Laboratory Experiment

pubs.acs.org/jchemeduc

Biodiesel from Seeds: An Experiment for Organic Chemistry

Steven W. Goldstein*
Department of Chemistry, University of Saint Joseph, West Hartford, Connecticut 06117-2791, United States
*
S Supporting Information

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

KEYWORDS: First-Year Undergraduate/General, Second-Year Undergraduate, Biochemistry, Environmental Chemistry,

Laboratory Instruction, Organic Chemistry, Agricultural Chemistry, Bioorganic Chemistry, Lipids, Nutrition, Spectroscopy

T he interest in biofuels in public and scientific forums has

escalated over the past few years. Whether this is due to
the thought that the world has reached peak oil extraction1 or a
search for a “friendlier” fuel, the general public is keenly aware
of biologically based additives and replacements for home
heating oil and fuels to power vehicles. The topic continues to
be the subject of much scientific investigation, with 100 or
more research articles being published by American Chemical
Society journals over each of the past four years. Although
much of the research has focused on the physical properties of
biodiesel, other investigations focus on the conversion of
various feedstock into fuel. One aspect of these studies that has
not been covered in depth is isolation of the energy rich
triglyceride raw material from natural sources. When students
see the direct connection between the naturally derived plant
energy source and the synthetic biofuel, a more complete
picture of the process is presented.
Plants and animals produce and store lipids, predominantly
as triglycerides, in order to access the energy potential of those
molecules at a later time. Seeds, which require energy to grow
in the time between germination and active photosynthesis,
store reserves of triglycerides in specific locations within the
structure of the seed. Accordingly, a significant proportion of
the cellular mass of seeds can be triglycerides.2 Upon
germination, the activation of various lipase3 and oxygenase4
enzymes within the seed begins to provide the developing
plants with the necessary nutrients for growth. The triglyceride
composition of seeds varies dramatically; some have virtually
none, while those seeds (and nuts) known for high oil content
can contain more than 65% lipids by mass.5 The fatty acid
constituents of triglycerides vary as well. Flax seeds contain a
high proportion of the triply unsaturated α-linolenic acid,6
whereas coconut oil is composed of predominantly saturated
acid (lauric, myristic, and palmitic acid) components.7 The
individual molecules of triglycerides (Figure 1) are typically
mixtures of one, two, or three different esterified fatty acids.8
© XXXX American Chemical Society and
Division of Chemical Education, Inc.
Figure 1. Variability in triglyceride structure.
Isolation of these oils from the plant components typically
involves pressing and/or solvent extraction.9,10
Ever since Rudolph Diesel investigated fueling his engine
with peanut oil,11 the use of vegetable oils to power combustion
engines has been of interest. While the energy to fuel engines
certainly exists with heavier-weight plant- and animal-derived
oils, other factors, especially viscosity,12,13 have limited their use
in modern engines. Biodiesel, a term that encompasses the
short chain (predominantly methyl and ethyl) ester of long
chain fatty acids, overcomes this initial issue. The characteristics
of these ester mixtures, such as degree of overall unsaturation,
quality of combustion, caloric value, viscosity, and residual
glycerol esters,12,13 identify their suitability for utility as fuel.
While not the focus of this study, the transesterification of
triglycerides into biodiesel has been thoroughly investigated.14
Base-catalyzed methods include the use of NaOH, KOH, and
other metal oxides,15−18 although care must be taken to ensure
that any free fatty acid in the oil does not neutralize the basic
catalyst (Scheme I). Acid-mediated transesterification19 gen-
erally utilizes H2SO4 or HCl. Conversion of the triglycerides
may also be accomplished with a variety of other salts20−23 or
substrate-supported enzymes.24−27 Critical to these processes is
the need for water-free triglycerides; any water in the
transesterification reaction may result in the production of
free fatty acids, thus complicating the separation of product
layers and/or contamination of the final product.
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Journal of Chemical Education
Scheme I. KOH/Methanol Conversion of Triglyceride into
Biodiesel
