Antioxidative capacity evaluation of imine compounds as metal ions chelators and free radical scavengers in biodiesel

Abstract Factors affecting the oxidation stability of commercially available biodiesel were primarily investigated using the acid value (AV), peroxide value (PV), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, and thermogravimetric (TG) analysis in the presence of imine antioxidants. In this study, the imine compounds N,N′-bis-(4-octadecanate)-salicyl ethylenediamine (stearic dhben) and N,N′-bis-(4-hexadecanate)-salicyl ethylenediamine (palmitic dhben) were synthesized to evaluate their abilities as metal chelators and oxygen scavengers. The AV analysis showed a slight difference between the samples with and without copper. The PV tests demonstrated that both imines were efficient free radical scavengers and copper ion chelators at both room temperature and 50 °C, outperforming the commercial antioxidants butylhydroxytoluene (BHT) and tert-butylhydroquinone (TBHQ). The Derivative Thermogravimetry (DTG) curves indicated that both imines were more effective compared to commercial antioxidants. In the DPPH assay, it was observed that the palmitic dhben imine exhibited the best performance, with an half maximal inhibitory concentration (IC50) of 12.4·10−5 mol L−1. Therefore, both stearic and palmitic dhben imines act as efficient biodiesel antioxidants at room temperature and 50 °C, functioning as excellent metal chelators and free radical scavengers. However, stearic dhben demonstrated better performance as a metal chelator, whereas palmitic dhben was more effective as a free radical scavenger.


