1

PROPERTIES OF OIL METHYL ESTERS PRODUCED

FROM QUASSIA UNDULATA SEEDS BY A TWO-STEP

ESTERIFICATION AND TRANSESTERIFICATION

PROCESS

BY

IKO, WANEN

(PG/M.Sc/10/58167)

DEPARTMENT OF BIOCHEMISTRY

UNIVERSITY OF NIGERIA

NSUKKA

MARCH, 2013

CERTIFICATION

2

IKO, WANEN, a postgraduate student of the Department of Biochemistry with the Reg. No.

PG/M.Sc/10/58167 has satisfactorily completed the requirement of research work, for the degree

of Master of Science (M.Sc) in Industrial Biochemistry and Biotechnology. The work embodied

in this project (dissertation) is original and has not been submitted in part or full for any other

diploma or degree of this or any other university.

DR. S. O. O EZE PROF. L.U. S. EZEANYIKA

(Supervisor) (Head of Department)

EXTERNAL EXAMINER

DEDICATION

3

This dissertation is dedicated to my Parents. All I am, or can be, I owe to God, who has provided

my parents, Mr. Yotam Iko Tsumba and Mrs. Dooshima Anas Tsumba, as pillars of my everyday

need and support.

ACKNOWLEDGEMENT

4

I owe unfeigned gratitude and appreciation to my supervisor Dr. S. O. O. Eze, for the valuable

guidance and advice. He inspired me greatly to work in this project. His willingness to motivate

me contributed tremendously to the successful completion of my project. I am very grateful to

the Head of Department, Prof. L. U. S. Ezeanyika and the entire staff of the Department of

Biochemistry. My heart is delighted to specially appreciate Dr. C. Ubani, my project reader,

whose efforts brought out the beauty of this work.

I am indebted to my parents for their unconditional love, encouragement, advice, support

(financially and otherwise) and immense contributions towards my academic pursuit. They have

shown me a life of love and I am forever grateful.

I give sincere thanks to my course mates and the entire postgraduate students of the Department

of Biochemistry. This study was worthwhile because of the numerous contributions of the above

mentioned. Everyone’s help was timely and pivotal. I am most grateful.

ABSTRACT

5

Plant oils are normally extracted from oil containing seeds that use oil rather than starch as an

energy store. A high oil yield of 42.36% was obtained from Quassia undulate seed through

soxhlet extraction method. A two-step esterification followed by transesterification process were

used to produce the fatty methyl esters (biodiesel) from QUSO. The yield of QUSO biodiesel

was 94.18%. The seed oil and biodiesel from Quassia undulata were analysed for their

physicochemical properties such as saponification value, acid value, iodine value, refractive

index, relative density, kinematic viscosity, flash point, cloud point, and peroxide value,. Higher

kinematic viscosity, cloud point, flash point and relative density values were obtained in

comparison to petroleum diesel. The observed values were in agreement with values obtained for

similar vegetable oils such as jatropha, linseed and shea butter oil. The fatty acid and fatty acid

methyl ester composition of QUSO were analysed using gas chromatography/mass spectrometric

(GC/MS) method. octadecanoic acid (33.44%), 6-octadecenoic acid (46.36%) and hexadecanoic

acid (10.45%) where found to be the major fatty acids in QUSO. The iodine and saponification

values testified to the nature of fatty acids obtained from GC/MS analysis. Octadecenoic acid,

which has been reported as a suitable major component for improving biodiesel fuel properties,

was the most abundant ester. Considering the values of the parameters, the high oil and biodiesel

yields and the non edible nature, QUSO has enormous potential for the industrial production of

biodiesel.

TABLE OF CONTENTS

PAGE

6

Title Page ... .. .. .. .. .. .. .. .. .. i

Certification ... .. .. .. .. .. .. .. .. .. ii

Dedication ... .. .. .. .. .. .. .. .. .. iii

Acknowledgements. .. .. .. .. .. .. .. .. .. iv

Abstract ... .. .. .. .. .. .. .. .. .. v

Table of Contents ... .. .. .. .. .. .. .. .. vi

List of Figures ... .. .. .. .. .. .. .. .. .. xii

List of Tables ... .. .. .. .. .. .. .. .. .. xiii

List of Abbreviations ... .. .. .. .. .. .. .. .. xiv

CHAPTER ONE: INTRODUCTION

1.1 Historical development of biodiesel … … … … … … 2

1.2 Methods of biodiesel production … … … … … … 4

1.2.1 Pyrolysis (Thermal cracking) … … … … … 4

1.2.2 Microemulsification … … … … … … 5

1.2.3 Transesterification … … … … … … 6

1.2.3.1 Esterification … … … … … … 7

1.2.3.2 Side reactions: Saponification and Hydrolysis … … … 8

1.2.3.3 Two-step acid-base transesterification … … … 8

1.3 Variables that influence the process of transesterification … … … 10

1.3.1 Catalyst … … … … … 10

1.3.1.1 Alkalis … … ... … … … 10

1.3.1.1.1 Mechanism of alkali catalyzed reaction … … … … … 11

1.3.1.2 Acid … … … … … … 12

1.3.1.2.1 Mechanism of acid catalyzed reaction … … … … 13

1.3.1.3 Enzyme … … … … … … 13

1.3.2 Effect of molar ratio … … … … … 14

1.3.3 Effect of reaction time and temperature … … … … 15

1.3.4 Effect of moisture and free fatty acids … … … … 15

1.4 Feedstock for biodiesel production … … … … … 16

7

1.4.1 Oilseeds … … … … … … … 17

1.4.2 Microbial and Alga oils … … … … … … 17

1.4.3 Used cooking oils/Waste vegetable oil … … … … 18

1.5 Benefits of biodiesel … … … … … … 18

1.5.1 Emissions of gases and particulate matter… … … … … … 19

1.5.2 Lubricity of biodiesel … … … … … … … 19

1.5.3 Biodegradability and Toxicity of biodiesel … … … … … 20

1.5.4 Safety and Stability of biodiesel … … … … … … 20

1.6 Disadvantages of biodiesel use … … … … … … 20

1.7 Fuel Properties and Quality Standards of Biodiesel … … … 21

1.7.1 Kinematic viscosity … … … … … … 22

1.7.2 Flash point temperature … … … … … … 23

1.7.3 Acid value … … … … … … … 23

1.7.4 Iodine value … … … … … … … 24

1.7.5 Cetane number … … … … … … … 24

1.7.6 Saponification ... … … … … … … 25

1.7.7 Refractive index … … … … … … … 26

1.7.8 Moisture content … … … … … … … 26

1.7.9 Cloud point … … … … … … … 27

1.7.10 Peroxide value … … … … … … … 27

1.8 Biodiesel use (Blending of esters) … … … … … … 28

1.9 Other industrial applications of biodiesel … … … … … 29

1.10 Fuel composition … … … … … … … 30

1.10.1 Biodiesel … … … … … … … 30

1.10.2 Petroleum diesel … … … … … … 32

1.11 Non-edible oils ... … … … … … … 33

1.11.1 Quassia undulata … … … … … .. … 34

1.12 Aim and objectives … … … .. … … … 35

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CHAPTER TWO: MATERIALS AND METHODS

2.1 Materials … … … … … … … … 34

2.1.1 Collection and processing of sample … … … … … 34

2.1.2 Chemicals/Reagents … … … … … … … 34

2.1.3 Instruments/Equipment … … … … … … 35

2.2 Preparation of reagents ... … … … … … … 36

2.2.1 Wij’s reagent … … … … … … … 36

2.2.2 0.5M Alcoholic KOH … … … … … … … 36

2.2.3 0.01M Sodium thiosulphate (N2S2O3) … … … … 36

2.2.4 1M Tetraoxosulphate (iv) (H2SO4) … … … … …. 36

2.2.5 Glacial acetic acid:Chloroform solution (3:2 v/v) … .. … 36

2.2.6 Saturated Potasium iodide (SKI) solution … … … … 36

2.2.7 0.5M Tetraoxosulphate (iv) acid (H2SO4) … … … … … 36

2.2.8 Hot neutralized ethanol … … .. … … … 36

2.2.9 Phenolphthalein indicator … … … … … … 36

2.2.10 0.1M disodium thiosulphate (N2S2O3) … … … … … 37

2.2.11 Saturated starch … … … … … … … 37

2.2.12 Potassium iodide (KI) solution … … … … … 37

2.3 Methods

2.3.1 Extraction of oil from Quassia undulata seeds … … … … 37

2.3.2 Physicochemical characterization of Quassia undulata seed oil … … 37

2.3.2.1 Determination of saponification value … … … … 37

2.3.2.2 Determination of acid value … … … … … 38

2.3.2.3 Determination of iodine value … … …. … 38

2.3.2.4 Determination of refractive index … … … … 39

2.3.2.5 Determination of relative density … … … … 39

2.3.2.6 Determination of kinematic viscosity …. … …. … 39

2.3.2.7 Determination of flash point … … … … … 40

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2.3.2.8 Determination of cloud point … … … … … 40

2.3.2.9 Determination of peroxide value … … … … 40

2.4 Two-step acid-base transesterification process … … … … 41

2.4.1 Acid-catalyzed esterification …. … … … … 42

2.4.2 Base-catalyzed transesterification … … …. …. … 42

2.5 Physicochemical characterization of Quassia undulata seed oil methyl esters 42

2.5.1 Determination of saponification value … … … … 42

2.5.2 Determination of acid value … … … … … … 43

2.5.3 Determination of iodine value … … … … … 43

2.5.4 Determination of relative density … … … … … 44

2.5.5 Determination of kinematic viscosity … … … … … 44

2.5.6 Determination of flash point … … … … … 45

2.5.7 Determination of cloud point … … … … … 45

2.5.8 Determination of refractive index … …. … … … 45

2.5.9 Determination of peroxide value … … … … … 45

2.6 Gas chromatography mass spectrometry (GC MS) analysis of QUSO … 46

CHAPTER THREE: RESULTS

3.0 Results … … … … … …. … … … 47

3.1 The physicochemical characterization of QUSO … … … … 47

3.2 Biodiesel yield from the two-step acid-base catalyzed transesterification … 47

3.3 The physicochemical characterization of QUSO methyl esters … … 48

3.4 Comparison of the properties of QUSO, FAME, Diesel and ASTM standard.. 49

3.5 GC MS analysis of QUSO and FAME … … … … … 50

3.5.1 Chromatogram of QUSO and FAME … … … … 50

3.5.2 GC MS mass spectra of QUSO … … … … … 52

3.5.3 GC MS mass spectra of QUSO FAME … … … … 54

3.6 Schematic representation of mass fragmentation pattern … … … 56

3.6.1 Mass fragmentation of fatty acids in QUSO … … …. … 58

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3.6.2 Mass fragmentation of fatty methyl esters in QUSO … … … 62

CHAPTER FOUR: DISCUSSION

4.1 Conclusion … … … … … … … … 72

REFERENCES … … … … … … … … 74

LIST OF FIGURES

Fig 1. The mechanism of thermal decomposition of triglycerides.. .. … … … 5

Fig 2. Transesterification of triglycerides…. … … … … … 7

Fig 3. Acid-catalyzed esterification…. … … … … … … … 7

Fig 4. (1): Formation of soap from reaction of FFAs with catalyst.. … … … 8

(2): Hydrolysis of biodiesel to yield FFA and methanol… … … … 8

Fig 5. Mechanism of base-catalyzed transesterification…. … … … … … 11

Fig 6. Mechanism of acid-catalyzed transesterification…. … … … … … 13

11

Fig 7. Some applications of biodiesel… … … … … … … … 30

Fig 8. Composition of petroleum diesel… …. … … … … … 33

Fig 9. The leaves, stem, fruit and seeds of Quassia undulata … … …. … … 35

Fig 10. Experimental setup for the preparation of alkyl esters from QUSO … … … 43

Fig 11. Total ion chromatogram of QUSO fatty acids… … … … … … 52

Fig 12. Total ion chromatogram of QUSO FAME… …. …. … …. … 53

Fig 13(a-h): Mass spectra of QUSO fatty acids… … …. … … … … 54

Fig 14(a-j): Mass spectra of QUSO FAME… …. … … … … … 55

Fig 15. Fragmentation by Lafferty rearrangement … … … … … … 58

Fig 16(a-d): Schematics of fragmentation pattern of component fatty acids in QUSO … 59

Fig 17(a-d): Schematics of fragmentation pattern of QUSO methyl ester … … … 63

LIST OF TABLES

Table 1. Examples of biodiesel produced from feedstocks high in FFAs… … … …10

Table 2. Production of biodiesel from various feedstocks… … … … … …16

Table 3. International standards on biodiesel… … … … … … …21

Table 4. FFA composition of QUSO and other vegetable oils… … … …32

Table 5. Non-edible oil seeds… … … … … … … … …34

Table 6. Result of the physicochemical characterization of QUSO… … … … 49

Table 7. Result of the physicochemical characterization of QUSO FAME… … … 50

Table 8. Properties of QUSO FAME in comparison to ASTM biodiesel standards … … 51

12

Table 9. Result of QUSO by GC MS analysis… … … .. … … … 53

Table 10. Result of QUSO FAME by GC MS analysis… … … … … … 54

LIST OF ABBREVIATIONS

AMU Atomic mass units (equals m/z when z= 1)

ASTM American Standard for Testing Materials

AV Acid Value

B100 Pure biodiesel

B20 20% biodiesel and 80% petroleum diesel

CI Compression ignition

CN Cetane Number

CO Carbon Monoxide

CP Cloud Point

DIN Deutsches Institut für Normung

EI Electron impact, ionization in mass spectrometry

13

EN European Norm

FAME Fatty Acid Methyl Ester

FFA Free Fatty Acid

FP Flash Point

FTD Flame thermoionic detector

GC Gas chromatography

HC Hydrocarbons

IV Iodine Value

KOH Potassium Hydroxide

KV Kinematic Viscosity

M/S Mass/Charge ratio

M+ Molecular ion

Max Maximum

Min Minimum

MS Mass spectrometry

MUFA Monounsaturated fatty acids

NOx Nitrogen oxides

PM Particulate Matter

PUFA Polyunsaturated fatty acids

PV Peroxide Value

QUSO Quassia undulata Seed Oil

RD Relative Density

RI Refractive Index

Rt Chromatographic retention time

SFA Saturated fatty acids

Sp.gr Specific gravity

SV Saponification Value

ULSD Ultra low sulpur diesel

V/v Volume/volume proportion

14

VO(s) Vegetable oils

15

CHAPTER ONE

1.0 INTRODUCTION

In the wake of rising prices and unstable supply besides environmental issues, renewed attention

has been paid to shifting away from the use of petroleum based fuels. The world’s energy

demand is commencing its dependency on alternative fuels. Such alternative fuels in use today

consist of bio-alcohols (such as ethanol), hydrogen, biomass, and natural oil/fat-derived fuels.

In search for new energy sources, much attention is focused on biodiesel as a reliable and

renewable resource that is to satisfy a significant part of the energy demands (Fan et al., 2009).

Currently, biodiesel is considered a promising alternative due to its renewability, better gas

emissions, non toxicity and its biodegradability (Akbar et al., 2009; Hossain et al., 2010).

Biodiesel is defined as mono alkyl esters of long chain fatty acids derived from vegetable oils or

animal fats (Knothe et al., 2002b). The term ‘mono alkyl esters’ indicates that biodiesel contains

only one ester linkage in each molecule. Plant oils and animal fats (triglycerides) contain three

ester linkages between fatty acids and glycerol which makes them more viscous.

Amongst the techniques applied to overcome the difficulties encountered with high fuel viscosity

is the chemical conversion (transesterification) of the oil to its corresponding fatty ester which is

the most promising solution (Zhang et al., 2003). Biodiesel production by transesterification can

be catalyzed with alkali, acid or enzyme. The chemically catalyzed processes, including alkali or

acid are more practical compared with enzymatic method (Shalaby and El-Gendy, 2012) because

enzymes are expensive and unable to provide the degree of reaction completion required to meet

the American Standard for Testing and Material (ASTM) fuel specification. More recently, a two

step transesterification process has been used to achieve high purity and yield of biodiesel

product in a short time. This two-step process is mostly employed when the feedstock has high

free fatty acid content.

A number of researchers have worked with feedstocks that have elevated free fatty acid (FFA)

levels (Zhang et al, 2003; Demirbas, 2008). However, in most cases, alkaline catalysts have been

used to remove free fatty acids (FFAs) from the process stream as soap (Canaki and Gerpen,

2001). As the FFA levels increase, this becomes undesirable because of the loss of feedstock as

well as the deleterious effect of soap on glycerol separation.

