Biodiesel production in Querétaro, México from used oil and analysis of the samples by nuclear magnetic resonance

and mass spectrometry.

Eugenia Josefina Aldeco-Pérez*, Saul López-Ordaz, Miguel Ángel Ramos-López, Adrián Sosa-Domínguez

Laboratorio de bioenergéticos, Facultad de Química, Universidad Autónoma de Querétaro.

Cerro de las campanas S/N, Col. Las Campanas, Querétaro, México. 76010.

*eugeniajosefina@[Link]

Abstract

In an effort to mitigate the impact of greenhouse gases in our planet, a biofuel laboratory located in Mexico has been
created. This laboratory is in charge of biodiesel production, using as staring material vegetal used oil obtained from
donations of alimentary businesses around. The purpose of this project is, first of all, to use a renewable fuel, and, in
second place, to promote the culture of reusing of vegetal oils, avoiding their inadequate disposal. Both reasons have the
aim of improving social issues where both academic staff and students are compromised with. Biodiesel has been analyzed
by nuclear magnetic resonance, where we observed the corresponding signals for methyl esters, as well as some
byproducts, the glycerides. Additionally, in mass spectrometry, we were able to appreciate the average methyl ester
composition, detecting that there is a variation from sample to sample. Results reveal that methodology for obtaining and
purifying biodiesel is appropriate, since most of the analyzed samples showed the characteristic signals and the by-
products are found just in low concentration. In general, biodiesel composes mostly on four methyl esters and we report
herein the proportion for each one.

Key words

Biofuel, biodiesel, used vegetal oil, nuclear magnetic resonance, mass spectrometry.

1. Introduction

In our days, fossil energetics is still a trend topic, being the main concern hydrocarbon depletion, but also gas and
particle emission released to the atmosphere. In order to decrease this fuel consumption, some other alternative
energies are being promoted like nuclear, solar, eolic, hydrogen, etc.; however, all these technologies are regularly
exploited in economically developed countries in behalf of requiring high initial investment for research but also
for implementation. The big opportunity for the developing countries, as México, is the biofuel adoption, due to
the fact that it is considered a renewable source, although its exploitation has been questioned, principally dealing
with the land use both for agricultural crops and human consumption [1]. Hereby, the idea of reusing vegetal oils
to produce a biofuel seems to be feasible and adjuvant with the environmental preservation since the process
allows residues exploitation generating an added value fuel, recycling by these means the CO2 between the
atmosphere and biosphere [2, 3]. Biodiesel as fuel, is relatively easy to obtain, besides possessing several
advantages like biodegradability, renewability, low toxicity, etc., as indicated in Figure 1.

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 Renewable
source

Decrease of
High flash
greenhouse
point
gases

Biodiesel
advantages

Low emission Biodegradable

of dangerous and low
particles toxicity

alternative to
fossil fuel

Figure 1. Advantages of using biodiesel in comparison with fossil fuel

Consequently, in an approach to reduce greenhouse gases, we established a biofuel laboratory, located at the
University of Querétaro in México (Universidad Autónoma de Querétaro). First essays were achieved on 2012 and
that year, we started to mix it up along with fossil diesel loading a University transportation bus. Now, we have a
100-liter pilot plant where, as first step, we have the picking up of the residues, generally coming from food
industry around (restaurants, hotels, University’s cafeterias). As second step, the use of semiautomatic reactors,
producing 72 liters per week, being immediately used to commute around 1200 students in daily bases, displaying
the importance of production and real application of biodiesel [4]. As side studies, some tests on thermal analysis
are being performed where is seen that gas emissions are reduced in quantity in comparison of using diesel fossil
when loaded in combustion engines [5,6].
After the transesterification reaction, several identification tests were practiced to the final product by means of
assuring the quality before being mixed and loaded into the buses. The measured properties are viscosity, density
and more recently, nuclear magnetic resonance and mass spectrometry, most of all nondestructive essays. Last
two techniques are not always used as a routine analysis; we discuss in this work some scopes of the them.

2. Material and methods

Transesterification reaction of used vegetal oils were performed in a 100-liter reactor, adding 80 liters of vegetal
used oil, methanol (20 liters), potassium hydroxide (quantity calculated from acid number, average 560 g) and
heating up to 60 OC during 2 hours. Biodiesel was decanted and washed two times with water and passed through
a desiccant filter; at the end, 72 liter of biodiesel were obtained. Glycerol obtained from the separation was
purified and used as reagent in the synthesis of alquidylic resins, a further project that also demonstrates
alternative applications of reaction by-products [7].

