Fort Hays State University

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Master's Theses Graduate School

Spring 2017

Soxhlet Extraction Of Avocado Endocarp and

Trituration Of Avocado Mesocarp For Biodiesel
Production.
Ifeoma Oraemesi
Fort Hays State University, [email protected]

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Oraemesi, Ifeoma, "Soxhlet Extraction Of Avocado Endocarp and Trituration Of Avocado Mesocarp For Biodiesel Production."
(2017). Master's Theses. 13.
https://scholars.fhsu.edu/theses/13

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 SOXHLET EXTRACTION OF AVOCADO ENDOCARP

AND TRITURATION OF AVOCADO MESOCARP

FOR BIODIESEL PRODUCTION.

being

A Thesis Presented to the Graduate Faculty

of the Fort Hays State University in

Partial Fulfillment of the Requirements for

the Degree of Master of Science

by

Oraemesi, Ifeoma Stella

B.S., Anambra State University

Date________________ Approved _________________

Major Professor

Approved ___________________
Chair, Graduate Council
 This thesis for

The Master of Science Degree

by

Ifeoma Stella Oraemesi

has been approved by

____________________________
Chair, Supervisory Committee

_____________________________
Supervisory Committee

_____________________________
Supervisory Committee

_____________________________
Supervisory Committee

_____________________________
Supervisory Committee

__________________________________
Chair, Department of Biological Sciences
 ABSTRACT

Finding alternative sources of renewable energy is on the rise globally. Renewable

sources of energy are advantageous because they are biodegradable, less toxic, and

combust efficiently. More importantly, raw materials for these sources can be

replenished. One alternative source of energy is biodiesel. Biodiesel is a fuel which

consists of mono-alkyl esters of long-chained fatty acids obtained from vegetable oil or

animal fats. They serve as efficient fuels to run diesel engines. Biodiesel is produced via

transesterification of oils wherein glycerine is a by-product. Avocado (Persea americana)

is a fleshy fruit with high lipid content, mostly monounsaturated fats, which amounts to

70% of its lipid content. These fruits serve as viable sources of biodiesel. In this research,

I used the soxhlet apparatus to extract oil from the stony endocarp and

trituration/geometric dilution to extract oil from fleshy mesocarp to produce biodiesel.

The solvent used in both methods was hexane. About 0.48 ml of oil per g tissue was

obtained from the avocado mesocarp via trituration extraction technique compared to

0.025 ml of oil per g tissue from avocado endocarp via soxhlet extraction. Oils extracted

were analysed using GC-MS and were composed of fatty acids like oleic acid,

palmitoleic acid, stearic acid, arachidonic acid, and myristic acid. These fatty acids were

transesterified to investigate potential for biodiesel production. Avocado’s high lipid

content can be explored in the area of renewable energy. The mixture of saturated and

unsaturated fatty acids can be advantageous in its use as biodiesel.

i
 ACKNOWLEDGEMENTS

This thesis was made possible through the help, advice and support of many

individuals. A very special thanks to Dr. Brian Maricle, my advisor, for helping me

through the toughest times, times when I got confused because of the difference in the

educational system I am used to, you were patient enough to help me understand and you

have been a source of encouragement and inspiration. A very special thanks to Dr. Arvin

Cruz, my co-advisor who worked tirelessly on this research and has been of great help.

Thanks also to the members of my graduate committee, Dr. Eric Gillock, Dr. Stephen

Donnelly, and Dr. Richard Packauskas, for reviewing my thesis and making

recommendations along the way.

I am immensely grateful to the Department of Biological Sciences and Fleharty

fellowship for providing financial support. Also to Kansas IDeA Network for Biomedical

Research Excellence (K-INBRE) P20GM103418 for the research grant which was used

to make this research possible. I wish to give special thanks to the Chemistry Department

for helping me in the areas involving chemistry during this research.

My special thanks go to the faculty and graduate students of the Department of

Biological Sciences for being supportive throughout every stage of my completing this

degree. I am thankful to Mr. Mehran Shahidi, Director of Intercultural Integration for

being attentive and encouraging me in my lowest times. I am grateful to the members of

Liberty Foursquare Church and Foursquare Gospel Church Mile 12, Nigeria for their

prayers, love and care.

ii
 And finally, I wish to thank my parents, Mr. and Mrs. Chinwuba, and my sisters,

Laura and Vivian Oraemesi, for always being there for me. I appreciate your love,

support, and prayers. I would not be where I am today without your help. From a

distance, your words find their way to me.

iii
 TABLE OF CONTENTS

Page

ABSTRACT...............................................................................................................i

ACKNOWLEDGEMENTS .......................................................................................ii

TABLE OF CONTENTS ...........................................................................................iv

LIST OF TABLES .....................................................................................................vi

LIST OF FIGURES ...................................................................................................viii

PREFACE ..................................................................................................................x

INTRODUCTION .....................................................................................................1

MATERIALS AND METHODS ...............................................................................9

Sample preparation ......................................................................................9

Soxhlet extraction of endocarp ....................................................................9

Trituration of mesocarp ...............................................................................10

Water degumming .......................................................................................11

Transesterification .......................................................................................12

GC-MS Analysis on Transesterified Oil .....................................................12

RESULTS ..................................................................................................................14

Effect of Drying ...........................................................................................14

GC-MS Analysis of Crude Substrate ..........................................................16

iv
DISCUSSION ............................................................................................................20

Oil Extraction Techniques ...........................................................................20

Avocado Oil.................................................................................................22

Conclusions and Future Directions .............................................................24

REFERENCES ..........................................................................................................27

TABLES ....................................................................................................................34

FIGURES ...................................................................................................................47

v
 LIST OF TABLES

Table Page .

