Teaching the Nature of Science Through Biodiesel Synthesis from Waste Cooking Oil: A Literature Review with Experimental Insights

Abstract

This work reviews the use of biodiesel synthesis experiments in science education, emphasising their potential for explicit nature of science (NOS) teaching. Through a literature review and experimental insights, it highlights how transesterification of waste cooking oil (WCO) with a basic catalyst can serve as an educational tool. While biodiesel reaction conditions are well-documented, this study presents them in a pedagogical context. Simple viscosity and density measurements illustrate empirical analysis, while a design of experiments (DoE) approach using a Hadamard matrix introduces systematic optimisation and scientific reasoning. By integrating biodiesel synthesis with explicit NOS instruction, this work provides educators with a framework to foster critical thinking and a deeper understanding of scientific inquiry. Additionally, this approach aligns with green chemistry principles and resource efficiency, reinforcing the broader relevance of sustainable chemistry.

1. Introduction

There is an economic and environmental problem caused by the uncontrolled disposal of waste cooking oil (WCO) [1,2], which is an oil-based substance consisting of edible vegetable matter that has been used in the preparation of foods and is no longer suitable for human consumption [3]. Due to unconscious behavior, inadequate regulations, or lack of enforcement, the majority of WCO is typically disposed of through the sewage system or as part of solid waste in landfills [4]. The possibility of recovering this waste through its chemical transformation into biofuel—specifically biodiesel, a type of liquid biofuel or bio-oil—has been studied in greater detail [5,6]. Biodiesel is currently defined in the European Union in the technical regulation EN 14214 [7] or in the USA in ASTM 6751-02 [8]. Thus, the American Society for Testing and Materials (ASTM) defines biodiesel as a mixture of mono-alkyl esters of long-chain fatty acids derived from renewable lipids, such as animal fats and vegetable oils, which is used in compression ignition engines (diesel engines) or heating boilers.

Biodiesel is primarily produced through a chemical process called transesterification (Figure 1), in which vegetable oils and animal fats react with an alcohol, such as methanol or ethanol, in the presence of a catalyst. This reaction results in a mixture of fatty acid methyl or ethyl esters—commonly known as biodiesel—with glycerol as a byproduct. Transesterification is the most widely used method due to its simplicity, low cost, and advantages over other techniques. The resulting fuel exhibits a higher cetane number, lower emissions, and greater combustion efficiency [9]. Notably, biodiesel shares similar physicochemical properties with petroleum-derived diesel [10], making it suitable for use in pure form or as a blend with petroleum diesel due to its complete miscibility [11]. In the light of this, biodiesel is proposed as a viable alternative fuel for diesel engines due to its non-toxic, biodegradable, and environmentally friendly characteristics [12].

Furthermore, biodiesel contains almost no sulfur and does not contribute to greenhouse gas emissions, owing to its closed carbon dioxide cycle. In this cycle, the CO₂ released during the combustion of biodiesel is offset by the CO₂ absorbed by the plants used to produce the vegetable oil that will be employed to make the fuel, making it a more sustainable option compared to fossil fuels [10]. However, in the case of biodiesel made from WCO, the quality of WCO is critical in producing acceptable biodiesel. WCO can contain various impurities that limit its use, making proper pre-treatment essential before the transesterification process [13]. Additionally, challenges concerning biodiesel such as higher nitrogen oxide emissions, elevated production costs, and poor cold flow properties must be addressed [14].

In addition, another key aspect to consider in biodiesel production via transesterification is the choice of catalyst, which significantly impacts reaction efficiency, yield, and product purity. Homogeneous catalysts, such as sulfuric acid, or bases, e.g., sodium hydroxide or potassium hydroxide, are widely used [11]. However, they require additional purification steps to remove residual catalysts and byproducts. Alternatively, heterogeneous catalysts, including solid acids, or enzymatic catalysts facilitate easier separation and potential reusability, though they may require more controlled reaction conditions [15]. To optimise reaction conditions, the use of design of experiments (DoE) is recommended, demonstrating its relevance in biodiesel synthesis [16].

2. Biodiesel and the Nature of Science

A biodiesel lab experience focused on converting used WCO into renewable fuel is a perfect opportunity to integrate the nature of science (NOS) principles into a hands-on learning experience. Ultimately, this should give students a better idea of how science is practiced by actual researchers [17].

NOS refers to the epistemology of science, science as a way of knowing, or the values and beliefs inherent to scientific knowledge and its development [18]. Given that, there are some key NOS aspects to be considered, such as understanding that scientific knowledge is tentative (subject to change); empirical (based on and/or derived from observations of the natural world); subjective (influenced by scientists’ background, experiences, and biases); imaginative and creative (involves the inventions of explanations); and socially and culturally embedded [19]. Understanding NOS is essential for science education, critical thinking, and informed decision-making in daily life [20,21,22]. On the other hand, the literature concludes that most of the students do not show adequate conceptions of NOS if not explicitly addressed. Explicit NOS instruction involves deliberately focusing on various aspects of NOS during classroom instruction, discussions, and/or questioning [23,24]. All in all, addressing NOS in an explicit manner has been shown to be beneficial [19].

2.1. Explicit vs. Implicit NOS Teaching

NOS should be a deliberate and structured component of science education. Teachers cannot assume that students will develop an understanding of NOS simply by engaging with scientific content. Instead, NOS principles must be explicitly stated, intentionally planned, and actively taught throughout science instruction [25].

In implicit NOS teaching, educators expect students to infer NOS concepts on their own while learning scientific content. This approach assumes that exposure to scientific theories, experiments, and historical cases will naturally lead to an understanding of how science operates. However, research indicates that students often fail to grasp key epistemological ideas—such as the tentative nature of scientific knowledge, the role of creativity in science, and the influence of social and cultural factors—without explicit guidance [26].