Introduction
The increasing awareness of the reduction of accessible fossil fuel reserves, associated with several environmental issues and high fossil fuel consumption, has increased interest in biofuels as a renewable and sustainable resource [1][2][3][4].
Fossil fuels release pollutants such as nitrogen oxides and carbon dioxide into the atmosphere, which causes environmental damage, interferes negatively with the ozone cycle, and plays an important role in the formation of photochemical oxidants and sulfur oxides [5,6].These greenhouse gas concentrations have rapidly increased, leading to unnatural warming that is driving other changes in our environment [7].
Biodiesel has been seen by many researchers as a viable alternative to conventional fuels.Biofuels are renewable energy sources because they are derived from organic matter that can be replenished.They release fewer pollutants during combustion compared to petroleum products, primarily due to their lower carbon intensity and reduced impurities.However, the sustainability of biofuels relies on responsible production practices and considering their entire life cycle [8].
Biodiesel consists of long-chain mono-alkyl esters of fatty acids derived from animal or vegetable oils, known as triacylglycerides.Its production occurs through the transesterification of these fatty acid esters with short-chain alcohols, such as methanol or ethanol [9].The unsaturation of the fatty acid chain is directly related to the chemical composition and physical characteristics of its products, such as biodiesel's oxidative stability [10,11].
The main advantages of biodiesel are that it is almost free of sulfur, aromatic compounds and other chemical substances that are harmful to the environment; it generates employment and income for rural farmers [12]; it requires less financial investment in comparison to fossil fuel studies; and it produces a lower amount of waste [13].
However, there are some disadvantages, such as oxidative degradation that modifies the fuel's characteristics.The susceptibility of biodiesel to oxidation is usually due to the high levels of unsaturation in the fatty acid chain, determined by the number and position of the double bonds [14,15].This makes biodiesel more susceptible to oxidation in the presence of O 2 , light, moisture, or elevated temperatures, or even in the presence of metals [14,16,17].
Biodiesel oxidation can contribute to the formation of insoluble products, degrade its fuel properties, and enhance its corrosive characteristics [18].This undesirable reaction changes biodiesel properties, such as viscosity, density, and total acid number, directly affecting its performance as an alternative fuel [19].One of the most significant causes of biodiesel oxidation is its poor storage stability, where it is exposed to metals and free radicals during transportation or storage in metallic tanks [20][21][22].
Antioxidant compounds are capable of retarding or inhibiting oxidation reactions and are typically added to biodiesel to increase its oxidative stability.Butylhydroxytoluene (BHT) and tert-butylhydroquinone (TBHQ) are examples of synthetic antioxidants used to improve the oxidation stability of biodiesel [23,24].Usually, synthetic antioxidants are commercially used because they show the best results and are added in much lower concentrations than oxidizable substrates.These compounds act by removing free radicals from the medium during autoxidation and preventing the formation of hydroperoxides in photooxidation [15].
The antioxidant mechanism involves the removal of reactive oxygen species or metal ions or the sequestration of free radicals [14,23].The antioxidant capacity is measured by the compound's ability to scavenge free radicals or chelate metal ions [25].Tang et al. [26] investigated antioxidants' effectiveness in biodiesel stability and observed that their activity increased with increasing concentration.They also noted the importance of biodiesel storage location in maintaining the fuel's properties over a long time.
We have been studying some synthetic Schiff base compounds with different structural features that exhibited potential antioxidant activity as radical scavengers or metal chelators, as well as anticorrosive activities in biodiesel [22,27].Imine derivatives of 2,4-dihydroxybenzaldehyde are part of a larger group of o-hydroxy Schiff bases.The imines synthesized in our group have aliphatic long chains in the structure of the Schiff base compounds, which can increase their solubility and dispersion in biodiesel.The imine bond present in long-chain Schiff base compounds gives them the ability to scavenge free radicals, which are highly reactive molecules involved in the oxidation process.Additionally, these compounds have four potential binding sites (two imine and two phenolic groups) that can lead to stable metallic complexes.The synthesis of new molecules is important for producing compounds that improve the quality of biodiesel and encourage its consumption.The main purpose of this work is to synthesize the new Schiff-base compound N,N'-Bis-(4-octadecanate)-salicyl ethylenediamine (stearic dhben) and investigate its abilities as a metal chelator and oxygen scavenger, in comparison to N,N'-bis-(4-hexadecanate)-salicyl ethylenediamine (palmitic dhben) and other commercial antioxidants.The palmitic dhben compound showed antioxidant activity as a metal chelator and oxygen scavenger.The latter property was responsible for reducing metal corrosion, as it decreases the amount of dissolved oxygen in the medium [22].Herein, experiments to determine peroxide values (PVs) and acid values (AVs), 2,2diphenyl-1-picrylhydrazyl (DPPH) assay, and thermogravimetric (TG) analysis were carried out with stearic and palmitic dhben, as well as other antioxidants, to better investigate their possible antioxidant activity.

Physical measurements
Elemental analyses using a Perkin-Elmer 2400 CHN Elemental Analyzer was performed at the Central Anal � ıtica of Universidade de São Paulo.Infrared (IR) spectra were recorded in Agilent Cary 630 Fourier-transform infrared spectroscopy (FTIR) instrument, in the range 4000-400 cm −1 , TG curves were obtained by using the thermal analysis equipment TGA-50 Shimadzu, while Ultraviolet-visible (UV-Vis) spectra were recorded on a double beam Q898UVDB machine from Quimis at our institution.The 1 H and 13 C Nuclear magnetic resonance (NMR) experiments were performed on a NMR spectrometer (Varian VNMRS 11.75 Tesla) at 500 and 125 MHz, respectively, using CDCl 3 as solvent at Laborat� orio Multiusu� ario de Ressonância Magn� etica Nuclear of Universidade Federal Fluminense.