For higher production yields of biodiesel, a two-step acid-base catalyzed reaction which converts

both the FFA and the triglyceride fractions to biodiesel is used. Typically, two types of reactions

16

are conducted sequentially in this process. The first is the esterification of the FFA to biodiesel,

followed by alkali-catalysed transesterification step to produce biodiesel from the triglycerides.

Generally, it has been observed that transesterification of triglycerides to alkyl esters (biodiesel)

generates a mixture that approximates the properties and performance of petroleum diesel fuel,

which allows it to be used directly as substitute fuel without modifications or as blending agents

for diesel fuel (Fukuda et al., 2001; Bello and Makanju, 2011).

Various vegetable oils are potential feedstocks for the production of fatty methyl esters or

biodiesel but the quality of the fuel is affected by the oil composition (Akbar et al., 2009).

Research results abound in literature on the production of biodiesel through transesterification of

edible and non edible oil from different parts of the world (Abayeh et al., 2007; Berchmans and

Hirata, 2008; Nayak and Patel, 2010). The production of biodiesel from edible vegetable oils has

progressively stressed food uses, price, production and availability of oils (Rashid et al., 2008).

New oil-seed crops that do not compete with traditional food crops are needed to meet existing

energy demands (Xu and Hanna, 2008). In Nigeria, there is an abundance of oil seeds that are

relatively unexplored (Abayeh et al., 2007; Eze, 2012), with no competing food uses.

The quality of biodiesel is monitored by the assessment of fuel properties such as iodine value,

acid value, peroxide value, cloud point, flash point, density and kinematic viscosity. The gas

chromatography mass spectrometry (GC MS) analysis reveals the fatty acid and fatty methyl

ester profiles of oil and biodiesel respectively. The knowledge of the fatty acid profile is

important because of the dependence of biodiesel properties on the structure of fatty acid alkyl

esters (Knothe, 2005).

1.1 Historical development of biodiesel

The idea to use vegetable oil as fuel is more than one hundred years old. In 1891, a German

engineer named Rudolf Diesel developed an internal combustion engine with the intention of

running it on a variety of fuels, including vegetable oil (Shereena and Thangaraj, 2009). Diesel,

conducted an engine test using peanuts at the Worlds Exhibition in Paris in 1900. The idea for

this testing apparently stemmed from the French Government, which was looking for a means of

domestic fuel production in its colonies. Several other European countries took up the idea after

the exhibition which resulted in a number of articles in different European countries. This

experimental research led to the first recorded production of a biodiesel-like fuel, an ethyl ester

17

made from West-African oil by G. Chavanne at the University of Brussels (Knothe, 2001). Early

experiments using vegetable oils in heavy farming equipments also occurred in South Africa in

the 1930’s and 1940’s (Demirbas, 2002).

Vegetable oils were used as diesel fuel from time to time in the 1930’s and 1940’s, usually only

in emergency. The first International Conference on plant and vegetable oils as fuels was held in

Fargo, North Dakota in August 1982. The primary concerns discussed were the cost of fuel, the

effect of vegetable oil fuels on engine performance and durability and fuel preparation

specification and additives. Oil production, oil seed processing and extraction were also

considered in the meeting (Ma and Hanna, 1999).

At the base of all the biodiesel experimentation, which has continued till date, was the realization

that, considering the evolution of the biodiesel engine towards petroleum diesel fuel, straight

vegetable oil was viscous for the fuel injectors to handle properly. A fuel of high viscosity tends

to form larger droplets upon injection, leading to poorer atomization during the spray thus

creating operation problems, such as increased carbon deposits which may enhance the

polymerization reaction, especially for oils of high degree of unsaturation (Alamu et al., 2008;

Freitas et al., 2010). It also leads to poor combustion and increased exhaust smoke and emissions

(Shereena and Thangaraj, 2009).

These problems can be solved by either adapting the engine to the fuel or by adapting the fuel to

the engine. Even though the alterations necessary to the fuel injectors were small and relatively

inexpensive, with tens of thousands of engines already in use, early experimentation focused on

reducing the viscosity of the vegetable oil fuels. Three processes are widely used in the

modification of plant oils and animal fats to produce fuels with approximately the properties and

performance of fossil fuel (Fukuda et al., 2001; Demirbas and Karslioglu, 2007).

1.2 Methods of biodiesel production

Three main processes have been investigated in an attempt to reduce the viscosity of vegetable

oils and animal fats to be utilized as an alternative fuel: pyrolysis, microemulsification and

transesterification.

18

1.3.1 Pyrolysis (Thermal cracking)

The pyrolysis of fats has been investigated for more than 100 years, especially in areas without

petroleum deposits. Pyrolysis is the heating of the biomass in the absence of air at temperatures

of 300 - 500°C with the aid of a catalyst (Singh and Singh, 2010). Under these conditions, the

components which remain are charcoal (solids) and volatiles. The volatiles can after treatment,

be used as biodiesel fuel (Scragg, 2003). The pyrolysed material can be vegetable oils, animal

fats, natural fatty acids and methyl esters of fatty acids. For example: soyabean oil had been

treated with an aluminum oxide catalyst and has yielded oil with properties close to those of

petroleum diesel

Many investigators have studied the pyrolysis of triglycerides with the aim of reducing the

viscosity and increasing the cetane number of vegetable oils to obtain products suitable for diesel

engines (Fukuda et al., 2001). The products contain alkanes, alkenes, alkyldienes, aromatic and

carboxylic acids (Fukuda et al., 2001; Leca et al., 2010) as shown in fig 1. Pyrolysed vegetable

oils generally have acceptable amounts of sulphur, water and sediment, as well as copper

corrosion values. However, they are unacceptable in terms of ash, carbon residues and pour point

(Fukuda et al., 2001: Pinto et al., 2005).

Pyrolysis has as disadvantages the expensive equipment and the need to separate the products by

distillation, which is also energy consuming (Leca et al., 2010). In addition, though the products

are chemically similar to petroleum derived gasoline and diesel fuel, the removal of oxygen

during thermal processing also eliminates any environmental benefits of using oxygenated fuel

(Fukuda et al., 2001).

1

2 3 2

1: Alkyl ester of fatty acid,

2: Alkane,

3: Aromatic compound

19

Fig 1: The mechanism of thermal decomposition of triglycerides. (Singh and Singh, 2010).

1.2.2 Microemulsification

The use of microemulsions with solvents such as methanol, ethanol and buthanol has been

studied as a means of solving the problem of high viscosity of vegetable oils (Fukuda et al.,

2001). Microemulsions are defined as colloidal equilibrium dispersions of optically isotropic

fluid microstructures with dimensions generally in the 1-50mm range formed spontaneously from

two normally immiscible liquids and one or more ionic or non ionic amphiphiles (Singh and

Singh, 2010).

Microemulsions are formed by the dispersion of a mixture of oil, water, surfactants and short-

chain alcohols such as methanol, ethanol and butanol (Scragg, 2003; Knothe, 2005). These fine

dispersions of water in an organic medium, stabilized by amphiphilic molecules, present an

interface through which the hydrophobic molecules are converted (Stamis et al., 1993). They

can improve spray characteristics by explosive vaporization of the low boiling constituents in

miscelles (Ma and Hanna, 1999: Pinto et al., 2005).

The microemulsification of soybean (Singh and Singh, 2010) and sunflower oil (Goering et al.,

1982) were reported to be nearly as good as petroleum diesel. However, irregular injector needle

sticking, heavy carbon deposits, incomplete combustion and increase in viscosity were reported

(Fukuda et al., 2010; Leca et al., 2010).

1.2.3 Transesterification

Tranesterification is the general term used to describe the important class of organic reactions

where an ester is transformed into another ester by interchange of the alkoxy moiety (Rafaat et

al., 2008). In this process, an alcohol reacts with triglycerides (TG) in the presence of catalyst.

The main purpose of transesterification is to reduce the viscosity of oil in order to achieve

properties that are more suitable for its function as a fuel (Hossain et al., 2010), a catalyst is

usually used to improve the reaction rate and yield (Singh and Singh, 2010). Excess alcohol is

used for shifting the equilibrium toward the product because of the reversible nature of the

reaction (Drapcho et al., 2008; Shereena and Thangaraj, 2009).

20

The alkyl esters produced depend on the alcohol used, where methanol and ethanol are mostly

used. Osai (2011), in his comparative studies on the effect of different alcohols on biodiesel yield

achieved high conversions of 90%, 85%, and 81% by reacting fluted pumpkin oil with methanol,

ethanol and propanol respectively. In another study by Berchmans and Hirata (2008), 90%

methyl ester yield was obtained through an alkali catalyzed transesterification process using

Jatropha curcas seed oil.

Transesterification has turned out to be an ideal modification process for biodiesel production

(Demirbas, 2009). The transesterification of triglycerides into methyl or ethyl esters reduces the

molecular weight to one-third that of the triglyceride and also reduces the viscosity by a factor of

about eight and increases the volatility marginally (Singh and Singh, 2010). This produces a

mixture (biodiesel) with suitable fuel properties.

The chemistry of transesterification is mainly centered on triglycerides because oil/fats contain

about 98% triglycerides (Ivanoiu, 2011). Therefore, the stoichiometric relationship requires 3mol

of alcohol per mol of TG (3:1) to form one mole of glycerol and three moles of the respective

fatty acid alkyl esters. In practice, the ratio needs to be higher to drive the equilibrium to a

maximum ester yield (Ma and Hanna, 1999). The transesterification of TG is a sequence of three

reversible reactions, in which the TG is first converted to monoglyceride (MG) and fatty acid.

Then, the diglyceride (DG) is converted to glycerol liberating an additional ester, and finally the

monoglyceride (MG) is converted to glycerol liberating the final fatty acid ester as shown in fig

2.

Fig 2: Transesterification of triglycerides

21

1.2.3.1 Esterification

The esterification process is a reversible reaction where free fatty acids (FFA) are converted to

alkyl esters through acid catalysis using HCL or more commonly H₂SO₄ (Scragg, 2003). When

oils are high in FFAs, the simultaneous esterification and transesterification reaction through the

acid catalysis is advantageous to potentially obtain nearly complete conversion to biodiesel

(Drapcho et al., 2008). The esterification process follows a similar reaction mechanism of acid-

catalysed transesterification as shown in fig 6. The reactants including FFA and alcohol are

catalyzed by acid to create the alkyl ester and water as shown in Fig 3. Berchmans and Hirata,

(2008) used 1% w/w of H₂SO₄ acid during the pretreatment step to reduce the FFA content to

less than 1% and obtain the methyl ester of 90% in the transesterification reaction.

Fig 3: Acid-catalysed esterification.

1.2.3.2 Side reactions: Saponification and Hydrolysis

The transesterification of oils and fats is often accompanied by 2 side reactions when the

feedstocks contain FFA and moisture. Influence of FFAs on the feedstock quality used in

biodiesel production in large part dictates what type of catalyst or process is needed to produce

fatty acid methyl esters (FAME) that will satisfy relevant biodiesel fuel standards such as

American standard for testing material (ASTM) or European norm (EN). The FFA and moisture

contents have significant effects on the transesterification of triglycerides with alcohol using base

as catalyst (Berchmans and Hirata, 2008). When the feedstock contains a significant percentage

of FFA (>3 wt. %), typical homogenous base catalysts such as sodium or potassium hydroxide or

methoxide will not be effective as a result of unwanted side reaction (reaction [1], fig. 4) in

which the catalyst will react with FFA to form soap and water (or methanol in the case of sodium

methoxide) until the catalyst is finally consumed and deactivated (Drapcho et al., 2008) resulting

in a mixture of FFA, unreacted TG, soap, DG, MG, biodiesel, glycerol, water, and/or methanol

(Moser, 2009). A second hydrolysis reaction causes the conversion of biodiesel, through base

catalysis, to FFAs as shown in reaction [2] fig 4.

22

Fig 4: Formation of soap from reaction of free fatty acids (FFA) with catalyst (reaction [1]) and

hydrolysis of biodiesel (reaction [2]) to yield FFA and methanol

1.2.3.3 Two-step acid-base transesterification

More recently, two-step transesterification process has been used to achieve high purity and yield

of biodiesel in short reaction time. This two-step process is mostly employed when the feedstock

has high FFA content. A number of researchers have worked with feedstocks that have elevated

FFA levels (Zhang et al., 2003; Demirbas, 2008). However, in most cases, alkaline catalysts have

been used and the FFAs were removed from the process stream as soap (Canaki and Van Gerpen,

2001). As the FFA level increases, this becomes undesirable because of the loss of feedstock as

well as the deleterious effect of soap on glycerol separation.

For higher production yields, a two-step acid-base catalyzed reaction which converts both the

FFA and the triglyceride fractions to biodiesel is used as shown in fig 1. Typically, two types of

reactions are conducted sequentially in this process. The first is the esterification of the FFA to

biodiesel esters, followed by alkali-catalyzed transesterification step to produce biodiesel from

the triglycerides. Alkali is a poor catalyst for FFA esterification, but mineral acids are efficient in

this capacity (Scragg, 2003). Due to its low cost and high ester yields, sulphuric acid is the

preferred catalyst used in the FFA esterification step of the two-step process (Demirbas, 2008) as

shown in table 1.

It was reported that to get complete FFA esterification in most vegetable oils, optimum

conditions of 65⁰C temperature, 2 hours reaction time and the acid to oil ratio of 5% w/w are

suitable (Singh and Pahdi, 2009). In the studies of Fan et al., (2009) they also concluded that a

two-step reaction was preferred to a single step or direct transesterification of the feedstock. High

biodiesel yields of 90% and 97% were achieved through two-step acid-base transesterification in

the studies of Berchmans and Hirata, (2008) and Singh and Pahdi, (2009) respectively.

- - - - - - [1]

- - - - - - [2]

23

Table 1: Examples of biodiesel produced from feedstocks high in free fatty acids (FFA)

Feedstock FFAs Pretreatment Catalyst for Ra Yield

Transesterification (wt%)

Pongamia pinnata Up to 20 H2SO4 KOH Me 97

Moringa oleifera 2.9/0.953b H2SO4 KOH Me n.f

c

Jatropha curcas 14/<1 H2SO4 KOH Me 99+

Mudhuca indica 20 None Pseudomonas cepacia Et 96+d

Nicotiana tabacum 35/<2 H2SO4 KOH Me 91

Calophyphylum inophyllum 22/<2 H2SO4 KOH Me 85

Zanthoxyhom inungeanum 45.5/1.16b

None H2SO4 Me 98

Heavea iraniliensis 17/<2 H2SO4 NaOH Me n.f

Heterotrophic microalgal 8.97b None H2SO4 Me n.f

Acid oil 59.3 None H2SO4 Me 95

Fat from meat and bone meal 11 H2SO4 KOH Me 45.7

Brown grease 40/<1 Diarylammonium catalystsNaOCH3 Me 98+d

Waste cooking oil 7.25/<1b H2SO4 NaOH Me 90

d

Water fryer grease 5.6 H2SO4 KOH Me/Et 90+

Tung oil 9.55/0.72b Amberlyst-15 KOH Me 90.2

Tall oil 100% None HCl Me n.f

Sorghum bug oil 10.5 None H2SO4 Me/Et 77.4 aR refers to ester head group. Me methyl, Et ethyl

b Acid value (mg KOH/g) was given instead of FFA. In cases where two values are given, the first value is

prior to pretreatment and the second is after. c Not reported

d Conversion to esters (wt %) is provided instead of yield.

Adapted from Moser (2009).

1.3 Variables that influence the process of transesterification

1.3.1 Catalyst

Catalysts used for the transesterification of triglycerides are classified as alkali, acid, or enzyme

(Vasudevan and Briggs, 2008; Shereena and Thangaraj, 2009; Singh and Singh, 2010).

1.3.1.1 Alkalis

Alkalis used for transesterification include NaOH, KOH, carbonates and alkoxides such as

sodium methoxide, sodium ethoxide, sodium propoxide and sodium butoxide (Fukuda et al.,

2001). Alkaline metal alkoxides ( as CH3ONa for methanolysis) are the most active catalysts,

since they give very high yields (> 98%) in short reaction times (30min) even if they are applied

at low molecular concentrations (0.5 mol%). However they are difficult to manipulate since they

are very hygroscopic (Pinto et al., 2005) and they require the absence of water which makes

them inappropriate for typical industrial processes (Schuchardt et al., 1998). Alkaline metal

24

hydroxides (KOH and NAOH) are cheaper than metal hydroxides, but less active. Nevertheless,

they are good alternative since they can give the same high conversions of vegetable oils just by

increasing the catalyst concentration to 1 or 2 mol% (Drapcho et al., 2008; Leca et al., 2010).