1. The quantities of avocado endocarp subjected to Soxhlet extraction and their

products ..........................................................................................................35

2. (a) First-fold trituration and (b) Second-fold trituration of oil data from avocado

mesocarp ........................................................................................................36

3. Fatty acid composition of oil analyzed from avocado mesocarp tissue and

avocado endocarp following trituration and Soxhlet extractions. Based on weight

(g) of endocarp and mesocarp from 13 replicate fruits that were 136.90g and

438.69g...........................................................................................................37

4. Retention times of fatty acid methyl ester components of oil analyzed from

avocado mesocarp tissue and avocado endocarp following trituration and Soxhlet

extractions. Based on weight (g) of endocarp and mesocarp from 13 replicate

fruits that were 136.90g and 438.69g.............................................................38

5. Mass to charge ratio (m/z) of fatty acid methyl ester components of oil analyzed

from avocado mesocarp tissue and avocado endocarp (a, b, c, d, e) following

trituration and Soxhlet extractions. Based on weight (g) of endocarp and mesocarp

from 13 replicate fruits that were 136.90g and

438.69g...........................................................................................................39

6. Additional components in oil analyzed from avocado mesocarp tissue following

trituration, endocarp tissue following Soxhlet extraction. Note: retention times of

the minor components are not listed due to their relative low

vi
 concentrations. ..............................................................................................44

7. Retention times of other fatty acid methyl ester components of oil analyzed from

avocado mesocarp tissue and avocado endocarp following trituration and Soxhlet

extractions. ....................................................................................................45

8. Retention times of some other fatty acid methyl ester components of oil analyzed

from avocado mesocarp tissue. ......................................................................46

vii
 LIST OF FIGURES

Figure Page

1. A typical Soxhlet extraction set-up ................................................................5

2. Transesterification reaction to produce biodiesel ..........................................7

3. The overall process of isolation, degumming, purification and

analysis of biodiesel .......................................................................................8

4. Moisture content of endocarp and mesocarp tissues of avocado, measured by

drying tissues in the oven at 45oC ..................................................................15

5. Volume of crude oil extracted from the endocarp and mesocarp tissues of avocado

after the extraction and rotovap process. .......................................................16

6. Structures of the fatty acids found in the avocado endocarp oil. ...................19

7. Structures of fatty acids found in the avocado mesocarp oil. ........................20

8. The Soxhlet extraction set up is comprised of a condenser (top), Soxhlet extractor

(middle), an extraction thimble to hold the sample (inside the Soxhlet extractor),

and a 200 ml round bottom flask (bottom) that holds the hexane extraction

solvent. Lubriseal was used to grease the glassware in parts where there is a

conjoining. .....................................................................................................47

9. The rotovap set up is comprised of a condenser (top), a receiving flask (flask on

the left), a round bottom flask that holds the sample (on the right), a heat source, a

vacuum source, a water source, and a regulator of the rotary

Movement ......................................................................................................48

viii
10. The trituration process used in the study, with avocado mesocarp dissolved in

hexane (a) and divided into five portions (b). ................................................49

ix
 PREFACE

This thesis follows the style of Journal of Environmental Science and Health.

x
 INTRODUCTION

Scarcity in fossil fuel sources remains an issue of concern (Shafiee and Topal,

2009). Although more studies are being conducted (Shafiee and Topal, 2009) to address

the issue on how much of these scarce resources are still left, some countries are already

embracing alternative sources of renewable energy (Wustenhage et al., 2007). Such

countries include Canada, Brazil, Turkey, and the United States of America (Apergis and

Payne, 2010). The energy crisis experienced by countries, particularly developing nations,

has heightened the need to turn towards alternative sources. In Nigeria, for example, the

energy crisis is caused by overconsumption of energy resources due to exponential growth

in population. Other factors such as poor infrastructure, lack of knowledge about

alternative sources of energy (Domac et al., 2005), energy waste, poor distribution of

energy, tax hikes, strikes by energy workers, and natural calamities (Ehinomen and

Adeleke, 2012) also contribute to the crisis. The economy of Nigeria is heavily dependent

on oil production and export (Sanusi, 2012). Unfortunately, most people in Nigeria do not

benefit from it. Electricity, fuel, diesel, and kerosene, which ought to be in excess within

the country, are actually scarce commodities (Ehinomen et al., 2012) because of corruption

and misappropriation of funds (Sanusi, 2012). Because the oil produced is non-renewable,

it is possible it will be used up some day (Shafiee and Topal, 2009) and that is the reason

for my study. To alleviate the issue, more studies need to be conducted in order to find

sources of energy that are environment friendly and renewable.

The motivation of this thesis is to explore alternative sources of renewable energy

that can be used worldwide. This includes my desire to help the Nigerian economy. I am

confident my study is beneficial to my fellow Nigerians by improving the standard of living

1
of the masses. If adopted in my country, results from this study will help create more job

opportunities, reduced incidence of oil spillage and other environmental hazards, with less

pollution, as well as improvement in the agricultural sector (Williams et al., 2011).

Environment friendly sources that are renewable tend to be carbon neutral,

biodegradable, less toxic and burns cleanly (Speight, 2014). Such fuel sources include

solar, wind, hydroelectric, geothermal, ocean tides and biomass (Johannson and Burnham,

1993). Demand on the use of biomass-based fuels have increased due to several factors

(Chen et al., 2012). First, they are abundant. Second, they help reduce the amount of waste

generated in landfills. Third, these materials are biodegradable; therefore, environment

friendly (Apergis and Payne, 2010). Also, exploitation of these alternative sources of

energy opens new avenues to create new job opportunities (Wei et al., 2010) to help sustain

human needs. This in turn help improve the economy (Domac et al., 2005).

Biodiesel is a renewable fuel consisting of mono-alkyl esters of long-chain fatty

acids from vegetable oil (Williams et al., 2011) or animal fats (Huber et al., 2006) that have

met the requirements of American Society for Testing and Materials (ASTM) D 6751 (Wu

et al., 2012) for use in diesel engines. Biodiesel also refers to pure fuel before blending

with diesel fuel (Giakoumis et al., 2014). Oil is converted through transesterification

(Anastopoulos et al., 2009) to methyl esters, also known as biodiesel (Baohua et al., 2012),

and glycerine. Biodiesel fuels are less toxic, have lower emission (Dang et al., 2012), less

air pollution (Chen et al., 2011), and are more environmentally friendly compared to fossil

fuels (Baohua et al., 2012).

Avocado (Persea americana Mill.) of the plant family Lauraceae produces a fruit

with high oil content (Mooz et al., 2012). The mesocarp (fleshy part of the fruit) makes up

2
60 to 75% of the total weight of avocado fruit (Costagli and Betti, 2015). Mesocarp is

composed of parenchyma cells that surround uniformly distributed specialized oil

containing idioblast cells (Reddy et al., 2012). The endocarp (stony part of the fruit) makes

up 13% of the total weight of the fruit (Avhad and Marchetti, 2015). Avocado is a tropical

fruit (Ogunwusi and Ibrahim, 2016) that stands out for its high nutritional value (Mooz et

al., 2012). It is also a good source of monounsaturated fatty acids, palmitic acid, and they

have low amounts of polyunsaturated linoleic acid and stearic acid (Ogunwusi and Ibrahim,

2016). Avocado oil has been used for cooking, cosmetics, and treating diseases, but has

not been widely studied as a good source of oil for renewable energy (Knothe, 2013).