Synthesis of the imines
All the imine compounds were prepared according to previously described methods, using P.A. grade reagents and characterized by different techniques [22].In this work, another imine compound derived from octadecanoic acid was synthesized.
Solutions of octadecanoic acid (1.28 g, 5 mmol, in 5 mL), 2,4-dihydroxibenzaldehyde (0.69, 5 mmol, in 5 mL), DCC (1.14 g, 5.5 mmol, in 10 mL) in dried chloroform (30 mL) along with solid DMAP (0.03 g, 0.25 mmol as a catalyst) were magnetically stirred at room temperature for 12 h in a 100 mL round-bottom flask.The byproduct was filtered off under suction and the solvent was removed using a rotavapor.The crude product was recrystallized from a hot solution of ethanol and the product obtained was a white solid.
The previously synthesized ester (0.1 g, 0.3 mmol, in 5 mL) was combined with a solution of ethylenediamine (10 mL, 0.15 mmol) in chloroform.The pH of the reaction mixture was 5.5, which was desirable, and it was refluxed for 4 h.The resulting solution was left overnight in the reaction flask at room temperature, producing a yellow product that was collected by suction-filtering [22,29].The imine yields were 43% and 40% for palmitic dhben and stearic dhben, respectively.Their chemical structures are shown in Figure 1.

Biodiesel oxidative stability analysis
To evaluate the oxidative stability of the biodiesel samples and the antioxidant efficiency of imines at different temperatures, in the presence or absence of metal contaminants and in comparison to commercial antioxidants, four tests were conducted: AV, PV, DPPH radical scavenging assay and TG analysis.

Acid value
The AV indicates the amount of free fatty acids in the oil, and high AVs suggest the occurrence of degradation reactions, which can be caused by various factors, such as oil type, heating temperature, presence of metals, and storage conditions [30].In this study, the AVs were determined using the acid-isooctane (AOCS) Ca 5a-40 method [31].Biodiesel samples, with and without Cu þ2 ions (60 ppm), were kept in beakers at room temperature.The assay was conducted for 30 days, and aliquots were analyzed once a week.For each analysis, 2 g of biodiesel sample and 25 mL of ethyl alcohol/diethyl ether mixture in a 1:2 ratio (v/v) were added to an Erlenmeyer flask.Then, two drops of a 1% phenolphthalein solution were added, and the solution was titrated with KOH (0.01 mol L −1 ).All experiments were performed in triplicate.The titration reaction is as follows: After the titration, the AV was calculated using Equation (1): where AV is the acid value measured in milligrams of KOH (mg) per gram of biodiesel (g); V t is the titration volume (L); Concentration KOH is the potassium hydroxide concentration (M); Molar Mass is given in g mol −1 ; and W sample is the sample weight (g).

Peroxide value
The PV measurement [32,33] was used to evaluate the oxidative stability of biodiesel.The PV was determined using the AOCS method, which quantifies all peroxide compounds that oxidize potassium iodide (KI) under test conditions in milliequivalents [34].Hydroperoxides present in biodiesel due to oxidation react with KI to form molecular iodine, which is then titrated using a sodium thiosulfate solution [35].The titration reaction is as follows: This study analyzed biodiesel samples both with and without metal ions and in the presence or absence of antioxidant compounds.Copper ions (60 ppm) or nickel ions (60 ppm) were added to the biodiesel to evaluate their catalytic action in the oxidation reaction, in the presence or absence of palmitic dhben (60 ppm), stearic dhben (60 ppm), TBHQ (60 ppm), or BHT (60 ppm) compounds, following the flowchart presented in Figure 2. The samples were placed in a beaker and kept at 50 � C for 120 days.
Biodiesel samples without metals and with copper ions (60 ppm), in the presence or absence of palmitic dhben (60 ppm) or stearic dhben (60 ppm) compounds, were also placed in a beaker.However, they were kept at room temperature for comparison purposes.
The assay was conducted once a week for 120 days.In each assay, 5 g of biodiesel was weighed into a 250 mL Erlenmeyer flask and dissolved in a 30 mL solution of acetic acid and anhydrous chloroform with a ratio of 3:2 (v/v).Once the sample was completely dissolved, 0.5 mL of saturated-KI solution was added, and the mixture was stirred for 1 min in the dark.Immediately afterward, 30 mL of distilled water was added, followed by the addition of 0.5 mL of a 1% starch solution as an indicator.Titration was then performed using a standard sodium thiosulfate solution (0.1 N).A separate blank analysis was conducted for each sample.The PV of the samples was calculated using Equation (2) [34]: where PV is the peroxide value measured in active oxygen milliequivalents (meq) per kilogram (kg), V t is the titration volume in milliliters (mL), Concentration Na2S2O3 is the concentration of sodium thiosulfate in normality (N), and W sample is the weight of the sample in kilograms (kg).All experiments were performed in triplicate.