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 Viscosity measurements were carried out at 40oC in a dial reading Brookfield viscometer LVT, using a bath
temperature regulator and 10 mL of biodiesel. Nuclear magnetic resonance (NMR) were carried out at Chemistry
Department of CINVESTAV, in a Jeol ECA500 and JEOL Eclipse 400; samples were prepared using 30 mg of the
biodiesel and 0.5 mL of CDCl3. Mass spectrometry was obtained in an Agilent LC/LCMSD 1969A model, TOF+ mode,
also located at CINVESTAV.

3. Results and discussion

Eight samples were taken when the 100-liter reactor was used. Viscosity, density, nuclear magnetic resonance
and mass spectrometry were carried out on all these samples.
First of all, it seems that nuclear magnetic resonance could not be a suitable analysis tool, since it is used principally
to characterize pure compounds, but as we could observe, is an easy, quick and non-destructive alternative
possessing high resolution both in 1H and 13C. We could then appreciate how transesterification reaction
proceeded and we could also check the purity of the final methyl esters [8].There are two important signals in
NMR: one corresponding to the methyl group and another due to the double bonds contained in some of the
esters. Particularly, signals of major importance that characterize biodiesel are at the following chemical shifts: 1H
NMR methyl ester signal is at around 3.6 ppm and 5.3 ppm for the double bond. In 13C NMR, at 174 ppm is located
the signal corresponding to the carbonyl ester group, at 130 ppm the double bond carbons and at 51 ppm the
methyl. Figures 2 and 3.

Figure 2. 1H NMR of sample (CDCl3, 500 MHz)

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 Figure 3. 13C NMR of sample 4 (CDCl3, 125 MHz)

In Table 1, we enlist the found values for the analyzed samples. In NMR, chemical shifts, multiplicity and
assignation according to DEPT, PENDANT, COSY and HECTOR experiments; we include as well viscosity and density
measurements, noting out that values are comprised in the accepted range mentioned in the European Standard
EN 14214 [9, 10].

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 Table 1. NMR, density and viscosity of the biodiesel analyzed samples
Sample Kinematic Relative 1H RMN 13C RMN
viscosity at density
40oC (mm2s-1) at 20oC
(Kgm-3)

1 4.65 879 5.28-5.20 (m, CH=CH), 3.56 (s, OCH3), 2.71-2.66 (m, CH2), 2.20 (t, 174.0 (CO), 130.0 (CH), 129.9 (CH), 128.0 (CH), 127.9
J= 7.22 Hz, -CH2), 1.99-1.92 (m, CH2), 1.59-1.51 (m, -CH2), 1.30- (CH), 51.3 (OCH3), 34.0, 31.9,31.5 29.7, 29.3, 29.2,
1.1 (m, CH2), 0.88 (t, J= 7.7 Hz, CH3), 0.86-0.78 (m, CH3) 27.2,25.6, 24.9, 22.7 (CH2), 14.1 (CH3)

2 4.43 890 5.28-5.20 (m, CH=CH), 3.55 (s, OCH3), 2.72-2.66 (m, CH2), 2.20 (t, 173.9 (CO), 131.8 (CH), 130.0 (CH), 129.8 (CH), 129.7
J= 7.6 Hz, -CH2), 1.99-1.90 (m, CH2), 1.58-1.51 (m, -CH2), 1.30- (CH),128.1, 128.0, 127.8, 127.7, 127.0 51.2 (OCH3),
1.1 (m, CH2), 0.88 (t, J= 7.5 Hz, CH3), 0.83-0.76 (m, CH3) 33.9, 31.9, 31.5 29.3, 29.2, 29.1, 29.0, 27.1, 25.6, 25.5,
24.9, 22.7, 22.6 (CH2), 14.0 (CH3)