This is focused on comparison of the oil extracted from both endocarp and

mesocarp tissues of avocado fruit. The oil from endocarp was extracted using the Soxhlet

extraction technique using hexane as solvent, which has been widely studied (Meyer and

Terry, 2008). The mesocarp underwent trituration with hexane as solvent for the oil

extraction. This trituration technique is mostly used in pharmaceutical industries

(Schachter and Harden, 1997) but has not been used in extraction of oil for energy purposes

(Nakamura et al., 2004 {b}). Most researchers have used Soxhlet extraction on the

mesocarp (Meyer and Terry, 2008; Mooz et al., 2012). My research compared the quantity

and quality of oil from both tissues, the moisture content, and the effectiveness of the

technique used.

Soxhlet extraction is a process used for liquid-solid extractions, especially for

compounds with limited solubility in the solvent (Meyer and Terry, 2008). Because

avocado endocarp has low solubility in the solvent (hexane), the Soxhlet was the most

suitable extraction technique. By contrast, mesocarp tissue was pasty after milling, where

3
using Soxhlet extraction technique would have been inappropriate. In Soxhlet method, the

sample is separated from the solvent based on different volatilities (Reddy et al., 2012).

Specifically, the sample is dried and milled into small particles and placed in a porous

thimble (Chemat et al., 2008). The thimble is placed in an extraction chamber, which is

suspended above a flask containing the solvent, and a condenser is placed on top of the

extraction chamber (Chemat et al., 2008). As the flask gets heated, the solvent evaporates

and moves up into the condenser, where it is converted into a liquid that trickles into the

extraction chamber containing the sample. Eventually, the solvent builds up in the

extraction chamber and completely surrounds the sample. The extraction chamber is

designed so that when the solvent surrounding the sample exceeds a certain level, it

overflows and trickles back down into the boiling flask (Chemat et al., 2008). As the

solvent passes through the sample it extracts the oil and carries them into the flask. The oil

then remains in the flask because of its low volatility. At the end of the extraction process,

which typically lasts a few hours, the flask containing the solvent and lipid is removed, the

solvent is evaporated and the oil is recovered (Chemat et al., 2008).

4
Figure 1. A typical Soxhlet extraction set-up.

Trituration technique is a solid – liquid extraction that involves the purification of

an impure compound by taking advantage of the solubility differences of the compound

and solvent mixture (Schachter and Harden, 1997). This extraction method is mostly used

in pharmaceutical industries (Nakamura et al., 2004a). Hexane was used as the solvent

since the mesocarp that was milled was sparingly soluble in it and impurities were highly

soluble. The mesocarp was suspended in the solvent so that all impurities were exposed to

solvent and had the opportunity to dissolve (Nakamura et al., 2004b). Then the liquid

portion was decanted for further extraction process (Schachter and Harden, 1997).

Trituration seemed appropriate for the mesocarp because of the nature of the mesocarp

before extraction.

5
 The oils obtained from both tissues were transesterified. Transesterification is a

chemical process that involves reaction of one (1) equivalent of triglyceride with three (3)

equivalents of an alcohol. The products of this reaction are one (1) equivalent of glycerol

and three (3) equivalents of the methyl esters (Lin and Lin, 2012). The alkyl chains of the

methyl esters will vary depending on the nature of the triglyceride present in the sample.

This reaction is accompanied by a base catalyst, [B], typically sodium hydroxide, NaOH.

Due to the solubility of glycerol in water, it can be easily removed by extraction. The fatty

acid methyl ester mixture obtained from transesterification is referred to as biodiesel

(Rachimoellah et al., 2009). Figure 2 shows the chemical equation that represents

transesterification.

6
 O

E -E
OCOR1 OH R1 OCH3
[B] +
OCOR2
+ 3 ROH OH O

OCOR3 OH
R2 OCH3

triglyceride alcohol glycerol R3 OCH3

(parent oil)

methyl esters (biodiesel)

Figure 2. Transesterification reaction to produce biodiesel.

Figure 3 summarizes all the steps involved with the isolation of biodiesel from

avocado fruit. The first step involves peeling and destoning of the avocado fruit. Destoning

is the process of separating the endocarp from the rest of the fruit. In this project, only the

mesocarp and endocarp were used to isolate biodiesel. The exocarp was discarded due to

its known low lipid content. Drying process is important in removing water contents of the

avocado. Lipids are impervious to water; therefore, any water in the sample will hinder the

isolation of triglycerides, which are the precursors needed to generate biodiesel.

7
Figure 3. The overall process of isolation, degumming, purification and analysis of

biodiesel.

Biodiesel consists of a mixture of methyl esters produced from transesterification

of triglycerides from the fruit sample (Figure 2). Triglycerides can be taken out of the

sample using various methods such as extraction with an organic solvent, supercritical fluid

extraction using CO2 and cold-press methods. In this project, the author uses Soxhlet

extraction for the isolation of triglycerides present in the endocarp. Due to the pasty

consistency of the mesocarp of the avocado, traditional methods of extraction may not be

feasible. Here, the author used trituration methods as a novel method of extracting the

triglyceride content of the mesocarp.

8
 Degumming is the process of removing water soluble impurities from a crude

sample of oily triglycerides. Impurities include pigments, metal complexes, peroxides,

proteins and other water soluble vitamins and minerals (Patel et al., 2016). After

degumming, the refined oil (triglycerides) was ready for transesterification to produce

biodiesel (Figure 2). Various methods of chemical analysis can be employed to analyze

biodiesel content, such as 1H-NMR (Proton Nuclear Magnetic Resonance) spectroscopy

and gas chromatograph coupled to mass spectrometry (GC-MS). In this research, GC-MS

was employed due to its high sensitivity and selectivity, low-cost and relative ease of

operation.

The aim of this study was to identify the difference in the oil content and moisture

content of oil from the mesocarp and endocarp tissues of avocado. Another objective was

to make a decision on which tissue produces suitable oil for biodiesel production. The

hypothesis tested was that the oil in both tissues would differ in lipid content and moisture

content. The mesocarp was hypothesized to have significantly more oil and moisture than

the endocarp, but the endocarp was hypothesized to have oil of better quality for production

of biodiesel.