By contrast, explicit NOS teaching ensures that these concepts are directly addressed. Simply covering scientific topics—such as the periodic table—does not guarantee that students will develop an awareness of how scientific knowledge is constructed, revised, and debated. Instead of assuming that students will uncover these ideas on their own, explicit instruction involves structured activities, guided discussions, and reflective exercises that make NOS principles visible and accessible.

Furthermore, NOS instruction should not be an isolated discussion about the philosophy of science [27]. When taught separately from core science content, NOS may seem disconnected from actual scientific practice, reducing its perceived relevance. Instead, NOS should be seamlessly integrated into science instruction, helping students see the connections between scientific concepts and the nature of scientific inquiry.

The distinction between explicit and implicit NOS teaching has profound implications for scientific literacy and socio-scientific issues (SSIs). Traditional science education, often rooted in a positivist view of knowledge, presents science as a collection of objective facts rather than a dynamic, evolving process. This limits students’ ability to engage in critical discussions about real-world issues that require an understanding of uncertainty, ethical considerations, and the interplay between science and society [28]. Sustainability education, for instance, requires students to critically evaluate how science interacts with social and environmental challenges.

Moreover, many socio-scientific issues that appear controversial to the general public—such as climate change, vaccinations, and biological evolution—are largely a result of poor NOS understanding [29]. A failure to recognise the tentative, evidence-based, and self-correcting nature of science can contribute to public skepticism toward well-established scientific theories. By embedding explicit NOS instruction within regular science lessons, educators can empower students to critically analyse scientific knowledge, appreciate its evolving nature, and engage meaningfully with socio-scientific challenges. It is important to note that merely engaging students in inquiry-based science experiences is unlikely to improve their understanding of NOS. Instead, these experiences should provide a context in which knowledgeable teachers can explicitly draw students’ attention to relevant NOS ideas [29].

2.2. Models for Explicit NOS Teaching

Over the years, various instructional models have been developed to effectively introduce the principles of NOS. One of the most influential frameworks is Lederman’s explicit-reflective approach [18], which argues that NOS concepts should be taught explicitly through direct instruction and reflectively through activities that encourage students to analyse their own assumptions. Lederman emphasises key NOS tenets, such as the tentative nature of scientific knowledge and the role of creativity in scientific inquiry. However, this perspective has been subject to criticism due to its limitations in capturing the complexity of scientific practice [30].

As a result, alternative approaches have emerged, including the Whole Science model [31], the Features of Science framework [32], and the Family Resemblance Approach (FRA) to NOS [33,34]. Focusing on the latter, FRA conceptualises NOS as a comprehensive framework that integrates scientific aims and values, practices, methodologies, and social norms, all of which should be incorporated into science education. Developed by Irzik and Nola [34] and inspired by Wittgenstein’s philosophy, this perspective argues that while all scientific disciplines share certain characteristics, no single characteristic can fully define science or demarcate it from other fields [30]. Instead of focusing on individual NOS aspects in isolation, FRA presents NOS holistically and contextually, offering a framework that captures both domain-general and domain-specific aspects of scientific disciplines [35]. This approach highlights both the similarities and differences among scientific fields, ensuring that students appreciate both the shared and unique features of different scientific disciplines [30].

Hence, while Lederman’s explicit-reflective model remains foundational, contemporary educators increasingly blend multiple approaches to enhance NOS instruction. For instance, combining explicit NOS instruction with inquiry-based laboratory experiences fosters a richer understanding of scientific practices. In this sense, a biodiesel synthesis experiment may provide an excellent opportunity to integrate FRA and explicit-reflective NOS instruction in a meaningful way.

Prior to conducting experiments, students can engage in guided discussions about fundamental NOS principles, such as the role of models in scientific explanations and the tentative nature of scientific knowledge. These discussions help make explicit the epistemic and social-institutional dimensions of science. For instance, during the experimental procedures, students can be encouraged to actively recognise and analyze NOS elements in action, such as examining how factors like catalyst concentration, reaction temperature, and reaction time influence experimental outcomes, illustrating the uncertainty and revisionary nature of science, or discussing ethical and economic considerations of biodiesel production, emphasising how scientific advancements are shaped by societal needs and constraints.

Moreover, following the experimental work, structured reflection activities could consolidate students’ NOS understanding, e.g., they can critically analyze their results, compare their laboratory approach to real-world industrial biodiesel production, and evaluate how experimental limitations influence scientific conclusions. As a result, this integrated approach may foster critical thinking, engagement with scientific practices, and a deeper understanding of the broader implications of scientific work.

2.3. Biodiesel Lab Experiences in Science Education

Biodiesel synthesis has become an effective hands-on approach for integrating green chemistry principles, scientific inquiry, and socio-scientific issues (SSI) into chemistry education. Across various educational levels, laboratory experiences centered on biodiesel production offer students the opportunity to engage in authentic scientific practices, fostering a deeper understanding of NOS while also addressing sustainability challenges. These experiences emphasise experimental design, data analysis, scientific communication, and decision-making in complex real-world contexts.

One major theme in biodiesel-based education is the use of inquiry-based learning (IBL) and DoE to promote scientific reasoning. For instance, Keppeler et al. [36] incorporated DoE principles to help students optimise biodiesel yield by varying reaction conditions systematically. Similarly, Leibfarth et al. [37] introduced an evidence-based decision-making approach through continuous-flow chemistry, allowing students to analyse reaction conditions in real time. These pedagogical strategies reinforce the iterative nature of scientific inquiry and help students develop critical thinking and problem-solving skills.

Another important aspect of biodiesel synthesis in education is its connection to SSI, which helps students recognise the broader societal implications of chemistry. Georgiou and Kyza [38] demonstrated how SSI-based learning interventions on biofuels foster students’ awareness of sustainability, ethical decision-making, and responsible citizenship. Similarly, Nida et al. [39] explored the controversial palm oil biodiesel industry in Indonesia, using it as a case study to engage students in debates about environmental responsibility and economic trade-offs. By framing chemistry education within SSI, these studies show how biodiesel synthesis can empower students to apply their scientific knowledge to real-world dilemmas.

