Biodiesel from Seeds: An Experiment for Organic Chemistry - Journal

Laboratory Experiment pubs.acs.org/jchemeduc

Biodiesel from Seeds: An Experiment for Organic Chemistry Steven W. Goldstein* Department of Chemistry, University of Saint Joseph, West Hartford, Connecticut 06117-2791, United States S Supporting Information *

ABSTRACT: Plants can store the chemical energy required by their developing offspring in the form of triglycerides. These lipids can be isolated from seeds and then converted into biodiesel through a transesterification reaction. This second-year undergraduate organic chemistry laboratory experiment exemplifies the conversion of an agricultural energy source directly into a mechanical energy source.

KEYWORDS: First-Year Undergraduate/General, Second-Year Undergraduate, Biochemistry, Environmental Chemistry, Laboratory Instruction, Organic Chemistry, Agricultural Chemistry, Bioorganic Chemistry, Lipids, Nutrition, Spectroscopy

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he interest in biofuels in public and scientific forums has escalated over the past few years. Whether this is due to the thought that the world has reached peak oil extraction1 or a search for a “friendlier” fuel, the general public is keenly aware of biologically based additives and replacements for home heating oil and fuels to power vehicles. The topic continues to be the subject of much scientific investigation, with 100 or more research articles being published by American Chemical Society journals over each of the past four years. Although much of the research has focused on the physical properties of biodiesel, other investigations focus on the conversion of various feedstock into fuel. One aspect of these studies that has not been covered in depth is isolation of the energy rich triglyceride raw material from natural sources. When students see the direct connection between the naturally derived plant energy source and the synthetic biofuel, a more complete picture of the process is presented. Plants and animals produce and store lipids, predominantly as triglycerides, in order to access the energy potential of those molecules at a later time. Seeds, which require energy to grow in the time between germination and active photosynthesis, store reserves of triglycerides in specific locations within the structure of the seed. Accordingly, a significant proportion of the cellular mass of seeds can be triglycerides.2 Upon germination, the activation of various lipase3 and oxygenase4 enzymes within the seed begins to provide the developing plants with the necessary nutrients for growth. The triglyceride composition of seeds varies dramatically; some have virtually none, while those seeds (and nuts) known for high oil content can contain more than 65% lipids by mass.5 The fatty acid constituents of triglycerides vary as well. Flax seeds contain a high proportion of the triply unsaturated α-linolenic acid,6 whereas coconut oil is composed of predominantly saturated acid (lauric, myristic, and palmitic acid) components.7 The individual molecules of triglycerides (Figure 1) are typically mixtures of one, two, or three different esterified fatty acids.8 © XXXX American Chemical Society and Division of Chemical Education, Inc.

Figure 1. Variability in triglyceride structure.

Isolation of these oils from the plant components typically involves pressing and/or solvent extraction.9,10 Ever since Rudolph Diesel investigated fueling his engine with peanut oil,11 the use of vegetable oils to power combustion engines has been of interest. While the energy to fuel engines certainly exists with heavier-weight plant- and animal-derived oils, other factors, especially viscosity,12,13 have limited their use in modern engines. Biodiesel, a term that encompasses the short chain (predominantly methyl and ethyl) ester of long chain fatty acids, overcomes this initial issue. The characteristics of these ester mixtures, such as degree of overall unsaturation, quality of combustion, caloric value, viscosity, and residual glycerol esters,12,13 identify their suitability for utility as fuel. While not the focus of this study, the transesterification of triglycerides into biodiesel has been thoroughly investigated.14 Base-catalyzed methods include the use of NaOH, KOH, and other metal oxides,15−18 although care must be taken to ensure that any free fatty acid in the oil does not neutralize the basic catalyst (Scheme I). Acid-mediated transesterification19 generally utilizes H2SO4 or HCl. Conversion of the triglycerides may also be accomplished with a variety of other salts20−23 or substrate-supported enzymes.24−27 Critical to these processes is the need for water-free triglycerides; any water in the transesterification reaction may result in the production of free fatty acids, thus complicating the separation of product layers and/or contamination of the final product.

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dx.doi.org/10.1021/ed4008974 | J. Chem. Educ. XXXX, XXX, XXX−XXX

Journal of Chemical Education

Laboratory Experiment

for 1 h. After it cools, the suspension is filtered and concentrated to yield the isolated vegetable oils; heptanes are recovered for reuse. The oil is characterized by mass, 1H NMR and IR spectroscopy, and relative viscosity. In week two, students convert the isolated triglycerides into biodiesel. Students transfer a sample of the isolated oil (1−5 g) to a test tube, add a solution29 of 40% KOH in methanol (1 mL per solution per g of oil), and place the test tube at 50−60 °C in a water bath; within a few minutes the phases separate and the less dense biodiesel rises to the top. The methyl esters are washed with saturated NaCl solution until neutral. Physical characteristics (e.g., mass, 1H NMR and IR spectroscopy, and relative viscosity) of the biodiesel are obtained for comparison with those of the isolated triglyceride. A detailed description of the experiment is in the Supporting Information.