The amount of catalyst used depends on the fatty acid content of the vegetable oil or animal fat

feedstock. Yields of methyl or ethyl esters from low-acid oil feedstocks often exceed 98% with

alkali catalysts typically in the concentration of nearly 1% for both NaOH and KOH in dry

conditions to avoid saponification side reactions (Drapcho et al., 2008). Fan et al. (2009)

reported that the two step catalyzed process using 1wt% KOH and a 97.02 % conversion of

biodiesel was obtained. In the studies of Alamu et al. (2008) alkali catalyzed transesterification

was adopted using NaOH and biodiesel yield of 95.8% was achieved. According to Fukuda et al.

(2001), alkali catalyzed transesterification proceeds approximately 4000 times faster than same

amount of acid catalyst and thus it is most often used.

1.3.1.2 Mechanism of alkali-catalyzed reaction

The reaction mechanism for alkali catalyzed transesterification as shown in fig 5 occurs in four

intermediate reversible steps that include:

1) The first step is the reaction of the base with the alcohol, producing an alkoxide and the

protonated catalyst.

2) Nucleophilic attack of the alkoxide at the carbonyl group of the TG generates a

tetrahedral intermediate,

3) The tetrahedral intermediate is then broken down, liberating the fatty acid ester

compound and the corresponding anion of the diglyceride.

4) The diglyceride deprotonates the catalyst, thus regenerating the active species, which is

now able to react with a second molecule of the alcohol to start another catalytic cycle.

Diglycerides and monoglycerides are converted by the same mechanism to a mixture of alkyl

25

esters and glycerol.

1.3.1.2 Acid catalyst

An alternative to base-catalysed transesterification is the use of acid catalyst (Berchmans and

Hirata, 2008) such as sulphuric acid, sulphonic acid, organic sulphonic acids, phosphoric acids

and hydrochloric acid (Ma and Hanna, 1999). Acid catalysts can be very effective in driving the

transesterification process when oils contain large amounts of FFA’s (Sing and Singh, 2010;

Drapcho et al., 2008). Acid catalysts convert FFA’s to biodiesel esters through esterification

while simultaneously catalyzing the transesterification of TG’s to biodiesel in a single step. Fan

et al. (2009) suggested two-step catalysis: first acid catalysis followed by alkaline catalysis when

the FFA content is very high. Thiruvengadaravi et al. (2009) reported that crude Pongamia

pinnata contains high FFA content and cannot be directly used with an alkaline catalyst.

Biodiesel produced from high FFA Pongamia has been carried out using H2SO4 acid and KOH

catalyst. The main disadvantage of acid transesterification is that the formation of esters is

Fig 5: Mechanism of base-catalyzed transesterification

Source: Demirbas, 2007

26

accompanied by that of water, which inhibits the process. At the same time, the high temperature

and long reaction time can burn part of the oil, which reduces the biodiesel yield and the acid,

being corrosive, deteriorates the equipment too (Gerpen and Knothe, 2005; Leca et al., 2010).

1.3.1.2.1Mechanism of acid-catalyzed reaction

The mechanism of acid catalyzed transesterification of vegetable oil begins with the protonation

of the carbonyl group of the ester. This leads to a carbocation, which after a nucleophilic attack

of the alcohol produces a tetrahedral intermediate. The tetrahedral intermediate breaks down to

give the ester as shown in fig 6.

Fig 6: Mechanism of acid catalyzed transesterification

1.3.1.3 Enzyme catalyst

In addition to the acid and base catalysts, enzyme catalysts are also considered for biodiesel

production. The enzymatic production of biodiesel through alcoholysis of TG has become more

attractive because it shows potential in overcoming the drawbacks of chemical processes

(Fjerbaek et al., 2009). Contrary to alkaline catalysts, enzymes do not form soaps and can

esterify both FFA and TG in one step without the need for a subsequent washing step. Thus,

enzymes have interesting prospect for industrial scale production and reduction of production

costs. Their potential for regioselective and especially for enantioselective synthesis makes them

valuable tools (Schuchardt et al 1998).

Lipases from bacteria and fungi are the most commonly used for transesterification and the

optimal parameters for the use of a specific lipase depends on the origin as well as the

formulation of the lipase (Fjerbaek et al., 2009). Enzymes sourced from various organisms such

as Candida antarctica (Zuhair, 2008), Pencillium simplicissimum (Stamatis, 1993),

27

Thermomyces lanuginosus (Turkan and Kalay, 2008) are used as biocatalysts for

transesterification of TG due to the high efficiency, more compatibility with variations in the

quality of the raw material and reusability (Leca et al., 2010).

Various studies have been conducted on the use of lipases as catalysts. In the transesterification

of rapeseed oil with 2-ethyl-1-hexanol, 95% conversion to esters was obtained using Candida

rugosa lipase powder (Singh and Singh, 2010). The transesterification of sunflower oil, fish oil

and grease with ethanol have been studied. In each case, high yields beyond 80% were achieved

using the lipases from M. meihei, Candida antarctica and Pseudomonas cepacia respectively

(Singh and Singh, 2010).

Enzyme catalysts are currently not feasible for commercial productions because the cost of lipase

is significantly greater than that of alkaline catalysts and the reaction yield as well as reaction

time is unfavourable compared to alkaline catalyzed reaction systems (Demirbas, 2008).

Immobilization of the enzyme and the use of multiple enzymes in sequence may provide future

opportunities in this area ( Shalabby and Al-Gendy, 2012).

1.3.2 Effect of molar ratio

Another important variable affecting the ester (biodiesel) yield is the molar ratio of alcohol to

vegetable oil. As indicated earlier, this reaction is reversible and the stoichiometry of the

transesterification reaction requires 3moles of alcohol per mole of triglyceride to yield 3moles of

fatty acid esters and 1mole of glycerol. Therefore, excess amounts of alcohol are needed to shift

the reaction equilibrium to the product side and higher molar ratios result in greater ester

conversion in a shorter time (Shereena and Thangaraj, 2009: Xu and Hanna, 2008). However, the

high molar ratio of alcohol to vegetable oil makes the recovery of glycerol difficult because there

is an increase in solubility (Demirba, 2008). When the glycerol remains in solution, it helps to

drive the equilibrium back to the left, lowering the yield of esters.

The molar ratio is associated with the type of catalyst used (Encinar et al., 2010). According to

Ma and Hanna (1999) an acid catalyzed reaction needed a 30:1 ratio of butanol to soybean oil,

while an alkali-catalyzed reaction required only a 6:1 ratio to achieve the same ester yield for a

given reaction time. Canakci and Van Gerpen (2001) determined that, increasing molar ratios of

alcohol to TG increased the rate of reaction and conversion yield as high as 98.4% at alcohol to

TG ratio of 30:1 was obtained, but the effect decreased sharply beyond 6:1. Thus a molar ratio of

28

6:1 is normally used to obtain biodiesel yields higher than 98% on a weight basis (Shereena and

Thangaraj, 2009: Singh and Padhi, 2009).

1.3.3 Effect of reaction time and temperature

The rate of reaction is strongly influenced by the reaction temperature. Higher reaction

temperatures speed up the reaction and shorten the reaction time. In the transesterification of

TG’s, the reaction is slow at the beginning for a short time and proceeds quickly and then slows

down again (Ma and Hanna, 1999).According to Xu and Hanna (2009), the methyl ester yield

increases with increasing reaction temperature. From the research of Xu and Hanna (2009), when

the reaction time was 40 minutes, methyl ester yield increased from 74% to 89% and 93% with

the reaction temperature increasing from 25 to 45 and then to 65⁰C. This is thought to be the

consequence of the favourable effect of the high temperature on diffusion of methanol molecules

and reaction with triglyceride molecules.

Hossain et al. (2010) reputed 2 hr reaction time gave better ester yield than 6 hour reaction time

for the production of biodiesel. It is generally reported that every reaction has a certain time of

completion. For the production of biodiesel, it takes about 90 to 120 minutes to complete the

conversion (Singh and Pahdi, 2009). The longer the reaction time, the more the hydrolysis of

ester would occur. It might produce many FFA’s at the end and these FFA’s would participate in

soap formation thus reducing the biodiesel yield. Thus excess reaction time does not promote the

conversion but favours the reduction in the ester yield.

1.3.4 Effect of moisture and free fatty acids

The quality of any feedstock has considerable effect on the level of biodiesel production

(Shereena and Thangaraj, 2009). For alkali-catalyzed transesterification, the TG and alcohol

must be substantially anhydrous and the FFA level of TG at minimal because these impurities

result to adverse reactions such as saponification and hydrolysis (Drapcho et al., 2008) as shown

in Fig 4 (1 and 2). The soap produced through saponification consumes the catalyst and reduces

the catalytic efficiency, as well as causing an increase in viscosity, the formation of gels and

difficulty in achieving separation of glycerol (Ma and Hanna, 1999). Fukuda et al. (1999) in their

research also noted the influence of feedstock quality (moisture and FFA) on the

transesterification reaction.

29

Excess amount of FFAs and water are common features of waste vegetable or animal-based oils.

A conversion yield of 65% to 84% esters using crude vegetable oil as compared to 94% to 97%

yield with refined oil under same reaction conditions has been obtained (Singh and Pahdi, 2009).

In many cases, feedstock quality deteriorates gradually due to improper handling and

inappropriate storage condition. Improper handling would cause the water content to increase. In

addition, exposing the oil to open air and sunlight for a longtime would cause the concentration

of FFA to increase significantly (Berchmans and Hirata, 2008).

1.4 Alternative Feedstocks for biodiesel production.

Generally, there are four major categories of biodiesel feedstock namely: oilseeds, algae, animal

fats, and the various low-value materials such as cooking oils, greases, and soapstocks.

Table 2: Production of biodiesel from various alternative feedstocks Feedstock Oil yield Catalyst Temp (ºC) MeOH Yield (wt. %)

Sunflower frying oil n KOH 25ºC 6:1 90

Rice bran n H2SO4/KOH 60ºC 10:1 92

Peanut oil n NaOH 50ºC 90

Sesame indicum 44-58 NaOH 60ºC 6:1 74

Camelina sativa 31 KOH n 6:1 98

Soybean soapstock 50 H2SO4 35ºC 30:1 99

Cynara curdunculus 25 NaOH 75ºC 12:1 94

Balanites aegyptiana 47 KOH n 6:1 90

Pork lard n KOH 65 7.5:1 97.8

Used frying oil n NaOH 60 7:1 88.8

.

1.4.1 Oilseeds

Oils are produced from plants throughout the world in substantial quantities. Plant oils are

normally extracted from oil-containing seeds where the plant uses oil rather than starch as an

energy store for the seed (Scragg et al., 2003). Many research publications have concluded that

vegetable oils have potential properties for biodiesel production. In view of this, considerable

efforts have gone into developing vegetable oil derivatives that approximate the properties and

performance of petroleum diesel fuels.

Adapted from Moser, 2009

n refers to not reported

30

Depending on the climate and soil conditions, different countries are looking for different types

of oilseeds as substitutes for diesel fuels (Shereena and Thangaraj, 2009). Traditional oilseed

feedstocks for biodiesel production include; soybean in USA, rapeseed/canola in Europe, Palm in

Indonesia and Malaysia, and coconut in Philipines (Rodriques-Acosta, et al, 2010). In tropical

countries, palm or coconut oil is mostly considered for biodiesel production (Knothe, 2005).

As indicated in table 2 above, biodiesel production is very promising because of the high yields

recorded from various seeds. Alternative feedstocks normally arise out of necessity from regions

of the world where the above materials are not locally available or as part of concerted effort to

reduce dependence on imported petroleum.

1.4.2 Microbial and Algae oils

Microalgae exhibit versatile growing conditions. Their versatility can serve many purposes with

respect to overproduction of oils for potential use in biodiesel. The amount of oil that cells,

whether yeasts, fungi or algae, can accumulate varies with the species concerned. Algae have

shown to accumulate an impressive amount of lipids, over 80% of their dry weight (Drapcho et

al., 2008), one of which is Botrycocus braunii (Scragg et al., 2003). Algae have been suggested

as a good candidate for fuel production because of its higher photosynthetic efficiency, higher

biomass production and faster growth rate compared to other energy crops.

Accumulation of oils, in the form of triglycerides, is not a feature of most microbial cells but is

confined to a relatively small number of yeasts, fungi and algae. Oil accumulation occurs as a

result of unbalanced metabolism. When all nutrients are present in the growth medium, the

synthesis of new cells, with minimal level of lipid, will proceed (Ratledge et al., 2008).

The storage of energy as oil rather than as carbohydrates however slows the reproduction rate of

any algae such that higher oil strains generally grow slower than low oil strains (Vasudevan and

Briggs, 2008). An additional challenge, when trying to maximize oil production with algae, is the

unfortunate fact that higher oil concentrations are achieved only when the algae are stressed, in

particular due to nutrient restrictions (Vasudevan and Briggs, 2008). Low nitrogen conditions can

stimulate lipid accumulation, some of which can be as high as 50 – 60% (Scragg et al., 2003).

The disadvantages of growing algae from natural settings and the risk of contamination of

species that would ultimately overtake the oleaginous (oily) cultures of microalgae due to many

uncontrollable environmental factors prevents the wide use of this oil source.

31

1.4.3 Waste vegetable oil (used cooking oil)

The recycling of waste frying oils offers a significant potential as low cost raw material for

biodiesel production (Singh and Singh, 2010). Research abound in literature on the use of waste

oil under acid, alkaline and enzyme catalysis for the production of biodiesel. Fan et al. (2009)

reported the use recycled canola oil via acid pretreatment to achieve high ester yields. In another

studies using a high FFA containing feedstock, Berchmanns and Hirata (2008) obtained up to

90% methyl ester yield through two step acid-base transesterification.

Used cooking oils exhibit properties different from those of refined or crude oils. This may be

due to the degradation products of TG and foreign materials that are found in the used oils

(Knothe, 2005). The high temperatures of a particular cooking process and the water from the

foods accelerate the hydrolysis of triglycerides and increased FFA content in the oil (Fan et al.,

2009). The relatively high amount of FFAs and water in this feedstock results in the production

of soap in the presence of alkali catalyst, hence additional steps to remove any water and either

the FFAs or soap from the reaction mixture are required (Vasudevan and Briggs, 2008).

1.5 Benefits of biodiesel

Biodiesel is superior to conventional petroleum-based diesel fuel in many ways. Among the

attractive features of biodiesel are:

1.5.1 Emission of gases and particulate matter

The current trajectory of fossil fuel use and its related emission of greenhouse gases are

unsustainable. Fuel combustion produces many pollutants among which are unburnt HC’s, CO

and NOx (NO and NO2). Unburnt HC’s and CO are products of incomplete combustion whereas

NOx are products of the high combustion temperatures (Scragg et al., 2003). Biodiesel reduces

tailpipe particulate matter (TPM), hydrocarbons (HC), and carbon monoxide (CO) emissions

from most modern diesel engines (Rao, 2008).

These benefits occur because biodiesel contains 11% oxygen by weight. The fuel oxygen allows

the fuel to burn more completely, thus fewer unburned fuel emissions result (Fukuda et al.,

2001). According to Rao (2011), the oxygen content of a fuel improves its combustion efficiency

due to an increase in the homogeneity of oxygen with the fuel during combustion. The same

phenomenon reduces air toxics, which are associated with the unburnt HC and PM emissions.

32

With the relatively low emission profile of biodiesel, it is an ideal fuel for use in sensitive

environment such as heavily polluted cities (Vasudevan and Briggs, 2008).

Due to its agricultural source, biodiesel essentially contains no or very little sulphur and thus

offers promise to reduce particulate and toxic emissions, which is one of the primary objections

to diesel engine (Bello and Makanju, 2011). However, some investigations show a slight increase

in the emission of nitrogen oxides (NOx) with biodiesel use. Biodiesel may emit some pollutants

when combusted, but they generally burn cleaner than corresponding fossil fuels used in similar

applications.

1.5.2 Lubricity of biodiesel fuel

Lubricity refers to the properties within a said liquid which reduces friction and wear between

moving parts. The injection systems of many diesel engines rely on the fuel to lubricate its parts

(Da Silva et al., 2006). Biodiesel provides excellent lubricity to the fuel injection system by

allowing the engine moving parts to move more easily (Gerpen and Knothe, 2005).