3 4.36 876 5.38-5.24 (m, CH=CH), 3.62 (s, OCH3), 2.76-2.71 (m, CH2), 2.26 (t, 174.2 (CO), 130.26, 130.20, 130.0, 129.7, 128.0, 127.9
J= 7.4 Hz, -CH2), 2.03-1.93 (m, CH2), 1.62-1.54 (m, -CH2), 1.36- (CH), 51.4 (OCH3), 34.1, 31.9, 31.5 29.5, 29.7, 29.6, 29.5,
1.2 (m, CH2), 0.93 (t, J= 7.8 Hz, CH3), 0.87-0.82 (m, CH3) 29.4, 29.3, 29.2, 25.0, 24.9, 22.7, 22.6 (CH2), 14.1 (CH3)

4 4.30 866 5.37-5.22 (m, CH=CH), 3.61 (s, OCH3), 2.79-2.70 (m, CH2), 2.25 (t, 174.21 (CO),131.9 130.2, 130.1, 129.9, 129.8, 129.7,
J= 7.6 Hz, -CH2), 2.04-1.93 (m, CH2), 1.61-1.53 (m, -CH2), 1.34- 128.0, 127.9 (CH), 51.36 (OCH3), 34.1, 31.9, 31.5, 29.8,
1.17(m, CH2), 0.93 (t, J= 7.6 Hz, CH3), 0.87-0.81 (m, CH3) 29.7, 29.6, 29.5, 29.3, 29.2, 29.1, 27.2, 25.6, 24.9, 22.7,
22.6 (CH2) 14.12, 14.0 (CH3)*HETCOR

5 4.25 872 5.39-5.21 (m, CH=CH), 3.63 (s, OCH3), 2.79-2.72 (m, CH2), 2.34-2.24 174.3, 173.3 (CO),131.9 130.2, 130.0, 129.7, 127.9,
(m, -CH2), 2.08-1.95 (m, CH2), 1.67-1.53 (m, -CH2), 1.36-1.19 (m, 127.1 (CH), 51.4 (OCH3), 34.1, 31.9, 31.6, 29.8, 29.7,
CH2), 0.95 (t, J= 7.6 Hz, CH3), 0.88-0.83 (m, CH3) 29.6, 29.4, 29.2, 29.1, 27.2, 25.6, 25.0, 24.9, 22.7, 22.6
(CH2), 14.1 (CH3)#

6 4.70 878 5.39-5.25 (m, CH=CH), 3.63 (s, OCH3), 2.81-2.71 (t, J=6.6 Hz, CH2), 174.3 (CO),130.3, 130.2, 130.0, 129.9, 129.7, 128.1,
2.27 (t, J= 7.6 Hz, -CH2), 2.06-1.95 (m, CH2), 1.64-1.55 (m, -CH2), 127.9 (CH), 51.4 (OCH3), 34.1, 31.9, 31.6, 29.8, 29.7,
1.36-1.18 (m, CH2), 0.95 (t, J= 7.6 Hz, CH3), 0.88-0.83 (m, CH3) 29.6, 29.5, 29.4, 29.3, 29.2, 29.1, 27.2, 25.6, 25.0, 22.7,
22.6 (CH2), 14.1 (CH3)

7 4.52 879 5.40-5.27 (m, CH=CH), 3.64 (s, OCH3), 2.75 (t, J=6.4 Hz, CH2), 2.28 174.3 (CO),130.2, 130.1, 130.0, 129.8, 128.3, 128.1,
(t, J= 7.6 Hz, -CH2), 2.06-1.96 (m, CH2), 1.60-1.57 (m, -CH2), 1.36- 127.9, 127.8 (CH), 51.5 (OCH3), 34.1, 32.0, 29.8, 29.7,
1.22 (m, CH2), 0.95 (t, J= 7.4 Hz, CH3), 0.89-0.84 (m, CH3) 29.6, 29.4, 29.3, 29.2, 29.1, 27.2, 25.7, 25.0, 22.7, 22.6
(CH2), 14.1 (CH3)

