Advances in Chemical Engi neering and Science , 20 1 1, 1, 96-101
doi:10.4236/aces.2011.13016 Published Online July 2011 (
Copyright © 2011 SciRes. ACES
Chemical Extraction and Property Analyses of Marula Nut
Oil for Biodiesel Production
Jerekias Gandure*, Clever Ketlogetswe
Mechanical Engineering Department, University of Botswana, Gaborone, Botswana
E-mail: {*gandurej, ketloget}
Received May 24, 201 1; revised June 19, 2011; accepted June 25, 2011
To identify and develop alternative and renewable sources of fuel for the transport sector is a present chal-
lenge for engineers and researchers. This work was carried out to assess yield of marula (Sclero carrya/birrea)
nut and chemical properties of crude marula nut oil for biodiesel production in Botswana. Chemical extrac-
tion of marula oil was done to establish actual oil content by use of hexane/iso-propyl alcohol solvent in a
soxhlet set up. Distillation was carried out on a Rotavapor system prior to oil purging using nitrogen gas. The
results indicated that marula nuts have about 58.6% oil content. Characterisation of the extracted crude oil
was carried out to determine its chemical composition using the Waters GCT Premier Time of Flight (TOF)
Mass Spectrometer (MS) coupled to the Agilent 6890 N Gas Chromatography (GC) system. Ethyl oleate
(ethyl ester) was found to be the dominant fatty acid. Trans-Oleic acid was also abundant but could not be
quantified because it was not found in the standard mixture. Crude marula oil was also found to have an ester
content of 93.7%, acid value of 1.4 mg KOH/g, and free fatty acid content of 0.7%. These results are mar-
ginally out of specifications for biodiesel by international standards, implying that crude marula oil is a po-
tential substrate for biodiesel production.
Keywords: Marula Oil, Chemical Extraction, Chemical Properties
1. Introduction
The persuit for biodiesel production in Botswana is mo-
tivated by factors including volatile oil prices, potential
for job creation, fuel security and economic diversifica-
tion. The desire to establish energy self-reliance and to
develop alternatives to finite fossil fuel resources has sti-
mulated development of fuel technologies that are based
on use of renewable agriculture based materials as feed-
stocks. In the case of renewable fuels for compression
ignition (diesel) engines, the majority of efforts to date
have focused on biodiesel, which consists of alkyl esters
of fatty acids. Biodiesel has been shown to give engine
performance that is generally comparable to that of con-
ventional diesel fuel while reducing engine emissions of
particulates, hydrocarbons and carbon monoxide [1-3].
Biodiesel can be produced from any material that con-
tains fatty acids, bonded to other molecules or present as
free fatty acids. As a result various vegetable oils can be
used as feedstocks for biodiesel production. The choice
of feedstock is based on such variables as local availa-
bility, cost, government support and performance as a
fuel [4].
This work seeks to establish yield and properties of
crude marula nut oil of Botswana’s climatic conditions
as a potential substrate for biodiesel production. Marula
tree is indigenous to most parts of Southern Africa. In
Botswana, it is widely distributed all over the country,
but it is concentrated in the north eastern part of the
country. The tree grows in warm and dry climatic condi-
tions, and produ ce oval fruits that turn p ale yellow when
ripe. The fruit consists of a hard woody seed covered by
pulp and juice which makes the fleshy part of the fruit.
The hard seed contains mostly two oil rich nuts (kernel)
which can be eaten as a snack. However, in some parts of
Botswana the nut oil is currently being used to produce
cosmetic ointments by small groups of rural communities.
There is now a worldwide trend to explore wild plants
for oil to augment the already existing sources of o il. The
fact that the marula tree grows in drier parts where
common oil seeds cannot thrive has stirred interest in
marula nut oil as a valuable renewable source of energy.
Copyright © 2011 SciRes. ACES
This has led to the evaluation of marula nut oil as a po-
tential substrate for biodiesel produ ction.
