Article pubs.acs.org/EF
Free and Total Glycerin in Biodiesel: Accurate Quantitation by Easy Ambient Sonic-Spray Ionization Mass Spectrometry Anna Maria A. P. Fernandes,† David U. Tega,‡ Jose L. P. Jara,† Ildenize B. S. Cunha,† Gilberto F. de Sá,§ Romeu J. Daroda,∥ Marcos N. Eberlin,† and Rosana M. Alberici*,† †
ThoMSon Mass Spectrometry Laboratory, and ‡Analitical Center, Institute of Chemistry, University of Campinas, UNICAMP, 13083-970 Campinas, São Paulo (SP), Brazil § Department of Fundamental Chemistry, Federal University of Pernambuco, 50590-470 Recife, Pernambuco (PE), Brazil ∥ National Institute of Metrology, Quality and Technology (INMETRO), 25250-020 Duque de Caxias, Rio de Janeiro (RJ), Brazil ABSTRACT: Quantitation of free and total glycerin content in biodiesel is performed via a simple and direct ambient desorption/ ionization mass spectrometric technique. Easy ambient sonic-spray ionization mass spectrometry (EASI−MS) is applied to either the crude or derivatized sample spiked with an internal standard allowing for immediate quantitation of free and total glycerin, a major quality parameter for biodiesel. The linear range obtained by EASI−MS for free glycerin was from 0.005 to 0.100% (m/m), and that for bound glycerin (mono-, di-, and triacylglycerides) was from 0.01 to 0.50% (m/m). These values are in concordance with those required by major regulatory agencies for biodiesel quality control, which are, for free glycerin, a 0.02% (m/m) maximum and, for total glycerin, a 0.25% (m/m) maximum. The simple and fast EASI(+)−MS protocol seems to offer a proper substitute or alternative for the more demanding and time-consuming gas chromatography with flame ionization detection (GC−FID) standard methodology.
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INTRODUCTION Biodiesel is a renewable energy source of current worldwide interest and one of the most attractive alternatives for replacing the rapidly depleting and increasingly expensive petrofuels.1,2 Biodiesel consists of fatty acid alkyl esters produced by transesterification of vegetable oils or animal fats with a short-chain alcohol. Glycerin is therefore formed during the biodiesel production process as a side product. The content of free and bound glycerin, sum of monoacylglyceride (MAG), diacylglyceride (DAG), and triacylglyceride (TAG) residuals, is a major parameter used to measure biodiesel quality. High total glycerin (free + bound glycerin) content is known to cause problems during storage because of the separation of the glycerin, which form deposits at injection nozzles, pistons, and valves because of incomplete fuel burning.3−6 For biodiesel to be commercialized as a pure biofuel or blending stock for diesel fuels, it must meet a set of requirements defined worldwide by standard specifications, such as ASTM D6751 and EN 14214.7,8 In Brazil, these specifications are established by the ́ Agência Nacional do Petróleo, Gás Natural e Biocombustiveis (ANP) through Resolution number 7, 2008, which is based on ASTM and EN specifications.9 Gas chromatography with flame ionization detection (GC−FID) is “the official method” to characterize biodiesel (B100) according to the ASTM D6584 and EN 14105.10,11 GC−FID analysis is however time-consuming and requiring also derivatization procedures for quantitation of glycerin and mono-, di-, and triacylglycerides residuals. In addition, dependent upon the feedstock used for biodiesel production, the GC still encounters coelution problems. A less-demanding, reliable, simple, and fast method able to quantitate free and total glycerin in biodiesel samples from various feedstocks would therefore greatly contribute to the quality control of biodiesel. Here, we report on the use of easy ambient sonic-spray ionization mass spectrometry (EASI−MS)12−14 to quantitate © 2012 American Chemical Society
