Journal of Chromatography B, 983–984 (2015) 94–100
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Ultrahigh performance supercritical fluid chromatography of lipophilic compounds with application to synthetic and commercial biodiesel M. Ashraf-Khorassani a , J. Yang b , P. Rainville b , M.D. Jones b , K.J. Fountain b , G. Isaac b , L.T. Taylor a,∗ a b
Department of Chemistry, Virginia Tech, Blacksburg, VA 24061, United States Waters Corporation, 34 Maple Street, Milford, MA 01757, United States
a r t i c l e
i n f o
Article history: Received 2 September 2014 Accepted 14 December 2014 Available online 6 January 2015 Keywords: Ultrahigh performance supercritical fluid chromatography Tobacco seed oil Normal phase chromatography Evaporative light scattering detection Biodiesel purity test Octa-decyl bonded silica particles
a b s t r a c t Ultrahigh performance supercritical fluid chromatography (UHPSFC) in combination with sub-2 m particles and either diode array ultraviolet (UV), evaporative light scattering, (ELSD), or mass spectrometric (MS) detection has been shown to be a valuable technique for the determination of acylglycerols in soybean, corn, sesame, and tobacco seed oils. Excellent resolution on an un-endcapped single C18 column (3.0 mm × 150 mm) with a mobile phase gradient of acetonitrile and carbon dioxide in as little as 10 min served greatly as an improvement on first generation packed column SFC instrumentation. Unlike high resolution gas chromatography and high performance liquid chromatography with mass spectrometric detection, UHPSFC/MS was determined to be a superior analytical tool for both separation and detection of mono-, di-, and tri-acylglycerols as well as free glycerol itself in biodiesel without derivatization. Baseline separation of residual tri-, di-, and mono-acylglycerols alongside glycerol at 0.05% (w/w) was easily obtained employing packed column SFC. The new analytical methodology was applied to both commercial B100 biodiesel (i.e. fatty acid methyl esters) derived from vegetable oil and to an “in-house” synthetic biodiesel (i.e. fatty acid ethyl esters) derived from tobacco seed oil and ethanol both before and after purification via column chromatography on bare silica. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The development of new technologies that afford the production of fuels that are obtained from renewable resources is driven by both environmental concerns and fossil fuel deficiency. Biofuels, such as the biodiesel produced via base catalyzed transesterification of vegetable oils and animal fats using either ethanol or methanol in the U.S. is a promising alternative fuel source [1]. Biodiesel has similar chemical structure and energy content to petro-diesel in terms of fuel quality. Compared to petroleum diesel, however, biodiesel can reduce CO2 emissions approximately 78%. As an energy source, biodiesel (i.e. fatty acid alkyl esters) should have certain criteria [2]. The feed-stocks available for producing biodiesel are highly dependent upon the region, climate, soil conditions, and geography. For example, rapeseed is the dominant
∗ Corresponding author. Tel.: +1 540 231 6680; fax: +1 540 232 3255; mobile: +1 540 239 3944. E-mail address: [email protected] (L.T. Taylor). http://dx.doi.org/10.1016/j.jchromb.2014.12.012 1570-0232/© 2015 Elsevier B.V. All rights reserved.
feedstock for biodiesel production in Europe, soybean in the United States and palm oil in tropical countries such as Malaysia and the Philippines. Coconut is another feedstock used for biodiesel production in the Pacific Rim region. The advantages of biodiesel in addition to being considered more environmentally friendly than petro diesel are higher octane number and higher combustion efficiency. Biodiesel is usually commercially blended with petro-diesel to 5% biodiesel. A major problem related to the use of biodiesel is its inherent instability to oxidation. For example, biodiesel degradation can change the fuel properties by formation of gums, acids, insolubles, and other oxidation products. The level of impurities in commercial biodiesel B100 as cited in ASTM D 6751 for (a) tri-acylglycerol which is unreacted starting material, (b) di-acylglycerol and mono-acylglycerol which are reaction intermediates, and (c) free glycerol which is a by-product should be 0.02 wt% for glycerol itself and 0.24 wt% total glycerol which includes mono-, di-, and tri-acylglycerols as well as glycerol itself. It is interesting to note that each year producers of biodiesel create more than a million tons of “just” glycerol worldwide much of which goes to waste [3]. The presence and
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concentration of these impurities obviously lead to biodiesel products that vary in complexity, polarity, solubility, and volatility. Analysis of acylglycerols typically follows two routes: (1) analysis of the whole molecule which can be a mixture of glycerol itself, mono-acylglycerol, di-acylglycerol, and tri-acylglycerol and (2) analysis of the range of fatty acids that originally esterify the glycerol molecule [4]. Analyses of whole acylglycerol molecules have been reported in the literature and in ASTM D 6584. Such techniques use GC, HPLC, TLC or a combination of these [5]. Most GC techniques employ flame ionization detection; while HPLC frequently uses evaporative light scattering [6] and mass spectrometric [7] detection. For glycerol, mono-, and di-acylglycerols, the analytical method is based on conversion of the molecular hydroxyl function into silyl derivatives in the presence of pyridine and N-trimethylsilyltrifluoroacetamide followed by high temperature GC on a short capillary column with thin film thickness using on-column injection and flame ionization detection [4]. Most techniques, on the other hand, dedicated to HPLC are designed to deal with tri-acylglycerols (TAGs). In most of these cases, the methodology employs multi-dimensional chromatography wherein argentation chromatography is one of the components designed to achieve separation of unsaturated acylglycerols Packed column supercritical fluid chromatography (SFC), on the other hand, can be essentially considered to be a more powerful tool for lipid analyses [8] than either HRGC or HPLC. In comparison to GC, acylglycerols are separated via SFC at a much lower temperature and compared to HPLC, different selectivity and shorter analysis times are obtained with SFC. Contrary to numerous references that have appeared in the older literature, polar solutes such as free glycerol can be analyzed via SFC without derivatization. For example, TAGs have been analyzed by packed column SFC using for example 2-ethylpyridine [9] and silver-ion exchange [10] stationary phases. Using a reversed phase stationary phase such as octadecylsilica [11], the SFC separation may be based upon carbon number (i.e. total number of carbons in all fatty acids) and/or on the number of double bonds. In a more recent reversed phase separation [12], three Zorbax SB-C18 columns (4.6 mm × 250 mm, 5 m) were coupled in series. Temperature was 25 ◦ C with an acetonitrile/methanol modifier gradient. With a flow rate of 2.5 mL/min, a mixture of eight TAGs eluted between 45 and 60 min, although they were not baseline resolved. On the other hand, separation of selected unsaturated vegetable oils on the silver loaded column was mainly based on the number of double bonds. Evaporative light scattering detection results were reproducible and provided enhanced sensitivity compared to UV detection. An improvement in chromatographic performance for similar lipophilic analytes is reported here. Fast determination of residual glycerol, free fatty acids, and mono-, di-, and tri-acylglycerols in both synthetic biodiesel prepared “in-house” and commercial B100 biodiesel (e.g. typically referred to as fatty acid alkyl esters) is described here using ultrahigh performance supercritical fluid chromatography (UHPSFC). UHPSFC is a relatively new separation technique that uses compressed carbon dioxide as the primary mobile phase [13]. It takes advantage of sub-2 m particle chromatography columns and advanced chromatography systems that are designed to achieve fast and reproducible separation with high efficiencies and unique selectivity. The established UHPSFC/MS approach has potential application in lipidomics and food testing as a complementary method alongside HPLC–MS and HRGC–MS, as the former can separate both polar and non-polar lipids in a single run to improve both detection limits and peak shape. Unlike HRGC/MS, low volatile and very long chain fatty acids (>24 carbon atoms) can be easily analyzed without concern for analyte degradation. In this regard, the possibilities for lipid analyses via UHPSFC were recently illustrated by Jones et al. [14] who performed the separation of neutral and amphipathetic lipids using
