-hexane were used to prepare a series of standard solutions containing six concentration levels [16] for each component. The concentration of methyl oleate, monoolein, diolein and triolein in the standard solutions varied from 0.010 to 0.100 mg mL)1 and for oleic acid from 0.010 to 0.104 mg mL)1.

HPLC Instrumentation

GC analyses were performed with a Hewlett-Packard 6890 gas chromatograph (Wilmington, DE, USA). The

HPLC analyses were performed with a HP1050 (Agilent Technologies, Wilmington, DE, USA) series liquid chromatograph equipped with helium degasser, auto-sampler, and quaternary pump module. Lipid class separations were obtained on a Phenomenex (Torrance, CA, USA) cyanopropyl column (CN, 250 4.6 mm i.d., 5 lm) with guard column (30 4.6 mm i.d., 5 lm) of the same phase using a binary solvent mixture of n-hexane (solvent A) and MTBE (solvent B), each fortied with 0.4% acetic acid at a column ow rate of 1.0 mL min)1 [16]. The modied binary solvent prole used was: after an initial Original

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isocratic period for 5 min at 100% A, the solvent composition was changed to 20% A:80% B over 10 min, held for 2 min then returned ballisticly to 100% A. The column was equilibrated for 10 min before the next injection, for a total run time of 29 min. A Varex (Burtonsville, MD, USA) Model III evaporative light scattering detector (ELSD) at a drift tube temperature of 30 C and a nebulizer nitrogen ow of 1.51 SLPM was used for detection. Sample injection volume was 20 lL. Acquisition and processing of data was made using an HP Vectra VL computer equipped with HP Chemstation software.
Liquid Chromatography-Mass Spectra Analysis (LC-MS)

Table 1. Analysis of triacylglycerols and partial glycerides added to puried soy and rape oil methyl esters (biodiesel) by HTGC and HPLC Soy Methyl Esters (SME) wt% MAG Series 1 Theoryc HTGCd HTGCe HPLC Series 2 Theoryc HTGCd HTGCe HPLC Series 3 Theoryc HTGCd HTGCe HPLC Series 4 Theoryc HTGCd HTGCe HPLC 1.95 1.76 1.96 1.98 0.96 0.87 0.98 1.01 0.48 0.46 0.48 0.52 0.24 0.24 0.24 0.26
ab

Rape Methyl Esters (RME) wt%

ab

DAG 1.96 1.70 2.09 2.14 0.97 0.82 1.02 1.15 0.48 0.42 0.48 0.64 0.24 0.22 0.23 0.38

ab

TAG 2.04 2.01 1.99 2.22 1.01 0.94 0.98 1.27 0.50 0.44 0.47 0.75 0.25 0.21 0.23 0.38

MAGab 1.83 1.87 1.95 1.78 0.91 0.98 0.97 0.84 0.45 0.49 0.48 0.42 0.22 0.27 0.25 0.20 a a a a b b b b c c c c d d d d

DAGab 1.81 1.47 1.75 1.80 0.85 0.75 0.86 0.89 0.42 0.38 0.43 0.52 0.21 0.20 0.21 0.27 a a a a b b b b c c c c d d d d

TAGab 1.82 1.80 1.88 1.83 0.90 0.88 0.93 0.88 0.45 0.41 0.39 0.47 0.22 0.21 0.22 0.24 a a a a b b b b c c c c d d d d

ab b a a c c c c d d d d e e e e

ab b a a cd d c c e e e e f f f f

a a a a b b b b c c c c d d d d

HPLC with mass spectrometric detection (LC-MS) was conducted with a Waters HPLC 2690 Separation Module (Waters Co., Milford, MA, USA) connected in series to a Waters 996 Photodiode Array Detector (PDA) and a Waters Thermabeam Mass Detector (MS-EI, Integrity System). The PDA was set to scan from 200 to 400 nm, and the MS-EI detector was set to scan the mass range m/z 90 600 at 1 scan s)1 with an ionization energy of 70 eV. Ionization source temperature was 200 C, nebulizer temperature was 42 C, and expansion region temperature was 75 C. Separations were obtained on a Phenomenex LUNA cyanopropyl column (150 3 mm, 3 lm) with a mobile phase of n-hexane and MTBE (95:5 v/v) each fortied with 0.4% acetic acid) and run isocraticly at a ow rate of 0.43 mL min)1.

