The work was supported by Author YL&#39;s start-up fund and Pedagogy Enhancement Award (PEA) at California State University, Sacramento.

1. Oil source and preparation
<ol>
	<li>Add 2.0 mL of HPLC-grade methanol into a 15 mL centrifuge tube. 
	CAUTION: Methanol is a highly flammable liquid. It is toxic if swallowed, in contact with skin, or if inhaled, and it causes damage to the eyes. Ensure to wear personal protective equipment (PPE) when working with methanol and use it in the fume hood.</li>
	<li>Add one pellet of KOH (~0.10 g) to the centrifuge tube and dissolve the KOH solid using the ultrasonic cleaner (40 kHz) by just turning on the ultrasonic. 
	CAUTION: KOH is harmful if swallowed. It causes severe skin burns, eye damage, and serious eye damage. Ensure to wear PPE when working with this substance. 
	NOTE: For optimal results, place the centrifuge tube inside a beaker filled with water and then position the beaker within the ultrasonic bath. This immersed configuration guarantees thorough exposure of the reaction mixture to the ultrasonic irradiation, maximizing its effectiveness.</li>
</ol>
2. Transesterification process
<ol>
	<li>Add 8.0 mL of extra virgin olive oil to the centrifuge tube.</li>
	<li>Cap and close the centrifuge tube tight and shake the centrifuge tube vigorously to mix the oil and the potassium methoxide&#160;solution. 
	NOTE: Keep the centrifuge cap tight when shaking the centrifuge tube.</li>
	<li>Loosen the cap and put the centrifuge tube into the ultrasonic bath. Turn on the ultrasonic bath for 1 min.</li>
	<li>After the first 1 min, close the centrifuge cap tight and shake the centrifuge tube vigorously again.</li>
	<li>Loosen the cap and put the reaction mixture into the ultrasonic bath for another 14 min.</li>
	<li>Transfer the reaction mixture to a separatory funnel and drain the bottom glycerin layer.</li>
	<li>Wash the top layer with 15 mL of saturated NaCl solution 3x, to wash the excess methanol and residual catalyst out of the ester. Ensure the pH of the final washing is neutral by testing with a pH paper.</li>
	<li>Transfer the top biodiesel&#160;layer into a dry, clean vial, add anhydrous&#160;Na2SO4&#160;to the vial, swirl the mixture, and let the&#160;mixture stand for about 15 min till the biodiesel&#160;is clear. Use the clear biodiesel product for&#160;characterization.</li>
</ol>
3. Characterization of biodiesel
<ol>
	<li>Fourier-transform infrared (FT-IR) analysis
	<ol>
		<li>Record the FT-IR spectra across a wide range of 4000-400 cm-1. Measure each sample by co-adding 16 scans at a resolution of 4 cm-1. Perform background subtraction by acquiring a fresh air spectrum before each sample scan. This is to ensure accurate baseline correction and minimized sample contamination. Before each new sample, clean the ATR plate using methanol then dry with lint-free wipes.</li>
	</ol>
	</li>
	<li>Proton nuclear magnetic resonance (1H NMR) analysis
	<ol>
		<li>To analyze the chemical composition of the biodiesel product, record the nuclear magnetic resonance (NMR) spectra of biodiesel and extra virgin olive oil on a 500 MHz NMR spectrometer at room temperature. Utilizing a high-resolution 5 mm double gradient probe, prepare each sample by dissolving 50 mg of the sample in 0.7 mL of deuterated chloroform (CDCl3) containing 0.05% tetramethylsilane (TMS) as an internal standard. Acquire 1H NMR spectra using the TOPSPIN program with 16 scans and referenced to the TMS standard at 0.0 ppm. 
		&#8203;CAUTION: CDCl3 is harmful if swallowed and toxic if inhaled. It causes skin irritation and serious eye irritation. Wear PPE when working with this substance.</li>
	</ol>
	</li>
	<li>Viscosity analysis
	<ol>
		<li>Prepare two 5.75 inch glass Pasteur pipettes and one pipette pump.</li>
		<li>Make two marks on each pipette with a pen. The top mark is on the body of the pipette, and the second mark is on the narrow stem, approximately 2 cm up from the tip.</li>
		<li>Use a pipette pump to fill the pipette with extra virgin olive oil with the meniscus at the top mark.</li>
		<li>Remove the pipette pump and start the stopwatch. Stop the stopwatch as extra virgin olive oil reaches the lower mark.</li>
		<li>Repeat steps 3.3.3 and 3.3.4 with biodiesel product instead of extra virgin olive oil.</li>
		<li>Determine the relative viscosities of biodiesel versus the extra virgin olive oil by timing their passage through a glass pipette. 
		Relative Viscosity = (oil time)/(biodiesel time).</li>
	</ol>
	</li>
	<li>Flammability tests
	<ol>
		<li>Immerse a cotton string of approximately 2 cm in length into biodiesel and another cotton string into extra virgin olive oil. Ensure complete saturation of the string with the respective liquid. Place the coated cotton strings on aluminum foil.</li>
		<li>In a designated laboratory area, away from flammable solvents, assess the ease of igniting each cotton string and observe the quality of the flame produced. Determine if one cotton string ignites more readily than the other. Evaluate which liquid exhibits superior wicking capabilities and which sustains a stronger burn.</li>
	</ol>
	</li>
</ol>