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both BEH (unbonded organic/inorganic silica hybrid) and HSS C18 (un-endcapped silica) columns packed with sub-2 m particles. Studies using UHPSFC coupled with photodiode array (DAD), mass spectrometry (MS), and evaporative light scattering (ELSD) detection for the separation of TAGs isolated from tobacco, corn, sesame, and soybean seed oils are presented here. A single unendcapped C18 column with acetonitrile-modified carbon dioxide was employed for separation of all seed oils. Excellent chromatography of associated, hydroxyl-containing di-acylglycerols, mono-acylglycerols, and glycerol wherein methanol was employed as a secondary modifier in addition to acetonitrile was also achieved. 2. Experimental 2.1. Sample description Pure fatty acid ethyl esters (C16 , C18 , C18:1 , C18:2 , and C18:3 ) were purchased from Sigma–Aldrich (St. Louis, MO) and mixed to form a model biodiesel. Pure mono-, di-, and trioctadecylglycerol plus free glycerol and soybean oil were also obtained from Sigma–Aldrich. All standards were prepared in a 50/50 dichloromethane/methanol (DCM/MeOH) mixture. The model biodiesel was prepared in the same mixture as a 5% (v/v) solution. Tobacco seed oil was obtained from R.J. Reynolds Tobacco Co. (Winston-Salem, NC). Soybean oil, corn oil, and sesame seed oil were obtained from Sigma–Aldrich (St. Louis, MO). Five percent of the different oils was dissolved in DCM/MeOH (1/1, v/v) for both UV and ELS detection and then 0.1% for the Xevo G2 QTof MS. 2.2. Chromatographic conditions Supercritical fluid chromatography experiments were performed using a Waters Acquity UPC2 system (Milford, MA, USA) equipped with (a) high pressure mixing binary solvent delivery manager, (b) fixed loop design autosampler, (c) active back pressure regulator, (d) column compartment with active heating and column switching control, (e) photodiode array (PDA) and evaporative light scattering (ELS) detectors. Experiments were carried out using an ACQUITY UPC2 HSS C18 SB column (150 mm × 3.0 mm, 1.8 m) at a temperature of 25 ◦ C. The mobile phase consisted of compressed CO2 (component A) and either 100% acetonitrile or acetonitrile/methanol (90:10) (component B). The mobile phase flow rate was maintained at 1–2 mL/min under all different gradient conditions. Backpressure was maintained isobarically and automatically by a backpressure regulator (ACQUITY CCM) at a pressure of 1500 psi. The injection volume was varied 2–8 L for both PDA and ELSD; while, 0.1 L was the volume for MS detection. Photodiode array detection was monitored at a wavelength range 190–400 nm with a reference of 400–500 nm. The Waters ACQUITY ELSD detector was operated with nebulizer cooling, drift tube: 50 ◦ C, gas pressure: 40 psi and gain 10, make up flow (isopropyl alcohol) was added at 0.2 mL/min before the ELSD. The solvent flow was split prior to the back pressure regulator for ELSD (split ratio 1:3) and MS electrospray probe. 2.3. Mass spectrometric conditions Mass spectrometry was performed using Xevo G2 QTof (Waters Corp., Milford, MA, USA). The solvent flow was split post PDA detection using a pre-BPR flow Upchurch cross 1/16 PEEK splitter. CO2 -miscible make-up solvent (MeOH in 10 mM NH4 OAc), delivered by a HPLC 515 make-up pump (Waters Corp., Milford, MA, USA), was added at a flow rate of 0.2 mL/min and mixed with the chromatographic effluent to aid ionization. A fraction of the total flow was directed from the union to the ESI source through
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a transfer line, whereas the remaining mobile phase was directed to the BPR PEEK connection. The ESI source was operated in positive ionization mode with capillary and cone voltages of +3 kV and 30 V, respectively. The source temperature, cone gas flow, desolvation temperature, and desolvation gas flow were set at 150 ◦ C, 10 L/h, 500 ◦ C and 600 L/h, respectively. MS data were acquired in the range of 400–1200. Data handling and instrument control were performed with Masslynx 4.1 (Waters Corp., Milford, MA, USA).
3. Results and discussion 3.1. UHPSFC of tri-acylglycerols SFC is useful for the separation of lipids such as carotenoids and tri-acylglycerols (TAGs) which have numerous analogs with similar structures. Currently, there is much interest in rapid characterization of TAGs which are natural compounds produced by the esterification of glycerol with fatty acids. While first generation supercritical fluid chromatographic (SFC) instrumentation has been successfully applied to the characterization of TAGs in various seed oils, the application of ultrahigh performance supercritical fluid chromatography (UHPSFC) to the range of byproducts that are produced during the conversion of TAGs to fatty acid alkyl esters (i.e. biodiesel) has not been considered. Separation and detection of the wide analyte polarity range afforded by underivatized mono-, di-, and tri-acylglycerols, glycerol, fatty acid alkyl esters, and free fatty acids by UHPSFC with three different detectors is discussed here. Fig. 1 shows the UV (i.e. diode array), ELSD, and MS chromatograms for the separation of TAGs in soybean oil. All of the chromatographic effluent first passes through the DAD flow cell then via a 3/1 split to the back pressure regulator and either ELSD or MS. Each gradient separation was performed at 25◦ C with an ACQUITY UPC2 HSS C18 SB column (HSS = high strength silica) and a mobile phase of acetonitrile-modified carbon dioxide. Near baseline peak resolution was observed with a single 3.0 mm × 150 mm column packed with 1.8 m bonded silica particles in approximately 16 min. Baseline stability was excellent even under gradient conditions. The resulting chromatographic system allowed reproducible separations and possible quantification of analytes at low concentrations in various samples. These optimized conditions were used to separate and profile TAGs in tobacco seed oil, soybean oil, corn oil, and sesame seed oil. Data were acquired in UHPSFC-MSE mode, which is an unbiased Tof acquisition method in which the mass spectrometer switches between low and high energy on alternate scans for structural elucidation and identification. In all cases, distinct profiles and excellent separations were obtained for all oil types when using UHPSFC with both MS and ELSD detection. Fig. 2 shows the UHPSFC/MS separation and detection of various TAGs in different oils. TAGs were identified using accurate mass spectra collected by QTof MS with MSE and the Waters Progenesis QI. In positive ion mode MSE low energy, TAGs produced intact ammonium ion adducts [M+NH4 ]+ precursor exact mass when ammonium acetate was present in the make-up solvent. In MSE high energy, abundant fragment ions were produced corresponding to the neutral loss of one of the sn-1, sn2 or sn-3 fatty acids plus ammonia. For example, the ion at m/z 874.7823 corresponding to 52:3 TAG (calculated fatty acid carbon atom: total number of double bonds), can be identified as POL (i.e. palmitic–oleic–linoleic) due to the presence of abundant fragment ions at m/z 575.5038, 577.5201 and 601.5186. These ions correspond to the neutral loss of fatty acyl groups P, O, and L plus ammonia, respectively [4]. Table 1 shows a list of all identified TAGs in the different oils based on the low energy precursor exact mass and corresponding high energy fragment ions.