9
 MATERIALS AND METHODS

Sample preparation

Thirteen avocado fruits were bought from a local grocery store (Dillon’s, Kroger

Company) with a total mass of 2,180.22 g. The avocados were peeled (the exocarp was

removed), followed by a separation of the endocarp (stony portion) from the mesocarp

(fleshy portion) (Costalgi and Betti, 2015). Endocarps were cut into smaller sizes ranging

from 0.60 g to 3.41 g to aid drying. The cut endocarp and mesocarp were portioned into

aluminum foil weighing boats for drying.

Endocarp and mesocarp were dried initially at 25°C for two weeks, after which the

temperature was increased to 45°C for one week (Reddy et al., 2012). Dried endocarp and

mesocarp were milled (Reddy et al., 2012) in a Wiley mill (model 3383-L10; Thomas

Scientific; Swedesboro, NJ, USA), and then stored in a refrigerator until extraction (Reddy

et al., 2012). Total weight of endocarp after milling was 136.90 g, and total weight of

mesocarp was 438.69 g.

Soxhlet extraction of endocarp

Extraction of oil from endocarp tissue was carried out using the Soxhlet extractor

(Fig. 1) (Reddy et al., 2012). Milled endocarp tissue was divided into four batches and

subjected to the Soxhlet extraction process (Table 1). These batches had the endocarp

sample in the thimble ranging from 31.00 to 37.00 g and extraction solvent (hexane)

10
ranging from 100.0 to 150.0 ml. The extraction was carried out at 65°C for 72 hours, with

constant running water throughout the process. The extract obtained from each batch

ranged from 52.0 to 110.0 ml. Each batch was left to undergo Soxhlet extraction for 72

hours. After 72 hours, the extraction was stopped, and the extract was transferred to an

Erlenmeyer flask sealed with parafilm.

To remove the sediments still present, the extract was vacuum filtered with a

Buchner funnel and filter paper that was wet with hexane. The filtrate was transferred to a

500 ml round bottom flask for rotovap using Rotavapor – R, Büchi. In rotovap, the oil was

separated from the solvent (Chen et al., 2012). The solvent evaporates but is not lost. It is

instead collected in the receiving flask (Fig. 9). The round bottom flask is half immersed

in a bath containing ethylene glycol and the rotovap is set to approximately 70 rpm.

Evaporation was done under reduced pressure, and heat was applied at 45oC. Filtrate was

rotovapped until the solvent distilled into the receiving flask, typically requiring 1 hour.

Trituration of mesocarp

Dried and milled mesocarp tissue was divided into small portions and hexane was

used as the solvent (Bora et al., 2001). The mesocarp was dissolved in hexane and left to

stand for 48 hours at room temperature while sealed with parafilm (Fig. 10). After 48 hours,

a heterogeneous mixture was obtained. A pipette was used to carefully transfer the liquid

hexane (with oil extract) without disturbing the mixture. After the first trituration on all

five beakers, a second trituration was carried out on the same tissue. The beakers had the

mesocarp samples unequally distributed among them. The mesocarp in the beakers were

11
dissolved in hexane ranging from 80.0 to 155.0 ml (Table 2). Extract volumes within the

range 20 to 80 ml were obtained in both trituration processes (Bora et al., 2001).

Crude extracts were subjected to analysis using a Shimadzu GCMS-QP2010SE Gas

Chromatography Mass Spectrometer (GC-MS) (Shimadzu Corp., Kyoto, Japan). The crude

extracts from the endocarp and mesocarp were analyzed using ether and methanol as

solvents that in dissolving the samples. At the start of the analysis, The parameters included

ion source temperature 200°C, interface temperature 280°C, solvent cut time of 3, 5

minutes, column oven temperature 50°C, injection time 270°C, injection mode: split,

number of rinses: 1, gas: helium, pressure 26.7 kPa, total flow 22.7 ml/min, column flow

0.68 ml/min, purge flow 1.5 ml/min, linear velocity 30 cm/sec., retention time 27 min,

thickness 0.25 µm, length 30 m, diameter 0.25 mm, microscan width 0.Oven temperature

increased to 200°C and the pressure increased to 64 kPa at 16 min retention time to the end

of the analysis. The retention time for each analysis was 27 min. The time during the

analysis when peaks were seen for each of these fatty acids is the retention time. This was

recorded for the fatty acids identified.

Water degumming

The soluble impurities in crude avocado oil were identified as gums which consist

of phospholipids and metal complexes, free fatty acids, peroxides with their

breakdown products, and pigments (Marenchino et al., 2006). Gums and metal complexes

were removed by degumming (Aly, 1992) or chemical refining. The mesocarp crude

extract was divided into three portions and degummed with 3.6 to 4 ml of water, stirred,

12
and refluxed for 30 min at a temperature of 75.0 to 85.0°C (Zufarov et al., 2008). The

degummed crude extract was centrifuged at a speed of 3200 rpm for 30 min using Clay

Adams Compact II centrifuge (Becton Dickinson and Company; Sparks, MD). After

centrifugation, the sludge was separated from the centrifugate (supernatant) by decantation.

The oil was in the supernatant.

Transesterification

0.50 g of sodium hydroxide pellets were crushed in a mortar. The crushed solid was

transferred to an Erlenmeyer flask containing 14 ml methanol. This was stirred until

sodium hydroxide dissolved. A 60.0 ml of oil was added to the solution and heated between

45.0 to 50.0oC and refluxed for 1 hour. The mixture was allowed to cool and transferred to

separatory funnel where it was left to separate into two distinct layers. The bottom layer

was collected in a beaker. 10 ml of water was added to the top layer and left to stand to

remove unreacted methanol and glycerine that might be present. 3 ml hexane was also

added to aid separation. The mixture was left to stand until distinct layers were seen. The

bottom layer was discarded while the top layer contained biodiesel.

GC-MS Analysis on Transesterified Oil

Transesterified oil from the endocarp and mesocarp were subjected to analysis

using a Shimadzu GCMS-QP2010SE Gas Chromatography Mass Spectrometer (GC-MS).

The parameters included ion source temperature 200.0°C, interface temperature 270.0°C,

13
solvent cut time of 3, 5, 10 minutes, threshold 300, column oven temperature 100.0°C,

injection time 270.0°C, injection mode: split, number of rinses: 1, gas: helium, pressure

48.7 kPa, total flow 28.1 ml/min, column flow 0.74 ml/min, purge flow 1.5 ml/min, linear

velocity 32 cm/sec., retention time 60 min, thickness 0.25 µm, length 30 m, diameter 0.25

mm, microscan width 0.