Biodiesel Synthesis by Ultrasonic-Assisted Transesterification of Vegetable Oils

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+ultrasound+to+the+synthesis+of+nanostructured+materials.">Applications of ultrasound to the synthesis of nanostructured materials.</a> Adv Mater. 22 (10), 1039-1059 (2010).</li><li>Kaur, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound-assisted+green+synthesis+of+five-membered+O-+and+S-heterocycles.">Ultrasound-assisted green synthesis of five-membered O- and S-heterocycles.</a> Syn Comm. 48 (14), 1715-1738 (2018).</li><li>Liu, Y., Myers, E. J., Rydahl, S. A., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasonic-assisted+synthesis,+characterization,+and+application+of+a+metal-organic+framework:+A+green+general+chemistry+laboratory+project.">Ultrasonic-assisted synthesis, characterization, and application of a metal-organic framework: A green general chemistry laboratory project.</a> J Chem Edu. 96 (10), 2286-2291 (2019).</li><li>Tan, S. X., Lim, S., Ong, H. C., Pang, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=State+of+the+art+review+on+development+of+ultrasound-assisted+catalytic+transesterification+process+for+biodiesel+production.">State of the art review on development of ultrasound-assisted catalytic transesterification process for biodiesel production.</a> Fuel. 235, 886-907 (2019).</li><li>Mahamuni, N. N., Adewuyi, Y. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fourier+transform+infrared+spectroscopy+(FTIR)+method+to+monitor+soy+biodiesel+and+soybean+oil+in+transesterification+reactions,+petrodiesel&#8722;+biodiesel+blends,+and+blend+adulteration+with+soy+oil.">Fourier transform infrared spectroscopy (FTIR) method to monitor soy biodiesel and soybean oil in transesterification reactions, petrodiesel&#8722; biodiesel blends, and blend adulteration with soy oil.</a> Ener Fuels. 23 (7), 3773-3782 (2009).</li><li>Castej&#243;n, D., Fricke, P., Cambero, M. I., Herrera, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+1H-NMR+screening+of+fatty+acid+composition+in+edible+oils.">Automatic 1H-NMR screening of fatty acid composition in edible oils.</a> Nutrients. 8 (2), 93 (2016).</li><li>Doudin, K. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+and+qualitative+analysis+of+biodiesel+by+NMR+spectroscopic+methods.">Quantitative and qualitative analysis of biodiesel by NMR spectroscopic methods.</a> Fuel. 284, 119114 (2021).</li><li>Prat, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chem21+selection+guide+of+classical-and+less+classical-solvents.">Chem21 selection guide of classical-and less classical-solvents.</a> Green Chem. 18 (1), 288-296 (2016).</li><li>Ameen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospects+of+catalysis+for+process+sustainability+of+eco-green+biodiesel+synthesis+via+transesterification:+A+state-of-the-art+review.">Prospects of catalysis for process sustainability of eco-green biodiesel synthesis via transesterification: A state-of-the-art review.</a> Sustainability. 14 (12), 7032 (2022).</li><li>Malek, M. 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The authors have nothing to disclose.