Table 1 List of detected TAGs in each seed oil with their retention time via UHPSFC/MS. Ret. time (min)
Soybean oil
Corn oil
Sesame oil
Tobacco seed oil
10.86 11.11 11.40 11.60 11.78 11.90 12.11 12.32 12.49 12.71 13.00 13.08 13.22 13.40 13.93 14.26 14.73 15.14 15.70
LnLnL LnLL LLL OLLn PLLn SLLn OLL POLn OOL POL PPL OOO SLL POO SOL PSL SSL PSO PSA
– LnLL LLL OLLn – SLLn OLL POLn OOL POL PPL OOO SLL POO SOL PSL SSL PSO PSA
– LnLL LLL OLLn – SLLn OLL POLn OOL POL PPL OOO SLL – SOL PSL SSL PSO PSA
– LnLL LLL – SLLn OLL – OOL POL PPL – SLL POO SOL PSL SSL – –
– not detected.
3.2. Feasibility of impurities assay A single ACQUITY HSS C18 SB column (3.0 mm × 150 mm) packed with 1.8 m particles was used to develop the separation of a model mixture of anticipated biodiesel components such as free glycerol, acylglycerols, and fatty alkyl esters. When a faster gradient of modifier was employed, all the components were eluted in less than 9 minutes without a significant loss in resolution even among the mono-, di-, and tri-acylglycerols. Application of a steeper modifier gradient ramp in mobile phase under the same chromatographic conditions further decreased the analysis time to 4 min with again minimal loss in peak resolution. These data indicated that the gradient profile, flow rate, and mobile phase modifier can be readily changed in order to optimize the separation regardless of the biodiesel analysis required. Furthermore, all the listed parameters are compatible with MS detection, such that positive identification of all acylglycerol species plus glycerol itself is possible. Finally a faster separation without significant loss in analyte resolution could translate into higher sample throughput when monitoring product quality. Equally important is the observation that baseline separation of all compounds was achieved. It is also imperative that one must also use a method whereby the impurities do not interfere with proper quantification of residual acylglycerols in the sample. This situation can become critical since most biodiesels have minor components which elute early but after elution of the fatty acid ethyl esters (i.e. biodiesel components). For selected analyses, flow was lowered from 1.2 to 1.0 mL/min to minimize these interferences. It should be noted that reproducibility was excellent for all analytes. To demonstrate this feature, five replicate chromatographic injections of our model biodiesel mixture that contained the four spiked impurities using ELSD were performed. Percent RSD for all peak areas was between 1 and 4%, and the RSD for retention time of all peaks was less than 0.1%. Next a series of model biodiesel mixtures were prepared wherein the compounds varied in concentration. In each case, FAEE, soybean oil TAGs, and free glycerol were mixed at different concentrations and the resulting mixture was injected into the ACQUITY HSS C18 SB column (3.0 mm × 150 mm). A series of chromatograms were thus generated wherein the spiking percentage varied from 0.2% to 5.0% (v/v). At the lowest concentration (0.2%), the glycerol peak was clearly seen and well resolved from the other component peaks for quantification purposes. In order to estimate system detection limit, larger injection volumes (4 and 8 L) of the
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Fig. 1. Sequential UHPSFC of soybean oil with UV, ELSD, and MS detection using CO2 with CH3 CN as modifier. Chromatography conditions: gradient elution: 2–20% CH3 CN in 18 min. Flow: 1.5 mL/min, 25 ◦ C. Column ACQUITY UPC2 HSS C18 SB column (150 mm × 3.0 mm, 1.8 m).
Fig. 2. UHPSFC/MS of different oils using CO2 with CH3 CN as modifier. Gradient elution: 2–20% CH3 CN in 18 min. Flow: 1.5 mL/min, 25 ◦ C. Column ACQUITY UPC2 HSS C18 SB column (150 mm × 3.0 mm, 1.8 m).
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Fig. 3. UPSFC/ELSD detection of glycerol at 0.05% level at two different injection volume (4 and 8 L). Chromatography conditions: T = 0 min, 98/2 CO2 /modifier (90/10 CH3 CN/CH3 OH), T = 10 min, 80/20, T = 12 min, 60/40, T = 15 min, 60/40, flow = 1.2 mL/min., 25 ◦ C. Column ACQUITY UPC2 HSS C18 SB column (150 mm × 3.0 mm, 1.8 m) volume.
Fig. 4. UHPSFC/ELSD of 3 different batches of synthetic biodiesel. Chromatography conditions: T = 0 min, 90/10 CO2 /modifier, T = 10 min, 50/50, T = 11 min, 50/50, T = 11.1 min, 90/10, T = 12 min, 90/10, flow = 1.0 mL/min., 25◦ C. Column ACQUITY UPC2 HSS C18 SB column (150 mm × 3.0 mm, 1.8 m).
mixture spiked with impurities at 0.05% of glycerol yielded comparable results (Fig. 3). The S/N ratio for glycerol using ELSD was about 10/1 at the 0.05% level with a 4 L injection volume which was equivalent to 2 g mass injected and 40/1 at the
same level with an 8 L injection which was equivalent to 4 g injected on-column. The detection limit demonstrated here via this method is compatible with ASTM 6751for biodiesel impurities analysis.
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Fig. 5. Injection of commercial biodiesel B100 and synthetic biodiesel spiked with different impurities. Chromatography conditions: T = 0 min, 90/10 CO2 /modifier, T = 10 min, 50/50, T = 11 min, 50/50, T = 11.1 min, 90/10, T = 12 min, 90/10, flow = 1.0 mL/min., 25 ◦ C. Column ACQUITY UPC2 HSS C18 SB column (150 mm × 3.0 mm, 1.8 m).
Fig. 6. UHPSFC/ELSD separation of synthetic biodiesel before and after purification. Chromatography conditions: T = 0 min, 90/10 CO2 /modifier, T = 10 min, 50/50, T = 11 min, 50/50, T = 11.1 min, 90/10, T = 12 min, 90/10, flow = 1.0 mL/min., 25◦ C. Column ACQUITY UPC2 HSS C18 SB column (150 mm × 3.0 mm, 1.8 m).