14
 RESULTS

Effect of Drying

Mass of the endocarp and mesocarp were reduced after drying at 25.0°C for two

weeks and 45.0°C for one week (P < 0.001). Avocado mesocarp replicates that were dried

had masses ranging from 85.00 to 162.00 g with an average mass of 148.4 g prior to drying.

Mesocarp tissue had an average moisture content of 75.1% (Fig. 4). Mass of the mesocarp

replicates were significantly reduced after drying and had an average mass of 36.2 g.

Avocado endocarp replicates had masses ranging from 73 to 140 g with an average of 106.9

g before drying. Endocarp tissue had an average moisture content of 37.8%. After drying,

there was an average mass of 60.6 g. Mesocarp tissue had significantly higher water content

compared to endocarp (T-test, P < 0.001; Fig. 4).

100

-
)
%
0
80
t
n
te 60
n
0
c
e
r 40
u
t
s
0 20
E
0
endocarp mesocarp
avocado tissues

Figure 4. Moisture content of endocarp and mesocarp tissues of avocado, measured by

drying tissues in the oven at 45.0oC.

15
 Oil extracted from mesocarp tissue produced a total of 225.0 ml of oil from 13

fruits, significantly more than the endocarp, which produced a total of 3.0 ml of oil from

the same 13 fruits (T-test, P < 0.001). Different techniques were used for different tissues

of avocado (mesocarp and endocarp), where these represent the crude extract from both

tissues (Fig. 5). On a per-mass basis, mesocarp produced 0.478 ml oil/g tissue whereas

endocarp produced 0.0248 ml oil/g tissue (Fig. 5).

250
L) a.
(mL)
L
E 200
e
E 150
u
0
> 100
oil
taa 50
to0
0
e
u
0.5 b.
(/)

tis
(/)
0.4
g
r
e 0.3
p

0 0.2
L
E 0.1
0.0
endocarp mesocarp
avocado tissues

Figure 5. Volume of crude oil extracted from the endocarp and mesocarp tissues of avocado

after the extraction and rotovap process.

16
GC-MS Analysis of crude substrate

For the oil analysed from the mesocarp tissue, fatty acids like Δ9-oleic acid, Δ9-

palmitoleic acid, Δ9,12-linoleic acid, Δ5,8,11,14-arachidonic acid, myristic acid, Δ8,11,14-

linolenic acid, Δ9,12,15- linolenic acid, palmitic acid, and stearic acid were identified (Table

3). Δ9,12,15- linolenic acid had the highest concentration. Myristic acid and Δ8,11,14- linolenic

acid were present in amounts greater than the other fatty acids identified. For the oil

analyzed from the endocarp tissue, fatty acids like Δ9-palmitoleic acid, Δ9,12-linoleic acid,

Δ5,8,11,14-arachidonic acid, myristic acid, Δ8,11,14- linolenic acid, Δ9,12,15- linolenic acid,

palmitic acid and stearic acid were identified (Table 3). Endocarp had lauric acid, Δ6-oleic

acid, Δ5,8,11,14-arachidonic acid, Δ9,12,15- linolenic acid, Δ9,12- linoleic acid, and Δ9-oleic acid

(Table 3). Arachidonic acid had the highest concentration. Δ9,12- linoleic acid and Δ9-oleic

acid were present in greater amounts when compared to the remaining fatty acids

identified. Structures of the fatty acids identified in endocarp can be seen in figure 6. Both

the endocarp and mesocarp tissues of avocado had oleic acid, Δ9,12,15- linolenic acid and

arachidonic acid present, but in varying amounts. Mesocarp had 26.8% saturated fatty acids

and 73.4% unsaturated fatty acids. Endocarp had 9% saturated fatty acids and 91%

unsaturated fatty acids. The fatty acid methyl esters from the mesocarp tissue included Δ9-

oleic acid methyl ester, Δ9-palmitoleic acid methyl ester, Δ9,12-linoleic acid methyl ester,

Δ5,8,11,14-arachidonic acid methyl ester, myristic acid methyl ester, Δ8,11,14- linolenic acid

methyl ester, Δ9,12,15- linolenic acid methyl ester, palmitic acid methyl ester, and stearic

acid methyl ester were identified (Table 3). Fatty acid methyl esters from the endocarp had

lauric acid methyl ester, Δ6-oleic acid methyl ester, Δ5,8,11,14-arachidonic acid methyl ester,

Δ9,12,15- linolenic acid methyl ester, Δ9,12- linoleic acid methyl ester, and Δ9-oleic acid

17
methyl ester (Table 3). These fatty acid methyl esters had retention times ranging from 4.5

to 46.7 minutes. The mass to charge ratio (m/z) of these fatty acid methyl esters are found

in Table 5.

Numerous other compounds were present in the oil, including ascorbic acid,

ethylisoallocholate, 7- hexadecadienol, cyclopropaneoctanoic acid, and others (Table 6).

Among these compounds are other fatty acids that are not common esters (Zhao, 2012),

like saturated branched chain fatty acid methyl esters, monoenoic, dienoic and trienoic fatty

acid methyl esters, halogenated fatty acids, methyl esters, and others (Christie, 2017).

These can be converted to methyl esters during the transesterification process. The methyl

esters that were identified included hexanoic acid methyl esters, nonanoic acid methyl

esters, hexanedioic acid methyl esters, and others (Table 7), their retention times were

recorded (Table 7).

18
 O

HO

∆9 – oleic acid

O
HO

∆9,12 – linoleic acid

O

HO

∆5,8,11,14 – arachidonic acid

O

HO

∆6 – oleic acid
O

HO

∆9,12,15 – linolenic acid

O

HO

lauric acid

Figure 6. Structures of the fatty acids found in the avocado endocarp oil.

19
 O

HO

myristic acid

HO

∆9 – palmitoleic acid

HO

∆9 – oleic acid

HO

∆9,12 – linoleic acid

O

HO

∆9,12,15 – linolenic acid

O

HO

∆8,11,14 – linolenic acid

HO

stearic acid

20
 O

HO

palmitic acid
O
HO

∆9,12 – linoleic acid

Figure 7. Structures of fatty acids found in the avocado mesocarp oil.