a Wt % of monoacylglycerols (MAG); diacylglycerols (DAG); triacylglycerols (TAG) determined in SME and RME esters. HTGC and HPLC wt % values of partial glycerides found in the SME and RME esters are the average of two samples with two determinations (n = 4). Variability in each determination 0.02%. b Means within a series in the same column with no letter in common (a, b, c, d) are signicantly dierent (p 0.05). c Theory is wt % of partial glycerides added to the puried SME and RME esters, which contained < 0.05 MAG, DAG, and TAG. d HTGC data obtained for SME and RME sample set 1. e HTGC data obtained for SME and RME sample set 2 after purication of the SME by column chromatography.

Statistical Analyses
Analysis of variance between the GC and HPLC methods and lipid class determination by each method was done with the Statistical Analysis System (SAS, 1996) protocol. All values for the glycerides listed in the tables are the average of two samples with duplicate determinations (n 4). A Student t test and Bonferroni (Dunn) t test were performed on the means of values and the tested signicance level was p 0.05.

Results and Discussion

The oil seed feedstock most commonly used for the production of biodiesel in the Original

United States and Europe is soybean oil and rapeseed oil, respectively. Accordingly, a comparison of the HTGC [14] and HPLC [16] methods for determining bound glycerol in biodiesel (fatty acid methyl esters, FAME) produced from these oils was undertaken. The oils of interest were converted to fatty acid methyl esters (FAME) by alkali transesterication [4] of the rened oils and after distillation met the ASTM biodiesel fuel standards. Chromatographic analyses of the SME esters (TLC, HTGC, and HPLC), however, showed traces of partial glycerides that were removed by passing the esters through a silica column. In this manner, highly rened methyl ester of soy (SME) and rape (RME) were obtained that contained no detectable traces of glycerides or free glycerol using the techniques of HTGC, and HPLC (Table 1). The two puried biodiesel fuels were then spiked with weighed amounts (approximately 2% by weight) of pure mono-, di-, and triolein (MAG, DAG, and TAG,) and these solutions were serially diluted with the puried soy and rapeseed oil FAME to

produce a series of SME and RME ester samples with known weight percentages (from 0.25 through 2.05 wt%) of MAG, DAG, and TAG. Both series of standard biodiesel samples were then analyzed for their free and bound glycerol content following the HTGC protocol outlined in the ASTM standard [20]. The separation of the fatty acid and neutral lipid standards obtained with the HPLC method reported herein is shown in Fig. 1. As seen, a baseline separation of all lipid classes was obtained within 20 min. Moreover, under the conditions used there also is resolution of the 1,2 and 1,3-diolein isomers and the 1 and 2monoolein isomers. While the HPLC method does not measure free glycerol, as does HTGC, it does measure FFA, which HTGC does not [16]. A representative analysis of the SME prepared in this work by the two methods is shown in Figs. 2 and 3, respectively. As seen in Fig. 2, the HTGC method not only separates the ester samples based on lipid class retention time but also speciates the FAME and residual partial glycerides based on fatty acyl chain length. In

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Fig. 1. HPLC separation of neutral lipid standards: Peak no., lipid component: 1 squalene; 2 cholesteryl oleate; 3 methyl oleate; 4 oleic acid; 5 triolein; 6 gamma-tocopherol; 7 beta-tocopherol; 8 delta-tocopherol; 9 1,3-diolein; 10 1,2-diolein; 11 1-monoolein; and 12 2-monoolein