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This study yielded a surprising result regarding the retention times of the various components in the model mixture. An increase in amount of soybean oil tri-acylglycerols injected from 0.2% to 5.0% had little or no effect upon retention time of specific lipophilic analytes. The result insofar as glycerol was quite different. Retention time of glycerol for 4 g injected was 14.63 min. An increase in amount injected from 4 g to 100 g injected caused the retention time to reproducibly go from14.63 min to 14.57 min to 14.44 min to 14.28 min. Since all of the separations in the total study were normal phase as evidenced by the observation that analyte retention increased with the number of non-derivatized hydroxyl groups (i.e. elution order is tri-acylglycerol with no hydroxyls, followed by di-acylglycerols with one hydroxyl, followed by mono-acylglycerol with two hydroxyls, followed by glycerol with three hydroxyls). Thus glycerol appears to become less polar as the mass of glycerol increases in the experiment. Since the mass injected has no effect on the soybean acylglycerol retention tine, it would appear that the effect with glycerol is unrelated to a change in mobile phase physical properties or to a change from supercritical to subcritical conditions. On the other hand, it does seem reasonable that a highly polar molecule like glycerol with three hydroxyl groups would exhibit greater intermolecular interaction at a greater analyte concentration in a basically nonpolar medium of 98% CO2 . In other words, the decrease in retention time could be explained by a reduction in effective polarity for glycerol. 3.3. Biodiesel purity test The above method was used to determine the purity of three different synthetic biodiesels from tobacco seed oil prepared “inhouse” (submitted for publication: M. Ashraf-Khorassani, W.M. Coleman III, M.F. Dube, L.T. Taylor, “Synthesis, Purification, and Quantification of Fatty Acid Ethyl Esters After trans-Esterification of Large Batches of Tobacco Seed Oil”). Due to the complexity of the biodiesel, the method was further optimized by lowering the mobile phase flow rate from 1.2 to 1.0 mL/min in order to enhance the separation of impurities that eluted after biodiesel (Fig. 4). As can be observed, all three biodiesel samples showed the presence of multiple impurities as well as mono-acylglycerols at a retention time of 6.2 min. These impurities were highest in batch 2 and least in batch 3. For comparison, B100 biodiesel from a commercial source was obtained and also analyzed, Fig. 5A. It is interesting to note that mono-acylglycerols were observed in the commercial B100 biodiesel based on comparison of retention time. Other impurities, however, were not detected. Fig. 5B shows the separation of model (i.e. pre-mixed fatty acid alkyl esters) biodiesel spiked with different, but known, impurities for comparison. No quantification of impurities was attempted in this study In order to remove the impurities from synthesized biodiesel, a simple two-step column chromatography process was developed. In this process, tobacco seed oil biodiesel with associated impurities was passed through a bare silica column and eluted via gravity first using hexane and followed by elution of the remaining analytes using ethanol. Solvent from each fraction was evaporated and the proper amount of each sample was dissolved in MeOH/DCM (1/1, v/v). Analysis of the fractions was performed using the UHPSFC method developed previously. Fig. 6 shows the separation of biodiesel (a) before purification, (b) after purification when hexane was the eluting solvent, and (c) when ethanol was
the initial eluting solvent. The results suggested that nearly pure synthetic biodiesel was obtained in the hexane fraction. Monoacylgycerols were again easily separated and detected in these biodiesel samples using UHPSFC/ELSD. See Fig. 6 caption for silica chromatography conditions. 4. Conclusions UHPSFC with ELSD was determined to be much faster and easier than either high temperature GC or GC with preliminary derivatization as an analytical tool for determination of biodiesel impurities. A complete separation of tri-acylglycerols, di-acylglycerols, monoacylglycerols, and free glycerol from model biodiesel was obtained in less than 15 min. Faster separation was obtained by increasing the gradient elution. The ELSD S/N ratio for glycerol for an injected mass of 2 g was about 10/1. This level of detection could easily be lowered with higher injection volume (10 L) to be compatible with ASTM D 6751 for detection and analysis of glycerol (0.02%). Low volatile and very long chain fatty acids (>24 carbon atoms) could be easily analyzed with UHPSFC although it was not demonstrated here. With GC, high MW components or impurities may elute in the same chromatographic region as the mono-, di-, and tri-acylglycerols, which can cause poor accuracy in quantification. With UHPSFC, complete baseline separation of mixtures of FAME, FFA, mono-, di-, and tri-acylglycerols and free glycerol affords quantitative results with good accuracy and precision. References [1] J.K. Rodriguez-Guerrero, R.M. Filho, P.T.V. Rosa, Production of biodiesel from castor oil using sub and supercritical ethanol: effect of sodium hydroxide on the ethyl ester production, J. Supercrit. Fluids 83 (2013) 124–132. [2] J.W. Diehl, F.P. DiSanzo, Determination of total biodiesel fatty acid methyl ethyl esters, and hydrocarbon types in diesel fuels by supercritical fluid chromatography-flame ionization detection, J. Chromatogr. Sci. 45 (2007) 690–693. [3] http://cenm.ag/two14 [4] S.C. Moldoveanu, Y. Chang, Dual analysis of triglycerides from certain common lipids and seed extracts, J. Agric. Food Chem. 59 (2011) 2137–2147. [5] J.L. Bernal, M.T. Martin, L. Toribio, Supercritical fluid chromatography in food analysis, J. Chromatogr. A 1313 (2013) 24–36. [6] M. Buchgraber, F. Ulberth, E. Anklam, Comparison of HPLC and GLC techniques for the determination of the triglyceride profile of cocoa butter, J. Agric. Food Chem. 48 (2000) 3359–3363. [7] C.A. Dorschel, Characterization of the TAG of peanut oil by electrospray LC–MS–MS, J. Am. Oil Chem. Soc. 79 (2002) 749–753. [8] T. Bamba, J.W. Lee, A. Matsubara, E. Fukusaki, Metabolic profiling of lipids by supercritical fluid chromatography/mass spectrometry, J. Chromatogr. A 1250 (2012) 212–219. [9] E. Lesellier, A. Tchapla, Retention behavior of triglycerides in octadecyl packed subcritical fluid chromatography with CO2 /modifier mobile phases, Anal. Chem. 71 (1999) 5372–5378. [10] Q. Zhou, B. Gao, X. Zhang, Y. Xu, H. Shi, L. Yu, Chemical profiling of triacylglycerols and di-acylglycerols in cow mile fat by ultra-performance convergence chromatography combined with a quadrupole time-of-flight mass spectrometry, Food Chem. 143 (2014) 199–204. [11] A. Dermaux, M. Medvedovici, E. Ksir, M. Van Hove, P. Talbi, Sandra, Elucidation of the triglycerides in fish oil by packed column supercritical fluid chromatography fractionation followed by capillary electrochromatography and electrospray mass spectrometry, J. Microcol. Sep. 11 (1999) 451–459. [12] E. Lesellier, A. Tchapla, Supercritical fluid chromatography with packed columns, techniques and applications, in: K. Anton, C. Berger (Eds.), Chromatographic Science Series, vol. 75, Marcel Dekker Inc., New York, 1998, p. 195. [13] A.G.-G. Perrenoud, J.-L. Veuthey, D. Guillarme, Comparison of ultra-high performance supercritical fluid chromatography and ultra-high performance liquid chromatography for the analysis of pharmaceutical compounds, J. Chromatogr. A 1266 (2012) 158–167. [14] M.D. Jones, G. Isaac, G. Astarita, A. Aubin, J. Shockor, V Shulaev, Lipid Class Separation Using UPC2 /MS, Waters Corp., Application Note 720004579.