Low lubricity petrooleum diesel fuel can cause premature failure of injection system components

and decreased performance (Zhang et al., 2003). Petroleum diesel fuel was once lubricated

primarily with sulphur. When fuel containing sulphur is burned, it provides SO₂, the primary

component of acid rain. Recently, with the introduction of low sulphur and ultra low sulphur

diesel fuel, many of the compounds which previously provide lubricating properties to petroleum

fuel have been removed. By blending biodiesel in amounts as little as 5%, the lubricity of ultra

low sulphur diesel can be drastically improved and the life of an engine’s fuel injection system

extended (Vasudevan and Briggs, 2008).

1.5.3 Biodegradability and toxicity of biodiesel

Biodiesel is made entirely from vegetable oil or animal fats, therefore it is biodegradable and

nontoxic (Vasudevan and Briggs, 2008). Studies have shown biodiesel to biodegrade up to 4

times faster than petroleum diesel fuel, with about 98% biodegradation in 3 weeks (Gerpen and

Knothe, 2005). This property reduces environmental risk of spills because biodiesel breaks down

naturally while petroleum products tend to coat surfaces in the event of a spill.

33

1.5.4 Safety and Stability of biodiesel

Biodiesel is safer to handle than petroleum diesel fuel because of its low volatility. Due to the

high energy content of all liquid fuels, there is a danger of accidental ignition when the fuel is

being stored, transported or transferred. The possibility of having an accidental ignition is related

in part to the temperature at which the fuel will create enough vapours to ignite, known as the

flash point temperature. The lower the flash point of a fuel, the lower the temperature at which it

will form a combustible mixture (Adebayo et al., 2011). For example, petroleum diesel has a

flash point of 64 ⁰C, which means that it can form a combustible mixture at temperature as low

as 64⁰C. Biodiesel on the other hand has a flash point of over 150⁰C, meaning it cannot form a

combustible mixture until it is heated well above the boiling point of water (Rodriques-Acosta et

al., 2010). It is rare that biodiesel fuel is subjected to these types of condition, making biodiesel

quite safe to store, handle and transport. Biodiesel is therefore classified as a non-flammable

liquid.

1.6 Disadvantages of biodiesel use

Although the advantages make biodiesel seem very appealing, there are also some disadvantages

when using biodiesel. Due to the high oxygen content, it releases relatively high NOx levels

during combustion. But this can be reduced to below petroleum diesel levels by adjusting engine

timing and using a catalytic converter (Rao, 2011). Storage conditions of biodiesel must be

monitored strictly as biodiesel has a lower oxidative stability (Afolabi, 2008). Biodiesel has

lower temperature flow properties than petroleum diesel which means it will crystallize into a gel

at low temperatures when used in its pure form (Abayeh et al., 2007). Biodiesel is also more

susceptible to degradation, which is promoted by the presence of oxygen, high temperatures, and

the presence of certain metals (Leiner, 1980).

1.7 Fuel properties and quality standards of biodiesel

Specifications for biodiesel require particularly close attention due to the large variety of

vegetable oils that can be used for biodiesel production and the variability in fuel characteristics

that can occur with fuel produced from this feedstock. Today, biodiesel has much stricter

definitions in the form of quality standards established to gain wider acceptance from engine

manufacturers, distributors, retailers and users (Johnston, 2006). Numerous biodiesel standards

are currently in force in a number of countries including the EN 14214 in the European countries

34

such as Germany, Italy, France, and the Czech Republic and the ASTM in the USA. These

standards provide fuel property values required for a mixture of alkyl esters to be considered as

biodiesel. If these limits are met then the biodiesel can be used in modern engines with little or

no modification (Abayeh et al., 2007).

Table 3: International standards on biodiesel

Source: National standard for biodiesel, 2003

1.7.1 Kinematic viscosity

Kinematic viscosity is a coefficient defined as the ratio of the dynamic viscosity of a fluid to its

density (Yoon et al., 2008). While dynamic viscosity is a measure of the resistance to flow of a

fluid under gravity (Gerpen and Shank, 2001). A fuel of high viscosity tends to form larger

droplets upon injection, leading to poorer atomization during the spray and creating operational

problems, such as increased carbon droplets and may enhance the polymerization reaction

(Freitas et al., 2011).

Parameters Austria

(ON)

Czech

(CSN)

France Germany

(DIN)

Italy USA

(ASTM) Density @ 15%

g/cm³

0.85-0.89 0.87-0.89 0.87-0.89 0.875-0.89 0.86-0.90 -

Viscosity @ 37.

mm²/s

3.5-5.0 3.5-5.0 3.5-5.0 3.5-5.0 3.5-5.0 1.9-6.0

Flash point

(⁰C)

100 100 100 100 100 130

Cetane number ≥49 ≥49 ≥49 ≥49 - ≥47

Iodine number

gI2/100g

≤120 - ≤115 ≤115 - -

Acid value

mgKOH/g

≤0.8 ≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.5

Water and

sediment (%).

- - - - - ≤0.05

35

The difference in the viscosity of fuels is attributed to the different fatty acid composition as

described by Xu et al. (2009). Viscosity is influenced by structural features of the alkyl esters

like chain length, degree of unsaturation and chain branching (Abayeh et al., 2007). Generally

viscosity increases with the number of CH2 moieties in the fatty ester chain and decreases with

an increasing unsaturation (Knothe, 2008).

For oils exposed to oxidizing conditions and high temperatures, degradation of the oil is

normally accompanied by an increase in viscosity (Popovich and Hering, 1959). Changes

occurring in the oil under these conditions can be followed by viscosity measurement. The

kinetic viscosity of biodiesel according to ASTM is within the range of 1.6 – 6.0 as shown in

table 3. Lower viscosity may also indicate the presence of methanol in the biodiesel.

1.7.2 Flash point temperature

Flash point is the minimum temperature at which the fuel will give off enough vapours to

produce an inflammable mixture (fuel vapour and air) above the fuel surface, when the fuel is

heated under standard test conditions (Rao, 2011).

The fundamental reason for measuring flash point is to assess the safety\hazard of a liquid with

regards to its flammability and then classify the liquid into a recognized hazard group. This

classification is used to warn of a risk and to enable the correct precautions to be taken when

manufacturing, storing, transporting or using the liquid (Belewu et al., 2010). Tests have shown

that as little as 1% methanol in biodiesel can lower the flash point from 170⁰C to less than 40⁰C

(Van Gerpen, 2004). Therefore by including a flash point specification of 130⁰C, the ASTM

standard limits the amount of alcohol to a very low level (<0.1%).

The flash point is used as a safety index for biofuels because it correlates to the fuel ignitability

and varies inversely with the fuel’s volatility. Biodiesel with a flash point of 150⁰C falls under

the non hazardous category and it is safe for usage. Specifications also quote flash point for

quality control purposes. It indicates the level of purification the fuel has undergone; as the

presence of a very small amount of alcohol in the biodiesel leads to a significant drop in the flash

point (Abayeh et al., 2007). Also, a change in flash point may indicate the presence of potentially

dangerous volatile contaminants or the adulteration of one product by another.

36

1.7.3 Acid value

The acid value is the quantity of base, expressed as milligrams of potassium hydroxide per gram

of sample, required to titrate a sample to a specified end point (Van Gerpen et al., 2004). It is a

direct measure of FFA’s in a sample. The lower the acid value of oil, the fewer FFA’s it contains

which makes it less exposed to the phenomenon of rancidity (Asuquo et al., 2010). The acid

value increases with an increase in peroxides because the esters first oxidize to form peroxides

which undergo complex reactions, including a split into more reactive aldehydes, which further

oxidize into acids (Hossain et al., 2010). The FFA’s can lead to corrosion and may be a symptom

of water in the fuel.

Acid value is required to determine the amount of catalyst needed for transesterification or

whether to pre-treat the feedstock to first accomplish acid-catalyzed esterification before

conducting the much faster base-catalyzed transesterification procedure (Drapcho et al., 2008).

The FFA and moisture contents have significant effects on the transesterification of TG with

alcohol using a base catalyst (Berchmans and Hirata, 2008). Acid value is also used as a guide in

the quality control as well as monitoring oil degradation during storage (Fan et al., 2009;

Afolabi, 2008). The acceptable FFA of a feedstock should be less or equal to 0.5% of the oil

according to ASTM specifications.

1.7.4 Iodine value

Iodine value is a measure of the average amount of unsaturation of fats and oils and is expressed

in terms of the number of grams of iodine absorbed by 100 grams of sample (Akbar et al., 2009).

Iodine absorption occurs at the double bond positions. Iodine values are useful for determination

of overall degree of saturation of oil. High degrees of unsaturation are not desirable for fuels

because their oxidation reactions generally found at high temperatures during combustion may

result in irreversible polymerization to plastic like substances (Rao, 2011). Polyunsaturated fatty

acids are very susceptible to polymerization and gum formation caused by oxidation during

storage, or by a complex oxidation and thermal polymerization at higher temperature and

pressure of combustion (Afolabi, 2008).

The chemical changes in the fuel associated with oxidation usually produce hydroperoxides that

can, in turn, produce short chain fatty acids, aldehydes and ketones. Under the right conditions,

the hydroperoxides can also polymerize. Iodine value is applied for monitoring progress to

37

hydrogenation, degree of fractionation and for identity characterization of fats (Hoffmann, 1986).

The DIN specification for biodiesel is less or equal to 120 as shown in table 3.

1.7.5 Cetane number

Cetane number (CN) is a prime indicator of the quality of fuel in compression ignition engines. It

is a relative measure of the interval between the beginning of injection and auto-ignition of the

fuel (Rao, 2011). It is generally dependent on the composition of the fuel and can impact the

engine’s startability, noise level, and exhaust emissions (Van Gerpen, 2004).

Higher Cetane values are desired. Biodiesel esters tend to show relatively higher cetane values

averaging near 50 (Drapcho et al., 2008). The higher the CN, the shorter the delay interval and

the greater its combustibility (Gerpen et al., 2004). Branching and chain length influence the CN.

The CN becomes smaller with decreasing chain length and increasing branching (Rao, 2011).

In general, diesel engines will operate better on fuels with CN’s above 50. Too high or low CN’s

can cause operational problems. If a CN is too high, combustion can occur before the fuel and air

is properly mixed, resulting in incomplete combustion and smoke. If CN is too low, engine

roughness, misfiring, higher air temperatures, slower engine warm up, and also incomplete

combustion occur (Demirbas, 2005). Therefore cetane number is also indicative of the relative

fuel stability. The specification for biodiesel according to ASTM is 47 as shown in table 3.

1.7.6 Saponification value

The saponification value is the number of milligrams of potassium hydroxide required to

neutralize the FAs liberated on complete hydrolysis or saponification of 1g of the oil (Igwenyi et

al, 2011). Saponification value is an index of the average size of fatty acid present, which

depends upon the molecular weight and percentage concentration of fatty acid components in the

oil (Parthiban et al., 20011).

An increase in saponification value in oil increases the volatility of the oil and this enhances the

quality of the oil because it shows the presence of lower molecular weight components in 1g of

the oil. This is in agreement with the report of Afolabi 2011, that oil fractions with saponification

values of 200mgKOH/g and above possess low molecular weight fatty acids. Since 1g of oil/fat

containing low molecular weight fatty acids will have more molecules than oil/fat containing

higher molecular weight fatty acids (Igwenyi et al., 2011). This principle reveals that the number

of milligrams of KOH required to saponify the oil will be greater in the former than in the latter

38

case. Therefore, the higher the saponification value, the lower the molecular weight of the fatty

acids and the better the quality of the oil.

1.7.7 Refractive index

The refractive index is the quotient of the sine of the incidence angle of light in the air and the

sine of the angle of refraction of light in the substance (Hoffmann, 1986). It employs the

principle of critical angle using diffused light. It is used in measuring the concentration of

solutions because when the concentration or density of a substance increases, its refractive index

increases proportionally (Parthiban et al., 2011).

The refractive index is characteristic of each kind of oil/fat. Its value varies with the degree and

type of unsaturation of component fatty acids in an oil/fat sample. Refractive index increases

with the increase in unsaturation and with the chain length of fatty acid (Nayak and Patel, 2010).

Therefore the refractive index of oils is subject to change during processing (hydrogenation) and

use (polymerization during heat treatments) hence it can be used successfully in quality control.

Refractive index and specific gravity measurements rarely provide sufficient information to

quantitatively identify a pure analyte, but are highly useful to check oil

contamination/adulteration (Parthiban et al., 2011).

1.7.8 Moisture content

Biodiesel water content is an important parameter because it affects biodiesel oxidation stability,

therefore influencing the storage life of the fuel (Dias et al., 2008). Water can be present in two

forms, either as dissolved water or as suspended water droplets. While biodiesel is generally

considered to be insoluble in water, it actually takes up (hygroscopic) considerably more water

than petroleum diesel. Biodiesel contains as much as 0.15% of dissolved water while petroleum

diesel usually takes up about 0.005% (Van Gerpen, 2004). The standards for petroleum diesel

fuel (ASTM D975) and biodiesel (ASTM D6751) both limit the amount of water to 0.05% as

shown in table 3. Water promotes adverse reaction to transesterification which will convert

biodiesel back into FFA’s as shown in reaction [2] of fig 4. Suspended water is a problem in fuel

injection equipment because it contributes to the corrosion of the closely fitting parts in the fuel

injection system.

The moisture content is particularly important when applied to oils and fuels since it will provide

a measure of deterioration and contamination (Popovich and Hering, 1959). Water can also

contribute to microbial growth in the fuel. This problem can occur in both biodiesel and

39

petroleum diesel fuel and can result in acidic fuel and sludge’s that plug fuel filters (Van Gerpen,

2004). The water in biodiesel plays an important role in predicting quality performance of the

fuel. In commercial practice, the level of moisture and impurities is one of the most important

quality characteristics limited by norms and standards (Hoffmann, 1986). The low moisture

content often shows that a fuel is good and could not be easily subjected to

contamination/rancidity.

1.7.9 Cloud point

Cloud point (CP) is the temperature at which some of the molecules in the fuel first begin to

freeze, resulting in the appearance of crystals in the fuel, which gives it an initial cloudy

appearance (Abayeh et al., 2007). A major problem of biodiesel is poor temperature flow

properties indicated by relatively high CP. This makes CP a critical factor in the cold weather

performance of diesel fuel. Therefore, it is an index of the lowest temperature of the fuel’s

usability for certain applications. Operating at temperatures below the CP of a biodiesel fuel can

result in filter clogging due to wax crystals (Abayeh et al., 2007). Since the saturated methyl

esters are the first to precipitate, the amounts of these esters, methyl palmitate and methyl

stearate, are the determining factors for the CP.

The cloud point of a fuel can be modified in two ways. One is through the use of additives that

retard the formation of solid crystals in the biodiesel. The cloud point can also be modified by

blending feedstocks that are relatively high in saturated fatty acids with feedstocks that have

lower saturated fatty acid content. The result is a net lower CP for the mixture. Thus, the lower

the CP, the higher the quality of the fuel since a high CP limits the flow properties of biodiesel,

which influences its use in a cold environment (Xu and Hanna, 2009). According to Popovich

and Hering (1959) the cloud point may also be used in identifying the source of the oil/fat.

1.7.10 Peroxide value

The primary products that appear during the autoxidation of fats/oils are hydroperoxides (Liener,

1980). These hydroperoxides contain ‘active or peroxide oxygen’, which if decomposed in a

medium (acid), can be measured and the amount of hydroperoxides calculated. Peroxide value is

the amount of substances in the sample, expressed in terms of milliequivalent of ‘active or

peroxide oxygen’ per kilogram fat which oxidize potassium iodide under the operating

conditions (Hoffman, 1986).

40

Metals present in trace amounts are often responsible for the primary initiation, and metals that

are oxidized by one electron transfer are the most active. Accordingly, cobalt, copper, iron,

nickel, manganese and other such metals have been found to be potent lipid peroxidants (Liener,

1980). The nature and extent of the changes that take place in fats/oils in storage or upon heating

and oxidation depend very much on the kind of fat/oil used and the conditions under which it has

been heated. The usual method of assessing hydroperoxide is by the determination of peroxide

value (Gunstone,1999).

Peroxide value is used to monitor the development of rancidity through the evaluation of the

quantity of peroxides generated in the initial product of oxidation. Regarding the nature of the

fatty acids involved, the results of most studies demonstrate that in nearly all cases, the

unsaturated fatty acids were the most susceptible of the fatty acid in question to these effects

(Igwenyi et al., 2011).

1.8 Biodiesel Use (Blending Of Esters)

Biodiesel can be used as a blend component in petroleum in any proportion because it is

completely miscible with ultra low sulphur diesel fuel (ULSD). Once mixed, the blend will

exhibit properties different from neat biodiesel or petroleum fuels. Specifically, the most

important fuel properties influenced by blending of biodiesel with petroleum are lubricity,

exhaust emissions, CN, flash point, oxidative stability, low-temperature operability, kinematic

viscosity, and energy content (Moser, 2009).