Fig. 2. HTGC chromatogram of standards and soy methyl esters: (a) neutral lipid standards: Peak no., standard: 1 glycerol; 2 1,2,4-butanetriol (internal standard); 3 1-monoolein; 4 prin (internal standard); 5 1,3-diolein; 6 triolein; (b) soy methyl esters: Peak no., component: 3 monoglycerides; 5 diglycerides; 6 triglycerides; 7 methyl esters

contrast, the HPLC method separates the lipid classes based on polarity rather than molecular weight but there can be some speciation of acyl groups within a lipid class, such as those that contain mixed long and short-chain acyl moieties, such as butterfat. This problem, however, is not encountered with FAME samples prepared from the more common feedstocks used to prepare biodiesel, namely

soy and rape oils and tallow [21]. Moreover, the HPLC method (Fig. 3) oers the advantage that it can measure the presence of FFA in the samples whereas the HTGC method does not. The major advantage of the HTGC method is that it can measure the residual free glycerol in biodiesel samples (Fig. 2), which the HPLC method at this time is unable to do quantitatively. Another apparent advan-

tage of the HPLC method is that it does not require derivatization of the samples as does the HTGC method and the HPLC method is much less operatordependent in identifying chromatographic peaks for inclusion in data analysis since the elution windows are narrower in the HPLC method because of the limited molecular species separation of FAME of carbon chain-length of 1618 carbons. Other advantages of the HPLC method are that it can analyze free fatty acids and ester derivatives of fats and oils other than FAME [16, 21, 22] and therefore is useful for analyzing biodiesel fuels derived from mixed feedstocks. Table 1 lists the results obtained for the analysis of the SME and RME sample series, respectively to which the partial glycerides were added. All samples were analyzed following the ASTM HTGC protocol [20] in two separate laboratories. Analyses of the two SME sample sets by HTGC showed that at the highest spike level (Series 1, HTGCd and HTGCe, Table 1) the amounts of MAG and DAG in the samples diered (p < .05) from each other but neither analysis diered from the theoretical. For the Series 2 determinations only the DAG values diered from each other but again not with the theoretical. For the other SME determinations (Series 34, Table 1) the MAG, DAG and TAG amounts determined for each sample set agreed with each other and theory. The statistical dierences found for the HTGC Series 1 and 2 determinations (Table 1) can be attributed to the fact that the SME in HTGCe was passed through a silica column before addition of the spiked glycerides, which may have removed interfering contaminants. For the RME sample sets (Series 14, Table 1) the HTGC data at all spike levels were in statistical agreement with each other as well as the theoretical amounts of the glycerides added to the sample. The SME and RME sample sets subsequently were analyzed by HPLC and the data obtained for the bound glyceride content compared to the HTGC data. For the RME samples, all the HPLC determinations were statistically the same as the theoretical and the HTGC values at all glycerides levels over the wt% range added (Sets 14, Table 1). On the other hand, for the SME sample sets, the HPLC amounts for the DAG found in both Series 1 and 2 (Table 1) diered Original

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from the HTGCd amounts but not the HTGCe and theory amounts. All other HPLC determinations were in statistical agreement with the theoretical and both HTGC values. This dierence in DAG determination between the HTGC and HPLC methods also was found for a third series of SME samples to which known weighed amounts the glycerides were added (data not shown). In a third RME sample the data again were found to be statistically the same. A comparison of the precision of the HTGC and HPLC methods in quantitating the glyceride classes in SME and RME samples, as well as other fatty esters, was made from an analysis of the repeatability of the values determined for each series in Table 1. This analysis showed that the coecient of variation (%), which ranged between 0.52.5 and 0.53.5%, for the HTGC and HPLC methods, respectively was similar. An analysis of the amounts recovered, namely relative percent of experimental value to the theoretical also was made to assure the accuracy of each method. In general recoveries for the HTGC method were 10% of theory for all the glyceride levels studied. For the HPLC method, recoveries for Series 1 and 2 (Table 1) also were on the order of 10% but at the lower levels (Series 4, Table 1) the variation in DAG and TAG recoveries was higher (20%). Finally, the data were analyzed to gain insight on the limits of quantitation (LOQ) of the glycerides in the SME and RME samples using the HTGC and HPLC methods described herein. This was done following the protocol described by Thomsen et al. [23], which dierentiates the concepts of limits of detection from quantitation. In general the LOQ for MAG, DAG and TAG in the SME and RME biodiesel samples was 2.0, 0.25, and 0.05 lg, respectively for the HTGC method and 10, 20, and 180 lg, respectively for the HPLC method. The above observed anomaly of higher than expected DAG content by HPLC and HTGC in the spiked SME samples listed in Table 1 also has been observed for biodiesel samples that we analyzed from several laboratory and commercial sources but not for all samples analyzed. To address this we took one SME sample with a relatively high DAG content as determined by HPLC (compared to HTGC) and fractionated the sample by silica chromatography and Original