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Ultrahigh performance supercritical fluid chromatography of lipophilic compounds with application to synthetic and commercial biodiesel M. Ashraf-Khorassani a , J. Yang b , P. Rainville b , M.D. Jones b , K.J. Fountain b , G. Isaac b , L.T. Taylor a,∗ a b
Department of Chemistry, Virginia Tech, Blacksburg, VA 24061, United States Waters Corporation, 34 Maple Street, Milford, MA 01757, United States
a r t i c l e
i n f o
Article history: Received 2 September 2014 Accepted 14 December 2014 Available online 6 January 2015 Keywords: Ultrahigh performance supercritical fluid chromatography Tobacco seed oil Normal phase chromatography Evaporative light scattering detection Biodiesel purity test Octa-decyl bonded silica particles
a b s t r a c t Ultrahigh performance supercritical fluid chromatography (UHPSFC) in combination with sub-2 m particles and either diode array ultraviolet (UV), evaporative light scattering, (ELSD), or mass spectrometric (MS) detection has been shown to be a valuable technique for the determination of acylglycerols in soybean, corn, sesame, and tobacco seed oils. Excellent resolution on an un-endcapped single C18 column (3.0 mm × 150 mm) with a mobile phase gradient of acetonitrile and carbon dioxide in as little as 10 min served greatly as an improvement on first generation packed column SFC instrumentation. Unlike high resolution gas chromatography and high performance liquid chromatography with mass spectrometric detection, UHPSFC/MS was determined to be a superior analytical tool for both separation and detection of mono-, di-, and tri-acylglycerols as well as free glycerol itself in biodiesel without derivatization. Baseline separation of residual tri-, di-, and mono-acylglycerols alongside glycerol at 0.05% (w/w) was easily obtained employing packed column SFC. The new analytical methodology was applied to both commercial B100 biodiesel (i.e. fatty acid methyl esters) derived from vegetable oil and to an “in-house” synthetic biodiesel (i.e. fatty acid ethyl esters) derived from tobacco seed oil and ethanol both before and after purification via column chromatography on bare silica. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The development of new technologies that afford the production of fuels that are obtained from renewable resources is driven by both environmental concerns and fossil fuel deficiency. Biofuels, such as the biodiesel produced via base catalyzed transesterification of vegetable oils and animal fats using either ethanol or methanol in the U.S. is a promising alternative fuel source [1]. Biodiesel has similar chemical structure and energy content to petro-diesel in terms of fuel quality. Compared to petroleum diesel, however, biodiesel can reduce CO2 emissions approximately 78%. As an energy source, biodiesel (i.e. fatty acid alkyl esters) should have certain criteria [2]. The feed-stocks available for producing biodiesel are highly dependent upon the region, climate, soil conditions, and geography. For example, rapeseed is the dominant
∗ Corresponding author. Tel.: +1 540 231 6680; fax: +1 540 232 3255; mobile: +1 540 239 3944. E-mail address: [email protected] (L.T. Taylor). http://dx.doi.org/10.1016/j.jchromb.2014.12.012 1570-0232/© 2015 Elsevier B.V. All rights reserved.
feedstock for biodiesel production in Europe, soybean in the United States and palm oil in tropical countries such as Malaysia and the Philippines. Coconut is another feedstock used for biodiesel production in the Pacific Rim region. The advantages of biodiesel in addition to being considered more environmentally friendly than petro diesel are higher octane number and higher combustion efficiency. Biodiesel is usually commercially blended with petro-diesel to 5% biodiesel. A major problem related to the use of biodiesel is its inherent instability to oxidation. For example, biodiesel degradation can change the fuel properties by formation of gums, acids, insolubles, and other oxidation products. The level of impurities in commercial biodiesel B100 as cited in ASTM D 6751 for (a) tri-acylglycerol which is unreacted starting material, (b) di-acylglycerol and mono-acylglycerol which are reaction intermediates, and (c) free glycerol which is a by-product should be 0.02 wt% for glycerol itself and 0.24 wt% total glycerol which includes mono-, di-, and tri-acylglycerols as well as glycerol itself. It is interesting to note that each year producers of biodiesel create more than a million tons of “just” glycerol worldwide much of which goes to waste [3]. The presence and
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concentration of these impurities obviously lead to biodiesel products that vary in complexity, polarity, solubility, and volatility. Analysis of acylglycerols typically follows two routes: (1) analysis of the whole molecule which can be a mixture of glycerol itself, mono-acylglycerol, di-acylglycerol, and tri-acylglycerol and (2) analysis of the range of fatty acids that originally esterify the glycerol molecule [4]. Analyses of whole acylglycerol molecules have been reported in the literature and in ASTM D 6584. Such techniques use GC, HPLC, TLC or a combination of these [5]. Most GC techniques employ flame ionization detection; while HPLC frequently uses evaporative light scattering [6] and mass spectrometric [7] detection. For glycerol, mono-, and di-acylglycerols, the analytical method is based on conversion of the molecular hydroxyl function into silyl derivatives in the presence of pyridine and N-trimethylsilyltrifluoroacetamide followed by high temperature GC on a short capillary column with thin film thickness using on-column injection and flame ionization detection [4]. Most techniques, on the other hand, dedicated to HPLC are designed to deal with tri-acylglycerols (TAGs). In most of these cases, the methodology employs multi-dimensional chromatography wherein argentation chromatography is one of the components designed to achieve separation of unsaturated acylglycerols Packed column supercritical fluid chromatography (SFC), on the other hand, can be essentially considered to be a more powerful tool for lipid analyses [8] than either HRGC or HPLC. In comparison to GC, acylglycerols are separated via SFC at a much lower temperature and compared to HPLC, different selectivity and shorter analysis times are obtained with SFC. Contrary to numerous references that have appeared in the older literature, polar solutes such as free glycerol can be analyzed via SFC without derivatization. For example, TAGs have been analyzed by packed column SFC using for example 2-ethylpyridine [9] and silver-ion exchange [10] stationary phases. Using a reversed phase stationary phase such as octadecylsilica [11], the SFC separation may be based upon carbon number (i.e. total number of carbons in all fatty acids) and/or on the number of double bonds. In a more recent reversed phase separation [12], three Zorbax SB-C18 columns (4.6 mm × 250 mm, 5 m) were coupled in series. Temperature was 25 ◦ C with an acetonitrile/methanol modifier gradient. With a flow rate of 2.5 mL/min, a mixture of eight TAGs eluted between 45 and 60 min, although they were not baseline resolved. On the other hand, separation of selected unsaturated vegetable oils on the silver loaded column was mainly based on the number of double bonds. Evaporative light scattering detection results were reproducible and provided enhanced sensitivity compared to UV detection. An improvement in chromatographic performance for similar lipophilic analytes is reported here. Fast determination of residual glycerol, free fatty acids, and mono-, di-, and tri-acylglycerols in both synthetic biodiesel prepared “in-house” and commercial B100 biodiesel (e.g. typically referred to as fatty acid alkyl esters) is described here using ultrahigh performance supercritical fluid chromatography (UHPSFC). UHPSFC is a relatively new separation technique that uses compressed carbon dioxide as the primary mobile phase [13]. It takes advantage of sub-2 m particle chromatography columns and advanced chromatography systems that are designed to achieve fast and reproducible separation with high efficiencies and unique selectivity. The established UHPSFC/MS approach has potential application in lipidomics and food testing as a complementary method alongside HPLC–MS and HRGC–MS, as the former can separate both polar and non-polar lipids in a single run to improve both detection limits and peak shape. Unlike HRGC/MS, low volatile and very long chain fatty acids (>24 carbon atoms) can be easily analyzed without concern for analyte degradation. In this regard, the possibilities for lipid analyses via UHPSFC were recently illustrated by Jones et al. [14] who performed the separation of neutral and amphipathetic lipids using