The choice of substrate for the production of biodiesel on a
commodity scale is fraught with technical as well as ethical
issues. The use of edible oils (e.g., corn, soybean, peanut,
sunflower) in their raw state would likely cause a demand-
driven market and subsequent rise in price of these critically
important foodstuffs. The utilization of these oils after primary
usage, especially by restaurant and industrial fryers, requires
extensive removal of free fatty acid contaminants, a result of
prolonged heating.27 Animal fat also contains a large amount of
these impurities and makes subsequent processing problematic.
Some authors have claimed the future of economically viable
biodiesel production relies on the use of a nonedible vegetable
oil (e.g., Jatropha or Pongamia) with low free fatty acid
content.13 It is through the isolation of various common oils
and subsequent conversion into biodiesel that students have a
more complete understanding of the process.
Experiments in this Journal have certainly provided readers
with procedures to convert cooking oil into biodiesel16−19
wherein the substrates are both used and virgin vegetable oils. It
is a concern that students may not identify a central theme of
this experiment; the energy stored by the plants for its
developing offspring may be utilized by humans for other uses.
To date, there is only one experiment in which the triglyceride
starting material is isolated from its natural source.28 While that
work is unique in its perspective, it requires a six-week
inoculation and culturing of an algae broth, beyond the scope of
many undergraduate laboratories. Herein is provided a two-
laboratory session experiment that isolates triglycerides from
seeds, followed by a transesterification reaction to prepare the
biodiesel product. Successful completion of this experiment
allows students to quantify the amount of triglycerides in seeds
and convert it into a methyl ester that could be used as fuel.
They will also understand what observable changes accompany
this transformation (e.g., viscosity, NMR spectroscopy) and
that the original source of energy can be utilized by the plant or
by humans.
■
THE EXPERIMENT
Students work alone or with a single lab partner. This
laboratory exercise can easily be done in two 3- to 4-h lab
sessions. Seeds available are soybeans, safflower, flax, sunflower,
and coffee (used); nut seeds (coconuts, peanuts, almonds,
cashews, and pistachios) can be used, but allergies to nut seeds
by some students could present potential issues with
aerosolized nut powder from grinding. A very moist seed
(soybean) or a seed processed with water (coffee),29 is dried in
an oven prior to grinding.
In week one, students isolate the triglyceride fraction from a
seed. Students grind seeds to roughly an “espresso” particle size
that is free-flowing, taking care not to form a peanut butter
consistency with oilier seeds (e.g., sunflower and flax). The
ground material is suspended in heptanes and heated to reflux
B dx.doi.org/10.1021/ed4008974 | J. Chem. Educ. XXXX, XXX, XXX−XXX
Laboratory Experiment
for 1 h. After it cools, the suspension is filtered and
concentrated to yield the isolated vegetable oils; heptanes are
recovered for reuse. The oil is characterized by mass, 1H NMR
and IR spectroscopy, and relative viscosity.
In week two, students convert the isolated triglycerides into
biodiesel. Students transfer a sample of the isolated oil (1−5 g)
to a test tube, add a solution29 of 40% KOH in methanol (1 mL
per solution per g of oil), and place the test tube at 50−60 °C
in a water bath; within a few minutes the phases separate and
the less dense biodiesel rises to the top. The methyl esters are
washed with saturated NaCl solution until neutral. Physical
characteristics (e.g., mass, 1H NMR and IR spectroscopy, and
relative viscosity) of the biodiesel are obtained for comparison
with those of the isolated triglyceride.
A detailed description of the experiment is in the Supporting
Information.
■ HAZARDS
Heptanes and methanol are flammable, as well as inhalation and
contact hazards. Potassium hydroxide is very caustic; chemical
splash goggles and other forms of PPE (e.g., gloves, aprons)
should be worn at all times. Triglycerides and the analogous
biodiesel are flammable and should be disposed of following
guidelines for organic waste disposal. Contact with CDCl3
should be avoided; it is an eye and skin irritant and hazardous.
■ RESULTS AND DISCUSSION
This experiment has been completed by a total of 44 students
in four sections of a second-semester introductory organic
chemistry course. Consideration of the type of seed to be