Fig. 3. HPLC chromatograms of neutral lipid standards and of soy methyl esters (SME): (a), Peak no., neutral lipid standard: 1 methyl oleate; 2 oleic acid; 3 triolein; 4 1,3 and 1,2-diolein, respectively; and 5 1 and 2-monoolein, respectively; (b) soy methyl esters, Peak no., lipid class: 1 methyl esters; 2 free fatty acids; 3 triacylglycerols; and 4 diacylglycerols

Fig. 4. HPLC chromatogram of sample, enriched in diacylglycerols, isolated from soy methyl esters by silica chromatography: Peak no., lipid class: 1 methyl esters; 2 triacylglycerols; and 3 diacylglycerols and monohydroxy fatty esters

isolated a SME fraction that was enriched in DAG (8 wt% by HPLC). This fraction subsequently was analyzed by HPLC (Fig. 4) in which the column

euent was routed through a UV and mass spectra detector connected in series. The peaks eluting in the DAG region of the chromatogram showed a UV

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Table 2. Analysis of free fatty acids and residual glycerides present in simple alkyl esters produced from various feedstocks by HPLC and HTGC Alkyl estersa HYSEE REE SME SEE IPT Method HTGC HPLC HTGC HPLC HTGC HPLC HPLC HPLC FFAb 0.40 0.25 0.22 0.86 1.46 MAGb 0.97 0.95 1.19 0.93 0.52 0.34 0.22 0.38 DAGb 0.56 0.43 0.39 0.49 0.14 0.35 0.89 0.47 TAGb 0.11 ndc 0.46 0.61 nd nd nd 0.80

a Alkyl esters: hydrogenated soy ethyl esters (HYSEE); rapeseed ethyl esters (REE); soy methyl esters (SME); soy ethyl esters (SEE); tallow isopropyl esters (IPT). b Wt % ( 0.02%) of free fatty acids (FFA), monoacylglycerols (MAG), diacylglycerols (DAG), and triacylglycerols (TAG) found in alkyl ester samples. c Not detected.

Fig. 5. Chromatograms of biodiesel and biodiesel/diesel fuel blends. (a) HPLC chromatogram of B20 blend of soy methyl esters and diesel fuel; Peak no., component: 1 diesel fuel; and 2 methyl esters. (b) HTGC chromatogram of same B20 blend; Peak no., component: 1 diesel fuel; 2 biodiesel fuel; 3 tricaprin (internal standard)

spectrum with maxima at 234 and 230 nm, which are characteristic for a conjugated diene structure [24]. The mass spectrum (LC-EI-MS) of this peak grouping gave a prominent molecular ion at m/e 310 and fragmentation ions at 293, 277, and 207, which are characteristic for a hydroxy substituted conjugated diene fatty methyl ester [23], such as shown in Fig. 4. That this product is present in SME samples in low levels is not surprising since it is a simple oxidation

product of methyl linoleate, which typically accounts for > 50% of the SME fatty esters. That this oxidation product may or may not be present in the SME sample indicates the extent that the product is handled and subjected to oxidation. One possible reason that it can be detected by HPLC and not always by HTGC is that under typical HTGC operating temperatures, hydroxy substituted fatty esters can readily dehydrate to alkenes, which in this instance would