A third key dimension of biodiesel education is its role in teaching green chemistry principles. Many studies have emphasised the environmental benefits of biodiesel production, particularly through the use of renewable feedstocks, atom economy, catalysis, and energy-efficient reaction conditions [40]. Jin and Bierma [41] highlighted the practical implementation of green chemistry by integrating service-learning projects, where students converted campus waste vegetable oil into biodiesel for university vehicles. This approach not only reinforced chemistry concepts but also connected students with their community, making the learning experience more impactful.

Scientific communication and collaboration also play a crucial role in these laboratory experiences. Neiles et al. [42] designed a scaffolded biodiesel laboratory experience that required students to engage in multiple forms of scientific writing, including formal lab reports and stakeholder communication. By requiring students to explain their findings to audiences with varying levels of scientific knowledge, this approach helped them develop communication skills essential for professional scientific practice. Furthermore, Hupp [43] demonstrated how analytical chemistry students applied chemometric methods to biodiesel analysis, reinforcing interdisciplinary connections in science education.

The design of biodiesel experiments also varies based on the educational level of students. High school experiments tend to focus on simplified visual methods for product detection, such as observing phase separation [44] or using thin-layer chromatography for qualitative analysis [45]. These techniques make biodiesel synthesis accessible to younger students while reinforcing foundational chemical concepts like esterification and transesterification. At the undergraduate level, more advanced approaches, such as microwave-assisted transesterification [42] and catalyst optimisation using chemometrics [36], expose students to modern research techniques and statistical data analysis.

In summary, biodiesel synthesis provides a versatile and engaging platform for chemistry education, aligning with multiple facets of scientific literacy and NOS.

Table 1. Diversity of student engagement and pedagogical value of biodiesel experiments.

Students’ Role	Experimental Involvement	Pedagogical Aspects	Refs.
Passive experimenters	Perform synthesis (follow structured protocols), basic tests; no design or decision-making	Observation, empirical basis, properties of materials	[44,45,46]
Active experimenters	Modify reaction conditions, conduct synthesis and analysis; guided inquiry	Inquiry, control of variables, scientific methods	[37,47,48]
Experimental designers	Literature research, propose variables, validate results with further tests	Modeling, DoE, creative thinking	[42,48,49]
Data analysts	Analyze GC-MS data sets using chemometric methods	Data interpretation, pattern recognition, chemometrics	[43]
Socio-scientific debaters	Simulations, discussions, role-play, choose biodiesel type (limited lab work)	SSI, ethical reasoning, argumentation	[38,39]
Service-based participants	Collect local WCO, synthesise biodiesel for real-world application	Community engagement	[41]
Scientific communicators	Write reports, create communications for various audiences, peer collaboration	Communication, argument from evidence, interdisciplinary learning	[42]

provides an overview of the different student roles and their involvement in biodiesel education activities across the literature. Whether through inquiry-based experimental design, sustainability discussions, or interdisciplinary analytical methods, these educational experiences help students understand chemistry as a dynamic and socially relevant science. The integration of biodiesel experiments into science curricula not only enhances students’ technical and analytical skills but also prepares them to make informed decisions as responsible global citizens.

2.4. Assessment Strategies, Student Feedback and Learning Outcomes

To address the educational value of biodiesel-related activities, a variety of assessment approaches have been reported in the literature. These include formal evaluations of students’ understanding of scientific content, application of the principles of green chemistry, and competence in DoE, as well as informal assessments such as questionnaires, interviews, course feedback, and instructor observations.

El Seoud et al. [48] employed a comprehensive evaluation scheme, including lab notebooks, seminar participation, and a final presentation, with average scores suggesting high student competency.

Other projects focused assessment on students’ application of green chemistry principles [36], critical analysis through literature discussions [43], and synthesis of learning in the form of scenario-based reports [47]. Informal methods were also common, such as student reflections [41], Likert-scale surveys gauging engagement and satisfaction [45], and post-activity interviews [39], which revealed positive perceptions of ethical, environmental, and societal dimensions.

Some studies combined multiple approaches, including observations of student behaviors tied to inquiry-based practices [42] and performance on quizzes, lab reports, and group presentations [49]. These varied assessment strategies reinforce the multifaceted educational impact of biodiesel-related projects and suggest promising avenues for gauging student understanding of NOS and DoE in future implementations.

In addition to assessing specific competencies, many studies reported positive learning outcomes that contrasted with more traditional, lecture-based instruction. Kim [49] noted that students were highly motivated and engaged throughout the biodiesel activity and gained confidence in doing and learning chemistry. Similarly, El Seoud et al. [48] found that students not only demonstrated strong performance in diverse assessment categories—ranging from experimental decision-making to seminar participation—but also evaluated the project positively, emphasising the value of active learning and interdisciplinary collaboration.

The authentic and socially relevant context of biodiesel production appears to contribute to students’ deeper understanding of chemistry’s role in real-world problems. According to Jin and Bierma [41], students felt a strong sense of responsibility in producing a functional biodiesel product, experiencing a rewarding connection between their laboratory efforts and potential community impact. Other reports observed that students became more aware of sustainability issues and developed a broader understanding of chemistry’s ethical and societal dimensions [39,45]. These affective and cognitive learning outcomes underscore the pedagogical value of context-based and inquiry-driven approaches, which have been shown to enhance student engagement, promote critical thinking, and promote student autonomy, particularly when compared to more conventional teaching formats [37,42,44].