2. Materials and Methods
2.1. Chemical Extraction
Chemical extra ction was carri ed out to estab lish the actual
oil content (yield) of marula nut. The process had five
stages namely seed grinding, solvent extraction, filtration,
distillation and purging. 200 g of marula nuts were ground
into powder using a coffee grinder. The grinder was
loaded with 50 g of m a rul a nuts at a time and run for about
30 seconds. The operation was repeated to increase the
degree of fineness of the powder. The powder was then
used in the solvent extraction process. The solvent was
prepared by mixing 300 ml of hexane and 100 ml of iso-
propyl alcohol in a 500 ml flask. This formula ensures
total extraction of all lipids as hexane extracts all non-
polar lipids and iso-propyl alcohol polar lipids. 3 g of
anti-bumping stones (boiling stones) were added to the
mixture to ensure non-violent boiling of the solvent during
oil extraction. 74.6 g of powdered sample was loaded in a
thimble and placed inside a soxhlet. A soxhlet cover, con-
denser and heating mantle were mounted to complete the
soxhlet solvent extraction set. The solvent was heated to
boiling temperature and maintained in that phase for the
entire extraction process (6 hours). After 7 syphones, the
extracted liquid became clear, indicating that there was no
more oil in the sample. The process was stopped and sol-
vent/oil mixture was allowed to cool for about 3 hours.
Filtration was done to eliminate any possibility of solid
particles in the oil rich solvent. The separation of solvent
from the oil was achieved through distillation under vac-
uum pressure using a Rotavapor (rotary evaporator)
shown in Figure 1.
The heating bath of the rotavapor used distilled water
maintained at 40˚C for the entire sep aration process. The
condenser used water that is slightly above freezing tem-
perature and was maintained at that temperature by ice
blocks. This process shou ld ideally extract all the solven t,
starting with hexane and then iso-propanol (due to the
double bond). To ensure that no trace of solvent remains
in the oil, the oil was purg ed with n itrogen (nitrogen dry-
ing) for approximately 40 minutes. Nitrogen is used for
this purpose because it is inert and does not react with oil
2.2. Oil Characterization
Crude marula oil extract from solvent extraction was
used in its natural state for fatty acid profile analysis. The
oil will be converted into biodiesel by transesterification
process in the second phase of the study, and the fatty
acid profile analysis will be repeated to compare chang es
in properties. The composition of marula nut oil was
analysed using the Waters GCT premier Time of Flight
(TOF) mass spectrometer coupled to the Agilent 68 90 N
gas chromatograph system. The instrument has high sen-
sitivity and fast acquisition rates. In addition, the Na-
tional Institute for Standards and Technology (NIST)
developed Automated Mass Spectral Deconvolution and
Identification System (AMDIS) software package, (che- was used for peak iden-
tification. AMDIS extracts spectra for individual com-
ponents in a GC-MS data file and identifies target com-
pounds by matching these spectra against a reference
library, in this case the NIST library. AMDIS also allows
Figure 1. Rotavapor.
Copyright © 2011 SciRes. ACES
creation of personal libraries where routine analyses of
compounds is encountered.
2.2.1. Gas Chromatogr aph Conditi ons
1 µL of the sample extract was injected into the system
using an auto-injector. The injector temperature was set
at 260˚C in the splitless mode. Helium was used as the
carrier gas at a flow rate of 1ml/min. The separation was
achieved using a 30 meter DB5—MS column. The oven
temperature was kept at the initial 100˚C for 2 minutes,
and then gradually increased from 100˚C to 290˚C at a
rate of 10˚C per minute. The total run time was 35 min-
2.2.2. Mass Sp ectrome ter Conditions
The Mass Spectrometer (MS) conditions that were em-
ployed were a positive polarity of electron ionization
(EI), a source temperature of 180˚C, an emission current
of 359 uA. Other MS conditions including electron en-
ergy and resolution were set by the system auto tune
function. Detection was by the Micro Channel Plate de-
tector (MCP) whose voltage was set at 2700 V. The oil
composition was identified and quantified using the
NIST (2005) mass spectral library using a combination
of the Masslynx acquisition /data analysis software and
the AMDIS by NIST.
2.3. Oil Acidity
Acid value measurement of crude marula oil was done
by titration according to ASTM method D664 [5]. 125
ml of solvent, consisting of 50% isopropyl alcohol and
50% toluene, was prepared in a 600 ml beaker. 5 g of
marula oil was added to the beaker, followed by 2 ml of
phenolphthalein indicator. Beaker contents were titrated
with 0.1 M KOH to the first permanent pink color.
3. Results and Discussions
3.1. Marula Nut Oil Content (Yield)
The purged marula oil was weighed and had a weight of
43.6839 g. This was expressed as a percentage of the
weight of the original sample, 74.6000 g, used for sox-
hlet extraction. This yielded an oil content of 58.558%.
The efficiency of mechanical equipment used for large
scale oil extraction can thefore be benchmarked and im-
proved towards reaching the yield from chemically ex-
traction. Marula nut’s high oil content, coupled with
good chemical properties for biodiesel production, indi-
cates that it deserves consideration for biodiesel feed-
stock in Botswana.