Biodiesel can be used in its pure form, also known as neat biodiesel or B100. This is the

approach that provides the most reduction in exhaust particles, unburned hydrocarbons and

carbon monoxide. This approach is used in countries like Austria and Germany. It is the best way

to use biodiesel when its non-toxicity and biodegradability are important.

Biodiesel can also be used as a blend. Typically this can range from 5% to 50% biodiesel in 95%

to 50% petroleum diesel and is known as B5, B10 etc depending on the blend. Blends reduce the

cost impact of biodiesel while retaining some of the emission reductions. Most of these

reductions appear to be proportional to the percentage of biodiesel used (Friedrich, 2003).

Biodiesel can also be used as an additive (1% - 2%) and is known as B1 or B2. Tests for lubricity

have shown that biodiesel is a very effective lubricity enhancer. Even as little as 0.25% can have

a measurable impact and 1% - 2% is enough to convert a very poor lubricity fuel into an

41

acceptable fuel. Although these levels are too low to have any impact on the CN of the fuel or the

emissions from the engine, the lubricity provides a significant advantage at a modest cost

(Friedrich, 2003).

Blending petroleum diesel fuel with esters of vegetable oils is presently the most common form

of biodiesel. The most common ratio is 80% petroleum diesel and 20% biodiesel also termed

“B20”, indicating 20% level of biodiesel. There are numerous reports that significant emission

reductions are achieved with these blends (Knothe, 2001).

1.9 Other industrial applications of biodiesel

Fatty acid methyl esters can be transformed into a lot of useful chemicals, and raw materials for

further synthesis as shown in fig 7. Fatty acid alkyl esters can serve as valuable starting materials

or intermediates in the synthesis of fatty alcohols. The fatty alcohols are applied as

pharmaceutical and cosmetics additives (C16-C18), as well as lubricants and plastifying agents

(C6-C12), depending on the length of their carbon chain (Schuchard et al., 2008). The isopropyl

esters are also applied as plastifying agents and emollients.

Biodiesel, in conjunction with certain surfactants, can act as a contact herbicide for killing

broadleaf weeds in turfgrass (Moser, 2009). The alkanolamides have a direct application as non-

ionic surfactants, emulsifying, thickening and plastifying agents (Schuchard et al., 2008).

Another important non-fuel application of biodiesel is as an industrial environmentally friendly

solvent. Since they are biodegradable, they have high flash points and have very low volatilities

(Moser, 2009). The high solvent strength of biodiesel makes it attractive as a substitute for a

number of conventional and harmful organic solvents in applications such as industrial cleaning

and degreasing, resin cleaning and removal and as a medium in site bioremediation of crude

petroleum spills.

The fatty acid methyl esters are further used in the manufacture of carbohydrate fatty acid esters

(sucrose polyesters), which can be applied as non-ionic surfactants or edible non-calorific oils.

Glycerol, a by-product in biodiesel production, also has important applications, in cosmetics,

toothpastes, pharmaceuticals, food, lacquers, plastics, alkyl resins, tobacco, explosives, cellulose

42

processing (Drapcho et al., 2008).

Fig 7: Some applications of Biodiesel (Moser, 2009)

1.10 Fuel Composition

Biodiesel and petroleum diesel are not chemically similar. Biodiesel is composed of long-chain

methyl esters, whereas petroleum diesel is a mixture of aliphatic and aromatic hydrocarbons that

contain approximately 10 – 15 carbons. Because biodiesel and petroleum diesel have differing

chemical compositions, they have differing fuel properties.

1.10.1 Biodiesel

The performance of an ester as diesel fuel depends on the chemical composition of the ester,

particularly on the length of carbon chain and the degree of saturation (and unsaturation) of fatty

acid molecules (Rao, 2011). There are three main types of fatty acids that can be present in a

triglyceride which are saturated (Cn:0), monounsaturated (Cn:1), and polyunsaturated with two or

three double bonds (Cn:2,3).

From a chemical point of view, oils from different sources have different fatty acid compositions.

The fatty acids are different in relation to the chain length, degree of unsaturation or presence of

other chemical functions (Pinto et al., 2005). Generally only five fatty acyl chains are common in

most vegetable oils and animal fats (others are present in small amounts) as shown in table 4.

The relative amounts of the five fatty acids (palmitic, stearic, oleic, linoleic and linolenic)

43

determine the physical and chemical properties of the oils and the fuel derived from them

(Gerpen, 2004).

Various vegetable oils are potential feedstock for the production of biodiesel but the quality of

the fuel will be affected by the oil composition (Akbar et al., 2009). An earlier report by Louppe

et al. (2008) gave the fatty acid profile of Quassia undulata seed oil as having 46 – 61% oleic

acid, 20 – 26% stearic acid, 8 – 11% palmitic acid and 8 – 10% linoleic acid as its main fatty

acids. This shows that Quassia undulata biodiesel will have a mixture of 34.55% saturated

methyl esters, 56.02% monounsaturated methyl esters and 9.42% polyunsaturated methyl esters.

Ideally the vegetable oil for biodiesel production should have low saturation and low

polyunsaturation with higher monounsaturated fatty acids (Gunstone, 1999).

Transesterification does not alter the fatty acid composition of the feedstocks and this

composition plays an important role in some critical parameters of biodiesel, such as cetane

number and cold flow properties (Akbar et al., 2009). The carbon chain length of Quassia

undulata fatty acids, which includes only the long chain fatty acids, varies from 16 – 18. From

the chemical point of view, good oil for biodiesel production must be rich in long chain and low

unsaturation level fatty acids (Pinto et al., 2005).

Table 4: Free Fatty Acid Composition of QUSO and other vegetable oils.

a: (Bello and Makanju, 2011); b: (Louppe et al., 2008); c: (Pinto et al., 2005)

Fatty acid

Class

Jatrophaa

Groundnutc

Soyabeana

Safflowera

Castora

Quassia

undulatab

Palmitic 16:0 18.22 8.5 10.3 - 1.0 9.5

Stearic 18:0 5.14 6.0 4.7 3.3 1.0 23

Oleic 18:1 28.46 51.6 22.5 14.4 3.0 53.5

linoleic 18:2 48.18 26 54.1 75.5 4.2 9

Linolenic 18:3 - - 8.3 0.1 0.3 -

Ricinoloic 18:1 - - - - 89.5 -

Eicosanoic 20:0 - - - - 0.3 -

Total saturated - 23.36 15.74 15.10 3.30 2.30 34.21

Total

unsaturated

- 76.64 84.26 84.90 96.70 97.70 65.79

44

1.10.2 Petroleum Diesel

Conventional diesel is produced by the distillation of crude oil collecting middle distillate

fractions in the range of 175 – 370° C (Scragg, 2003). Diesel fuel typically contains over 400

distinct types of organic compounds which includes approximately 80% (vol.) of saturated

hydrocarbons (primarily paraffin’s, the straight chain hydrocarbons) and 20% of aromatic

hydrocarbons (naphthalene’s, the cyclic hydrocarbons and alkyl benzenes) (Rao, 2011). The

saturated hydrocarbons include approximately 44% of n-paraffin, 29% of i-paraffin and 7% of

naphthalene as shown in fig 8. Carbon numbers of these hydrocarbons range from 12 – 18 (Singh

and Singh, 2010). The aromatics are a class of hydrocarbons (HCs) that are characterized by

stable chemical structures. The aromatics containing multiple benzene rings are known as poly-

aromatic hydrocarbons (PAH’s). The aromatics include polycyclic aromatic compounds

containing 2,3 4 and 5 fused benzene rings and the benzene will act as nuclei for the growth of

undesirable shoot. Aromatics are considered desirable by compression ignition (CI) engine

operators because they provide greater energy per litre of diesel fuel, however they may

contribute to higher emissions of particulate matter (PM), and NOx, and have lower CN.

Fig 8: Composition of petroleum diesel fuel.

Source: Rao, 2011

1.11.3 Non edible oils

There are growing concerns about the utilization of non-edible oils for the production of

biodiesel. Jatropha curcas has good potential for biodiesel production. Other non-edible oils like

castor, Pongamia pinnata (karanja), argemone and sal are also being investigated (Vasudevan

and Briggs, 2008), yet the demand far exceeds the current and future production capacities of the

vegetable oil and animal fat industries (Xu et al., 2009).

45

New oil seed crops that do not compete with traditional food crops are needed to meet existing

energy demands. Hence careful sourcing of non-edible seeds/nuts is necessary (Gungstone,

1999). Fortunately, non-edible vegetable oils, mostly produced by seed bearing trees and shrubs

can provide an alternative. In Nigeria, there is an abundance of oil seeds that are relatively

unexplored (Abayeh et al., 2007; Eze, 2012). With no competing food uses, this characteristic

turns our attention to Quassia undulata. The high prices of commodity vegetable oils and animal

fats have made exploration of economical alternative, non food feedstocks an important research

topic.

Table 5: Non edible oil seeds

Species Oil fraction (%)

Tttttttt Castor 45 – 50

Jatropha curcas 50 – 60

Muhua 35 – 40

Sal 10 – 12

Linseed 35 – 45

Neem (Azadiracta indica) 20 – 30

Pongamia pinnata (Karanja) 30 – 40

Adapted from Singh and Singh (2010).

1.11.4 Quassia undulata

Quassia undulata is a perennial shrub distributed in tropical and subtropical Africa, America,

Asia and Australia. In Nigeria, Quassia undulata is called Gbur by the Tiv people. Quassia

undulata is a fast growing member of simaroubaceae family that has been variously reported to

have poisonous fresh seeds which may be due to its rich quassinoids (seco-triterpernoid

compounds), including quassin responsible for the bitter taste (50 times more bitter than quinine)

and certain indole alkaloids (derivatives of canthin-6-one) [Louppe et al., 2008]. According to

Harbourne (1973), alkaloids are often toxic to man and may have dramatic physiological

activities, hence their wide use in medicine. Fig 8 shows the stem, leaves, fruit and seeds of

Quassia undulata plant.

The stem, stem bark and root bark extracts and various quassinoids isolated from the plant have

shown antimalaria activity against Plasmodium falciparium (Adesanwo et al., 2009) and

Plasmodium berghei (Ajaiyeoba et al., 1999) and other interesting properties and may have

pharmacological potential. For example the quassinoid 15-desacetylundulatone, isolated from the

46

root bark has shown anti tumour activity against P388 mouse lymphocytic leukaemia cells and

colon 38 adenocarcinoma (Louppe et al., 2008). Louppe et al. 2008 also reported that a seed

extract of Quassia undulata showed insecticidal and arachnidal properties. Because the

production of biodiesel from edible oils has progressively stressed food uses, price, production

and availability of oils, Quassia undulata therefore occupies a special place among the oil plants

that grow in Nigeria.

1.12 RESEACH AIM AND OBJECTIVES

Worldwide, biodiesel production has been adjusted to the available crops in each region. An oil

seed crop amenable to Nigeria’s environmental condition is still in search. The aim of this study

was to investigate the properties of oil methyl esters produced from Quassia undulata seeds by a

two step esterification and transesterification process. The specific objectives include:

i. Extraction of oil from Quassia undulata seeds using n-hexane as solvent.

ii. Physicochemical characterization of Quassia undulata seed oil.

iii. Transesterification of Quassia undulata seed oil through methanolysis with an acid-

catalyzed pretreatment.

iv. Physicochemical characterization of Quassia undulata seed oil alkyl esters.

v. Determination and identification of the fatty acids in the oil and FAME in the biodiesel

using Gas chromatography mass spectrometric analysis.

vi. Comparison of the fuel properties of Quassia undulata seed oil alkyl esters with those of

international standard biodiesel and the properties of petroleum diesel.

Leaves Stem Fruit Seeds

Fig 9: The leaves, stem, fruit and seeds of Quassia undulata

47

CHAPTER TWO

MATERIALS AND METHODS

2.1 MATERIALS

2.1.1 Collection and processing of sample

The Quassia undulata nuts were collected from Yandev, Benue State of Nigeria and were

identified by Mr. Solomon Igah of the Department of Forestry, Akperan Orshi College of

Agriculture, Yandev. The nuts were cleaned of adhering soil and unwanted materials were

handpicked. The nuts were broken and the seeds removed. The seeds were ground with a blender

into fine particles and were ready for oil extraction.

2.1.2 Chemicals/Reagents used include:

Methanol Sigma Aldrich, Germany

Ethanol Sigma Aldrich, Germany

Tetraoxosulphate (vi) acid Sigma Aldrich, Germany

Potassium hydroxide pellets Riedel-de Haen, Czech Republic

Acetic acid May and Baker, England

Chloroform Sigma Aldrich, Germany

Sodium hydroxide Merck, Germany

Sodium thiosulphate

Carbon tetrachloride

Potassium iodide solution

Wijs solution

Iodine hydroxide

Starch

Phenolphthalein

48

Sodium chloride

2.1.3 Instruments/Equipment

Hotplate Gallenkamp, England

Hand Refractometer Atago, Tokyo Japan

Measuring cylinder Pyrex, England

Digital balance G and GR

Water bath Model K

Stirrer Gallenkamp, England

Water pump Atman powerhead, USA

Three neck round bottom flask Pyrex

Reflux condenser

Viscometer Cannon Fenske

GC-MS Shimadzu QP2010

2.2 Preparation of Reagents

2.2.1 Wijs Reagent

Wijs reagent was prepared by weighing iodine trichloride (2.0g) into an amber coloured bottle

followed by the addition of 17M acetic acid (700ml) and tetrachloromethane (300ml). Then,

iodine Crystals (10g) were added and the solution left to stand for 24 hours to equilibrate after

which the reagent was stored in an amber bottle at room temperature.

2.2.2 0.5M Alcoholic KOH (Alcoholic potassium hydroxide)

KOH pellets (30g) were dissolved in a conical flask using absolute ethanol and the solution was

made up to the one litre mark using the same solvent.

49

2.2.3 0.01M Sodium thiosulphate (Na2S2O3)

This was prepared by dissolving sodium thiosulphate (2.5g) in a conical flask and the solution

made up to 1 litre using distilled water.

2.2.4 1M H2SO4

Conc. H2SO4 (18M, 10ml) was measured out and diluted using 170ml of distilled water.

2.2.5 Glacial acetic acid: Chloroform Solution (3:2v/v)

This solution was prepared by mixing glacial acetic acid (300ml) and chloroform (200ml) in a

1000ml conical flask.

2.2.6 Saturated KI Solution

This was prepared by dissolving KI salt in a given amount of distilled water until the given

volume of water no longer dissolved more solute.

2.2.7 0.5M H2SO4

This was prepared by mixing 18 M H2SO4 (1ml) with distilled water (35 ml).

2.2.8 Hot Neutralized Ethanol

Hot neutralized ethanol was prepared by heating absolute ethanol (10ml) in a water bath at 400C

after which few drops of phenolphthalein were added (2–3 drops). The hot ethanol was then

titrated with 0.1M KOH.

2.2.9 Phenolphthalein Indicator

Phenolphthalein indicator was prepared by dissolving phenolphthalein (1g) in a given quantity of

distilled water after which the solution was made up to 100ml mark using the same solvent

(distilled water).

2.2.10 0.1M Sodium thiosulphate (Na2S2O3)

This was prepared by dissolving sodium thiosulphate (approximately 25g) in a conical flask and

the solution made up to one litre using distilled water.

2.2.11 Starch Indicator

This was prepared by dissolving soluble starch powder (1g) in 100ml of boiling water.

2.2.12 KI Solution (Potassium Iodide)

This was prepared by dissolving KI (100g) in a conical flask and the solution made up to one

litre using distilled water.