21
 DISCUSSION

Oil was extracted from avocado endocarp and mesocarp tissues. Soxhlet extraction

was done on the avocado endocarp whereas trituration extraction was done on the avocado

mesocarp. Significantly more oil was extracted from mesocarp than from endocarp. Oil

from both tissues was degummed (Halder et al., 2009) and transesterified (Joshi and Pegg,

2007). At the end of transesterification, GC-MS was used in the analysis of the oil to

determine the fatty acid methyl esters present. A mixture of saturated and unsaturated fatty

acids made up the oil that was transesterified. The oil from avocado endocarp is better

suited because of its low moisture content, and better oxidative stability. The endocarp is

usually thrown away, so using it for a source of oil would be cost saving.

Oil Extraction Techniques

The techniques used for extraction of oil from avocado mesocarp and endocarp

tissues were suitable based on the state of the tissues after they were milled (Qin and Zhong,

2016). Endocarp was a coarse solid. The Soxhlet extraction technique was best for the

endocarp because oil can be extracted without direct contact of the sample and solvent

(Laurens et al., 2012). In the Soxhlet extraction, the sample is separated from the solvent

based on different volatilities (Laurens et al., 2012). Specifically, the sample is dried and

milled into small particles and placed in a porous thimble (Chemat et al., 2008). The solvent

is in a flask below the Soxhlet extractor and condenser. As the solvent passes through the

sample it extracts the oil and carries them into the flask (Laurens et al., 2012). The oil then

remains in the flask because of their low volatility (Mooz et al., 2012). At the end of the

22
extraction process, which typically lasts a few hours, the flask containing the solvent and

lipid is removed, the solvent is evaporated and the oil is recovered (Chemat et al., 2008).

This method was suitable for endocarp because the sample in the thimble does not dissolve

when subjected to the solvent. Only the needed oil and solvent gets carried to the flask

containing the solvent. The thimble serves as a filter to make sure only extract mixed in

solvent was condensed to the receiving flask (Luque de Castro and Garcia Ayuso, 2000).

Trituration is a new technique for the extraction of oil from avocado mesocarp.

Trituration involves the purification of an impure compound (Shachter and Harden, 1997)

by taking advantage of the solubility differences of the compound and solvent mixture

(Nakamura et al., 2004a). The mesocarp was suspended in the solvent (hexane) so that all

impurities were exposed to solvent and had the opportunity to dissolve (Topare et al.,

2011). Then the liquid portion was decanted for further extraction process. Most previous

studies used the Soxhlet for both the endocarp and mesocarp tissues of avocado (Mooz et

al., 2012). Trituration technique was much more suitable for the mesocarp tissue because

mesocarp tissue was pasty after milling. Using the Soxhlet extraction for mesocarp would

have given a yield that would not be pure oil extract. This is true because milled mesocarp

tissue was sparingly soluble in hexane. Since the reaction would have been run for 72 hours

for Soxhlet extraction, the whole mesocarp would have dissolved in the solvent, especially

since heat was involved.

An additional benefit of both extraction techniques is that the solvent used can be

fully recovered at the end of the rotovap process. The solvent recovered was pure and could

be reused. The Soxhlet extraction took 72 hours in this research. An area for improving

this technique might be shortening the time to find the optimum time it takes to run the

23
Soxhlet extraction fully. The reaction duration in my process ensured that the extraction

was completely done.

Avocado Oil

Both extraction techniques worked well for the tissues. According to Kaiser et al.

(1992), avocado mesocarp has 25% total lipids and avocado endocarp has 1% lipid on a

fresh mass basis. This supports my results, which show significantly more oil extraction

from avocado mesocarp than avocado endocarp.

There has been little work on avocado oil as a source of oil for biodiesel production

(Rachimoellah et al., 2009). Both endocarp and mesocarp tissues are made of a mixture of

saturated and unsaturated fatty acids (Ma and Hanna, 1999). Saturated fatty acids have a

high melting and freezing point. This makes them stable at high temperature, but they have

better oxidative stability Bowen, 2010). Unsaturated fatty acids have lower gel point

(freezing point) and this makes them excellent for cold weather conditions (Gopinath et al.,

2010). Yet, unsaturated fatty acids are prone to oxidation (Gopinath et al., 2010). Based on

their composition, endocarp had 9% saturated fatty acids, and 91% unsaturated fatty acids

while mesocarp had 26.8% saturated fatty acids and 73.4% unsaturated fatty acid (Table

3). Methyl esters identified in the mesocarp tissue included Δ9-oleic acid methyl ester, Δ9-

palmitoleic acid methyl ester, Δ9,12-linoleic acid methyl ester, Δ5,8,11,14-arachidonic acid

methyl ester, myristic acid methyl ester, Δ8,11,14- linolenic acid methyl ester, Δ9,12,15-

linolenic acid methyl ester, palmitic acid methyl ester, and stearic acid methyl ester were

identified. Fatty acid methyl esters identified in the endocarp had lauric acid methyl ester,

24
Δ6-oleic acid methyl ester, Δ5,8,11,14-arachidonic acid methyl ester, Δ9,12,15- linolenic acid

methyl ester, Δ9,12- linoleic acid methyl ester, and Δ9-oleic acid methyl ester. Mesocarp had

26.8% saturated fatty acid methyl esters and 73.4% unsaturated fatty acid methyl esters.

Endocarp had 9% saturated fatty acid methyl esters and 91% unsaturated fatty acid methyl

esters. Having a mixture of both fatty acids might be complementary. Biodiesel fuel with

more unsaturated fatty acids has more density but less viscosity, lower cetane number

(Bamgboye and Hansen, 2008) and heating value (Berasategi et al., 2012), lower thermal

efficiency, lower hydrocarbon and carbon monoxide emission, and maximum gas pressure

(Gopinath et al., 2010). Considering their relative fatty acids, oil from mesocarp tissue

might have a higher chance of oxidative stability when compared with endocarp, and oil

from endocarp might be preferable in cold weather when compared to oil from mesocarp

(Gopinath et al., 2010). Most of the fatty acids in the oil from both tissues were similar to

studies conducted by Knothe (2013) and Rachimoellah et al. (2009) on avocado.

Oil from both tissues had other fatty acids that were different from the common

fatty acids. These included nonanoic acid methyl esters, hexanedioic acid methyl esters,

heptanoic acid methyl ester, and others (Table 7), their retention times were included in

Table 7. Fatty acids on triglycerides are converted to methyl esters through

transesterification (Bowen, 2010). The primary factor of oil conversion to biodiesel is

triglyceride (Bowen, 2010).