2.5. Practical Considerations for School Implementation

Implementing biodiesel activities in school laboratories requires careful consideration of safety, sourcing of materials, and infrastructure constraints. The main safety concerns relate to handling flammable and caustic reagents such as methanol, ethanol, and sodium or potassium hydroxide. The use of appropriate personal protective equipment—including safety goggles, gloves, lab coats—as well as working in ventilated fume hoods when handling volatile solvents or corrosive bases, is strongly recommended [37,45,47,48,49]. Methanol and biodiesel are flammable and should be kept away from open flames, and bases like NaOH and KOH are highly corrosive, requiring cautious handling and appropriate disposal protocols [42].

In alignment with best practices in laboratory training, several of the reviewed studies emphasised the importance of providing safety instructions to students prior to the activity. These include reviewing material safety data sheets, conducting pre-laboratory safety briefings, and requiring students to complete hazard analyses or safety checklists [42,47]. Such practices not only minimise risks but also foster a culture of safety and responsibility in the chemistry classroom.

Regarding the raw materials, to encourage community engagement, many educational implementations source WCO from local cafeterias, restaurants, or students’ homes [41,47,49]. This approach not only provides a cost-effective and sustainable feedstock but also creates opportunities for inquiry-based learning and outreach, as students gather contextual information on oil usage and waste practices [49].

With regard to the infrastructure requirements, these vary depending on the complexity of the biodiesel synthesis. While some experiments involve reflux setups with electromagnetic heaters and glassware [44,48], others demonstrate that simplified protocols—in which the oil is first heated in a beaker using an electric cooking plate and then transfer to a plastic bottle for mixing with other reactants—can be effective and accessible for schools with limited resources [45]. Where analytical equipment such as gas chromatography is unavailable, qualitative techniques like thin-layer chromatography are recommended as low-cost alternatives for biodiesel verification [45].

In addition, for school-level replication, simplified procedures can be adopted to measure the key physical properties of biodiesel such as viscosity and density. Viscosity can be determined using a basic glass capillary viscometer (e.g., Ostwald type), measuring the time required for a fixed volume of biodiesel to flow between two calibrated marks at a controlled temperature, typically 40 °C. This approach, while inspired by ASTM (American Society for Testing and Materials) D446 [50], is feasible in high school settings and provides acceptable reproducibility [44,46]. Similarly, density may be estimated by weighing a known volume of biodiesel using a standard 10 mL graduated cylinder and an electronic balance, following the general approach of ASTM D1298 [51] but adapted for educational purposes. These simplified methods require only basic lab equipment and can be safely integrated into the curriculum with proper teacher supervision.

In summary, despite occasional logistical challenges, such as the time required for certain processes or the absence of specific lab equipment [42], the literature illustrates that biodiesel activities are adaptable to a wide range of educational environments when safety and practicality are thoughtfully addressed.

3. Materials and Methods

This section provides experimental insights into the synthesis of biodiesel from WCO using basic homogeneous catalysis. While the reaction conditions and their impact on biodiesel yield and quality are well-established, the primary purpose of presenting this experimental background is pedagogical. It aims to offer educators a practical framework for developing lesson plans that explicitly introduce the teaching of NOS. For example, viscosity and density measurements are conducted as a straightforward and accessible method to assess biodiesel properties.

Furthermore, a DoE approach, utilizing a Hadamard matrix, is introduced to provide students with a systematic approach to optimise reaction parameters. This strategy not only enhances understanding of the variables involved but also serves as a foundation for incorporating NOS principles into the educational context of biodiesel production.

3.1. Experimental Considerations

The empirical part of this proposal of explicit NOS teaching based on a homogeneous-catalyst biodiesel lab experience begins with the experimental design. This includes the preliminary identification of the system, the design of the experimental setup, and the working procedure, covering the necessary reagents and the testing protocol.

In the system identification phase, the physical and chemical properties of the products and reagents involved in the proposed reaction are considered, including possible reaction mechanisms and their kinetics. It is worth mentioning that the transesterification reaction can be catalysed by acids or bases, and the reaction mechanisms described in the literature vary depending on the medium. The experimental process is designed to evaluate variables that could influence the system’s progress, based on prior research found in the literature.

The objective of the experimental design is to determine how various factors influence the most problematic property of WCO when considering its direct use as fuel in automotive engines or heating boilers: viscosity. To achieve this, changes in viscosity and density are analysed in the biodiesel or organic phase obtained from different trials. The schematic in Figure 2 illustrates the laboratory-scale procedure for the chemical transformation of vegetable oils.

In this work, a batch reactor with a constant volume has been used (see Figure 3a). The reactor is equipped with a rod agitator with adjustable speed and maintained at a constant temperature using an oil bath with controlled heating. WCO is filtered through a metal mesh with a pore diameter of 0.5 mm. For experiments with clean oil, filtration is unnecessary since commercial oil contains no impurities or suspended contaminants.

The separation system primarily consists of a separatory funnel (Figure 3b); however, additional rotary evaporation to break emulsions formed during reaction mass washing is strongly recommended. Through this process, unreacted methanol can be recovered and recirculated into the mixing vessel, which can be a simple beaker with magnetic stirring. Once the different phases are separated, the organic phase containing the methyl esters is purified. The final biodiesel is then analyzed for viscosity and density using ASTM standards. Viscosity measurements follow ASTM 446 [50] standards, while density is determined according to ASTM D1298 [51].

3.2. Homogeneous Basic Catalysis Key Conditions

The proposed experimentation involves studying the transesterification reaction under homogeneous conditions using basic catalysis. In light of this, homogeneous base catalysis is undoubtedly the most extensively studied transesterification reaction at the laboratory scale [52]. Its kinetics have been extensively analysed [53]. In short, a conversion rate of 90% to 98% from triglyceride to methyl ester can be achieved within 90 min, and the recommended conditions include a methanol-to-oil molar ratio of 6:1 (twice the stoichiometric ratio), a reaction temperature close to methanol’s boiling point at atmospheric pressure (e.g., 60–65 °C), and an agitation speed above 600 rpm. Furthermore, the recommended amount of catalyst varies according to bibliographic references. Coteron et al. [54] suggest using between 0.5% and 1.5% by weight of catalyst (NaOH). Above this range, saponification issues arise, while below it, conversion rates are low. Darnoko and Cheryan [53] estimate the optimal catalyst concentration (KOH) at 1% by weight. Regardless of the specific proportion, commonly used catalysts include alkalis such as sodium hydroxide (NaOH) and potassium hydroxide (KOH), carbonates, and their corresponding alkoxides, like sodium or potassium methoxide.