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both BEH (unbonded organic/inorganic silica hybrid) and HSS C18 (un-endcapped silica) columns packed with sub-2 m particles. Studies using UHPSFC coupled with photodiode array (DAD), mass spectrometry (MS), and evaporative light scattering (ELSD) detection for the separation of TAGs isolated from tobacco, corn, sesame, and soybean seed oils are presented here. A single unendcapped C18 column with acetonitrile-modified carbon dioxide was employed for separation of all seed oils. Excellent chromatography of associated, hydroxyl-containing di-acylglycerols, mono-acylglycerols, and glycerol wherein methanol was employed as a secondary modifier in addition to acetonitrile was also achieved. 2. Experimental 2.1. Sample description Pure fatty acid ethyl esters (C16 , C18 , C18:1 , C18:2 , and C18:3 ) were purchased from Sigma–Aldrich (St. Louis, MO) and mixed to form a model biodiesel. Pure mono-, di-, and trioctadecylglycerol plus free glycerol and soybean oil were also obtained from Sigma–Aldrich. All standards were prepared in a 50/50 dichloromethane/methanol (DCM/MeOH) mixture. The model biodiesel was prepared in the same mixture as a 5% (v/v) solution. Tobacco seed oil was obtained from R.J. Reynolds Tobacco Co. (Winston-Salem, NC). Soybean oil, corn oil, and sesame seed oil were obtained from Sigma–Aldrich (St. Louis, MO). Five percent of the different oils was dissolved in DCM/MeOH (1/1, v/v) for both UV and ELS detection and then 0.1% for the Xevo G2 QTof MS. 2.2. Chromatographic conditions Supercritical fluid chromatography experiments were performed using a Waters Acquity UPC2 system (Milford, MA, USA) equipped with (a) high pressure mixing binary solvent delivery manager, (b) fixed loop design autosampler, (c) active back pressure regulator, (d) column compartment with active heating and column switching control, (e) photodiode array (PDA) and evaporative light scattering (ELS) detectors. Experiments were carried out using an ACQUITY UPC2 HSS C18 SB column (150 mm × 3.0 mm, 1.8 m) at a temperature of 25 ◦ C. The mobile phase consisted of compressed CO2 (component A) and either 100% acetonitrile or acetonitrile/methanol (90:10) (component B). The mobile phase flow rate was maintained at 1–2 mL/min under all different gradient conditions. Backpressure was maintained isobarically and automatically by a backpressure regulator (ACQUITY CCM) at a pressure of 1500 psi. The injection volume was varied 2–8 L for both PDA and ELSD; while, 0.1 L was the volume for MS detection. Photodiode array detection was monitored at a wavelength range 190–400 nm with a reference of 400–500 nm. The Waters ACQUITY ELSD detector was operated with nebulizer cooling, drift tube: 50 ◦ C, gas pressure: 40 psi and gain 10, make up flow (isopropyl alcohol) was added at 0.2 mL/min before the ELSD. The solvent flow was split prior to the back pressure regulator for ELSD (split ratio 1:3) and MS electrospray probe. 2.3. Mass spectrometric conditions Mass spectrometry was performed using Xevo G2 QTof (Waters Corp., Milford, MA, USA). The solvent flow was split post PDA detection using a pre-BPR flow Upchurch cross 1/16 PEEK splitter. CO2 -miscible make-up solvent (MeOH in 10 mM NH4 OAc), delivered by a HPLC 515 make-up pump (Waters Corp., Milford, MA, USA), was added at a flow rate of 0.2 mL/min and mixed with the chromatographic effluent to aid ionization. A fraction of the total flow was directed from the union to the ESI source through
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a transfer line, whereas the remaining mobile phase was directed to the BPR PEEK connection. The ESI source was operated in positive ionization mode with capillary and cone voltages of +3 kV and 30 V, respectively. The source temperature, cone gas flow, desolvation temperature, and desolvation gas flow were set at 150 ◦ C, 10 L/h, 500 ◦ C and 600 L/h, respectively. MS data were acquired in the range of 400–1200. Data handling and instrument control were performed with Masslynx 4.1 (Waters Corp., Milford, MA, USA).
3. Results and discussion 3.1. UHPSFC of tri-acylglycerols SFC is useful for the separation of lipids such as carotenoids and tri-acylglycerols (TAGs) which have numerous analogs with similar structures. Currently, there is much interest in rapid characterization of TAGs which are natural compounds produced by the esterification of glycerol with fatty acids. While first generation supercritical fluid chromatographic (SFC) instrumentation has been successfully applied to the characterization of TAGs in various seed oils, the application of ultrahigh performance supercritical fluid chromatography (UHPSFC) to the range of byproducts that are produced during the conversion of TAGs to fatty acid alkyl esters (i.e. biodiesel) has not been considered. Separation and detection of the wide analyte polarity range afforded by underivatized mono-, di-, and tri-acylglycerols, glycerol, fatty acid alkyl esters, and free fatty acids by UHPSFC with three different detectors is discussed here. Fig. 1 shows the UV (i.e. diode array), ELSD, and MS chromatograms for the separation of TAGs in soybean oil. All of the chromatographic effluent first passes through the DAD flow cell then via a 3/1 split to the back pressure regulator and either ELSD or MS. Each gradient separation was performed at 25◦ C with an ACQUITY UPC2 HSS C18 SB column (HSS = high strength silica) and a mobile phase of acetonitrile-modified carbon dioxide. Near baseline peak resolution was observed with a single 3.0 mm × 150 mm column packed with 1.8 m bonded silica particles in approximately 16 min. Baseline stability was excellent even under gradient conditions. The resulting chromatographic system allowed reproducible separations and possible quantification of analytes at low concentrations in various samples. These optimized conditions were used to separate and profile TAGs in tobacco seed oil, soybean oil, corn oil, and sesame seed oil. Data were acquired in UHPSFC-MSE mode, which is an unbiased Tof acquisition method in which the mass spectrometer switches between low and high energy on alternate scans for structural elucidation and identification. In all cases, distinct profiles and excellent separations were obtained for all oil types when using UHPSFC with both MS and ELSD detection. Fig. 2 shows the UHPSFC/MS separation and detection of various TAGs in different oils. TAGs were identified using accurate mass spectra collected by QTof MS with MSE and the Waters Progenesis QI. In positive ion mode MSE low energy, TAGs produced intact ammonium ion adducts [M+NH4 ]+ precursor exact mass when ammonium acetate was present in the make-up solvent. In MSE high energy, abundant fragment ions were produced corresponding to the neutral loss of one of the sn-1, sn2 or sn-3 fatty acids plus ammonia. For example, the ion at m/z 874.7823 corresponding to 52:3 TAG (calculated fatty acid carbon atom: total number of double bonds), can be identified as POL (i.e. palmitic–oleic–linoleic) due to the presence of abundant fragment ions at m/z 575.5038, 577.5201 and 601.5186. These ions correspond to the neutral loss of fatty acyl groups P, O, and L plus ammonia, respectively [4]. Table 1 shows a list of all identified TAGs in the different oils based on the low energy precursor exact mass and corresponding high energy fragment ions.