3.2. Characterization of Crude Marula Oil
Tests were conducted in a systematic study to establish
the fatty acid profile of crude marula oil using procedure
and conditions described in Section 2.2. The results for
marula oil composition were compared with that for a
standard sample of vegetable oils prepared by Accu-
Standard. For simplicity, only a single set of sample re-
sults obtained from these experiments are presented and
discussed in this Section. This enables the main findings
of the study to be identified. The fatty acids and esters
detected, which are naturally present in the oil, are pre-
sented in Table 1.
The fatty acid profile of crude marula oil indicates a
good number of fatty acids and esters, half of which were
not found in the standard sample. The observation sug-
gests the uniqueness of this indigenous oil. The fatty acid
that was detected to be most abundant is ethyl oleate
(ethyl ester) with a concentration of 223.5 ppm (by wei-
ght per unit volume). Other compounds had substantial
presence which could improve if concentrated through
processing. Figure 2 represents the mass spectrum of
ethyl oleate.
The spectrum in Figure 2 indicates major peaks of the
compound, together with minor peaks that may be due to
fragmentation of the same compound resulting from la-
bile ester linkages possibly present in the compound, or
presence of another compound that is not fully resolved.
The mass spectrum of ethyl oleate is an intensity/relative
abundance (%) versus mass-to-charge ratio (m/z) plot
representing a chemical analysis of the compound. Gene-
Table 1. Crude marula oil fatty acid profile.
No. Fatty Acid Detected Status (in standard
mixture) Concentration
(ppm w/v) Comments
1 Palmitic acid No - Nil
2 Palmitic acid (methyl ester) Yes 17.4 Nil
3 Palmitic acid (ethyl ester) Yes 13.3 Nil
4 Trans-oleic acid No - Nil
5 Ethyl Oleate (Ethyl ester) Yes 223.5 Nil
6 Oleic acid, 3-hydroxypropyl ester No - Nil
7 Olein, 1-mono No - 2 isomers detected
Copyright © 2011 SciRes. ACES
Figure 2. Ethyl oleate.
rally, the mass spectrum of a sample is a pattern
representing the distribution of ions by mass (mass-to-
charge ratio) in a sample, usually acquired using a mass
spectrometer. Not all mass spectra of a given substance
are the same. Some mass spectrometers break the analyte
molecules into fragments; others observe the intact
molecular masses with little fragmentation. A mass
spectrum can represent many different types of informa-
tion based on the type of mass spectrometer and the spe-
cific experiment applied; but all plots of intensity versus
mass-to-charge are referred to as mass spectra.
Based on the data in Table 1 and the results in Figure 2,
crude marula nut oil composition depicts characteristics
of a good fuel, even before it is transesterified into bio-
diesel. According to Kyamuhangire [6], vegetable oil can
be used either directly in internal combustion engines or
converted into blended biodiesel. In response to the above
observations, the crude marula oil will be tested for per-
formance as a fuel in a variable compression ignition en-
gine in the second phase of the investigation.
3.3. Ester Content of Crude Marula Oil
According to European Standards for Biodiesel, EN
14214, a fuel should have a minimum ester content of
96.5% in order to be used as biodiesel [7]. To establish
the ester content in crude marula oil, the retention times
for the fatty acids listed in Table 1 were detected by use
of Masslynx software tools and were used to identify
compound peaks in the chromatogram. This enabled the
peak areas of the esters in the marula sample to be
summed up and expressed as a percentage of total sum of
peak areas of all compounds in crude marula oil sample.
Table 2 and Figure 3 represent fatty acid retentio n times
and peak areas, and chromatogram respectively.
The data summarised in Table 2 yields an ester con-
tent of 93.7% in crude marula oil. This value could im-
prove if peak areas for trans-oleic acid, palmitic acid and
its esters were considered, but their retention times could
not be matched with their peaks. However, an ester con-
tent of 93.7% for crude marula oil is quite close to the
recommended minimum of 96.5%, implying that trans-
esterifying the oil into biodiesel is likely to improve the
ester content to above the minimum recommended value.
3.4. Acidity of Crude Marula Oil
Three titrations were carried out and the average titration
value was 1.25 mL. The acid value of the oil was deter-
mined using equation 1.
Acid Value, AV = [(56.1 × N)/W] × Titration value (1)
where 56.1 = m olecu lar weight of KOH
N = molarity of the base
W = weight of sample (marula oil) in grams
Titration value = number of mL of KOH that neutral-
ised sample beaker.