25
 CONCLUSIONS AND FUTURE DIRECTIONS

Renewable energy is an area that is becoming desirable because of its numerous

benefits. Avocado as a source of biodiesel should be explored. Although avocado mesocarp

produces lots of oil, the endocarp oil seemed more suitable because the oil does not need

much processing for color or odor. Endocarp tissue has a lot of fatty compounds apart from

the known fatty acids that can be converted to esters. In addition, endocarp tissue is

typically considered as waste because it is not eaten. Mesocarp tissue contains lots of water

and it is not certain that all the water content can be removed, therefore to avoid emulsion

formation during the extraction process, endocarp tissue is preferred. Due to the chance of

withstanding cold weather conditions, oil from endocarp is suitable for biodiesel

production. Since endocarp is considered as waste, it is preferred for conversion to

biodiesel.

An area for future research will be working on how to increase the amount of oil

extracted from endocarp tissue. If there is enough oil extractable from endocarp tissue,

there would be no need to use edible portions of the fruit for oil production for renewable

energy. Another area to explore is using rotten avocados for biodiesel production. When

rotten, fruits cannot be sold, therefore, turning waste into energy would be beneficial. The

oil composition of a rotten avocado and fresh avocado is expected to remain the same (Qin

and Zhong, 2016), so this oil can be put into good use in renewable energy. The exocarp

can also be studied to determine if oil can be gotten from it. It will be another way of

converting waste to fuel.

26
 Avocado as a source of renewable energy could be beneficial to a developing

country like Nigeria, who depend solely on fossil fuels (Oyejide and Adewuyi, 2011).

Energy produced by photosynthesis carried out by plants years ago is responsible for fossil

fuels (Bassham et al., 1950). Unfortunately, modern civilization is using up in a few

centuries the excess of photosynthetic production accumulated over millions of years

(Bassham et al., 1950). When the fossil fuel is used up, the only oil that will be available

in Nigeria will be from contemporary sources like plants or algae (Bassham et al., 1950).

Therefore, the need to look into alternative source of energy is essential. Since 2015,

Nigeria has been in economic recession due to declines in the oil market. This has affected

the economy of the country (Sanusi, 2012). Nigeria and other countries whose economy is

dependent on oil (Ehinomen and Adeleke, 2012) should look into adopting renewable

energy. Countries that have adopted renewable energy have experienced economic growth

(Wei et al., 2010). The need for using biocomponents in our environment for the production

of alternative sources of renewable energy and a cleaner environment is increasing globally

because of its renewable tendencies (Apergis and Payne, 2010). Using avocado as a source

of renewable energy will be beneficial. Renewable energy helps to create job opportunities

(Wei et al., 2010) and can help improve a country’s economy (Domac et al., 2005).

Biodiesel is totally biodegradable, non-toxic, relatively low flammability and has a higher

flash point than fossil diesel, and reduced harmful emissions (Berggmann et al., 2006).

In conclusion, avocado as a source of renewable energy has lots of potential.

Among its many applications, avocado has been used for food, in skin care, and it also has

great potential in oil production. It is necessary to explore further of biodiesel production

from avocado.

27
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34
 Mass of

Volume of Volume Endocarp Mass of

Hexane Extract Wet with Dried

Endocarp Solvent Obtained Hexane Endocarp

Batch (ml) (ml) (g) (g)

1 100.00 87.00 52.23 32.38

2 150.00 52.00 43.80 35.75

3 150.00 110.00 40.72 37.48

4 150.00 88.00 37.54 31.25

Table 1. The quantities of avocado endocarp subjected to Soxhlet extraction and their

products.

35
 Volume of Hexane

Solvent Volume Extract

Beaker Number (ml) Obtained (ml)

1 100.00 30.00

2 95.00 50.00

3 155.00 80.00

4 100.00 70.00

5 115.00 65.00

(a)

Volume of Hexane

Solvent Volume Extract

Beaker Number (ml) Obtained (ml)

1 80.00 20.00

2 100.00 75.00

3 120.00 50.00

4 100.00 50.00

5 100.00 65.00

(b)

Table 2. (a) First-fold trituration and (b) Second-fold trituration of oil data from avocado

mesocarp.

36
 Mesocarp Composition Endocarp Composition

Components (%) Components (%)

Δ9-oleic acid 9.3 lauric acid 9.0

6.1 9.0
9
Δ -palmitoleic acid
Δ6-oleic acid

Δ9,12-linoleic acid 9.7 Δ5,8,11,14-arachidonic acid 46.7

Δ5,8,11,14-arachidonic acid 11.3 Δ9,12,15- linolenic acid 3.3

myristic acid 17.4 Δ9,12- linoleic acid 16.4

15.0 Δ9-oleic acid 15.6

Δ8,11,14- linolenic acid

Δ9,12,15- linolenic acid 22.0 NA NA

palmitic acid 4.5 NA NA

stearic acid 4.9 NA NA

Table 3. Fatty acid composition of oil analyzed from avocado mesocarp tissue and avocado

endocarp following trituration and Soxhlet extractions. Based on weight (g) of endocarp

and mesocarp from 13 replicate fruits that were 136.90g and 438.69g.

37
 Mesocarp Retention Endocarp Retention

Components Times Components Times

(min) (min)

Δ9-oleic acid methyl ester 16.2 stearic acid methyl ester 16.2

Δ9-palmitoleic acid methyl ester 16.3 lauric acid methyl ester 22.2

Δ9,12-linoleic acid methyl ester 18.4 Δ6-oleic acid methyl ester 16.2

Δ5,8,11,14-arachidonic acid methyl 20.0 Δ5,8,11,14-arachidonic acid 20.0

ester methyl ester

myristic acid methyl ester 17.5 Δ9,12,15- linolenic acid methyl 17.9

ester

Δ8,11,14- linolenic acid methyl ester 18.8 Δ9,12- linoleic acid methyl 17.5

ester

Δ9,12,15- linolenic acid methyl ester 17.9 NA NA

palmitic acid methyl ester 22.6 NA NA

stearic acid methyl ester 9.8 NA NA

Table 4. Retention times of fatty acid methyl ester components of oil analyzed from

avocado mesocarp tissue and avocado endocarp following trituration and Soxhlet

extractions. Based on weight (g) of endocarp and mesocarp from 13 replicate fruits that

were 136.90g and 438.69g.