Table 1 List of detected TAGs in each seed oil with their retention time via UHPSFC/MS. Ret. time (min)
Soybean oil
Corn oil
Sesame oil
Tobacco seed oil
10.86 11.11 11.40 11.60 11.78 11.90 12.11 12.32 12.49 12.71 13.00 13.08 13.22 13.40 13.93 14.26 14.73 15.14 15.70
LnLnL LnLL LLL OLLn PLLn SLLn OLL POLn OOL POL PPL OOO SLL POO SOL PSL SSL PSO PSA
– LnLL LLL OLLn – SLLn OLL POLn OOL POL PPL OOO SLL POO SOL PSL SSL PSO PSA
– LnLL LLL OLLn – SLLn OLL POLn OOL POL PPL OOO SLL – SOL PSL SSL PSO PSA
– LnLL LLL – SLLn OLL – OOL POL PPL – SLL POO SOL PSL SSL – –
– not detected.
3.2. Feasibility of impurities assay A single ACQUITY HSS C18 SB column (3.0 mm × 150 mm) packed with 1.8 m particles was used to develop the separation of a model mixture of anticipated biodiesel components such as free glycerol, acylglycerols, and fatty alkyl esters. When a faster gradient of modifier was employed, all the components were eluted in less than 9 minutes without a significant loss in resolution even among the mono-, di-, and tri-acylglycerols. Application of a steeper modifier gradient ramp in mobile phase under the same chromatographic conditions further decreased the analysis time to 4 min with again minimal loss in peak resolution. These data indicated that the gradient profile, flow rate, and mobile phase modifier can be readily changed in order to optimize the separation regardless of the biodiesel analysis required. Furthermore, all the listed parameters are compatible with MS detection, such that positive identification of all acylglycerol species plus glycerol itself is possible. Finally a faster separation without significant loss in analyte resolution could translate into higher sample throughput when monitoring product quality. Equally important is the observation that baseline separation of all compounds was achieved. It is also imperative that one must also use a method whereby the impurities do not interfere with proper quantification of residual acylglycerols in the sample. This situation can become critical since most biodiesels have minor components which elute early but after elution of the fatty acid ethyl esters (i.e. biodiesel components). For selected analyses, flow was lowered from 1.2 to 1.0 mL/min to minimize these interferences. It should be noted that reproducibility was excellent for all analytes. To demonstrate this feature, five replicate chromatographic injections of our model biodiesel mixture that contained the four spiked impurities using ELSD were performed. Percent RSD for all peak areas was between 1 and 4%, and the RSD for retention time of all peaks was less than 0.1%. Next a series of model biodiesel mixtures were prepared wherein the compounds varied in concentration. In each case, FAEE, soybean oil TAGs, and free glycerol were mixed at different concentrations and the resulting mixture was injected into the ACQUITY HSS C18 SB column (3.0 mm × 150 mm). A series of chromatograms were thus generated wherein the spiking percentage varied from 0.2% to 5.0% (v/v). At the lowest concentration (0.2%), the glycerol peak was clearly seen and well resolved from the other component peaks for quantification purposes. In order to estimate system detection limit, larger injection volumes (4 and 8 L) of the
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Fig. 1. Sequential UHPSFC of soybean oil with UV, ELSD, and MS detection using CO2 with CH3 CN as modifier. Chromatography conditions: gradient elution: 2–20% CH3 CN in 18 min. Flow: 1.5 mL/min, 25 ◦ C. Column ACQUITY UPC2 HSS C18 SB column (150 mm × 3.0 mm, 1.8 m).
Fig. 2. UHPSFC/MS of different oils using CO2 with CH3 CN as modifier. Gradient elution: 2–20% CH3 CN in 18 min. Flow: 1.5 mL/min, 25 ◦ C. Column ACQUITY UPC2 HSS C18 SB column (150 mm × 3.0 mm, 1.8 m).
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Fig. 3. UPSFC/ELSD detection of glycerol at 0.05% level at two different injection volume (4 and 8 L). Chromatography conditions: T = 0 min, 98/2 CO2 /modifier (90/10 CH3 CN/CH3 OH), T = 10 min, 80/20, T = 12 min, 60/40, T = 15 min, 60/40, flow = 1.2 mL/min., 25 ◦ C. Column ACQUITY UPC2 HSS C18 SB column (150 mm × 3.0 mm, 1.8 m) volume.
Fig. 4. UHPSFC/ELSD of 3 different batches of synthetic biodiesel. Chromatography conditions: T = 0 min, 90/10 CO2 /modifier, T = 10 min, 50/50, T = 11 min, 50/50, T = 11.1 min, 90/10, T = 12 min, 90/10, flow = 1.0 mL/min., 25◦ C. Column ACQUITY UPC2 HSS C18 SB column (150 mm × 3.0 mm, 1.8 m).
mixture spiked with impurities at 0.05% of glycerol yielded comparable results (Fig. 3). The S/N ratio for glycerol using ELSD was about 10/1 at the 0.05% level with a 4 L injection volume which was equivalent to 2 g mass injected and 40/1 at the
same level with an 8 L injection which was equivalent to 4 g injected on-column. The detection limit demonstrated here via this method is compatible with ASTM 6751for biodiesel impurities analysis.
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Fig. 5. Injection of commercial biodiesel B100 and synthetic biodiesel spiked with different impurities. Chromatography conditions: T = 0 min, 90/10 CO2 /modifier, T = 10 min, 50/50, T = 11 min, 50/50, T = 11.1 min, 90/10, T = 12 min, 90/10, flow = 1.0 mL/min., 25 ◦ C. Column ACQUITY UPC2 HSS C18 SB column (150 mm × 3.0 mm, 1.8 m).
Fig. 6. UHPSFC/ELSD separation of synthetic biodiesel before and after purification. Chromatography conditions: T = 0 min, 90/10 CO2 /modifier, T = 10 min, 50/50, T = 11 min, 50/50, T = 11.1 min, 90/10, T = 12 min, 90/10, flow = 1.0 mL/min., 25◦ C. Column ACQUITY UPC2 HSS C18 SB column (150 mm × 3.0 mm, 1.8 m).