50

2.3 METHODS

2.3.1 Extraction of oil from Quassia undulata seeds

The extraction of oil was done using soxhlet technique according to the method of Bello and

Makanju (2011). The powdered seeds (892.16g) were packed into the extraction chamber and

normal hexane poured into the round bottom flask of the soxhlet extractor. The mantle heater

was set at about 60⁰C and the oil in the seeds was leached for 24 – 48 hours in each case until all

the powdered seed was extracted. An exhaustive oil extraction was considered to be achieved

when no more oil was obtained. The extracted oil was oven dried at 40⁰C for 72 hours. The seed

oil was filtered through a Whatman filter paper No. 1 to remove foreign particles. The pure oil

preserved in cold storage. The percentage oil yield was calculated as follows:

Percentage oil yield = weight of oil obtained in gm x 100

Weight of seed taken in gm

2.3.2 Characterization of the Vegetable Oils

2.3.2.1 Determination of the Saponification Value

American Standard for Testing Material (ASTM) method D 5558-95 (2002) was used for the

determination of the saponification value of the vegetable oil. Approximately 2g of Quassia

undulata seed oil (QUSO) was weighed into a conical flask containing 25ml of 0.5M ethanolic

KOH and the resulting mixture was refluxed for 60 minutes. The resulting solution was

subsequently titrated against 0.5M HCl with phenolphthalein as indicator. The end point was

obtained when the pink colour changed into colourless. The same procedure was used for the

blank. The saponification value (SV) was then calculated using the expression;

Saponification value (S.V.) = 56.1 (B-S) x M of HCl

Weight of sample

Where;

B = ml of HCl required by blank

S = ml of HCl required by sample

M = Molarity of HCl

56.1= Molar mass of KOH

2.3.2.2 Determination of acid value

Acid value of QUSO was determined by ASTM method ASTM – D 974 00 (2002). About 0.5g

of sample was weighed into 250ml conical flask and 50ml of neutralized ethanol was added. The

51

mixture was heated on a water bath to dissolve the sample. The solution was titrated against

0.1M KOH using phenolphthalein as indicator. The acid value was determined after which the

free fatty acid was calculated respectively as follows;

Acid Value = A x M x 56.1 W

Where, A = ml of 0.1M KOH consumed by sample

M = Molarity of KOH

W = weight in grams of the sample

Then

Free fatty acid = Acid Value

2

2.3.2.3 Determination of Iodine Value

About 0.5g of QUSO was weighed into conical flask and 20ml of carbon tetrachloride was

added to dissolve the oil. Wij’s reagent (25ml) was added to the flask using a measuring

cylinder. A stopper was inserted and the content of the flask was vigorously swirled and the flask

was then placed in the dark for 1 hour. At the end of this period, 20ml of 10% aqueous potassium

iodide and 100ml of water were added using a measuring cylinder. The content was titrated with

0.1M sodium thiosulphate solution. Few drops of 1% starch indicator were added and the

titration continued by adding the sodium thiosulphate drop wise until coloration disappeared

after vigorously shaking. The same procedure was used for the blank test as described by

Hamilton and Hamilton (1992). The Iodine Value (I.V) was calculated

Iodine Value (I.V) = 12.69C (V1 – V2)

M

Where

C = concentration of sodium thiosulphate

V1 = volume of sodium thiosulphate used for blank

V2 = volume of sodium thiosulphate used for determination

M = mass of sample

12.69= Constant.

2.3.2.4 Determination of Refractive index

Atago hand refractometer was used for this determination. The QUSO sample was smeared on

the lower prism of the instrument and the lid was closed. A light source was passed by means of

the angled mirrow and the reflected light appeared in form of a dark background. Using a fine

52

adjustment knob, the telescope tube was moved until the black show appeared central in the

cross wire indicator. The refractive index was then read as described by Nayak and Patel (2010).

2.3.2.5 Determination of Relative density

Density bottle was used to determine the density of QUSO. A clean and dry bottle of 25ml

capacity was weighed (W0) and then filled with the oil, stopper inserted and reweighed to give

(W1). The oil was substituted with water after washing and drying the bottle and weighed to give

(W2), as described by Bagali et al (2010). The expression for relative density is given below.

Relative density = (W1 – W0) (W2 – W0)

Where W0 = weight of empty specific gravity bottle.

W1 = weight of test sample + specific gravity bottle.

W2 = weight of water + specific gravity bottle.

2.3.2.6 Determination of Kinematic Viscosity

A clean, dried viscometer with a flow time above 200 seconds for the fluid to be tested was used.

The sample was filtered through Whatmann No 1 filter paper to eliminate dust and other solid

material in the liquid sample. The viscosity meter was charged with the sample by inverting the

tube’s thinner arm into the liquid sample and suction force was drawn up to the upper timing

mark of the viscometer, after which the instrument was turned to it’s normal vertical position.

The viscometer was placed into a holder that was maintained at a constant temperature set at

29°C and allowed approximately 10 minutes for the sample to come to the bath temperature at

29°C. The suction force was then applied to the thinner arm to draw the sample slightly above

the upper timing mark. The afflux time by timing the flow of the sample as it flowed freely from

the upper timing mark to the lower timing mark was recorded as determined by Bagali et al.,

2010. The kinematic viscosity was calculated using the following expression:

L Water = L oil

T water T oil

Where

L = Dynamic viscosity

T = Time of flow

Kinematic viscosity = Dynamic viscosity

Density of sample

53

2.3.2.7 Determination of Flash Point

This was determined using a crucible, thermometer and hot-plate. The QUSO sample (5ml) was

poured in the crucible and placed (uncovered) on the hot-plate, which was turned on. The

thermometer was then inserted into the oil and the temperature rise observed carefully. The

temperature at which the fuel just started to burn while on the naked red-hot filament of the hot-

plate was immediately noted and taken as the Flash Point. The observed flash points were

recorded from the thermometer after triplicate run and the mean value reported. The above

procedure was repeated for the various samples as described by ASTM method (2002).

2.3.2.8 Determination of Cloud Point

The cloud point of QUSO was determined by the ASTM method D 2500 (2002). A cooling bath

was prepared using a 4L jar. Salt was added to the ice in the jar to obtain temperatures in the

range of -3 to -1°C. About 15 mL of QUSO was placed in a glass jar and the temperature was

lowered until clouds of crystals appeared at the bottom of the jar. The temperature at which a

cloud of crystals first appeared was recorded as the cloud point.

2.3.2.9 Determination of Peroxide Value

About 30ml of acetic acid:chloroform (3:2) solution was added into a conical flask containing

5g of QUSO. Saturated KI solution (0.5ml) was then added and the solution swirled in the dark

for one min after which 30ml of distilled water was then added. The mixture was titrated with

0.01M sodium thiosulphate solution with vigorous shaking until the yellow colour disappeared.

Approximately 0.5ml of starch indicator solution was added and titration continued until all the

blue colour had disappeared. A blank experiment was carried out in which no oil was added to

the flask, as described by Hamilton and Hamilton (1992). The peroxide value was caiculated

from the formula:

Peroxide value = (Vs – Vb) M x 100

W

Where Vs = is the volume in milliliters of the sodium thiosulfate.

Vb = is the volume of the blank.

M = is the molarity.

W = is the mass of the oil sample.

54

2.4 Two-Step Biodiesel Production Process

In this research, Quassia undulata seed oil was converted into monoalkyl esters by a two-step

acid-base reaction process according to the method of Singh and Singh (2009).

A

B

C

D

E

2.4.1 Acid-Catalyzed Esterification

About 200g QUSO was transferred into the three neck round bottom flask and preheated to the

temperature of 65⁰C using a heating mantle. The sulphuric acid methanol solution was prepared

by adding 5% w/w of sulphuric acid (to oil ratio) in 40g of methanol. The methanolic solution

was then added to the QUSO in the reaction flask and stirred continuously maintaining a steady

temperature of 65⁰C, and at this point, the measurement of reaction time started. The reaction

continued for two hours. After the required reaction time, the reaction mixture was poured into

the separating funnel and was allowed settled for 1 hour. The excess methanol together with

sulphuric acid and impurities was moved to the top layer after settlement. The lower layer (oil

phase) was ready for alkali-catalyzed transesterification. The oil yield was about 377.9g.

2.4.2 Alkali-Catalyzed Transesterification

The esterified QUSO (lower oil phase) was transferred to the round bottom flask and preheated

to the reaction temperature of 65⁰C. Sodium hydroxide concentration of 0.7 weight percentage of

oil was dissolved in about 22ml of methanol (6:1 oil methanol mole ratio). The mixture of

sodium hydroxide in methanol was added to the QUSO in the round bottom flask, while stirring

the material of the flask. The required reaction temperature was maintained by controlling the

A. Stirrer, B. Thermo-well with thermometer,

C. Three neck flask, D. Heating mantle,

E. Condenser.

Fig 10: Experimental setup for the preparation of alkyl esters from QUSO.

55

electrical heating till the reaction was complete. After the reaction, the reaction mixture was

poured into a separating funnel and allowed to settle overnight. After separation, the methyl

esters (upper phase) was washed with water.

2.5 Physicochemical Properties of Fatty Methyl Esters (Biodiesel)

2.5.1 Determination of the Saponification Value

American Standard for Testing Material (ASTM) method D 5558-95 (2002) was used for the

determination of the Saponification value of QUSO methyl ester. Approximately 2g of Quassia

undulata seed oil (QUSO) biodiesel was weighed into a conical flask containing 25ml of 0.5M

ethanolic KOH and the resulting mixture was refluxed for 60 minutes. The resulting solution was

subsequently titrated against 0.5M HCl with phenolphthalein as indicator. The end point was

obtained when the pink colour changed into colourless. The same procedure was used for the

blank. The Saponification value (SV) was then calculated using the expression;

Saponification value (S.V.) = 56.1 (B-S) x M of HCl

Weight of sample

Where;

B = ml of HCl required by blank

S = ml of HCl required by sample

M = Molarity of HCl

56.1= Molar mass of KOH

2.5.2 Determination of acid value

Acid value of QUSO was determined by ASTM method D 974 00 (2002). About 0.2g of sample

was weighed into 250ml conical flask and 50ml of neutralized ethanol was added. The mixture

was heated on a water bath to dissolve the sample. The solution was titrated against 0.1M KOH

using phenolphthalein as indicator. The acid value was determined after which the free fatty acid

was calculated respectively as follows;

Acid Value = A x M x 56.1 W

Where,

A = ml of 0.1M KOH consumed by sample

M = Molarity of KOH

W = weight in grams of the sample

Free Fatty Acid = Acid Value

2

56

2.5.3 Determination of Iodine Value

About 0.5g of QUSO methyl ester was weighed into conical flask and 20ml of carbon

tetrachloride was added to dissolve the oil. Wij’s reagent (25ml) was added to the flask using a

measuring cylinder. A stopper was inserted and the content of the flask was vigorously swirled

and the flask was then placed in the dark for 1hour. At the end of this period, 20ml of 10%

aqueous potassium iodide and 100ml of water were added using a measuring cylinder. The

content was titrated with 0.1M sodium thiosulphate solution. Few drops of 1% starch indicator

were added and the titration continued by adding the sodium thiosulphate drop wise until

coloration disappeared after vigorously shaking. The same procedure was used for the blank test

as described by Hamilton and Hamilton (1992). The Iodine Value (I.V) was calculated from the

the expression:

Iodine Value (I.V) = 12.69C (V1 – V2)

M

Where

C = concentration of sodium thiosulphate

V1 = volume of sodium thiosulphate used for blank

V2 = volume of sodium thiosulphate used for determination

M = mass of sample

12.69= Constant.

2.5.8 Determination of Refractive index

Atago hand refractometer was used in this determination. The QUSO biodiesel was smeared on

the lower prism of the instrument and the lid was closed. A light source was passed by means of

the angled mirrow and the reflected light appeared in form of a dark background. Using a fine

adjustment knob, the telescope tube was moved until the black show appeared central in the

cross wire indicator. The refractive index was then read as determined by Nayak and Patel

(2010).

2.5.4 Determination of Relative density

Density bottle was used to determine the density of QUSO methyl ester. A clean and dry bottle

of 25ml capacity was weighed (W0) and then filled with the oil, stopper inserted and reweighed

to give (W1). The oil was substituted with water after washing and drying the bottle and weighed

57

to give (W2), as described by Bagali et al (2010). The relative density was calculated from the

equation:

Relative density = (W1 – W0)

(W2 – W0)

Where

W0 = weight of empty specific gravity bottle.

W1 = weight of test sample + specific gravity bottle.

W2 = weight of water + specific gravity bottle.

2.5.5 Determination of Kinematic Viscosity

A clean, dried Cannon fenske viscometer with a flow time above 200 seconds for the fluid to be

tested was elected. The sample was filtered through a Whatmann No 1 filter paper to eliminate

dust and other solid material in the liquid sample. The viscosity meter was charged with the

QUSO methyl ester sample by inverting the tube’s thinner arm into the liquid sample and suction

force was drawn up to the upper timing mark of the viscometer, after which the instrument was

turned to its normal vertical position. The viscometer was placed into a holder and inserted to a

constant temperature bath set at 29°C and allowed approximately 10 minutes for the sample to

come to the bath temperature at 29°C. The suction force was then applied to the thinner arm to

draw the sample slightly above the upper timing mark. The afflux time by timing the flow of the

sample as it flowed freely from the upper timing mark to the lower timing mark was recorded as

determined by Bagali et al., 2010. The kinematic viscosity is determined from the expression:

L Water = L oil

t water t oil

Kinematic viscosity = Dynamic viscosity

Density of sample

Where

L = Dynamic viscosity

T = Time of flow

2.5.6 Determination of Flash Point

This was determined using a crucible, thermometer and hot-plate. The QUSO methyl ester (5ml)

was poured in the crucible and placed (uncovered) in the hot-plate, which was turned on. The

thermometer was then inserted into the QUSO methyl ester and the temperature rise observed

carefully. The temperature at which the fuel just started to burn while on the naked red-hot

filament of the hot-plate was immediately noted and taken as the Flash Point. The observed flash

58

points were recorded from the thermometer after triplicate run and the mean value reported. The

above procedure was repeated for the various samples as described byh ASTM method (2010).

2.5.7 Determination of Cloud Point

The cloud point of QUSO methyl ester was determined by the ASTM method D2500 (2002). A

cooling bath was prepared using a 4L jar. Salt was added to the ice in the jar to obtain

temperatures in the range of -1 to -3°C. About 15 mL of QUSO were placed in a glass jar and the

temperature was lowered until clouds of crystals appeared at the bottom of the jar. The

temperature at which a cloud of crystals first appeared was recorded as the cloud point.

2.5.9 Determination of Peroxide Value

About 30ml of acetic acid:chloroform (3:2) solution was added into a conical flask containing

5g of QUSO methyl ester. Saturated KI solution (0.5ml) was then added and the solution swirled

in the dark for one min after which 30ml of distilled water was then added. The mixture was

titrated with 0.01M sodium thiosulphate solution with vigorous shaking until the yellow colour

disappeared. Approximately 0.5ml of starch indicator solution was added and titration continued

until all the blue colour had disappeared. A blank experiment was carried out in which no oil was

added to the flask, as described by Hamilton and Hamilton (1992). The peroxide value was

calculated from the equation:

Peroxide value = (Vs – Vb)M x 100

W

Where Vs = is the volume in milliliters of the sodium thiosulfate.

Vb = is the volume of the blank.

M = is the molarity.

W = is the mass of the oil sample.

2.6 Gas chromatography mass spectrometry analysis (GC MS) of QUSO

The GC MS analysis was performed with Shimadsu, GCMS-QP2010 series gas chromatography

mass spectrometer equipped with flame thermoionic detector (FTD). The column oven

temperature was initially set at 70°C. The column temperature was then increased to 280°C at

10°C/min with a hold time of 5min. The carrier gas was nitrogen at a column flow rate of

1.80mL/min and the total flow rate of 40.8mL/min. The analysis was performed at injector

temperature of 250°C and ion source temperature of 200°C and a split ratio of 20:0.

59

CHAPTER THREE

3.0 RESULTS

3.1 The yield of oil

Quassia undulata had an oil yield of 42.36%.

3.2 The physicochemical properties of QUSO

The physicochemical properties of QUSO are shown in table 6.

Table 6: Result of the physicochemical characterization of QUSO

S/n Parameters Units Value

1 Saponification value Mg KOH/gºº 207.57

2 Free fatty acid % 18.51

3 Iodine value mgI2/100g 42.68

4 Refractive index - 1.466

5 Relative density - 0.92

6 Kinematic viscosity Mm2/s 38.26

7 Flash point ºC 255

8 Cloud point ºC 23

9 Peroxide value mgEqI/kg 0.00

3.3 Yield of fatty methyl esters after transesterification

The biodiesel yield of 94.18% was obtained after the transesterification of the oil.

60

3.4 The physicochemical properties of QUSO Methyl Esters

The physicochemical properties of QUSO methyl esters are shown in table 7.