free and bound glycerin in soybean-oil-based biodiesel. We have recently demonstrated the use of direct MS analysis,15−18 most particularly ambient desorption/ionization MS,19−24 as a direct, easy, and rapid method to characterize, at the molecular level, both the oil25−29 feedstock and their biodiesel products30−33 via characteristic TAG and fatty acid methyl ester (FAME) profiles. For that, we used mainly EASI−MS, a method that allows for the direct and fast MS analysis of samples in the open atmosphere with no pre-separation and no or little sample preparation protocols. Despite its superior simplicity and speed, EASI−MS profiles for oils and biodiesel samples have been found to closely match those obtained via GC−MS or liquid chromatography (LC)−MS runs.25
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EXPERIMENTAL SECTION
Chemical Reagents and Samples. High-performance liquid chromatography (HPLC)-grade methanol was purchase from Merck SA (Rio de Janeiro, Brazil) and used without further purification. Chloroform (99.5%), tetrahydrofuran (anhydrous, 99.9%), and acetyl chloride (reagent grade, 98%) were from Sigma-Aldrich (St. Louis, MO). All lipid standards used to prepare the stock solutions were from Nu-Check Prep, Inc. (Elysian, MN). The stock solutions of glycerin and 1,2,4-butanetriol were prepared in ethanol. Monopalmitin (M16:0), monoolein (M18:1), monolinolein (M18:2), monononadecenoin (M19:1), dipalmitin (D16:0), diolein (D18:1), dilinolein (D18:2), dinonadecenoin (D19:1), triolein (T18:1), trilinolein (T18:2), and triheptadecenoin (T17:1) were dissolved in chloroform, while monostearin (M18:0) and distearin (D18:0) were dissolved in tetrahydrofuran (THF). Individual calibration solutions were prepared in high-purity biodiesel by dilutions of 5% (m/m) stock solutions.
Received: March 5, 2012 Revised: April 11, 2012 Published: May 1, 2012 3042
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Figure 1. EASI(+)−MS of crude soybean biodiesel. (a) Full mass spectrum showing mainly the [FAME + Na]+ ions from linoleic acid (m/z 317). (b) Spectrum expansion in the MAG region from m/z 350 to 450 showing characteristic [MAG + Na]+ ions: monopalmitin (m/z 353), monolinolein (m/z 377), monoolein (m/z 379), and monostearin (m/z 381). (c) Spectrum expansion in the DAG region from m/z 580 to 680 showing characteristic [DAG + Na]+ ions: dipalmitin (m/z 591), dilinolein (m/z 639), diolein (m/z 643), and distearin (m/z 647). (d) Spectrum expansion in the TAG region from m/z 850 to 1000 showing characteristic [TAG + Na]+ ions: trilinolein (m/z 901) and triolein (m/z 907). For MAG, DAG, and TAG quantitation, individual analytical curves were constructed using 0.01, 0.05, 0.10, 0.25, and 0.50% (m/m) of each standard in biodiesel. Monononadecenoin (0.10%, m/m), dinonadecenoin (0.20%, m/m) and triheptadecenoin (0.20%, m/m) were used as the internal standard (IS) for MAG, DAG, and TAG, respectively.