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This study yielded a surprising result regarding the retention times of the various components in the model mixture. An increase in amount of soybean oil tri-acylglycerols injected from 0.2% to 5.0% had little or no effect upon retention time of specific lipophilic analytes. The result insofar as glycerol was quite different. Retention time of glycerol for 4 g injected was 14.63 min. An increase in amount injected from 4 g to 100 g injected caused the retention time to reproducibly go from14.63 min to 14.57 min to 14.44 min to 14.28 min. Since all of the separations in the total study were normal phase as evidenced by the observation that analyte retention increased with the number of non-derivatized hydroxyl groups (i.e. elution order is tri-acylglycerol with no hydroxyls, followed by di-acylglycerols with one hydroxyl, followed by mono-acylglycerol with two hydroxyls, followed by glycerol with three hydroxyls). Thus glycerol appears to become less polar as the mass of glycerol increases in the experiment. Since the mass injected has no effect on the soybean acylglycerol retention tine, it would appear that the effect with glycerol is unrelated to a change in mobile phase physical properties or to a change from supercritical to subcritical conditions. On the other hand, it does seem reasonable that a highly polar molecule like glycerol with three hydroxyl groups would exhibit greater intermolecular interaction at a greater analyte concentration in a basically nonpolar medium of 98% CO2 . In other words, the decrease in retention time could be explained by a reduction in effective polarity for glycerol. 3.3. Biodiesel purity test The above method was used to determine the purity of three different synthetic biodiesels from tobacco seed oil prepared “inhouse” (submitted for publication: M. Ashraf-Khorassani, W.M. Coleman III, M.F. Dube, L.T. Taylor, “Synthesis, Purification, and Quantification of Fatty Acid Ethyl Esters After trans-Esterification of Large Batches of Tobacco Seed Oil”). Due to the complexity of the biodiesel, the method was further optimized by lowering the mobile phase flow rate from 1.2 to 1.0 mL/min in order to enhance the separation of impurities that eluted after biodiesel (Fig. 4). As can be observed, all three biodiesel samples showed the presence of multiple impurities as well as mono-acylglycerols at a retention time of 6.2 min. These impurities were highest in batch 2 and least in batch 3. For comparison, B100 biodiesel from a commercial source was obtained and also analyzed, Fig. 5A. It is interesting to note that mono-acylglycerols were observed in the commercial B100 biodiesel based on comparison of retention time. Other impurities, however, were not detected. Fig. 5B shows the separation of model (i.e. pre-mixed fatty acid alkyl esters) biodiesel spiked with different, but known, impurities for comparison. No quantification of impurities was attempted in this study In order to remove the impurities from synthesized biodiesel, a simple two-step column chromatography process was developed. In this process, tobacco seed oil biodiesel with associated impurities was passed through a bare silica column and eluted via gravity first using hexane and followed by elution of the remaining analytes using ethanol. Solvent from each fraction was evaporated and the proper amount of each sample was dissolved in MeOH/DCM (1/1, v/v). Analysis of the fractions was performed using the UHPSFC method developed previously. Fig. 6 shows the separation of biodiesel (a) before purification, (b) after purification when hexane was the eluting solvent, and (c) when ethanol was
the initial eluting solvent. The results suggested that nearly pure synthetic biodiesel was obtained in the hexane fraction. Monoacylgycerols were again easily separated and detected in these biodiesel samples using UHPSFC/ELSD. See Fig. 6 caption for silica chromatography conditions. 4. Conclusions UHPSFC with ELSD was determined to be much faster and easier than either high temperature GC or GC with preliminary derivatization as an analytical tool for determination of biodiesel impurities. A complete separation of tri-acylglycerols, di-acylglycerols, monoacylglycerols, and free glycerol from model biodiesel was obtained in less than 15 min. Faster separation was obtained by increasing the gradient elution. The ELSD S/N ratio for glycerol for an injected mass of 2 g was about 10/1. This level of detection could easily be lowered with higher injection volume (10 L) to be compatible with ASTM D 6751 for detection and analysis of glycerol (0.02%). Low volatile and very long chain fatty acids (>24 carbon atoms) could be easily analyzed with UHPSFC although it was not demonstrated here. With GC, high MW components or impurities may elute in the same chromatographic region as the mono-, di-, and tri-acylglycerols, which can cause poor accuracy in quantification. With UHPSFC, complete baseline separation of mixtures of FAME, FFA, mono-, di-, and tri-acylglycerols and free glycerol affords quantitative results with good accuracy and precision. References [1] J.K. Rodriguez-Guerrero, R.M. Filho, P.T.V. Rosa, Production of biodiesel from castor oil using sub and supercritical ethanol: effect of sodium hydroxide on the ethyl ester production, J. Supercrit. Fluids 83 (2013) 124–132. [2] J.W. Diehl, F.P. DiSanzo, Determination of total biodiesel fatty acid methyl ethyl esters, and hydrocarbon types in diesel fuels by supercritical fluid chromatography-flame ionization detection, J. Chromatogr. Sci. 45 (2007) 690–693. [3] http://cenm.ag/two14 [4] S.C. Moldoveanu, Y. Chang, Dual analysis of triglycerides from certain common lipids and seed extracts, J. Agric. Food Chem. 59 (2011) 2137–2147. [5] J.L. Bernal, M.T. Martin, L. Toribio, Supercritical fluid chromatography in food analysis, J. Chromatogr. A 1313 (2013) 24–36. [6] M. Buchgraber, F. Ulberth, E. Anklam, Comparison of HPLC and GLC techniques for the determination of the triglyceride profile of cocoa butter, J. Agric. Food Chem. 48 (2000) 3359–3363. [7] C.A. Dorschel, Characterization of the TAG of peanut oil by electrospray LC–MS–MS, J. Am. Oil Chem. Soc. 79 (2002) 749–753. [8] T. Bamba, J.W. Lee, A. Matsubara, E. Fukusaki, Metabolic profiling of lipids by supercritical fluid chromatography/mass spectrometry, J. Chromatogr. A 1250 (2012) 212–219. [9] E. Lesellier, A. Tchapla, Retention behavior of triglycerides in octadecyl packed subcritical fluid chromatography with CO2 /modifier mobile phases, Anal. Chem. 71 (1999) 5372–5378. [10] Q. Zhou, B. Gao, X. Zhang, Y. Xu, H. Shi, L. Yu, Chemical profiling of triacylglycerols and di-acylglycerols in cow mile fat by ultra-performance convergence chromatography combined with a quadrupole time-of-flight mass spectrometry, Food Chem. 143 (2014) 199–204. [11] A. Dermaux, M. Medvedovici, E. Ksir, M. Van Hove, P. Talbi, Sandra, Elucidation of the triglycerides in fish oil by packed column supercritical fluid chromatography fractionation followed by capillary electrochromatography and electrospray mass spectrometry, J. Microcol. Sep. 11 (1999) 451–459. [12] E. Lesellier, A. Tchapla, Supercritical fluid chromatography with packed columns, techniques and applications, in: K. Anton, C. Berger (Eds.), Chromatographic Science Series, vol. 75, Marcel Dekker Inc., New York, 1998, p. 195. [13] A.G.-G. Perrenoud, J.-L. Veuthey, D. Guillarme, Comparison of ultra-high performance supercritical fluid chromatography and ultra-high performance liquid chromatography for the analysis of pharmaceutical compounds, J. Chromatogr. A 1266 (2012) 158–167. [14] M.D. Jones, G. Isaac, G. Astarita, A. Aubin, J. Shockor, V Shulaev, Lipid Class Separation Using UPC2 /MS, Waters Corp., Application Note 720004579.