As mentioned before, one drawback of base-catalyzed processes is their sensitivity to the purity of reactants, water content, and free fatty acids in the oil [55]. Water can lead to ester saponification in a basic medium [11]. Saponification not only consumes the catalyst but also causes emulsion formation, which complicates biodiesel purification. For base catalysis, it is recommended to use oils with an acid value (free fatty acids) below 0.5% by weight of the starting oil. This presents a challenge when dealing with WCO, which typically has an acid value above 2%. A preliminary experiment under the operating conditions recommended in the literature may be convenient to identify potential issues such as saponification and emulsion formation.

Considering these considerations, a series of experiments have been envisaged to explore the effects of various reaction parameters on the transesterification process. These parameters include temperature, the methanol-to-oil molar ratio, reaction time, and the type and amount of catalyst (sodium methoxide or potassium methoxide). A summary of the experimental design, including the tested conditions and variables, can be found in

Table A1. Testing conditions for basic homogeneous catalysis.

Test	Catalyst	3 Percentage (%)	Catalyst Mass ±0.01 (g)	Temperature ±0.5 (°C)	4 Molar Ratio	Reaction Time (min)
01 1	NaOH	1.25	0.83	70.0	6/1	60
02	NaOH	1.25	0.83	70.0	6/1	60
03 2	-	-	-	70.0	6/1	60
04	KOH	5.00	3.30	70.0	6/1	60
05	NaOH	3.33	2.20	70.0	6/1	60
06	KOH	1.25	0.83	70.0	6/1	40
07	KOH	3.33	2.20	70.0	6/1	60
08	NaOH	1.25	0.83	60.0	6/1	60
09	NaOH	1.25	0.83	80.0	6/1	60
10	NaOH	1.25	0.83	25.0	6/1	60
11	NaOH	1.25	0.83	70.0	6/1	30
12	NaOH	1.25	0.83	70.0	6/1	15
13	NaOH	1.25	0.83	70.0	6/1	45
14	NaOH	1.25	0.83	70.0	6/1	75
15	NaOH	1.25	0.83	70.0	6/1	60
16	KOH	0.05	2.20	70.0	4/1	60
17	KOH	0.03	2.20	70.0	8/1	60
18	KOH	1.25	0.83	70.0	6/1	60
19	KOH	1.25	0.83	70.0	6/1	260
20	KOH	3.33	2.20	70.0	6/1	60
21	KOH	0.75	0.49	70.0	6/1	60
22	KOH	5.00	3.30	70.0	6/1	60
23	KOH	0.01	0.49	70.0	4/1	60
24	KOH	0.01	0.49	70.0	8/1	60
25	KOH	1.25	0.83	70.0	6/1	100
26	KOH	1.25	0.83	70.0	6/1	60
27	KOH	1.25	0.83	70.0	6/1	60
H-1	KOH	0.03	2.20	70.0	8/1	100
H-2	KOH	0.01	0.49	70.0	4/1	100
H-3	KOH	0.05	2.20	70.0	4/1	40
H-4	KOH	0.01	0.49	70.0	8/1	40

¹ Using clean oil; ² without catalyst; ³ relative to methanol; ⁴ methanol-to-oil molar ratio.

in the Appendix A.

3.3. Design of Experiments (DoE): Hadamard Optimisation Method

The Hadamard optimisation method is used to determine which variable had the most significant influence on the transesterification process via basic catalysis. This experimental design method is included in 2^k factorial designs, specifically a fractional factorial design of two levels. This approach allows the investigation of up to k = N − 1 factors with only N trials, where N is a multiple of 4 [56,57,58,59,60,61].

With the factorial design, it is possible to investigate all possible combinations of factor levels in each complete trial or experiment replication. The average effect of a factor is defined as the change in response produced by a change in that factor’s level, averaged over the levels of the other factors. The magnitude and direction of factor effects are examined to determine which variables are likely the most significant.

The main limitation of this method is the number of system variables that can be considered. Due to the structure of Hadamard matrices, the method allows studying systems with three variables, but the next available matrix (8 × 8) would require analysing seven variables, each at two levels.

3.4. Experimental Procedure

The empirical part of this proposal for explicit NOS teaching, based on a homogeneous-catalyst biodiesel production process, utilizes the following reagents: WCO provided by the university’s catering service. The WCO was characterized and has the following properties: viscosity at 40 °C of 37.38 ± 0.05 mm²/s and density at 15 °C of 921.0 ± 0.5 kg/m. Additionally, methanol (99.8% purity) was used as the alcohol for the transesterification reaction. The catalysts employed include sodium hydroxide (NaOH) and potassium hydroxide (KOH), both in analytical grade, with purities of 99% and 85%, respectively.

The experimental design follows a structured process, beginning with the preparation and introduction of reagents, followed by the transesterification reaction, phase separation, and purification of the biodiesel product. The protocol also includes characterisation of key properties such as viscosity, density, and acid value to assess the quality of the biodiesel. In addition to these characterisation techniques, thin-layer chromatography (TLC) was employed to identify the biodiesel phase and confirm the presence of methyl esters. The procedure involved applying a sample of the biodiesel phase, along with a standard of 99% pure methyl oleate, both diluted with 95% pure hexane, onto the baseline of the chromatogram. The elution process was carried out using ethyl acetate as the eluent, and phosphomolybdic acid was used as a detector to visualize the components.