38
 Mesocarp m/z Endocarp m/z

Components Components

397, 380, 399,378,

369, 344, 357, 346,

330, 314, 334, 304,

282, 264, 281, 270,

253, 234, lauric acid methyl ester 245, 236,

9
Δ -oleic acid methyl ester
222, 203, 207, 191,

175, 151, 169, 153,

137, 111, 138, 122,

97, 83, 55, 95, 83, 55,

41 44

398, 377, 282, 264,

357, 341, 246, 235,

311, 282, 222, 193,

264, 225, 180, 151,

9
Δ -palmitoleic acid
206, 187, 137, 111,
methyl ester
167, 152, Δ6-oleic acid methyl ester 97, 83, 55,

138, 111, 41

97, 83, 55,

44

(a)

39
 Mesocarp m/z Endocarp m/z

Components Components

264, 235, 318, 262,

207, 193, 249, 235,

179, 151, 217, 203,

137, 110, 175, 149,

Δ9,12-linoleic acid methyl 95, 81, 67, Δ5,8,11,14-arachidonic acid 135, 121,

ester 41 methyl ester 95, 79, 67,

41

318, 247, 387, 372,

233, 203, 351, 319,

175, 150, 289, 274,

133, 119, 248, 234,

91, 79, 67, Δ9,12,15- linolenic acid methyl 207, 195,

41 ester 165, 151,

Δ5,8,11,14-arachidonic acid 133, 117,

methyl ester 95, 81, 44,

41

(b)

40
 Mesocarp m/z Endocarp m/z

Components Components

399, 389, 280, 256,

358, 346, 222, 196,

327, 315, 167, 151,

281, 269, 137, 110,

253, 227, 95, 81, 67,

213, 199, 41

171, 157,

143, 115,

myristic acid methyl ester 101, 88, Δ9,12- linoleic acid methyl

55, 43 ester

306, 288, 395, 384,

249, 235, Δ9-oleic acid methyl ester 359, 341,

208, 189, 331, 318,

177, 150, 282, 264,

135, 121, 247, 232,

93, 79, 67, 207, 178,

Δ8,11,14- linolenic acid 41 152, 137,

methyl ester 111, 97,

83, 55, 41

(c)

41
Mesocarp m/z m/z
Endocarp
Components
Components

396, 384,

361, 342,

325, 310,

295, 281,

265, 244,

225, 217,

203, 193,

174, 161,

Δ9,12,15- linolenic acid 143, 135, NA NA

methyl ester 117, 110,

94, 81, 69,

44, 40, 35

256, 227,

213, 199,

171, 157,

129, 115,

palmitic acid methyl ester 97, 73, 60, NA NA

43

(d)

42
 Mesocarp m/z m/z
Endocarp
Components Components

400, 386,

364, 341,

307, 281,

269, 253,

238, 209,

189, 149,

135, 123,

stearic acid methyl ester 95, 85, 57, NA NA

43

(e)

Table 5. Mass to charge ratio (m/z) of fatty acid methyl ester components of oil analyzed

from avocado mesocarp tissue and avocado endocarp (a, b, c, d, e) following trituration

and Soxhlet extractions. Measures were based on weight (g) of endocarp and mesocarp

from 13 replicate fruits that were 136.90 g and 438.69 g.

43
 Endocarp Mesocarp

Minor Components Minor Components

Ascorbic acid 11- Hexadecynal

Tetrapentacontane Ethylisoallocholate

11- Hexadecynal Dichloracetic acid

Nonadecatriene 7- Tetradecenal

7-Heptadecene 7- Hexadecenal

9-Tetradecen-1-ol acetate 8, 11, 14 Docosatrienoic acid

Cis-7,10- hexadecadienal Bicyclo (5.2.0) nonane

Oct-5-en-2-ol Di (1-decynyl) mercury

Dodecedienylacetate 2 -cyclohexene-1-carboxaldehyde

1- Octadecyne 1,2 15,16 diepoxyhexadecane

Beta-santalol 2,6 Octadiene-1,8-diol

10-oxocyclodec-2-enecarboxylic acid Cyclopropaneoctanoic acid

7- hexadecadienol NA

14- Octadecenal NA

Cyclotetradecatriene NA

Trichloroacetic acid NA

Table 6. Additional components in oil analyzed from avocado mesocarp tissue following

trituration, endocarp tissue following Soxhlet extraction. Note: retention times of the minor

components are not listed due to their relative low concentrations.

44
 Retention Retention

Mesocarp Times Endocarp Times

Components (min) Components (min)

4.5 cyclopentaneundecanoic acid 3.1

heptanoic acid methyl ester methyl ester

6.8 hexanoic acid methyl ester 4

octanoic acid methyl ester

nonanoic acid methyl ester 11.7 NA NA

nonanedioic acid methyl 10

NA NA
ester

4- decenoic acid methyl ester 20

NA NA

decanoic acid methyl ester 20.7 NA NA

heptanedioic acid methyl 23.6

NA NA
ester

octanedioic acid methyl ester 43.7 NA NA

Table 7. Retention times of other fatty acid methyl ester components of oil analyzed from

avocado mesocarp tissue and avocado endocarp following trituration and Soxhlet

extractions.

45
 Retention

Mesocarp Times

Components (min)

hexanoic acid methyl ester 3.1

hexanedioic acid methyl 13.1

ester

heptenoic acid methyl ester 40

8- nonenoic acid methyl 11.2

ester

cyclopentaneundecanoic acid 20

methyl ester

undecanoic acid methyl ester 21

Table 8. Retention times of some other fatty acid methyl ester components of oil analyzed

from avocado mesocarp tissue.

46
 Condenser

Soxhlet
extractor

Extraction
thimble

Flask holding
solvent

Figure 8. The Soxhlet extraction set up is comprised of a condenser (top), Soxhlet extractor

(middle), an extraction thimble to hold the sample (inside the Soxhlet extractor), and a 200

ml round bottom flask (bottom) that holds the hexane extraction solvent. Lubriseal was

used to grease the glassware in parts where there is a conjoining.

47
 Vacuum
regulator

Condenser

Receiving
flask

Flask holding
sample

Water
source

Heat
source

Figure 9. The rotovap set up is comprised of a condenser (top), a receiving flask (flask on

the left), a round bottom flask that holds the sample (on the right), a heat source, a vacuum

source, a water source, and a regulator of the rotary movement.

48
 (a)

(b)

Figure 10. The trituration process used in the study, with avocado mesocarp dissolved in

hexane (a) and divided into five portions (b).

49