Scheme I. KOH/Methanol Conversion of Triglyceride into Biodiesel

The choice of substrate for the production of biodiesel on a commodity scale is fraught with technical as well as ethical issues. The use of edible oils (e.g., corn, soybean, peanut, sunflower) in their raw state would likely cause a demanddriven market and subsequent rise in price of these critically important foodstuffs. The utilization of these oils after primary usage, especially by restaurant and industrial fryers, requires extensive removal of free fatty acid contaminants, a result of prolonged heating.27 Animal fat also contains a large amount of these impurities and makes subsequent processing problematic. Some authors have claimed the future of economically viable biodiesel production relies on the use of a nonedible vegetable oil (e.g., Jatropha or Pongamia) with low free fatty acid content.13 It is through the isolation of various common oils and subsequent conversion into biodiesel that students have a more complete understanding of the process. Experiments in this Journal have certainly provided readers with procedures to convert cooking oil into biodiesel16−19 wherein the substrates are both used and virgin vegetable oils. It is a concern that students may not identify a central theme of this experiment; the energy stored by the plants for its developing offspring may be utilized by humans for other uses. To date, there is only one experiment in which the triglyceride starting material is isolated from its natural source.28 While that work is unique in its perspective, it requires a six-week inoculation and culturing of an algae broth, beyond the scope of many undergraduate laboratories. Herein is provided a twolaboratory session experiment that isolates triglycerides from seeds, followed by a transesterification reaction to prepare the biodiesel product. Successful completion of this experiment allows students to quantify the amount of triglycerides in seeds and convert it into a methyl ester that could be used as fuel. They will also understand what observable changes accompany this transformation (e.g., viscosity, NMR spectroscopy) and that the original source of energy can be utilized by the plant or by humans.

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HAZARDS Heptanes and methanol are flammable, as well as inhalation and contact hazards. Potassium hydroxide is very caustic; chemical splash goggles and other forms of PPE (e.g., gloves, aprons) should be worn at all times. Triglycerides and the analogous biodiesel are flammable and should be disposed of following guidelines for organic waste disposal. Contact with CDCl3 should be avoided; it is an eye and skin irritant and hazardous.

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RESULTS AND DISCUSSION This experiment has been completed by a total of 44 students in four sections of a second-semester introductory organic chemistry course. Consideration of the type of seed to be processed was important to the overall success of the experiment. If the material was a very moist seed (soybean) or was processed with water (coffee),29 it should be dried in an oven to avoid the transfer of water to the triglyceride extract. Grinding the substrate to roughly “espresso” particle size is necessary to increase the surface area and the rate of extraction. The recovery and reuse of the heptanes is a key exemplification of a green experiment and an important consideration. The percent mass recovery of the crude oils from the various seeds is shown in Table 1. Table 1. Range of Student Recovery of Triglycerides from Some Example Seeds

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THE EXPERIMENT Students work alone or with a single lab partner. This laboratory exercise can easily be done in two 3- to 4-h lab sessions. Seeds available are soybeans, safflower, flax, sunflower, and coffee (used); nut seeds (coconuts, peanuts, almonds, cashews, and pistachios) can be used, but allergies to nut seeds by some students could present potential issues with aerosolized nut powder from grinding. A very moist seed (soybean) or a seed processed with water (coffee),29 is dried in an oven prior to grinding. In week one, students isolate the triglyceride fraction from a seed. Students grind seeds to roughly an “espresso” particle size that is free-flowing, taking care not to form a peanut butter consistency with oilier seeds (e.g., sunflower and flax). The ground material is suspended in heptanes and heated to reflux

Seed Type

Oil Recovered (% mass)

Soybeans Safflower Flax Sunflower Coffee (used)

8−23 24−34 25−40 46−50 8−17

Conversion of the triglyceride into the corresponding methyl esters is a relatively simple process. The elevated temperature causes a lowering of the oil’s viscosity, allowing for better reagent mixing, and speeds the rate of methanolysis. The addition of a solution of 40% KOH in methanol29 causes a rapid transesterification; in general, a 50−75% mass recovery of the biodiesel product for this step is obtained. 1H NMR, even at 60 MHz, distinguished between some of the different types of oils (examples of student spectra in the Supporting Information). The methylenes of the triglyceride core formed an ABX pattern centered at 4.22 ppm with a geminal coupling constant of −11.8 Hz and two vicinal coupling constants of 4.4 B




information and feedback. Thanks also to Peter Markow for his editorial comments.