produce an isomer comparable to methyl linolenate and hence be measured as part of the SME total. That this minor oxidation product was found only in some SME samples and not RME samples remains unclear but its presence may indicate the relative susceptibility of these ester products to oxidation. The versatility of the HPLC method in analyzing biodiesel samples from dierent feedstocks and esters other than methyl esters is demonstrated by the data given in Table 2, which lists the compositional analyses of methyl, ethyl, and isopropyl esters of fats and oils. These analyses can be done since there is little or no speciation of fatty esters; the separation of alkyl esters and glycerides is based on their relative polarities rather than molecular weight as in HTGC. In addition, the HPLC method also is capable of measuring free fatty acids (FFA) that may be present in the biodiesel samples (Fig. 2). The FFA may arise in the fuel as a result of the alkalicatalyzed transesterication process used to produce the esters or are formed by hydrolysis of the esters during storage. Another potential advantage of the HPLC method is that it oers the possibility of the direct measurement of biodiesel (any of the simple alkyl esters of fats and oils) when blended into diesel fuel, which HTGC cannot without signicant modication. That this is possible is shown in Fig. 5 where we show the HPLC and HTGC chromatograms obtained for a 20/80 (v/v %) blend of SME in diesel fuel (Fig. 5a and 5b, respectively). The versatility of HPLC in measuring the levels of biodiesel in commercial samples of biodiesel also is found in the data in Table 3 where the levels of partial glycerides are readily determined in both neat biodiesel samples and their 20% blends in petroleum diesel. Unfortunately, the HPLC method, while capable of detecting glycerol, at this time cannot be used to quantitate free glycerol in biodiesel, whereas the HTGC method can for both neat biodiesel samples and the B-20 blends (Table 3). The HPLC method is presently being evaluated, and if needed modied, for its potential for the quantitation of both free and bound glycerol.

Conclusions
We have presented a statistical study on the accuracy of the HTGC and HPLC Original

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methods for ascertaining the bound glycerol in biodiesel fuels obtained from SME and RME. Analysis of variance showed that with the exception of MAG and DAG in SME at the higher spiked levels, there were no statistical dierences in bound glycerol determination for the SME and RME biodiesel samples analyzed or a dierence between the HTGC and HPLC methods. The discrepancy in MAG but not DAG determinations by HTGC was eliminated by passage of the SME through a silica column. The minor deviation between DAG values, found for some but not all SME biodiesel samples, between the methods arises from the presence of minor oxidation products in the samples that are not measured by HTGC but are by HPLC. These minor components coelute with the DAG in HPLC and hence give slightly higher DAG values than those found by HTGC. Operationally, the HPLC method is easier to conduct than the HTGC method in that it requires no sample derivatization, has shorter analysis time, is directly applicable to most biodiesel fuels, and provides a simpler chromatographic separation of biodiesel components.

Table 3. Free and total glycerol and residual glycerides in biodiesel and biodiesel blendsa (B-20) as determined by HTGC and HPLC Samplea Source Free glycerolb wt% 0.020 0.008 0.021 0.022 0.021 0.041 0.012 0.007 0.011 0.003 0.005 MAGc wt% DAGc wt% TAGc wt% Bound glycerold wt% 0.014 0.043 0.044 0.045 0.058 0.099 0.010 0.013 0.049 0.016 Total glycerole wt% 0.240 0.022 0.064 0.065 0.065 0.098 0.111 0.047 0.033 0.092 0.067

Limitsf B-100 B-100 B-100 B-100 B-100 B-100 B-20h B-20 B-20 B-20
a

A A B B B C C A A A

0.037 0.146 0.145 0.146 0.174 0.012 0.037 0.045 0.137 0.009

0.029 0.038 0.039 0.045 0.058 0.430 0.003 0.006 0.088 0.090

ndg nd nd nd 0.037 0.305 nd nd nd nd

B-100 is neat biodiesel (methyl esters) and B-20 is a 20:80 v//v blend of biodiesel in petroleum diesel as obtained from commercial suppliers A, B, and C. b Free glycerol determined by high temperature gas chromatography (HTGC). c Weight % ( 0.02%) of monoacylglycerols (MAG), diacylglycerols (DAG), and triacylglycerols (TAG) as determined by high performance liquid chromatography (HPLC). d Bound glycerol calculated as specied in ASTM method D6751-02 [20]. e Total glycerol is sum of free and bound glycerol. f Specications for biodiesel (B-100): ASTM D6751-02 [20]. g nd = not detected. h Bound glycerol in the B-20 blends determined by HPLC.

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