TG analysis
Fresh samples were prepared following the composition outlined in the flowchart presented in Figure 2.These samples were placed in an oven and stored at 50 � C for a duration of 25 days to observe the initial degradation of biodiesel.
After the storage period, the samples were subjected to analysis using a TG analyzer (Shimadzu DTG-60).The analyzer heated the samples from 20 � C to 400 � C, with a slow heating rate of 10 � C per minute, while maintaining an argon flux of 50 mL per minute.

DPPH radical scavenging assay
DPPH is a stable free radical that possesses an unpaired electron.The DPPH radical scavenging activity of imine compounds was evaluated using a previously reported method [36].
The imine compounds studied here absorb light at different wavelengths than DPPH.Therefore, solutions of palmitic and stearic dhben imines were prepared in DMSO at concentrations ranging from 15 to 62 mmol L −1 .Additionally, a DPPH solution (200 mmol L −1 ) was prepared in DMSO and kept in the dark.Subsequently, an aliquot of the DPPH stock solution was added to the imine solutions, resulting in a final DPPH concentration of 167 mmol L −1 .The reaction mixtures were vigorously agitated and incubated for 15 min at room temperature in the dark.The absorbance of aliquots was measured at 525 nm using a UV-Vis spectrophotometer, and the readings were compared against a blank consisting of 0.5 mL of DPPH methanolic solution mixed with 2 mL of DMSO.The half-maximal inhibitory concentration (IC 50 ) was determined and analyzed to assess the antioxidant potency of the imines in reducing DPPH.All experiments were conducted in triplicate.

Imine characterization
The infrared spectra of the imine compounds exhibited the expected characteristic imine m(C ¼ N) band, which appeared at 1642 cm −1 for both compounds.A band near 1756 cm −1 was assigned to the m(C ¼ O) vibration of the ester group.The range of 2840 to 2920 cm −1 displayed m(C-H) vibrations from the alkyl groups (CH 2 and CH 3 ).Another band in the region of 3290-3305 cm −1 corresponded to the m(C-H) vibration of the phenolic hydrogen that forms an intermolecular bond with the imine nitrogen.
The 1 H NMR and 13 C NMR spectra of stearic dhben showed well-resolved peaks, and chemical-shift assignments were made based on the analysis of proton resonances' multiplicity patterns and carbon assignments.Important assignments for this compound class included a singlet at d 8.32 ppm for the imine proton, a singlet at d 3.91 ppm for the methylene group, a triplet at d 2.15 ppm (J ¼ 7.0 Hz) corresponding to the methylene hydrogens adjacent to the carbonyl group (-CH 2 COO-), a singlet at 1.26 ppm representing the 28 hydrogens of the other methylene groups, and a triplet at 0.88 ppm (J ¼ 7.5 Hz) for the methyl group of the alkyl chain (-CH 2 CH 3 ).The 13 C NMR spectra confirmed the expected assignments, with the imine (-CH 5 N-CH2-) appearing at 162.6 ppm, the carbonyl group of the ester (-C 5 O) at 173.5 ppm, and the imine methylene group (-CH ¼ N-CH 2 -) at 58.5 ppm.