processed was important to the overall success of the
experiment. If the material was a very moist seed (soybean)
or was processed with water (coffee),29 it should be dried in an
oven to avoid the transfer of water to the triglyceride extract.
Grinding the substrate to roughly “espresso” particle size is
necessary to increase the surface area and the rate of extraction.
The recovery and reuse of the heptanes is a key exemplification
of a green experiment and an important consideration. The
percent mass recovery of the crude oils from the various seeds
is shown in Table 1.
Table 1. Range of Student Recovery of Triglycerides from
Some Example Seeds
Seed Type Oil Recovered (% mass)
Soybeans 8−23
Safflower 24−34
Flax 25−40
Sunflower 46−50
Coffee (used) 8−17
Conversion of the triglyceride into the corresponding methyl
esters is a relatively simple process. The elevated temperature
causes a lowering of the oil’s viscosity, allowing for better
reagent mixing, and speeds the rate of methanolysis. The
addition of a solution of 40% KOH in methanol29 causes a
rapid transesterification; in general, a 50−75% mass recovery of
the biodiesel product for this step is obtained. 1H NMR, even at
60 MHz, distinguished between some of the different types of
oils (examples of student spectra in the Supporting
Information). The methylenes of the triglyceride core formed
an ABX pattern centered at 4.22 ppm with a geminal coupling
constant of −11.8 Hz and two vicinal coupling constants of 4.4
Journal of Chemical Education
and 5.8 Hz. At this spectrometer frequency, the methine proton
was partially buried beneath the olefin absorbance at 5.3 ppm.
Triglycerides that have a significant amount of unsaturation in
the carbon chain, such as flax oil,6 had a large peak at 5.3 ppm,
whereas in those that contained predominantly saturated fatty
acids, the glycerine methine was easily distinguishable.
Following the conversion to biodiesel, an examination by 1H
NMR spectroscopy clearly showed loss of the signal at 4.22
ppm and a new methyl ester signal at 3.66 ppm. Any signals in
the 5.3 region were solely due to olefinic protons on the carbon
chain. Analysis of the isolated triglyceride by IR spectroscopy
was straightforward (example of a student spectrum in the
Supporting Information); an ester absorbance at 1746 cm−1
and methylene “rock” at 724 cm−1 were hallmarks for this type
of compound. There was little difference in the IR spectra when
examined across the different oil types or after conversion to
biodiesel.
Critical to the utilization of biodiesel is the understanding
that, although the caloric value is similar to that of the original
triglyceride, the relative viscosity is much lower. Using a
disposable glass pipet as a crude viscometer,18,28 students
visualized and quantitated this physical change. Students
measured the time it took for the isolated vegetable oil and
the corresponding methyl esters to flow between two points 3
cm apart on a pipet. At least a 5- to 6-fold decrease in the time
was required for the biodiesel to traverse the two points relative
to the analogous triglyceride.
■
SUMMARY
This student experiment allowed for the isolation of energy-rich
triglycerides from natural sources and the subsequent
conversion into biodiesel over the course of two 3- to 4-h lab
sessions. Students separated an energy-rich plant constituent
and readily converted it into a synthetic fuel. Although it was
used as an experiment in a second-semester introductory
organic chemistry course, it could also be used in a general
chemistry or environmental chemistry curriculum. Its successful
completion by every student group in four laboratory sections
underlies the robustness of the science. Anecdotally, students
reported that this was their favorite lab of the course. On the
basis of post experiment lab reports, over 80% of the students
met the goals of understanding the isolation and character-
ization of triglycerides the first week and over 90% thoroughly
understood the transformation to biodiesel.
■
ASSOCIATED CONTENT
*
S Supporting Information
A detailed student handout of the experiment and instructor
notes with 1H NMR and IR spectra. This material is available
via the Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: sgoldstein@usj.edu.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
I am grateful to Yen Le for initial experiments in this study and
to the CHEM210 − Spring 2013 laboratory sections for
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Laboratory Experiment
information and feedback. Thanks also to Peter Markow for his
editorial comments.
■
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