Table 7: Result of the physicochemical characterization of QUSO FAME

S/n Parameters Units Value

1 Saponification value Mg KOH/gºº 192.14

2 Free fatty acid % 0.33

3 Iodine value mgI2/100g 50.76

4 Refractive index - 1.447

5 Relative density - 0.89

6 Kinematic viscosity Mm2/s 6.06

7 Flash point ºC 209

8 Cloud point ºC 13

9 Peroxide value mgEqI/kg 0.00

61

3.3 Comparison of the properties of QUSO, QUSO FAME, Petroleum diesel with ASTM biodiesel

standards

Table 8: Properties of QUSO And FAME In Comparison to ASTM Biodiesel Standards

S/n Parameters QUSO QUSO

FAME

(Biodiesel)

Diesel* Biodiesel*

ASTM

Standards

1 Colour Golden

Yellow

Light yellow Light Yellow -

2 Physical state at room temperature

(°C)

liquid liquid liquid -

3 Percentage oil yield (%) 42.36 94.18 - -

4 Relative density 0.92 0.89 0.82 0.87-0.89

5 Kinematic viscosity (mm2/s) 38.26 6.06 2.25 1.9-6.0

6 Flash point (ºC) 255 209 66 130 min

7 Refractive index 1.466 1.447 - -

8 Cloud point (ºC) 23 13 - -

9 Iodine value (mgI2/100g) 42.68 50.76 38 120 max

10 Peroxide value (mgEqI/kg) 0.00 0.00 - -

11 Free fatty acid (%) 18.51 0.33 - 0.5 max

12 Saponification value (Mg KOH/g) 207.57 192.14 - -

*Source: Kinast, 2003

3.5 The fatty acids in QUSO as shown by GC/MS

The chemical composition of QUSO/FAME fractions were studied by gas chromatography.

Constituent compounds were identified and their composition recorded as shown in tables 9 and

10.

62

3.5.1 Chromatogram of QUSO

15.592

C17:0

16.150

C16:0

17.292

C19:1

17.492

C19:0

17.882

C18:1

18.025

C18:0

19.225

C21:0

20.542

C18:2

Table 9: Result Of fatty acids in Quassia Undulata Seed Oil By GC-MS Analysis

Name of compound Mol. Formula Mol. Weight Ret. time (min) Peak area (%)

Heptadecanoic acid C17H34O2 270 15.592 0.66

Hexadecanoic acid C16H32O2 256 16.150 10.45

11-Nonadecenoic acid C19H36O2 296 17.292 5.70

Nonadecanoic acid C19H38O2 298 17.492 1.63

6-Octadecenoic acid C18H34O2 282 17.882 46.36

Octadecanoic acid C18H36O2 284 18.025 33.44

Heneicosanoic acid C21H42O2 326 19.225 0.26

9, 11- Octadecenoic acid C18H32O2 298 20.542 2.17

Fig 11: Total Ion Chromatogram of QUSO

3.6 The fatty acids in QUSO methyl esters as shown by GC/MS

63

Table 10: Result Of Quassia Undulata Seed Oil Biodiesel By GC-MS Analysis

Name of compound Mol. Formula Mol. Weight Ret. time (min) Peak area (%)

Hexadecanoic acid methyl ester C17H34O2 270 15.625 14.46

Heptadecanoic acid methyl ester C18H36O2 284 16.567 0.50

9-Octadecenoic acid methyl ester C19H36O2 296 17.433 49.07

Octadecanoic acid methyl ester C19H38O2 298 17.575 24.13

9-Hexadecanal C16H30O 238 17.817 1.14

11-Eicosenoic acid methyl ester C21H40O2 324 19.058 1.29

Arachidic acid methyl ester C21H34O2 326 19.250 5.56

13-Octadecenal C18H34O 266 19.600 0.67

Behenic acid methyl ester C23H46O2 354 20.825 1.45

Fig 12: Total Ion Chromatogram of QUSO Fatty acid methyl esters

64

Heneicosanoic acid methyl ester C22H44O2 340 22.475 1.72

3.7 The fragmentation pattern of fatty acids as shown by Mass spectrometry in Fig 13.

Fig 13a: Mass spectrum at retention time (tR) 15.592

Fig 13b: Mass spectrum at tR 16.150

Fig 13c: Mass spectrum at tR 17.292

Fig 13d: Mass spectrum at tR 17.492

Fig 13e: Mass spectrum at tR 17.882

65

Fig 13f: Mass spectrum at tR 18.025

Fig 13g: Mass spectrum at tR 19.225

Fig 13h: Mass spectrum at tR 20.542

3.5.3 The fragmentation pattern of fatty methyl esters as shown by Mass spectrometry in

Fig 14.

Fig 14a: Mass spectrum at tR 15.625

Fig 14b: Mass spectrum at tR 16.567

66

Fig 14c: Mass spectrum at tR 17.433

Fig 14d: Mass spectrum at tR 17.575

Fig 14e: Mass spectrum at tR 17.817

Fig 14f: Mass spectrum at tR 19.058

Fig 14g: Mass spectrum at tR 19.250

67

Fig 14h: Mass spectrum at tR 19.600

Fig 14i: Mass spectrum at tR 20.825

Fig 14j: Mass spectrum at tR 22.475

3.8 Schematic Representation Of Fragmentation Patterns of QUSO/Methyl esters as shown

by MS

The fragmentation pattern of the major components in the oil and FAME were identified. These

mass features were consistent with known mass spectra stored in Shimadzu computer data base,

as well as literature reports (Kale et al., 2011; Fagerquist et al., 1999; Oyugi et al., 2011;

Ramamurthi et al., 1998; Aini et al., 2009). They were confirmed by the study of classical

fragmentation pattern, base peak and molecular ion peaks of the compounds.

3.8.1 Mass fragmentation of fatty acids in QUSO

The identity of the compound represented by the peak at retention time (t.R) 16.150 min was

determined by diagnostic ion peaks at m/z 73, 115, 129, 185, 213, 227 and 256. The ion at

mass/charge (m/z) 73 was the base peak obtained by Mc-Lafferty rearrangement as shown in fig

13b. The peaks at m/z 115, 129, 143, 157, 171, 185, 199, 213, 227, 239 arise from the loss of the

neutral aliphatic radicals with chain length of C3-C15. This uniform series of ions 14 atomic mass

units apart, representing the loss of successive methyl group indicates that no other functional

group occurs in the chain. Also, a diagnostic ion was observed at m/z 239 (M+- 17) which might

68

imply the loss of OH- from the carboxyl group (Kale et al., 2011). The fragment at m/z 115

indicated the loss C11H21 of mass 141; m/z 129 indicated the loss of 127 mass C9H29 ion; m/z 143

(M+- C8H17) of 113 mass while m/z indicated the loss of a butyl ion of mass 57. The molecular

ion appeared at m/z 256, agreeing with the condensed formula C16H32O2. Together, this

fragmentation pattern is typical of a saturated fatty acid called hexadecanoic acid (palmitic acid).

239

199

143

129

115

101

155

141

127

113

71

51

17

[CH3(CH2)14COO]*

[CH3(CH2)10COO]*

[CH3(CH2)9COO]*185

[CH3(CH2)7COO]*

[CH3(CH2)6COO]*

[CH3(CH2)5COO]*

[CH3(CH2)4COO]*

73

197

[CH2COOCH3]*

C14H29*

C11H23*

C9H19*

C8H17*

C5H11*

C4H9*

OH-

C10H21*

m/z 256m/z 256m/z 256m/z 256

Fig 15a: Fragmentation pattern of compound at m/z 256

Analysis of GC peak at t.R 17.883 min revealed prominent base peak at m/z 55 and other

unsaturated aliphatic radicals with m/z 69, 83, 97 which are indicative of the fragmentation

pattern in monoenoic series (Hartig et al., 2005). Ion at m/z 235 is typical of the loss of methoxy

radical (CH3O-) of mass 31. Ion at m/z 97 (C7H13)

+ is a heptyl radical as shown in fig 13e. The

spectrum also shows a uniform series of ions 14 atomic mass units apart at m/z 55, 69, 83, 97,

Fig 15: Fragmentation by Lafferty rearrangement (Kale et al., 2011)

69

111 and then a characteristic diagnostic fragment between m/z 125 and 137 of 12 atomic mass

units indicating the location of the double bond between C9 and C10, in contrast to the normal

fragmentation of 14 units for methyl groups in the fatty acyl chain. From the fragmentation

pattern, the compound was identified as 6-octadecenoic acid (oleic), of the formula C18H34O2.

[CH3(CH2)5COO]*

[CH3(CH2)3COO]*

C7H13*

C5H9*

C4H7*

C6H11*

C8H15*

C9H17*

C10H19*

C11H21*

C13H25*

m/z 282m/z 282m/z 282m/z 282

227

213

199

185

171

157

143

129

101

179

151

55

69

83

97

111

125

137

[CH3(CH2)12COO]*

[CH3(CH2)11COO]*

[CH3(CH2)10COO]*

[CH3(CH2)9COO]*

[CH3(CH2)8COO]*

[CH3(CH2)7COO]*

[CH3(CH2)6COO]*

O

O

Fig 15b: Fragmentation pattern of compound at m/z 282

The GC peak at 18.025 min displayed a molecular ion at m/z 284, suggesting the structural

C18H36O2. Loss of (C4H9)+ resulted in a 227 mass C14H27O2 fragment. Other characteristic ions

include m/z 185 (M+- C7H15) generated for loss of heptyl radical of mass 99; m/z 241 (M

+- C3H7)

for the loss of propyl radical of mass 43 which are typical of saturated fatty acids (Hartig et al.,

2005). The prominent fragment ion at m/z 73 conclusively proved that its formation was the

result of intramolecular γ-hydrogen transfer to the ionized carbonyl oxygen via the hexacyclic

transition state followed by Cα - Cβ bond cleavage (Fagerquist et al., 1999). This reaction is

70

generally referred to as Mc-Lafferty rearrangement as shown in fig 15. In addition, the spectrum

shows ions representing fragmentations between methyl groups of the form [HOOC(CH2)n]+

from m/z 73 to 255. An ion at m/z 267 (M+- 17) presumably reflected a loss of OH

- from the

carboxyl group (Kale et al., 2011). The molecular ion and fragmentation pattern confirmed the

identity of the compound as octadecanoic acid (stearic).

255

227

213

199

185

171

143

87

73

211

197

141

113

99

85

71

57

29

[(CH2)14COOH]*

[(CH2)13COOH]*

[(CH2)12COOH]*

[(CH2)11COOH]*

[(CH2)10COOH]*

[(CH2)9COOH]*

[(CH2)7COOH]*

[(CH2)3COOH]*

[(CH2)2COOH]*

C15H31*

C14H29*

C10H21*

C8H17*

C7H15*

C6H13*

C5H11*

C4H9*

C3H7*

m/z 284m/z 284m/z 284m/z 284 Fig 15c: Fragmentation pattern of compound at m/z 284

The mass spectrum of peak at t.R 17.292 min showed an ion at m/z 55 (base peak), characteristic

of unsaturated fatty acids. Other major diagnostic ions were at m/z 69, 83, and 97 which

indicates the loss of fragments in monoenoic series (Hartig et al., 2005). Fragments at m/z 55

(M+- C4H7) indicates the loss of a 241 mass ion C15H29O2; m/z 83 (M

+- C6H11) indicates the loss

of C13H25O2 of mass 213; m/z 95 (M+- C7H13) indicates the loss of C12H23O2 ion mass 197. The

peaks at m/z 264 and 265 corresponds to the loss of methanol (CH3OH) and methoxy (CH3O-)

radicals. The characteristic fragmentation ions observed at m/z 74 and 222 are the result of a

rearrangement and fragmentation of the molecular ion, i.e, Mc-Lafferty rearrangement

(Fagerquist et al., 1999). The spectrum also shows a uniform series of ions 14 atomic mass units

71

apart, representing loss of each successive methyl group from m/z 55 to 111. Then there is a gap

of 12 amu between m/z 111 and 123 which suggests the location of the double bond between C11

and C12 in the fatty acyl chain. The molecular ion for the compound occurred at m/z 296, a useful

confirmation that we have a C19 fatty acid with a double bond. This fragmentation pattern

confirmed the compound nonadecenoate of the formula C19H36O2.

55

69

83

97

137

151

165

222

74

131

145

159

199

213

227

241[(CH2)13COOCH3]*

[(CH2)12COOCH3]*

[(CH2)11COOCH3]*

[(CH2)10COOCH3]*

[(CH2)7COOCH3]*

[(CH2)6COOCH3]*

[(CH2)5COOCH3]*

[CH2C(OH)OCH3]*

C16H38*

C12H21*

C11H19*

C10H17*

C7H13*

C4H7*

C5H9*

C6H11*

m/z 296m/z 296m/z 296m/z 296 Fig 15d: Fragmentation pattern of compound at m/z 296

3.8.2 Mass fragmentation of Fatty methyl esters in QUSO

The identity of the compound represented at tR 15.626 min was based on major spectra ion

peaks. The molecular ion at m/z 270, as well as the ion m/z 239 (M+- 31) representing loss of a

methoxy group, are diagnostic of methyl ester ( Oyugi et al., 2011). The base peak of the

spectrum is m/z 74 as shown in fig 14a. This fragment results from a Mc-Lafferty rearrangement

(Fagerquist et al., 1999), which transfers a γ-hydrogen atom of the acid moiety to the carbonyl

oxygen through a cyclic transition state, and cleaves the C2 - C3 bond to give two fragments as

shown in fig 12. Fragment ions at m/z 227 (M+- 43) represents loss of a C3 unit (C3H7) ion.

Fragment at m/z 87 indicates loss of a 183 mass C13H27 ion; m/z 129 (M+- C10H 21) of 141 mass;

m/z 157 (M+- C8H17) of 113 mass; m/z 171 (M

+- C7H15); m/z 185 (M

+- C6H13); m/z 213 (M

+-

72

C4H9); m/z 227 (M+- C3H7). Then there is a uniform series of ions 14 atomic mass units (amu)

apart, representing loss of each successive methyl and group (Kale et al., 2011) from the terminal

end of the molecule. The spectrum was distinct, with intense molecular ion and characteristic

diagnostic ion identifying the compound as Hexadecanoic acid methyl ester, of formula

C17H34O2.

241

227

213

171

157

129

87

183

141

113

99

85

57

43

29

[(CH2)13COOCH3]*

[(CH2)12COOCH3]*

185

[(CH2)11COOHCH3]*

[(CH2)9COOCH3]*

[(CH2)8COOCH3]*

[(CH2)7COOCH3]*

C2H5*

C3H7*

C4H9*

C6H13*

C7H15*

C8H17*

C10H21*

C13HH27*

m/z 270m/z 270m/z 270m/z 270

Fig 16a: Fragmentation pattern of compound at m/z 270

The base peak for the compound represented at t.R 17.433 min is m/z 55. The fragmentation

pattern in this compound is dominated by unsaturated aliphatic radicals with mass 55 (base

peak), 69, 83 and 97. The molecular ion is seen at m/z 296. Again, there is a distinct fragment

with prominent ions for the loss of methanol (M+- 32) at peak ion m/z 264 and of a methoxy

radical (M+- 31) at peak ion m/z 265. The peak at m/z 43 in the spectra of fatty esters results

from the loss of a propyl radical from the molecular ion to form a fragment of the type

(CH2)nCO2CH3+ (Kale et al., 2011). The fragment at m/z 55 (C4H7) indicates the loss of

[(CH2)13COOCH3] mass 241; fragment at m/z 69 (C5H7) indicates the loss [(CH2)12COOCH3]

mass 227; fragment at m/z 83 C6H 11 indicates the loss of [(CH2)11COOCH3] mass 213; fragment

73

at m/z 97 (C7H13) indicates the loss of [(CH2)10COOCH3] mass 199. These fragments (m/z 55,

69, 83, 97) are diagnostic of monoenoic series (Hartig et al., 2005). The fragments at m/z 74 and

222 results from a Mc-Lafferty rearrangement via a hexacyclic transition state which is followed

by Cα - Cβ bond cleavage. There is a gap of 14 atomic mass units for loss of methyl group at m/z

97 and then characteristic diagnostic fragment ions at m/z 111 and 123, consistent with the loss

12 atomic mass units which indicates the location of a double bond between C9 and C10

(Ramamurthi et al., 2008). From the fragmentation pattern, the compound is identified as methyl

octadec-9-enoate, of formula C19H36O2.