The following step-by-step procedure provides a clear and reproducible framework for educators and students, integrating both experimental techniques and theoretical understanding:

• Set-up of the isothermal reactor: Start the oil bath and set the desired reaction temperature. Wait for the system to reach a steady-state temperature before proceeding with the reaction.
• Preparation of reagents: Weigh the required amounts of oil and methanol according to the molar ratio specified in the experiment. Weigh the necessary amount of NaOH or KOH regarding the design of experiments. Prepare the corresponding methoxide solution by stirring.
• Introduction of reagents into the reactor: Add the oil into the reactor and wait until it reaches the programmed temperature. Open the water-cooling line of the distillation column to prevent the evaporation of methanol when the reaction temperature exceeds its boiling point, ensuring reflux. Just before adding the remaining reagents, turn on the reactor stirrer, setting it to a speed of 600 rpm or higher for all trials. Then, add the prepared methoxide solution into the corresponding volume of methanol. Start the timer to record the reaction time.
• Reaction completion: Once the reaction time has passed, turn off the stirrer, the oil bath, and the timer. Allow the reactor to cool down sufficiently to prevent the loss of unreacted methanol by evaporation. The methanol can then be recovered by distillation using a rotary evaporator. Close the water inlet (cooling system) and transfer the reactor contents into a separatory funnel. You should observe the formation of two distinct phases: the lower phase containing glycerol and the upper phase containing the methyl esters (biodiesel). If both phases are not visible, it may indicate emulsion formation or that the transesterification did not occur. To break any emulsions, treat the mixture with a rotary evaporator at 60 °C for 30 min, then let the phases settle again.
• Purification of the separated phases (Figure 4): Once two distinct phases have formed, collect the lower phase (glycerol) in a beaker and send it to the rotary evaporator to recover any methanol. Weigh and label the glycerol phase with the experiment date and number. For the biodiesel (upper phase), wash it with two solutions: first with an acidified solution to neutralise the biodiesel phase and then with distilled water. In the first wash, add 20 mL of 5% phosphoric acid (by weight) for every 100 g of biodiesel phase. In the second wash, add 20 mL of distilled water. For both washes, agitate the solution, allow phases to separate, and discard the lower layer. Repeat the process for the second wash. After the washes, the biodiesel is separated, stored in an opaque container, and kept for further analysis.

6.

Characterisation of viscosity and density: The viscosity and density of the biodiesel are measured in some samples. In addition, TLC is performed to confirm the presence of methyl esters.

4. Results and Discussion

Following the experimental design, the tests outlined in Table A1 (see Appendix A) were conducted. The initial oil mass used was consistently 300.00 ± 0.01 g, with waste oil being used in all tests except for trial 01, which was carried out with clean oil. The reaction yield data and values for viscosity at 40 °C and density at 15 °C are summarised in

Table A2. Experimental results.

Test	Viscosity ±0.05 (mm2/s)	Density ±0.5 (kg/m3)	Mass Biodiesel ±0.01 (g)	Mass Glycerol ±0.01 (g)	Yield (%)
01 1	4.12	850.0	243.54	25.0	90.2
02	6.80	892.0	282.60	41.08	94.2
03 2	36.45	922.0	-	-	-
04	4.85	884.0	274.33	63.92	91.4
05	5.60	888.0	280.70	61.30	93.6
06	7.77	892.5	266.18	21.80	-
07	5.67	888.0	283.53	30.45	94.5
08	6.21	888.5	268.72	31.75	89.6
09	6.18	888.5	272.25	25.60	90.7
10	8.45	895.0	285.24	28.30	-
11	7.54	892.0	279.03	19.93	-
12	7.87	892.5	270.44	17.40	-
13	7.23	891.0	280.29	20.95	-
14	6.63	890.0	281.62	23.71	93.8
15	6.40	890.0	280.60	22.50	93.5
16	5.49	888.0	268.98	29.60	89.6
17	4.52	884.5	272.86	34.29	90.9
18	6.59	892.0	284.38	19.22	94.8
19	5.34	888.0	281.93	21.64	93.9
20	4.67	885.0	270.02	28.03	90.0
21	9.00	899.5	205.02	20.07	-
22	4.53	884.0	258.07	34.13	86.0
23	18.14	914.5	273.34	-	-
24	8.86	899.0	286.20	15.67	-
25	5.91	895.0	274.10	24.26	91.4
26	6.63	892.0	277.48	19.30	92.5
27	7.79	893.0	272.37	13.32	-
H-1	4.57	883.5	266.38	31.95	88.8
H-2	32.53	921.0	262.70	-	-
H-3	6.85	892.0	270.20	22.43	90.1
H-4	18.35	912.5	175.08	15.01	-

¹ Using clean oil; ² without catalyst.

in the Appendix A.

Regarding the catalyst, good reproducibility was observed, meaning that both sodium methoxide and potassium methoxide performed similarly under the same operating conditions with the same starting oil, aligning well with the literature [52]. Nevertheless, repeatability was lower, likely due to previously mentioned factors such as heterogeneity of the WCO and emulsion formation during the washing stage. For subsequent experiments investigating the influence of reaction variables in methanolysis, viscosity and density values were considered independent of the alkaline catalyst used.

Additionally, as mentioned previously, TLC was used to identify the biodiesel phase, confirming that the predominant component is methyl esters. Figure 5 displays three chromatograms, where both the biodiesel sample and a 99% pure methyl oleate standard were applied to the baseline. Phosphomolybdic acid was used as the detector, and ethyl acetate/hexane mixtures (95:5, 85:15, and 90:10) were used as eluents. In all three trials, the dominant spot in the biodiesel sample had an Rf value similar to the standard, confirming the presence of methyl esters. A second “tailing” spot observed across all chromatograms indicates the presence of free fatty acids.