In this demonstration, an ultrasonic assisted method of base-catalyzed production of biodiesel is elucidated for optimal efficacy. For optimal results, the centrifuge tube should be placed inside a beaker filled with water and then the beaker should be placed within the ultrasonic bath. This immersed configuration guarantees thorough exposure of the reaction mixture to the ultrasonic treatment, maximizing its effectiveness. If desired, a centrifuge rack can also be used to replace the beaker inside the ultrasonic bath, w...

Biodiesel, derived from common, plant-based oils and fats, emerges as a sustainable solution to mitigate reliance on petroleum1. This renewable substitute showcases reduced greenhouse gas emissions, notably carbon dioxide, while relying on sustainable resources. Furthermore, biodiesel presents distinct advantages over petroleum diesel, characterized by its sulfur-free composition, non-toxic nature, and biodegradability. As an alternative to conventional fossil fuels, biodiesel aligns with the United Nations&#39; (UN&#39;s) Net Zero policy by reducing our dependence on non-renewable fossil fuels and mitigating the adverse effects of climate change. Biodiesel offers a promising path to meeting current energy needs, making it a powerful choice for a greener future2.
The predominant method used for biodiesel production involves transesterification, a chemical process where triglycerides found in oils and fats react with an alcohol, typically methanol or ethanol, in the presence of a catalyst under elevated temperature conditions1,2,3,4.&#160;This reaction yields fatty acid alkyl esters, the principal component of biodiesel. Various types of vegetable oils serve as primary feedstocks for biodiesel production, including both edible5 (e.g., extra virgin olive oil and corn oil) and non-edible oils6,7,8 (e.g., caper seed oil), as well as waste oils9. Methanol is most commonly used for this transesterification process as it is a relatively inexpensive alcohol. Additionally, an array of catalysts such as sulfuric acid, phosphoric acid, potassium hydroxide, sodium hydroxide, or enzymes like lipase can be used to expedite the transesterification process1,2,3,4. Traditionally, the reaction mixture is heated under reflux for prolonged periods, typically 30 min or more. Heating is not as energy efficient as ultrasonication while also posing safety risks5. Consequently, there is a need for a safer, faster, and more energy efficient transesterification process.
Ultrasound irradiation emerges as a superior alternative to conventional energy sources such as heat, light, and electricity, primarily due to the phenomenon of acoustic cavitation10. This phenomenon, characterized by the formation, expansion, and violent collapse of bubbles, generating localized hotspots with temperatures reaching approximately 5000 K and pressures of 1000 atm. Such extreme conditions, coupled with rapid heating and cooling rates (over 1010 K/s), furnish the requisite energy for a wide array of chemical reactions to occur efficiently at room temperature, including those previously deemed unattainable by conventional means10. Ultrasonic-assisted synthesis is rapidly gaining ground across diverse research areas. Notably, interest in ultrasonic-assisted synthesis in organic synthesis and solid-state materials is driven by its environmentally friendly nature, energy efficiency, and abbreviated reaction times under ambient conditions5,11,12,13,14,15,16.&#160;A prompt and effective technique is introduced here for secure ultrasonic-assisted transesterification of vegetable oils using an alkaline catalyst yielding pure biodiesel products within a short time frame. While extra virgin olive oil serves as the demonstration medium in this study, it is imperative to note that the ultrasonic method holds applicability to a spectrum of vegetables oils5,17.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chloroform-d</td>
			<td>Fisher Scientific</td>
			<td>865-49-6</td>
			<td>&#8226; Harmful if swallowed. 
			&#8226; Causes skin irritation. 
			&#8226; Causes serious eye irritation. 
			&#8226; Toxic if inhaled. 
			&#8226; Suspected of causing cancer. 
			&#8226; Suspected of damaging fertility or the unborn child. 
			&#8226; Causes damage to organs through prolonged or repeated exposure</td>
		</tr>
		<tr>
			<td>Heated Ultrasonic Baths, Digital, Branson Ultrasonic</td>
			<td>Branson&#160;</td>
			<td>89375-492</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific Company</td>
			<td>67-56-1</td>
			<td>Highly flammable liquid and vapor. Toxic if swallowed, in contact with skin or if inhaled. Causes damage to organs (Eyes).</td>
		</tr>
		<tr>
			<td>Potassium hydroxide&#160;</td>
			<td>Fisher Scientific Company</td>
			<td>1310-58-3</td>
			<td>May be corrosive to metals. Harmful if swallowed. Causes severe skin burns and eye damage. Causes serious eye damage</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>7647-14-5</td>
			<td>Not hazardous</td>
		</tr>
		<tr>
			<td>Vegetable oils</td>
			<td></td>
			<td></td>
			<td>A commonly consumed food with a long history of safe use in pesticides.&#160;</td>
		</tr>
	</tbody>
</table>