The computed acid value of crude marula oil was 1.4
mg KOH/g, implying a percentage free fatty acid con-
Copyright © 2011 SciRes. ACES
Table 2. Fatty acid retention time s and pe ak areas.
No. Fatty Acid Detected Status (in stan-
dard mixture) Concentration
(ppm w/v) Retention Time
(Sec) Peak area
1 Palmitic acid No - 17.50 -
2 Palmitic acid (methyl ester) Yes 17.4 17.13 -
3 Palmitic acid (ethyl ester) Yes 13.3 17.86 -
4 Trans-oleic acid No - - -
5 Ethyl Oleate (Ethyl ester) Yes 223.5 19.40 55 635
6 Oleic acid, 3-hydroxypropyl ester No - 22.1393 7 389
7 Olein, 1-mono No - 25.30 20 414
Sum of Peak Areas 83 438
Total sum of all Peak Areas 89 094
Figure 3. Chromatogram.
tent of 0.7% according to equation 2. ASTM D6751
stipulates that the total acid number should be 0.50 mg
KOH/g max.
Percentage Free Fatty Acid (% FFA) = 0.5 × Acid Value
Generally, when the FFA level is less than 1%, and
certainly if it is less than 0.5%, the FFAs can be ignored.
A range between 2% - 3% of FFA may be the limit if
traces of water are present. The free fatty acid content of
0.7% obtained in this work for crude marula oil com-
pares very closely with specification for processed bio-
diesel. This indicates high potential of crude marula oil
for use as feedstock for production of biodiesel.
4. Conclusions
A study to establish the actual oil content (yield) of ma-
rula nuts was carried out chemically using soxhlet sol-
vent extraction. The study also examined chemical com-
position and some properties of crude marula oil to as-
sess suitability for use as feed stock for biodiesel produc-
From the results of the study, it can be concluded that.
1) The oil content of marula nut is about 59%. This is
a relatively high yield desirable for a potential feedstock
for biodiesel production.
2) Crude marula nut oil has chemical properties that
can enable it to function as biodiesel in IC diesel engines.
This implies that transesterifying crude marula nut oil
under standard conditions has potential to produce bio-
diesel of international quality standard.
3) Trans-Oleic acid is the major fatty acid in crude
marula nut oil. The second most abundan t free fatty acid
detected is Ethyl Oleate which is an ethyl ester. The ester
content of crude marula oil is about 94%. Other free fatty
acids detected include Oleic acid, 3-hydroxypropyl ester,
Palmitic acid (methyl ester), Palmitic acid (ethyl ester),
suggesting that crude marula nut oil has strong charac-
teristics required for biodiesel fuel.
4) The acid value of crude marula oil is about 1.4 mg
KOH/g, implying a free fatty acid (FFA) content of
about 0.7%. This suggests that crude marula nut oil
makes a good feedst ock for production of bio di e se l .
5. Acknowledgements
We acknowledge support of the University of Botswana,
and the Ministry of Wildlife, Tourism and Environment
who granted a research permit for this work.
6. References
[1] M. S. Graboski and R. L. McCormick, “Combustion of
Fat and Vegetable Oil Derived Fuels in Diesel Engines,”
Progress in Energy and Combustion Science, Vol. 24,
Copyright © 2011 SciRes. ACES
1998, pp. 125-164.
[2] P. M. Adriano, C. T. Richard, M. Luciano, C. Jonas and F.
A. Dejanira, “Biodegradability of Diesel and Biodiesel
Blends,” African Journal of Biotechnology, Vol. 7, No. 9,
2008, pp. 1323-1328.
[3] C. Shu-Mei, H. Yuh-Jeen, C. Shunn-Cheng and Y.
Hsi-Hsien, “Effects of Biodiesel Blending on Particulate
and Polycyclic Aromatic Hydrocarbon Emissions in
Nano/Ul-trafine/Fine/Coarse Ranges from Diesel Engine,”
Aerosol and Air Quality Research, Vol. 9, No. 1, 2009, pp.
[4] M. J. Haas, A. J. Mc Aloon, W. C. Yee, T. A. Foglia, “A
Process Model to Estimate Biodiesel Production Costs,”
Bioresource Technology, Vol. 97, 2006, pp. 671-678.
[5] J. D. Mac Farlane, “Determining Total Acid in Biodiese,”
[6] W. Kyamuhangire, “Perspective of Bioenergy and Jatro-
pha in Uganda,” International Consultation on Pro-Poor
Jatropha Development, Casa Son Bernadu, via Lawren-
tina, Rome, Vol. 289, 2008.
[7] Committee for Standardization, “European Standards for
Biodiesel,” 2010.