4.1. Reaction Yield

The crude yield, presented in Table A2 of Appendix A, was calculated for trials in which the biodiesel phase exhibited a kinematic viscosity below 7 mm²/s. In these cases, the yield was determined assuming that the entire mass of the biodiesel phase consisted of converted methyl esters from the original oil. The calculated yields ranged from 86% to 94.8%. This variation can be attributed to the heterogeneity of the WCO used as feedstock and the challenges associated with emulsion formation during the purification (washing) stage. The highest yield (94.8%) was obtained under the following conditions: 1.25 wt% sodium methoxide catalyst relative to methanol mass, a methanol-to-oil molar ratio of 6:1, and a reaction temperature of 70 °C. When the catalyst concentration was increased to 3.33 wt% under the same operating conditions, yields ranged from 90.0% to 94.5%. However, at 5 wt% catalyst, yields decreased, varying between 86% and 91.4%.

4.2. Viscosity and Density Reduction

4.2.1. Influence of Reaction Time

The variation in viscosity and density of the biodiesel phase was analysed under the following conditions: 1.25 wt% catalyst (relative to methanol mass), a methanol-to-oil molar ratio of 6:1, and a reaction temperature of 70 °C. A sharp decrease in viscosity (79%) was observed within the first 15 min of the reaction. After this initial phase, the reduction became more gradual, with an additional 6.8% decrease between 15 and 260 min. Similarly, density decreased by 3.1% within the first 15 min, reaching a total reduction of 3.59% after 260 min.

4.2.2. Influence of Catalyst Mass

The effect of catalyst mass was studied by varying its weight percentage relative to methanol while keeping temperature (70 °C), reaction time (60 min), and methanol-to-oil molar ratio (6:1) constant. As shown in Table A2 (Appendix A), when the catalyst concentration was 0.75 wt%, viscosity and density decreased by 75.3% and 2.4%, respectively. At 5 wt% catalyst, the reductions were more pronounced, with viscosity decreasing by 87.2% and density by 4.1%.

4.2.3. Influence of Methanol-to-Oil Molar Ratio and Reaction Temperature

An analysis of trials 16, 17, and 20 revealed that increasing the methanol-to-oil molar ratio, while keeping all other variables constant, resulted in a decrease in viscosity and density. This effect was more noticeable when lower catalyst concentrations were used, as observed in trials 21, 23, and 24 (see Table A2, Appendix A).

Regarding temperature, trials 8, 9, and 10 (Table A2, Appendix A) indicated that within the 60–80 °C range, viscosity and density values remained largely unchanged. However, in the 25–60 °C range, viscosity reduction was more significant, increasing from 77.4% to 83.4%.

4.3. Design of Experiments (DoE)

Assuming that temperature did not have a significant effect on the system within the 60–80 °C range (as previous findings showed no significant differences in viscosity and density values within this range), the Hadamard matrix could then be applied to evaluate which of the remaining three variables—catalyst content, methanol-to-oil molar ratio, or reaction time—had the greatest impact on reducing viscosity and density. Therefore, with the previous assumption regarding temperature, we had a system with three variables, each at two levels: high and low. These levels (see

Table 2. Settings/ranges of the system variables.

Variable	High Level	Low Level
Reaction time (min)	100	40
Catalyst mass (g)	2.20	0.49
Methanol-to-oil molar ratio	8:1	4:1

) for each variable were determined based on previous experimental data.

The Hadamard matrix for a system with three variables and two levels per variable is as follows:

$$\left(\begin{array}{cccc}1& \hspace{0.33em}\hspace{0.33em}1& \hspace{0.33em}\hspace{0.33em}1& \hspace{0.33em}\hspace{0.33em}1\\ 1& \hspace{0.33em}\hspace{0.33em}1& -1& -1\\ 1& -1& \hspace{0.33em}\hspace{0.33em}1& -1\\ 1& -1& -1& \hspace{0.33em}\hspace{0.33em}1\end{array}\right)$$

(1)

In this matrix, the first column identifies that we have four different trials, whereas columns 2–4 correspond to the different variables. In our study, the second column represents the reaction time variable, the third column represents the catalyst mass variable, and the fourth column represents the methanol/oil molar ratio variable. A value of 1 indicates the high level of the variable, while a value of −1 represents the low level.

The linear model derived from this design is expressed by the following equations:

$${\mathrm{Y}}_1={\mathsf{\theta }}_0+{\mathsf{\theta }}_1+{\mathsf{\theta }}_2+{\mathsf{\theta }}_3+{\mathsf{\epsilon }}_1$$

(2)

$${\mathrm{Y}}_2={\mathsf{\theta }}_0+{\mathsf{\theta }}_1-{\mathsf{\theta }}_2-{\mathsf{\theta }}_3+{\mathsf{\epsilon }}_2$$

(3)

$${\mathrm{Y}}_3={\mathsf{\theta }}_0-{\mathsf{\theta }}_1+{\mathsf{\theta }}_2-{\mathsf{\theta }}_3+{\mathsf{\epsilon }}_3$$

(4)

$${\mathrm{Y}}_4={\mathsf{\theta }}_0-{\mathsf{\theta }}_1-{\mathsf{\theta }}_2+{\mathsf{\theta }}_3+{\mathsf{\epsilon }}_4$$

(5)

where, Y₁, Y₂, Y₃, and Y₄ are the experimentally measured values of viscosity and density for each trial. The terms θ₁, θ₂, and θ₃ are associated with the estimated effects of reaction time, catalyst mass, and methanol-to-oil molar ratio, respectively, while θ₀ denotes the overall mean response. The error terms (ε) account for experimental variability.

Thus, the experimental design matrix with the combination of factor levels tested for each trial (H-1 to H-4) and measured responses are presented in

Table 3. Experimental design matrix and measured responses.