ultrasonic-assisted preparation of biodiesel products from vegetable oils

Utilizing vegetable oil as a sustainable feedstock, this study presents an innovative approach to ultrasonic-assisted transesterification for biodiesel synthesis. This alkaline-catalyzed procedure harnesses ultrasound as a potent energy input, facilitating the rapid conversion of extra virgin olive oil into biodiesel. In this demonstration, the reaction is run in an ultrasonic bath under ambient conditions for 15 min, requiring a 1:6 molar ratio of extra virgin olive oil to methanol and a minimum amount of KOH as the catalyst. The physiochemical properties of biodiesel are also reported. Emphasizing the remarkable advantages of ultrasonic-assisted transesterification, this method demonstrates notable reductions in reaction and separation times, achieving near-perfect purity (~100%), high yields, and negligible waste generation. Importantly, these benefits are achieved within a framework that prioritizes safety and environmental sustainability. These compelling findings underscore the effectiveness of this approach in converting vegetable oil into biodiesel, positioning it as a viable option for both research and practical applications.

Author Spotlight: Employing Green-Chemistry Principles for Safe and Sustainable Synthesis of Biodiesels

Ultrasonic-Assisted Preparation of Biodiesel Products from Vegetable Oils

Our research introduces an environmentally-friendly and a safe method to produce biodiesel via ultrasonic-assisted transesterification reaction. We are trying to answer how to employ green chemistry principles and use ultrasonic energy to promote reactions. We are currently using ultrasonic technologies to enhance the sustainability of reactions, following the principles of green chemistry.We have developed a safe, rapid, and efficient method for the synthesis of high-purity biodiesel from vegetable oils. In this method, biodiesel is synthesized under ambient temperature and atmospheric pressure, which is more energy efficient and safer than conventional heating methods. Moreover, the purity of the biodiesel product is close to 100%

In this demonstration, the transesterification reaction of extra virgin olive oil and methanol, catalyzed by KOH, produces biodiesel at room temperature in an ultrasonic bath (Figure 1)5. The starting materials in the centrifuge tube show the reactants are immiscible and divided into two layers as seen in Figure 2A. The upper layer is a mixture of methanol and KOH while the lower layer is composed of extra virgin olive oil. To promote hom...

Read this Scientific Article Protocol about Author Spotlight: Employing Green-Chemistry Principles for Safe and Sustainable Synthesis of Biodiesels at JoVE.com

A safe ultrasonic-assisted transesterification method for vegetable oils using an alkaline catalyst is presented here. The method is rapid and efficient for preparing pure biodiesel products.

Utilizing vegetable oil as a sustainable feedstock, this study presents an innovative approach to ultrasonic-assisted transesterification for biodiesel ...

ultrasonic-assisted-preparation-biodiesel-products-from-vegetable

Research

JoVE Journal

Chemistry

440 Views.  California State University, Sacramento. Our research introduces an environmentally-friendly and a safe method to produce biodiesel via ultrasonic-assisted transesterification reaction. We are trying to answer how to employ green chemistry principles and use ultrasonic energy to promote reactions. We are currently using ultrasonic technologies to enhance the sustainability of reactions, following the principles of green chemistry.We have developed a safe, rapid, and efficient method for the synthesis of high-purity biodiesel ...