8 4.42 867 5.42-5.26 (m, CH=CH), 3.64 (s, OCH3), 2.75 (t, J=6.4 Hz, CH2), 2.28 174.3 (CO),130.2, 130.1, 130.0, 129.8, 128.3, 128.1,
(t, J= 7.6 Hz, -CH2), 2.07-1.95 (m, CH2), 1.63-1.55 (m, -CH2), 1.36- 127.9, 127.8 (CH), 51.5 (OCH3), 34.1, 32.0, 31.6, 29.8,
1.19 (m, CH2), 0.95 (t, J= 7.6 Hz, CH3), 0.89-0.83 (m, CH3) 29.7, 29.6, 29.5, 29.4, 29.3, 29.2, 29.1, 27.2, 25.2, 25,
22.7, 22.6 (CH2), 14.2, 14.1 (CH3)

m=multiple, d=doublet, t=triplet, q=quartet

a. 400 MHz
b. 500 MHz
c. 100 MHz
d. 125 MHz
* HETCOR: in this sample signals at 131-127 ppm in 13C were found to have a correlation with the multiple at 5.3 ppm in 1H, and they correspond
to the olefin fragment. Signals at 51.3 ppm and 3.61 ppm have also a correlation and are due to the presence of methoxide of methyl ester
fragment. Some other signals, correspond to the aliphatic fragment, like the one at 34.1 ppm that correlates with the triplet at 2.25 ppm,
assigned to the CH2 in alpha position to the carbonyl group. Finally, the multiple at 0.84 ppm, correlates with the 14.1 ppm signal, for the methyl.
# CH corresponding to triglycerides are clearly observed (1H at 4.28-4.00 ppm with the corresponding multiplicity and at 62-68 ppm in 13C)

5
1
H NMR technique is even so sensitive that is possible to visualize three group of signals corresponding to the CH2
and CH of the glycerides, between 4.35 and 4.00 ppm (Figure 2), whose contents varies from 1.87 to 0.15%,
according to the calculated rate. This signal is clearly visible in sample 5 spectra, but so weak in sample 4. Figure
4.

Figure 4. Glycerides signal expansion for sample 5, 1H RMN (CDCl3, 500 MHz)

Glycerides are compounds formed between the reaction of glycerol and fatty acids, considered as by-products of
the transesterification, since they are impurities, it is desirable that they do not appear in biodiesel moreover if it
is going to be used in combustion engines [11]. In the 1H NMR spectra, we found a group of weak signals, except
on sample 5, maybe because of the origin of the oil mixture. Glycerides percentage was calculated as a rate
between the total area and the area corresponding to these signals in 1H NMR. In general, values are acceptable
low, according to EN14214, that reports maximum limits of monoglycerides of 0.8%, while diglycerides and
triglycerides are 0.2%. Four of the values are on the accepted range (samples2, 6, 7, 8) and four of them are out
(samples 1, 3, 4, 5), giving us an indication of the purity of the biofuel. Table 2.

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Table 2. Estimated percentage of glycerides contained in the biodiesel samples

Sample Total area Signal area between % glycerides

4.3-4.0 ppm

1 42.90 0.13 0.30

2 38.10 0.06 0.15

3 39.82 0.12 0.30

4 42.33 0.20 0.47

5 82.84 1.55 1.87

6 40.97 0.08 0.20

7 39.47 0.05 0.12

8 39.9 0.05 0.12

It is important to note that there are reported studies distinguishing mono, di and triglycerides by spectroscopic
methods with mixtures of them. The pattern found therein compared with the one obtained by us, correspond to
a mixture of both di and triglycerides, presenting a multiple signal (diglyceride) at 4.10 ppm and two doublets at
4.27 ppm (triglycerides) [12,13]. Figure 4.

In 13C we observed the glyceride carbons, at 68.9 ppm (CH), 68.3 ppm (CH), 65.0 ppm (CH2) and 62.1 ppm (CH2),
looking the same as in the reported pattern.
The size of glyceride signals is comparatively shorter than the ester signal, probing that reaction and purification
procedures were properly achieved and by-products are present only in minimum amount. Additionally, no
methanol or glycerol residues were detected in any sample by this technique.
On the other hand, mass spectrometry allows us to distinguish several components of the ester mixture, for
example, in samples 1 and 2, the major fragment correspond to the oleic acid methyl ester (54 and 55%,
respectively), while for the rest of the samples is the linolenic acid methyl ester (42-54%). Because of the method,
molecular mass has an extra unit contributing to the total mass.
Furthermore, we observe some other fragments corresponding to additional components, that are listed in Table
3, and also an approximate normalized percentage is presented, concerning to the relative intensity of parent
peak, found for methyl esters in each of the cases.