To measure free glycerin, a simple derivatization procedure using acetyl chloride was used to improve the limit of quantitation. The derivatization was carried out simply by adding 70 μL of acetyl chloride in 100 μL of biodiesel spiked with known amounts of glycerin and 1,2,4-butanetriol as the IS. The acetylation reaction occurs rapidly and produces products that are more readily ionized by EASI(+). 3043
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Analytical curves for free glycerin were constructed using 0.005, 0.010, 0.025, 0.050 and 0.100% (m/m) of glycerin in biodiesel and 0.025% (m/m) of 1,2,4-butanetriol as the IS. A standard sample of high-purity soybean biodiesel was prepared using methanol in the presence of sodium methoxide as the catalyst, according to an improved transesterification procedure described in detail elsewhere.34 To achieve the highest possible level of purity, the transesterification and purification protocols were repeated 3 times to ensure the least amount of residual glycerol as glycerin and mono-, di-, and triglycerides. This highly pure soybean biodiesel was used to prepare the analytical curves. Four commercial biodiesel samples were used to determinate via EASI(+)−MS the content of free and bound glycerin (MAG + DAG + TAG) in “real” samples. General Experiment Procedures. EASI−MS was performed in the positive-ion mode using a single-quadrupole mass spectrometer (Shimadzu) equipped with a homemade EASI source, which is described in detail elsewhere.12 A tiny droplet of the crude or derivatized biodiesel sample (2 μL) was placed directly into a paper surface (brown Kraft envelope paper). Major parameters used for EASI were a methanol flow rate of 20 μL min−1, N2 as the nebulizing gas at 3 L min−1, and a paper-entrance angle of ∼30°. Mass spectra were accumulated over 30 s and scanned over the m/z 50−1000 range. Analytical curves were plotted for quantitation of free glycerin and mono-, di-, and triacylglycerides in biodiesel. Each point on the analytical curves represents the average of three independent measurements. The analytical curves were made in triplicate, and the commercial biodiesel samples were analyzed at the same time. GC−MS Analysis. The GC analysis was performed according to the standard test method ASTM D6584.10
Figure 2. Analytical curves obtained by EASI(+)−MS for monolinolein (◇), dilinolein (■), and trilinolein (△) in soybean biodiesel.
Table 1. Analytical Factors
MAG 16:0 MAG 18:2 MAG 18:1 MAG 18:0 DAG 16:0 DAG 18:2 DAG 18:1 DAG 18:0 TAG 18:2 TAG 18:1 glycerin
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RESULTS AND DISCUSSION The main goal was to test the ability of the direct EASI(+)−MS analysis to properly quantitate free and bound glycerin (MAG + DAG + TAG) levels in soybean-oil-based biodiesel, and for that, a highly pure soybean-oil-based biodiesel was used as a test case. Via EASI(+)−MS, FAME from linoleic acid is mainly detected as its sodiated ion [FAME + Na]+ of m/z 317 with minor ions from esters of oleic acid of m/z 319 and linolenic acid of m/z 315 (Figure 1a). Monoacylglycerides in their [MAG + Na]+ forms (Figure 1b) were detected as ions of m/z 353 (M16:0), m/z 377 (M18:2), m/z 379 (M18:1), and m/z 381 (M18:0). Similarly, diacylglycerides were detected in their [DAG + Na]+ forms (Figure 1c) of m/z 591 (D16:0), m/z 639 (D18:2), m/z 643 (D18:1), and m/z 647 (D18:0). Residual triacylglycerides were also detected in their [TAG + Na]+ forms (Figure 1d) of m/z 901 (T18:1) and m/z 907 (T18:2). These ions closely reflect the expected composition of free fatty acids of soybean-based biodiesel. Linear calibration curves were obtained for mono-, di-, and triacylglycerides in soybean biodiesel (0.01−0.50%, m/m). Figure 2 shows a typical analytical curve for monolinolein, dilinolein, and trilinolein. Correlation coefficients mostly in the 0.98−0.99 range were obtained (5 points), demonstrating the good linearity of the EASI(+)−MS method. Table 1 lists major analytical factors. Note that the analyzed percent range is in accordance with those of standard protocols (ASTM D6584 and EN 14105). Note also that, in general, the worst R2 values are for glycerides derived from fully saturated fatty acids. These glycerides are less easily ionized via EASI ionization conditions similar to other MS techniques,35,36 but they are minor constituents in soybean-oil-based biodiesel and, hence, contribute less significantly in the total content of bound glycerin. Another interesting aspect of the EASI(+)−MS data is the average coefficients of variation (ACVs), which are usually high in the
[M + Na]+ (m/z)
R2
linear range (%, m/m)
ACVa (%)
353 377 379 381 591 639 643 647 901 907 241b
0.901 0.993 0.995 0.986 0.978 0.972 0.997 0.997 0.996 0.999 0.998
0.01−0.50 0.01−0.50 0.01−0.50 0.01−0.50 0.01−0.50 0.01−0.50 0.01−0.50 0.01−0.50 0.01−0.50 0.01−0.50 0.005−0.100
12.0 10.2 8.6 9.2 8.8 10.8 10.2 12.0 9.8 14.5 6.8
a ACV = average coefficient of variation. bm/z values of the sodium adduct of the triacetylated glycerin derivative.