Acid value
Assessing the AV of the samples under the specified conditions provides an indication of the level of degradation in oils.The AV results of the biodiesel samples, based on the storage time, are summarized in Table 1.
The AVs obtained from pure biodiesel and copper-contaminated biodiesel exhibited a slight difference.Even in the samples with metal contaminants, which are known to contribute to the formation of a larger quantity of peroxides, the presence of high levels of acid was not observed.This can be attributed to the decomposition of peroxides, which commonly occurs and leads to the formation of acids, ketones, esters, aldehydes, alcohols, and alkanes.
After 15 days, the AVs of both samples exceeded 0.50 mgKOH g −1 , which is the maximum recommended AV value for biodiesel according to American Society for Testing and Materials (ASTM) D6751 and European Standard (EN)14214 standards [21,37].Therefore, it is advised to utilize the fuel within 15 days of storage at room temperature (25 � C) in the dark to ensure its optimal quality.

Peroxide value
The storage stability tests were conducted by keeping the samples for about 4 months (120 days), and during this period some changes in PV caused by their oxidation were analyzed.The results for the samples stored at room temperature and those kept at 50 � C, both without and with metal contaminants, are depicted in Figure 3.
Samples without metal ions (Figure 3a) showed the lowest PVs during the storage period at room temperature, especially the sample with palmitic dhben.Thus, it was observed that the presence of metals at trace levels may act as potent catalysts for the oxidative degradation of biodiesel samples, producing primary and secondary oxidation products (Jain et al. 2014).
After approximately 40-60 days of storage, the PV of all samples decreased.This behavior represented an inflection point, where peroxide formation ceased and its consumption began due to secondary reactions generated by degradation products (aldehydes and ketones) caused by the breakdown of peroxides and hydroperoxides [38].
The two antioxidants act at different times, and it was possible to identify those that performed better in terms of biodiesel stability.In Figure 3a and b, the samples without imines had a larger peak, representing greater degradation, and both imines were able to reduce this degradation, as illustrated by the PVs.
Table 2 presents the order in which the samples kept at room temperature reached the maximum PV, indicating the shortest time or the highest PV.Both imine compounds were effective in preventing oxidative degradation of biodiesel with and without copper.However, the sample of palmitic dhben with copper started to show higher PVs after 10 days.
In general, palmitic dhben exhibited better performance at room temperature compared to stearic dhben, as its peroxide levels were lower, even in the sample that reached the peak in 10 days.The PV values of the samples stored at 50 � C are presented in Figure 3c-e.
Figure 3c-e illustrate that both samples with and without metal ions exhibited higher peroxide levels at 50 � C compared to those at room temperature, indicating that temperature directly influences oil degradation.Table 3 provides the sequence in which the samples attained the maximum PV, considering either the time (for samples without metal and with Ni 2þ ions) or their PV (for samples with Cu 2þ ions) at 50 � C.