Test	Response (Y)	Time (min)	Catalyst Mass (g)	Molar Ratio 1	Viscosity (mm2/s)	Density (kg/m3)
H-1	Y1	100	2.20	8:1	4.57	0.8835
H-2	Y2	100	0.49	4:1	32.53	0.9210
H-3	Y3	40	2.20	4:1	6.85	0.8920
H-4	Y4	40	0.49	8:1	18.35	0.9125

¹ Methanol-to-oil molar ratio.

. The small measurement uncertainties (±0.05 mm²/s for viscosity; ±0.5 kg/m³ for density) were assumed negligible for this screening study.

From Equations (2)–(5), the main effects θ₀, θ₁, θ₂, and θ₃ are calculated as follows:

$${\mathsf{\theta }}_1={\displaystyle \frac{{\mathrm{Y}}_1+{\mathrm{Y}}_2-{\mathrm{Y}}_3-{\mathrm{Y}}_4}{4}}$$

(6)

$${\mathsf{\theta }}_2={\displaystyle \frac{{\mathrm{Y}}_1-{\mathrm{Y}}_2+{\mathrm{Y}}_3-{\mathrm{Y}}_4}{4}}$$

(7)

$${\mathsf{\theta }}_3={\displaystyle \frac{{\mathrm{Y}}_1-{\mathrm{Y}}_2-{\mathrm{Y}}_3+{\mathrm{Y}}_4}{4}}$$

(8)

$${\mathsf{\theta }}_0={\displaystyle \frac{{\mathrm{Y}}_1+{\mathrm{Y}}_2+{\mathrm{Y}}_3+{\mathrm{Y}}_4}{4}}$$

(9)

These coefficients were used to construct predictive models such as

Y = θ₀ + θ₁·t + θ₂·C + θ₃·R

(10)

for viscosity and density, where t is the reaction time (min), C is the catalyst mass (g), and R is the methanol-to-oil molar ratio. By applying this to the variables targeted for optimisation—viscosity (μ) and density (ρ)—the following results were obtained:

μ = 15.575 + 2.975·t − 9.865·C − 4.115·R

(11)

ρ = 902.25 − 14.50·C − 4.25·R

(12)

Analysis revealed that catalyst mass exerted the strongest influence. The methanol-to-oil molar ratio showed secondary but significant effects. Reaction time demonstrated only marginal impact for viscosity and negligible effect on density.

These results demonstrate that while both increased catalyst loading and higher methanol-to-oil ratios improve biodiesel quality by reducing viscosity and density, adjusting catalyst proportion offers substantially greater effectiveness. Consequently, the Hadamard matrix approach identified increased catalyst input as the optimal strategy for enhancing biodiesel quality from heterogeneous used frying oil, particularly for achieving desirable reductions in both viscosity and density.

4.4. Comparison with Quality Standard Biodiesel

For instance, the European biodiesel quality standard, EN 14214 [7], specifies that kinematic viscosity at 40 °C must be in the range of 3.5–5.0 mm²/s and density at 15 °C in the range of 860–900 kg/m³. As shown in Table A2 (Appendix A), the trial performed with clean oil (number 01) produced a biodiesel phase with viscosity and density values within the aforementioned standard limits. However, the values from samples obtained using WCO deviated more from these limits, despite following bibliographic recommendations regarding catalyst amount, methanol excess, reaction time, and temperature. The collected data suggest that under the same reaction conditions as the clean oil trial (time, methanol-to-oil molar ratio, and temperature), a higher catalyst concentration is required to meet the standard viscosity range. This observation is further supported by the Hadamard matrix experimental design. Specifically, trials 5, 7, and 20, conducted with a catalyst concentration of 3.33% by weight relative to methanol, produced biodiesel with viscosity and density values close to or within the acceptable range. However, to consistently meet the standard, a slightly higher catalyst concentration may be necessary. Notably, trials 4 and 22, carried out with 5% catalyst, a methanol-to-oil molar ratio of 6:1, a reaction time of 60 min, and a temperature between 60 and 80 °C, resulted in a biodiesel phase primarily composed of methyl esters, with viscosity at 40 °C and density at 15 °C falling within the specified standard limits.

Beyond optimising biodiesel production, analyzing these key reaction parameters has provided valuable insights for developing explicit NOS teaching applications. The systematic study of factors such as reaction time, catalyst concentration, methanol-to-oil molar ratio, and temperature offers a strong foundation for integrating biodiesel synthesis into science education.

5. Conclusions

This work reviews the use of biodiesel synthesis from WCO as a potential educational strategy for explicitly teaching NOS. Through a literature review and experimental insights, it explores how this approach may integrate fundamental scientific principles, laboratory techniques, and real-world applications to support NOS instruction.

From an experimental perspective, the collected data highlight the influence of reaction parameters—catalyst concentration, reaction time, methanol-to-oil molar ratio, and temperature—on biodiesel quality, particularly viscosity and density. As an example of the application of a DoE approach, the use of a Hadamard matrix illustrates a systematic method for evaluating these variables. This strategy could serve as a valuable tool for fostering discussions on experimental design, variable control, and data analysis in science education.

Apart from the technical aspects, biodiesel synthesis may offer a meaningful context for engaging with key NOS concepts. The empirical nature of science can be explored through viscosity and density measurements, while variations in biodiesel yield due to WCO heterogeneity and emulsion formation highlight the role of uncertainty in scientific investigations. Additionally, the need to adjust reaction conditions to achieve optimal results aligns with the idea that scientific knowledge evolves through continuous refinement. The broader societal and environmental implications of biodiesel production also provide an opportunity to discuss the interconnectedness of science, technology, and sustainability.

This work aims to contribute to the discussion on how biodiesel synthesis may be integrated into chemistry education. While further studies are needed to assess its pedagogical effectiveness for explicitly teaching NOS, this approach appears to have the potential to enhance students’ understanding of scientific inquiry while fostering awareness of sustainable chemistry.