and 5.8 Hz. At this spectrometer frequency, the methine proton was partially buried beneath the olefin absorbance at 5.3 ppm. Triglycerides that have a significant amount of unsaturation in the carbon chain, such as flax oil,6 had a large peak at 5.3 ppm, whereas in those that contained predominantly saturated fatty acids, the glycerine methine was easily distinguishable. Following the conversion to biodiesel, an examination by 1H NMR spectroscopy clearly showed loss of the signal at 4.22 ppm and a new methyl ester signal at 3.66 ppm. Any signals in the 5.3 region were solely due to olefinic protons on the carbon chain. Analysis of the isolated triglyceride by IR spectroscopy was straightforward (example of a student spectrum in the Supporting Information); an ester absorbance at 1746 cm−1 and methylene “rock” at 724 cm−1 were hallmarks for this type of compound. There was little difference in the IR spectra when examined across the different oil types or after conversion to biodiesel. Critical to the utilization of biodiesel is the understanding that, although the caloric value is similar to that of the original triglyceride, the relative viscosity is much lower. Using a disposable glass pipet as a crude viscometer,18,28 students visualized and quantitated this physical change. Students measured the time it took for the isolated vegetable oil and the corresponding methyl esters to flow between two points 3 cm apart on a pipet. At least a 5- to 6-fold decrease in the time was required for the biodiesel to traverse the two points relative to the analogous triglyceride.

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(1) Campbell, C. J.; Laherrère, J. H. The End of Cheap Oil. Sci. Am. 1998, 278 (3), 78−83. (2) Laidman, D. L.; Tavener, R. J. Triglyceride Mobilization in the Germinating Wheat Grain. J. Biochem. 1971, 124 (2), 4−5. (3) Graham, I. A. Seed Storage Oil Mobilization. Annu. Rev. Plant Biol. 2008, 59, 115−142. (4) Penfield, S.; Graham, S.; Graham, I. A. Storage Reserve Mobilization in Germinating Oilseeds: Arabidopsis As a Model System. Biochem. Soc. Trans. 2005, 33 (Part2), 380−383. (5) Venkatachalam, M.; Sathe, K. Chemical Composition of Selected Edible Nut Seeds. J. Agric. Food Chem. 2006, 54 (13), 4705−4714. (6) Richter, E. V.; Spangenberg, J. E.; Kreuzer, M.; Leiber, F. Characterization of Rapeseed (Brassica napus) Oils by Bulk C, O, H, and Fatty Acid C Stable Isotope Analyses. J. Agric. Food Chem. 2010, 58 (13), 8048−8055. (7) Laureles, L. R.; Rodriguez, F. M.; Reaño, C. E.; Santos, G. A.; Laurena, A. C.; Mendoza, E. M. T. Variability in Fatty Acid and Triacylglycerol Composition of the Oil of Coconut (Cocos nucifera L.) Hybrids and Their Parentals. J. Agric. Food Chem. 2002, 50 (6), 1581−1586. (8) Moldoveanu, S. C.; Chang, Y. Dual Analysis of Triglycerides from Certain Common Lipids and Seed Extracts. J. Agric. Food Chem. 2011, 59 (6), 2137−2147. (9) Eisenmenger, M.; Dunford, N. T.; Eller, F.; Taylor, S.; Martinez, J. Pilot Scale Supercritical Carbon Dioxide Extraction and Fractionation of Wheat Germ Oil. J. Am. Oil Chem. Soc. 2006, 83 (10), 863− 868. (10) Bozan, B.; Temelli, F. Supercritical CO2 Extraction of Flaxseed. J. Am. Oil Chem. Soc. 2002, 79 (3), 231−235. (11) Shay, E. G. Diesel Fuel from Vegetable Oils: Status and Opportunities. Biomass Bioenergy 1993, 4 (4), 227−242. (12) Ma, F.; Hanna, M. A. Biodiesel Production: A Review. Bioresour. Technol. 1999, 70 (1), 1−15. (13) Pinzi, S.; Garcia, I. L.; Lopez-Gimenez, F. J.; Luque de Castro, M. D.; Dorado, G.; Dorado, M. P. The Ideal Vegetable Oil-Based Biodiesel Composition: A Review of Social, Economical and Technical Implications. Energy Fuels 2009, 23 (5), 2325−2341. (14) Demirbas, A. Biodiesel Fuels from Vegetable Oils via Catalytic and Non-Catalytic Supercritical Alcohol Transesterifications and Other Methods: A Survey. Energy Convers. Manage. 2003, 44 (13), 2093−2109. (15) Vicente, G.; Martinez, M.; Aracil, J. Integrated Biodiesel Production: A Comparison of Different Homogeneous Catalysts Systems. Bioresour. Technol. 2004, 92 (3), 297−305 and references therein. (16) Akers, S. M.; Conkle, J. L.; Thomas, S. N.; Rider, K. B. Determination of the Heat of Combustion of Biodiesel Using Bomb Calorimetry. A Multidisciplinary Undergraduate Chemistry Experiment. J. Chem. Educ. 2006, 83 (2), 260−262. (17) Bucholtz, E. C. Biodiesel Synthesis and Evaluation: An Organic Chemistry Experiment. J. Chem. Educ. 2007, 84 (2), 296−298. (18) Behnia, M. S.; Emerson, D. W.; Steinberg, S. M.; Alwis, R. M.; Duenas, J. A.; Serafino, J. O. A Simple, Safe Method for Preparation of Biodiesel. J. Chem. Educ. 2011, 88 (9), 1290−1292. (19) Bladt, D.; Murray, S.; Gitch, B.; Trout, H.; Liberko, C. AcidCatalyzed Preparation of Biodiesel from Waste Vegetable Oil: An Experiment for the Undergraduate Organic Chemistry Laboratory. J. Chem. Educ. 2011, 88 (2), 201−203. (20) Wang, Y.; Nie, J.; Zhao, M.; Ma, S.; Kuang, L.; Han, X.; Tang, S. Production of Biodiesel from Waste Cooking Oil via a Two-Step Catalyzed Process and Molecular Distillation. Energy Fuels 2010, 24 (3), 2104−2108. (21) Granados, M. L.; Alonso, D. M.; Alba-Rubio, A. C.; Mariscal, R.; Ojeda, M.; Brettes, P. Transesterification of Triglycerides by CaO:

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SUMMARY This student experiment allowed for the isolation of energy-rich triglycerides from natural sources and the subsequent conversion into biodiesel over the course of two 3- to 4-h lab sessions. Students separated an energy-rich plant constituent and readily converted it into a synthetic fuel. Although it was used as an experiment in a second-semester introductory organic chemistry course, it could also be used in a general chemistry or environmental chemistry curriculum. Its successful completion by every student group in four laboratory sections underlies the robustness of the science. Anecdotally, students reported that this was their favorite lab of the course. On the basis of post experiment lab reports, over 80% of the students met the goals of understanding the isolation and characterization of triglycerides the first week and over 90% thoroughly understood the transformation to biodiesel.

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ASSOCIATED CONTENT

S Supporting Information *

A detailed student handout of the experiment and instructor notes with 1H NMR and IR spectra. This material is available via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS I am grateful to Yen Le for initial experiments in this study and to the CHEM210 − Spring 2013 laboratory sections for C




Increase of the Reaction Rate by Biodiesel Addition. Energy Fuels 2009, 23 (4), 2259−2263. (22) Díaz, L.; Borges, M. E. Low-Quality Vegetable Oils As Feedstock for Biodiesel Production Using K-Pumice as Solid Catalyst. Tolerance of Water and Free Fatty Acids Contents. J. Agric. Food Chem. 2012, 60 (32), 7928−7933. (23) Einloft, S.; Magalhães, T. O.; Donato, A.; Dullius, J.; Ligabue, R. Biodiesel from Rice Bran Oil: Transesterification by Tin Compounds. Energy Fuels 2008, 22 (1), 671−674. (24) Li, W.; Du, W.; Liu, D. Rhizopus oryzae Whole-Cell-Catalyzed Biodiesel Production from Oleic Acid in tert-Butanol Medium. Energy Fuels 2008, 22 (1), 155−158. (25) Liu, Y.; Liu, T.; Wang, X.; Xu, L.; Yan, Y. Biodiesel Synthesis Catalyzed by Burkholderia cenocepacia Lipase Supported on Macroporous Resin NKA in Solvent-Free and Isooctane Systems. Energy Fuels 2011, 25 (3), 1206−1212. (26) Fu, B.; Vasudevan, P. T. Effect of Organic Solvents on EnzymeCatalyzed Synthesis of Biodiesel. Energy Fuels 2009, 23 (8), 4105− 4111. (27) Kulkarni, M. G.; Dalai, A. K. Waste Cooking OilAn Economical Source for Biodiesel: A Review. Ind. Eng. Chem. Res. 2006, 45 (9), 2901−2913. (28) A direct connection between algae lipid production and biodiesel is exemplified Blatti, J. L.; Burkart, M. D. Releasing Stored Solar Energy within Pond Scum: Biodiesel from Algal Lipids. J. Chem. Educ. 2012, 89 (2), 239−242. (29) Kondamudi, N.; Mohapatra, S. K.; Misra, M. Spent Coffee Grounds As a Versatile Source of Green Energy. J. Agric. Food Chem. 2008, 56 (24), 11757−11760.

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Biodiesel from Seeds: An Experiment for Organic Chemistry - Journal

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