Samples containing stearic and palmitic dhben exhibited the longest storage time to reach the maximum PV, demonstrating the effective ability of the imine compounds to scavenge free radicals and act as efficient antioxidants even in the absence of metal ions.
Figure 3e illustrates that all samples with Cu 2þ ions at 50 � C reached the maximum PV at the same time, but there was a significant difference in the values depending on the presence and type of antioxidant.
In general, the PVs of samples with Cu 2þ ions were approximately twice as high as those with Ni 2þ ions, with a maximum PV of 590 meq kg −1 for biodiesel without antioxidant, while the same sample of biodiesel with Ni 2þ ions reached a maximum PV of 359 meq kg −1 , as shown in Figure 3d and e.
Studies indicate that copper degrades more than nickel [39], and the effectiveness of antioxidants decreases depending on the type of metal.The presence of imine compounds significantly reduced biodiesel degradation, with copper-contaminated samples showing a maximum PV of 240 meq kg −1 for stearic dhben and 285 meq kg −1 for palmitic dhben.Nickel-contaminated samples exhibited the highest PV of 240 meq kg −1 for stearic dhben and 230 meq kg −1 for palmitic dhben.Moreover, PV values above 300 meq kg −1 are considered extremely high for fuels [14], and all values obtained in the presence of both imines were within suitable ranges for biodiesel fuel, meeting the standards.
Comparing the commercial antioxidants BHT and TBHQ under the same conditions, it was observed that they reached the maximum PV in a shorter time compared to samples with imines and without metal ions (Figure 3c and Table 3), indicating that imines have a better ability to maintain oil stability over a longer period.At around 40 days of storage, when BHT and TBHQ samples reached the PV peak of 200 meq kg −1 , the PVs of samples with palmitic and stearic dhben were about 90 meq kg −1 , indicating that imines were more efficient as free radical scavengers than the commercial antioxidants in the absence of metal contaminants, especially BHT.
The nickel-contaminated samples (Figure 3d and Table 3) exhibited low PVs in the presence of imines.The samples with BHT reached the degradation peak in 24 days, TBHQ in 38 days, palmitic dhben in 43 days, and stearic dhben in 50 days, which was the longest time.In addition to demonstrating longer oil stability, stearic dhben showed smaller PVs than palmitic dhben.Among the nickel-contaminated samples with antioxidants, the sample with TBHQ exhibited the highest PV.All copper-contaminated samples (Figure 3e and Table 3) reached the degradation peak at approximately the same time (around 30 days).The main difference among them was the PV value, with pure biodiesel reaching 590 meq kg −1 , the sample with BHT at 310 meq kg −1 , palmitic dhben at 290 meq kg −1 , stearic dhben at 230 meq kg −1 , and TBHQ at 210 meq kg −1 .Thus, the imines demonstrated better performance than BHT, which had a PV peak exceeding 300 meq kg −1 .However, TBHQ was more effective in chelating copper than imines, although its maximum PV was very close to the peak of stearic dhben.
Both imines exhibited good performance in preventing the oxidative degradation of biodiesel samples, significantly reducing the PV of all samples.This indicates that they were successful in chelating metal ions and scavenging free radicals at both room temperature and 50 � C. Overall, stearic dhben performed better at 50 � C, even in comparison to palmitic dhben, as its PV peaks were similar to or smaller than those of palmitic dhben degradation peaks.