ambient ionization technique37 but were reduced to an acceptable value of ca. 10% by the use of the IS.38 Free glycerin was also detected directly from the crude biodiesel samples by EASI(+)−MS as its sodiated adduct [Gly + Na]+ of m/z 115. However, the concentration of glycerin in biodiesel samples is normally very low (in the parts per million range); hence, the ion abundance is close to noise levels. Derivatization via acetylation (Scheme 1) was therefore tested to improve glycerin detectability. The derivatization procedure used was, however, fast and performed by the simple addition of a few droplets of acetyl chloride to the crude biodiesel sample. Acetylation was indeed found to significantly increase the sensitivity of EASI(+)−MS analysis to free glycerin in biodiesel. Figure 3 compares the EASI(+)−MS of a highly pure biodiesel sample spiked with 0.100% (m/m) of glycerin and 0.025% (m/m) of the IS before and after derivatization. Note in Figure 3a that free glycerin is not detected in the crude sample as either [Gly + H]+ of m/z 93 or [Gly + Na]+ of m/z 115. After derivatization with acetyl chloride (Figure 3b), however, acetylated glycerin was readily detected as sodiated adducts of m/z 199 (diacetin) and 241 (triacetin). The much more abundant ion of m/z 241 was therefore used to construct the analytical curve for free glycerin quantitation. Note that the ion of m/z 255 in Figure 3b corresponds to the triacetylated form of the IS (1,2,4-butanetriol). Figure 3c shows the gradual 3044
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Scheme 1. Sequencial Acetylation of Glycerin
Figure 3. Glycerin detection in soybean biodiesel. EASI(+)−MS profile (a) before and (b) after acetyl chloride derivatization. (c) Gradual changes in the relative abundance of analyte and IS ions for spectra used to construct the analytical curve (from left to right): 0.005, 0.010, 0.025, 0.050, and 0.100% (m/m) of glycerin and 0.025% of IS.
analytical curve for free glycerin quantitation as its triacetin form. Figure 4 shows that quite linear analytical curves could indeed be obtained for free glycerin quantitation (0.005−0.100% of glycerin in biodiesel), indicating that not only bound glycerin but also quite accurately quantitated free glycerin in biodiesel can be performed by direct EASI(+)−MS analysis. Table 1 summarizes the analytical factors for glycerin quantitation by EASI(+)−MS. To test the method for “real” samples, four commercial biodiesel samples were analyzed. For comparison, the free and bound glycerin levels were also measured by the classical GC−FID method. Calculation of total glycerin was made using eq 1, according to ASTM 658410 total glycerin=free glycerin+bound glycerin
(1)
where bound glycerin = 0.2591MAG + 0.1488DAG + 0.1044TAG. Table 2 compares the free and total glycerin content (%, m/m) for the four biodiesel samples as measured by either GC or EASI(+)−MS. The amount of free glycerin mainly reflects the
Figure 4. Analytical curve for EASI(+)−MS quantitation of free glycerin in soybean biodiesel as its triacetin derivative.