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 Table 3. Mass spectrometry data of biodiesel samples.
Sample Signal 1 (m/z) % Signal 2 (m/z) % Signal 3 (m/z) % Signal 4 (m/z) % Main methyl
C19H36O2 C17H34O2 C19H34O2 C19H32O2 ester
296.2715 270.2559 294.2559 292.2408
1 [M+H]+=297.2790 54.1 [M+H]+=271.2641 26.6 [M+H]+=295.2630 8.3 [M+H]+=293.2481 5 Oleic acid methyl
ester
2 [M+H]+=297.2789 55.9 [M+H]+=271.2640 6.5 [M+H]+=295.2632 29.1 [M+H]+=256.1247 3.7 Oleic acid methyl
ester
3 [M+H]+=297.2777 30 [M+H]+=271.2629 2.1 [M+H]+=294.2623 42.8 [M+H]+=293.2467 13.9 Linolenic acid
methyl ester
4 [M+H]+=297.2774 27.7 [M+H]+=271.2626 1.9 [M+H]+=295.2623 50 [M+H]+=293.2463 14.7 Linolenic acid
methyl ester
5 [M+H]+=297.2777 31.9 Not present 0 [M+H]+=295.2623 54.0 [M+H]+=293.2471 13.1 Linolenic acid
methyl ester
6 [M+H]+=297.2779 37.9 [M+H]+=271.2630 3.2 [M+H]+=295.2625 51.6 [M+H]+=293.2473 5.6 Linolenic acid
methyl ester
7 [M+H]+=297.2780 30 [M+H]+=271.2630 1.6 [M+H]+=295.2631 51.1 [M+H]+=293.2470 15.5 Linolenic acid
methyl ester
8 [M+H]+=297.2780 32.6 [M+H]+=271.2626 1.4 [M+H]+=295.2631 50.7 [M+H]+=293.2468 14.4 Linolenic acid
methyl ester

Isotopic patterns are well distinguished for each indicated signal, and parent peak of each was taken to compare
the relative intensities [14].
According to Georgogianni [3], the composition of frying oil performed in gas chromatography, reveals the
presence of the following fatty acids: palmitic acid (C16H32O2), stearic acid (C18H36O2), oleic acid (C18H34O2), linoleic
acid (C18H32O2), and linolenic acid (C19H30O2). Where major acids are linoleic, palmitic and oleic acid, in 57%, 18%
and 16%, respectively. In our specific case, after transesterification, we observe two methyl esters as main
products: for example, in sample 1, oleic acid methyl ester (54%), followed by palmitic acid methyl ester (26%).
By contrast, for samples 3-8, major ester is the linoleic acid methyl ester and oleic acid methyl ester. Table 3.
The variation could be because of the several sources of vegetal oil and also purity or contaminants present,
considering that oil donations are mixed together in order to have 80 liter of oil mixture for each run.

In sample 5, where glycerides are present in greater amount, ester composition is different to others,
comparatively; and also palmitic acid methyl ester is not present, being major components linoleic and oleic acid
methyl esters.

4. Conclusions

It was possible to identify methyl ester components and also the impurities present in biodiesel, obtained from
used vegetal oil subjected to transesterification, by means of proton and carbon NMR as well as by mass
spectrometry. These analyses give an idea of the approximate composition, important information since the final
product is fueled in combustion engines of the University buses. NMR is an easy, quick and non-destructive
technique that probed to be useful giving some important parameters of the methyl esters contained in biodiesel.
In 1H and 13C NMR, the presence of glycerides (di and triglycerides), was perceptible and the calculated rate was
from 0.12 to 1.87%, this latter value for sample 5, which presented some differences from others.

Major methyl esters, found in mass spectrometry, correspond to oleic acid methyl ester (for samples 1 and 2, 54
and 55%), and linolenic acid methyl ester (samples 3-8, 42-54%).

All these results showed that the strategy of reusing vegetal oil as source for biodiesel is appropriate and the
impurities are present just in some samples, as well as we could realize the approximate composition of the methyl
ester mixture.

8
Acknowledgements

This project was supported by SEDESU (Secretaría de Desarrollo Sustentable Querétaro) FCQ-2015-09, FOFI UAQ (Fondo
para el fortalecimiento de la Investigación) FCQ-2014-04.

Technicians at CINVESTAV-Zacatenco: Q.F.B. Ma. Teresa Cortez Picasso, Geiser Cuéllar Rivera.

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