Thermogravimetric analysis
Thermogravimetric analyses were conducted for all samples after 25 days of incubation at 50 � C. The samples were weighed and analyzed, and the results are presented in Table 4.
The TG analyses were transformed into derivative thermogravimetry (DTG) curves, which depict the mass loss rate as a function of temperature and provide a better visualization of the degradation process through peaks.These DTG curves are presented in Figure 4a-c, allowing for a more detailed observation of the degradation.
According to the literature [40], biodiesel degradation typically occurs between 175 � C and 300 � C.This range was confirmed in Figure 4a-c, which displayed degradation peaks ranging from 225 to 275 � C.
Figure 4a reveals that even without metal contamination, samples containing palmitic and stearic dhben exhibited degradation at higher temperatures, indicating their superior stability compared to BHT and TBHQ.The samples with BHT and TBHQ reached the degradation peak earlier than the pure biodiesel sample, followed by the sample with TBHQ, which also applies to the presence of nickel ions (Figure 4b).
For copper-contaminated samples with commercial antioxidants, the temperatures of degradation (Figure 4c) were very similar to those of the synthesized imines, suggesting their similar efficiency in chelating copper.Overall, stearic dhben demonstrated the best performance, surpassing palmitic dhben, under all conditions (Figure 4a-c).

DPPH radical scavenging assay
The free radical scavenging ability of stearic and palmitic dhben was evaluated using DPPH, and the corresponding UV-Vis spectra are displayed in Figure 5a and b.These spectra illustrate the absorbance of DPPH at 525 nm, which decreases with increasing concentrations of the imines.This observation indicates that both stearic and palmitic dhben possess the capability to scavenge DPPH free radicals.
Figure 5a and b clearly demonstrate that both stearic and palmitic dhben are capable of reducing DPPH absorbance as the concentration of the antioxidants increases.To better understand the imines' ability to reduce DPPH free radicals and absorbance, the IC 50 values can be analyzed.The IC 50 value represents the concentration of the antioxidant compound required to decrease the DPPH absorbance by 50%.This value can be determined by plotting the inhibition curve using the absorbance data against the concentration of the compound used (Figure 6a and b) and calculating it using the slope of the linear regression [41].
The IC 50 values for the solutions of stearic and palmitic dhben were calculated using the regression equations, by substituting the 'y' value with half of the DPPH absorbance.The calculated IC 50 values were found to be 14.6 � 10 −5 mol L −1 for stearic dhben and 12.4 � 10 −5 mol L −1 for palmitic dhben.In comparison, according to literature [41], the IC 50 value for the BHT compound under the same conditions was reported as 4.04 � 10 −4 mol L −1 .
Based on these findings, both stearic and palmitic dhben demonstrated significant antioxidative activity when compared to the BHT commercial antioxidant, as indicated by their lower IC 50 values.

Conclusion
The synthesized imines, stearic and palmitic dhben, were characterized to verify their expected structure and suitability for evaluating their antioxidant activity.
The acid value (AV) analysis revealed a slight difference between the samples with and without copper.This suggests that even though the sample had a significant amount of peroxides, it did not exhibit a high AV.After 15 days, the AV exceeded 0.5 mg KOH g −1 , which is the maximum allowable value for biodiesel.
The peroxide value (PV) tests demonstrated that both imines were efficient free radical scavengers and copper chelators at both room temperature and 50 � C, outperforming the commercial antioxidants.At room temperature, palmitic dhben exhibited better performance when combined with pure biodiesel, indicating its strong affinity for scavenging free radicals.At 50 � C, the imines generally performed better compared to the commercial antioxidants BHT and TBHQ.BHT was ineffective as a metal chelator, as its sample degraded more than the sample without antioxidants in the presence of nickel ions.For copper-contaminated samples, the degradation peak exceeded 300 meq kg −1 , which is unsuitable for fuels.On the other hand, among the copper-contaminated samples, TBHQ exhibited the best performance, slightly surpassing the performance of stearic dhben.The DTG curves demonstrated that both imines were more effective than the commercial antioxidants in delaying biodiesel degradation to higher temperatures, both with and without nickel and copper ions.Overall, stearic dhben exhibited better performance than palmitic dhben in all conditions.
The analysis of the imines' antioxidant activity indicated that both compounds were capable of reducing DPPH absorbance and efficiently capturing free radicals.Moreover, palmitic dhben demonstrated the best performance, with an IC 50 value of 12.4 � 10 −5 mol L −1 .
Therefore, both stearic and palmitic dhben imines acted as efficient antioxidants for biodiesel at room temperature and 50 � C, functioning as effective metal chelators and free radical scavengers.However, stearic dhben showed better performance as a metal chelator, while palmitic dhben was superior as a free radical scavenger.
It is important to note that research in this field is still ongoing, and the effectiveness of long-chain Schiff base compounds as antioxidants for biodiesel may vary depending on the specific production and usage conditions of the fuel.Nonetheless, these compounds represent a promising avenue for improving the quality and performance of biodiesel by providing protection against oxidation.

Figure 3 .
Figure 3. PV measurement: (a) without metals as a function of time at room temperature; (b) with Cu þ2 contaminant as a function of time at room temperature; (c) without metal contaminant as a function of time at 50 � C; (d) with Ni 2þ as a function of time at 50 � C; (e) with Cu 2þ as a function of time at 50 � C.

Figure 4 .
Figure 4. DTG curves: (a) without metal contaminants as a function of temperature; (b) with Ni 2þ as a function of temperature; (c) with Cu 2þ as a function of temperature.

Table 2 .
Order in which the samples reached the peak at room temperature.
OrderWithout metal (time analyzed) With copper ions (time analyzed) With copper ions (PV analyzed)

Table 3 .
Order in which the samples reached the peak at 50 � C.