changes in the relative abundances of the ions of m/z 241 (analyte) and 255 (IS) for the spectra used to construct the 3045
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0.002 0.002 0.030 0.012 ± ± ± ±
GC
0.154 0.178 0.280 0.276 0.038 0.034 0.035 0.027 ± ± ± ±
EASI
0.444 0.397 0.364 0.329 0.001 0.004 0.019 0.016 ± ± ± ±
GC
0.002 0.058 0.188 0.150 0.000 0.002 0.014 0.007 ± ± ± ± 0.156 0.162 0.231 0.210 ± ± ± ±
0.002 0.006 0.027 0.027
GC
± ± ± ±
GC
± ± ± ± ± ± ± ±
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CONCLUSION The EASI(+)−MS technique, when applied directly to the crude and derivatized sample, has been shown to provide very fast, quite linear, and accurate quantitation of free and total glycerin in biodiesel samples. The method therefore seems to offer a promising substitute or complementary technique for the more demanding and time-consuming GC−FID protocol currently required by major regulatory agencies for biodiesel quality control.
0.072 0.040 0.073 0.042 0.850 1.190 0.780 0.640 0.0002 0.0001 0.0001 0.0001
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AUTHOR INFORMATION
± ± ± ±
Corresponding Author
*Telephone/Fax: (+55) 19-35213073. E-mail: rmalberici@ hotmail.com.
0.0090 0.0100 0.0060 0.0060
GC
0.510 0.566 0.674 0.687 EASI
0.009 0.011 0.009 0.033
1.343 0.432 0.881 0.913
0.069 0.053 0.081 0.072
0.087 0.106 0.536 0.513
EASI
EASI
efficacy of the purification process, whereas the total glycerin reflects the efficacy of transesterification. According to EN 14214 and ASTM D6751, maximum total glycerin contents are 0.25 and 0.24%, respectively. The maximum value for free glycerin is 0.02% in both specification standards, as well as by ANP. Table 2 shows that the GC and EASI(+)−MS produced very similar results. Both methods should therefore be considered suitable and exact enough (p > 0.05, Student’s t test) to analyze free glycerin in biodiesel. For bound glycerin analysis (MAG + DAG + TAG), significant differences (p < 0.05, Student’s t test) are observed, with EASI(+)−MS values being generally higher. GC−FID is the “official technique” for glycerin analysis in biodiesel according to the international regulations, but such GC runs are known to be influenced by several factors, such as baseline drift, overlapping signals, and aging of standards and samples.3 These fluctuations may contribute to lower than real values for glyceride detection by GC−FID, and such factors are not always addressed in standards and reports. For example, the use of methyl heptadecanoate as the IS presents a problem for the analysis of animal-fat-based biodiesels because they display natural contents of this IS. The GC temperature program of EN 14103 also requires modification for biodiesel containing shorter-chain esters because, otherwise, erroneous results could be obtained.6 Additionally, the ASTM and EN protocols were developed for the analysis of soybean-based biodiesels; hence, GC−FID parameters and conditions optimized for this specific type of biodiesel may not be fully applicable to vegetable oil methyl esters obtained from lauric oils, such as for those using coconut and palm kernel oils. The Brazilian commercial soybean-based biodiesel samples analyzed in this study may legally contain up to 30% of animal-fat-based biodiesel. This accepted admixture could therefore have contributed to the lower levels of bound glycerin found via GC−FID in this study, because optimal GC−FID analysis conditions for animal-fat-based biodiesel are unknown. The limitations of the GC method have recently been reviewed elsewhere.39
The authors declare no competing financial interest.
0.0007 0.0007 0.0007 0.0007
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± ± ± ±
ACKNOWLEDGMENTS This work was supported by the following Brazilian Research Foundations: CNPq, FAPESP, and FINEP.
0.0083 0.0082 0.0070 0.0068
EASI
Notes
sample
TAG (%, m/m) DAG (%, m/m) MAG (%, m/m) free glycerin (%, m/m)
Table 2. Comparison of Results Obtained by GC and EASI Methods
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1 2 3 4
total glycerin (%, m/m)
Energy & Fuels
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