C9H17*137

159[CH3(CH2)6COOCH3]*

241

227

213

111

83

69

55

[(CH2)13COOCH3]*

[(CH2)12COOCH3]*

185

[(CH2)11COOCH3]*

[(CH2)9COOCH3]*

C4H7*

C5H9*

C6H11*

C8H15*

31 CH3O-

74

222

[CH2C(OH)OCH3]*

265C18H33O*

C16H30*

[(CH2)10COOCH3]*199

C7H13*97

m/z 296m/z 296m/z 296m/z 296 Fig 16b: Fragmentation pattern of compound at m/z 296

The compound represented at t.R 17.525 min has a base peak at m/z 74 and a molecular ion at

m/z 298. The prominent fragment at m/z 74 (base peak) is diagnostic of a long chain aliphatic

fatty acid of methyl ester (Oyugi et al., 2011) confirming that the formation of the compound

was a result of site-specific rearrangement of atoms, in which γ-hydrogen from portion 4 of

aliphatic chain is transferred to the carbo-methoxy group, through a sterically-favoured six-

74

membered transition state (Mc-Lafferty rearrangment) followed by Cα - Cβ bond cleavage as

shown in fig 12. Fragments at m/z 87 indicates loss of a 211 mass C15H31 ion; m/z 143 (M+-

C11H23) of 155 mass; m/z 199 (M+- C7H15) indicates the loss of a heptyl radical of 99 mass; m/z

255 (M+- C3H7) indicates loss of propyl radical of 43 mass; m/z 57 [M

+- (CH2)13COOCH3]

indicates the loss of butyl ion of 241 mass. The peaks at m/z 87, 143, 199, 255 arise from loss of

the neutral aliphatic radicals with chain lengths C3-C15. The cleavage at the carbonyl carbon and

loss of methoxy radical gives the peak at 267. The fragments at m/z 87, 101, 115, 129, 143, 157,

171, 185, 199, 213, 227, 241, 255 shows a uniform series of ions 14 atomic mass units apart,

formed by the loss of neutral aliphatic radicals of the general formula [(CH2)nCOOCH3]+

which

indicates no occurrence of other functional groups in the chain (Oyugi et al., 2011). This

fragmentation pattern and molecular ion are diagnostic of the compound identified as

Octadecanoic acid methyl of the formula C19H38O2.

[(CH2)3COOCH3]*

[(CH2)2COOCH3]*

197

143

255

241

185

101

87

211

155

113

85

57

43

[(CH2)13COOCH3]*

[(CH2)12COOCH3]*

199[(CH2)10COOCH3]*

[(CH2)9COOCH3]*

[(CH2)6COOCH3]*

C3H7*

C4H9*

C7H15*

C8H17*

C11H23*

C14H29*

C15H31*

m/z 298m/z 298m/z 298m/z 298

Fig 16c: Fragmentation pattern of compound at m/z 298

75

The mass spectrum of the compound represented at t.R 19.250 min shows a fragment at m/z 74

(base peak) which is due to the Mc-Lafferty rearrangement in the alkyl portion of the compound.

This fragment is characteristic of the base peak of saturated fatty acids (Fagerquist et al., 1999).

The spectrum also shows a particularly strong signal at m/z 87 [(CH2)2COOCH3]+ indicative of

the removal of the fragment (C15H31)+ ion of mass 211 which occurs in aliphatic methyl esters

(Bhagdeo, 2004). The molecular ion at 326, as well as ion at m/z 295 (M+- 31) representing a

methoxy group are diagnostic of methyl ester (Oyugi et al., 2011). Other distinct fragments

observed include m/z 87 which indicates loss of a 239 mass C17H33 ion; m/z 129 (M+- C14H29) of

197 mass; m/z 143 (M+- C13H27) of 183 mass; m/z 157 (M

+- C12H25) of 169 mass; m/z 171 (M

+-

C11H23) of mass 155; m/z 199 (M+- C9H19) of mass 127; m/z 255 (M

+- C5H11) of mass 71; m/z

269 (M+- C4H9) of mass 57; m/z 283 (M

+- C3H7) of mass 43. A homologous series of related ions

at 87, 101, 129, 143, 157, 171, 185, 199, 213, 227 etc shows the sequential loss of methylene

groups in the chain. This is diagnostic of the occurrence of no other functional group in the chain

(Oyugi et al., 2011). The fragmentation patterns suggested the presence of Arachidate methyl

ester of the formula C21H42O2.

283

269

255

199

185

157

143

129

115

101

87

239

225

211

197

183

169

141

127

71

57

43

[(CH2)16COOCH3]*

[(CH2)15COOCH3]*

[(CH2)14COOCH3]*

[(CH2)10COOCH3]*

[(CH2)19COOCH3]*

[(CH2)7COOCH3]*

[(CH2)6COOCH3]*

[(CH2)5COOCH3]*

[(CH2)4COOCH3]*

[(CH2)3COOCH3]*

[(CH2)2COOCH3]*

C17H35*

C16H33*

C15H31*

C14H29*

C13H27*

C12H25*

C10H21*

C9H19*

C5H11*

C4H9*

C3H7*

m/z 326m/z 326m/z 326m/z 326 Fig 16d: Fragmentation pattern of compound at m/z 326

76

CHAPTER FOUR

DISCUSSION AND CONCLUSION

4.1 DISCUSSION

Plant oils are normally extracted from oil-containing seeds that use oil rather than starch as an

energy store for the seeds. Quassia undulata seed oil gave an oil yield of 42.36%. The oil content

of Quassia undulata seed was found to be higher than those of well known oil seeds such as

linseed (33.33%), soybean(18.35%), [Gunstone, 1999], Sterculia foetida (32.44%) [Kale et al.,

2011] and Afzelia Africana (18.5%) [Igwenyi et al., 2011] but comparable to Sterculia setigera

oil(45.0%) [Kai and Pryde, 1982] and palm kernel oil (45.6%) [Eze, 2012]. The presence of the

alkaloid, quassin, makes Quassia undulata seed oil less useful for nutritional purposes and less

competitive for other applications. Quassia undulata seed oil is therefore suitable as a non-

edible, lower valued vegetable oil feedstock that can provide a commercial quantity of oil for the

production of biodiesel. An earlier report by Louppe et al (2008), gave the percentage oil content

of Quassia undulata seed oil as 56%. The difference in the yields of vegetable oils might be

attributed to environmental factors such as rainfall, soil fertility, maturation period, agronomic

practices and genetic substitutions (Asuquo et al., 2010).

Saponification value is an index of the average molecular weight of the fatty acids present in oil.

The saponification value obtained for QUSO 207.57mgKOH/g was comparable to those of

Jatropha andrieuxii (Rodriguez-Acosta et al., 2010), palm kernel seed, fresh groundnut oil

(Afolabi, 2008), and linseed (Singh and Singh, 2009) which are 202.5, 191.97, 199.19 and 195.0

respectively. Higher relative weights were reported for oils like 144.25 for Thevitia nerrifolia

(Abayeh et al., 2007), 163.39 for almond nut oil (Afolabi, 2008), and 151.48 for fluted pumpkin

(Osai, 2011). Saponification values have been reported to be inversely related to the average

molecular weight of the fatty acids in the oil fraction (Eze, 2012). The high saponification value

in QUSO indicates an increase in the volatility of the oil. It enhances the quality of the oil

because it shows the presence of lower molecular weight components in 1g of the oil which can

give high amount of energy on combustion (Igwenyi et al., 2011).

The acid value is a measure of quality. Acid value corresponds to the amount of potassium

hydroxide required to neutralize the free fatty acid. The acid value of QUSO 37.02mgKOH/g

was comparable to values reported for vegetable oils such as 36.46 for Jatropha curcas (Nayak

77

and Petal, 2010), and 36.14 for fresh groundnut oil (Afolabi et al., 2008). On the other hand, the

acid value of QUSO was much higher than the values for common vegetable oils such as 0.27 of

castor seed oil (Afolabi, 2008), 1.79 of shea butter oil (Asuquo et al., 2010) and 4.49 of Afzerlia

africana (Igwenyi et al., 2008). The the acid value of QUSO, however, falls below the value of

44.88 reported by Eze (2012) for Dacryotes oil. The high acid value of QUSO indicates that the

oil contains high amount of FFAs and will be more suited for two stage transesterification

process to obtain reasonable biodiesel yields.

Iodine value of oil is a measure of its degree of unsaturation and it is a useful decisive factor for

purity and identification (Igwenyi et al., 2011). The iodine value indicates the tendency of an oil

to be unstable as it measures the presence of C=C bonds that are prone to oxidation (Parthiban, et

al., 2011). The iodine value of QUSO 42.68mgI2/g was comparable to values of 54.24 for palm

oil (Da-Silva et al., 2006), 63.45 for shea butter (Asuquo et al., 2010) and 62.66 for Thevitia

nerrifolia (Abayeh et al., 2007). Higher iodine values were reported for vegetable oils like 145

for safflower oil, 131for soybean (Eromosele, 1993), 104.46 for Jatropha curcas (Archana et al.,

2011), and 159.0 for castor oil (Bello and Makanju, 2011). Low iodine value oils have higher

cetane values and are more efficient fuels than high iodine value oils. Biodiesel made from

QUSO, with a low iodine value, will also be less susceptible to oxidation and polymerization

reactions.

The refractive index of oil is the ratio of the speed of light in vacuum at a defined wavelength to

its speed in the oil. The refractive index 1.466 of Quassia undulata seed oil is comparable to the

value 1.470 for Afzelia africana (Igwenyi et al., 2011) and 1.49 for Jatropha curcas oil

(Archcana et al., 2011) while it falls below the value of 1.6 for shea butter oil (Asuquo et al.,

2010). The refractive index of oil increases with increase in unsaturation and chain length of fatty

acids (Nayek and Petal, 2010). Since the refractive index measurements vary during processing

(hydrogenation) and use (polymerization during heat treatments), it can be used successfully in

quality control. Refractive index measurements rarely provide sufficient information to

quantitatively identify a pure analyte, but are highly useful to check oil containment/adulteration

(Parthiban et al., 2011).

Relative density is the ratio of the weight of a given volume of oil to the weight of an equal

volume of water (Yoon et al., 2008). The differences between the relative densities of oil are

quite small, particularly among common vegetable oils. Jatropha curcas (Belewu et al., 2010),

78

fluted pumpin (Osai, 2011), and shea butter (Asuquo et al., 2010) have the specific gravities of

0.901, 0.908 and 0.92 respectively, which are comparable to the value of 0.901 for Quassia

undulata seed oil while rapeseed oil has a relative density of 0.882 which is lower than that of

QUSO. Generally, the relative density of oil decreases with molecular weight, but increases with

unsaturation level (Gunstone, 1999). According to Hoffmann (1986), if measured to four or five

decimals, relative density can be used for hydrogenation control since its value changes with the

degree of unsaturation. Quassia undulata seed oil has a specific gravity that is less than that of

water. Vegetable oils with high relative density values are more susceptible to oxidation and

polymerization reaction when exposed to high temperature and oxygen.

Kinematic viscosity is the ratio of the dynamic viscosity of a fluid to its density. The kinematic

viscosity of QUSO 38.26 mm2/s is lower than the values of 53.6 for crambe oil (Singh and Singh,

2010), 58.5 for sunflower oil, 50.1 for cotton seed oil, 65.4 for soybean oil and 46.3 for corn oil

as reported by Demirbas (2005). Viscosity increases with molecular weight of the oil and

decreases with increasing unsaturation level and temperature (Akbar et al., 2009). The high

viscosity value of QUSO indicates that it may not be suitable as a straight vegetable oil source.

The flash point is considered a rough measure of the tendency of the oil to vaporize (Popovich

and Hering, 1959). Flash point is normally used as a safety index for biodiesel. The flash point of

Quassia undulata seed oil (255°C) is lower than the value 214°C reported for Jatropha curcas

oil (Raja et al., 2011). The fundamental reason for measuring flash point is to assess the safety

hazard of a liquid with regards to its flammability and then classify the liquid into a recognized

hazard group. The high flash point of QUSO is important from the view point of safe handling,

storage and transportation.

Cloud point corresponds to the temperature at the onset of crystallization of the oil. It is

therefore, an index of the lowest temperature of the oil usability for certain applications. The

cloud point of QUSO (23°C) is higher than those of common vegetable oils such as the value of

18°C for safflower, 12.8°C for peanut, and 20°C for babassu oil, while the value of 31°C for

palm oil (Singh and Singh, 2010) is high compared to QUSO. This implies that operating at

temperatures below the cloud point of oils with high cloud point values can result in wax crystal

formation. QUSO has a better potential in warm climatic areas as a biodiesel source.

79

Peroxide value is used to monitor the development of rancidity through the evaluation of the

quantity of peroxide generated in the product. Peroxide value in QUSO is zero. Result for the

peroxide value of QUSO is similar to that reported by Eze (2012) for pumpkin and African bean.

Generally, low levels of PV are indicative of early stages of the rancidity due to oxidation and

may suggest the presence of potent antioxidants. Low peroxide value implies that the oil may not

be easily susceptible to deterioration.

The analysis of QUSO biodiesel revealed the saponification value of 192.14mgKOH/g which is

similar to the saponification value of 195.0 for Jatropha curcas reported by Raja et al, (2011).

Saponification value is used as a guide to monitor the average size of the fatty methyl esters

present in the fuel. According to Igwenyi et al. ( 2011) Saponification values are very important

in fuels as they show the amount of energy the fuel contains on combustion. It also indicates the

level of saturation of fatty methyl esters (Parthiban, 2011).

The acid value measure the content of FFAs in the sample. The presence of FFAs influence fuel

aging due to hydrolytic cleavage of ester bonds. The suggested ASTM standard for pure

biodiesel sets the maximum FFA value of 0.5%. Analysis of QUSO biodiesel gave the FFA

value of 0.33% which is within the specified range. The reduction in the FFA reported in this

research (from 18.51-0.33) via 2 step acid-base transesterification was similar to the results by

Canaki and Gerpen (2001) and Berchmann and Hirata (2008). The FFA contents were reduced to

less than 1% in the first process which served as pretreatment step. Acid value is affected by the

type of feedstock used for fuel production and its respective degree of refinement. This is an

important variable in considering the quality of fuel because the lower the value, the better the

quality of fuel.

Iodine values are useful for determination of overall degree of saturation of biodiesel. The iodine

value of QUSO biodiesel (50.76 gI2/100g) is similar to the valhue of 60 reported for biodiesel

produced from waste cooking oil (Shalaby and El-Gendy, 2012) and it is slightly lower than the

value of 80 for castor oil biodiesel (Bello and Makanju, 2011). ASTM specifies that iodine value

should be less than 120gI2/100g of sample. Biodiesels with low iodine values leave very small

carbon deposits on the injector and combustion chamber thus improving life of components. Eze

(2012) reported that, iodine value depends on the feedstock of origin and this greatly influences

fuel oxidation tendency. This implies that most of the fatty acids in QUSO biodiesel have low

unsaturation and should be relatively stable. The lower the iodine value, therefore, the better the

80

fuel as biodiesel. Fatty acids with high unsaturation are prone to air oxidation and polymerization

reactions which may lead to blockage of filters and fuel lines during storage (Abayeh et al.,

2007).

The relative density of biodiesel was determined as 0.872. The standard for biodiesel states that

the fuel should have a density between 0.870 – 0.89g/cm3. The density of oils utilized for

biodiesel production is usually higher than their biodiesel derivatives. QUSO has a density of

0.901 which was reduced after transesterification to 0.872. This is in agreement with the result

reported by Singh and Padhi (2009) for the transesterification of Jatropha curcas oil and

Demirbas (2005) for sunflower, cotton seed and soybean oil transesterification. This property is

important mainly in airless combustion systems because it influences the efficiency of

atomization of the fuel (Rao, 2009). The relative density of QUSO, therefore, would give a high

energy per unit volume of biodiesel fuel. The density might also serve as an indicator of the

completeness of the transesterification reaction (Dias et al., 2008).

The main reason for converting vegetable oils to biodiesel is to reduce the viscosity of the oil

since oils have higher viscosity relative to their corresponding biodiesel fuel (Abayeh et al.,

2007). The kinematic viscosity of QUSO methyl ester is lower than the viscosity of methyl esters

produced from common vegetable oils such as sunflower (10.3), cottonseed (11.1) and soybean

oil (11.1) as reported by Demirbas (2005). According to ASTM (2002) specification, for

biodiesel to be used in diesel engines, the kinematic viscosity must be between 1.9 and 6.0mm2/s.

The kinematic viscosity of QUSO biodiesel is 6.067. Viscosity is very much related to the

chemical structure of the fuel. It is inversely related to the number of double bonds and increases

with increasing length of fatty acid chain (Knothe, 2002b). The viscosity of QUSO stands at a

safe value with reference to the standard specification. High viscosities lead to negative impacts

on fuel injection performance leading to deposition on the injectors.

The flash point is used as a safety index for biodiesel fuel. It also indicates the level of

purification the fuel has undergone as the presence of a very small amount of alcohol in the

biodiesel leads to a significant drop in the flash point (Abayeh et al., 2007). The flash point

varies inversely with the fuel volatility (Rao, 2009). The flash point of QUSO biodiesel (209°C)

is higher than that of conventional diesel fuel (66°C) as shown in table 8 because QUSO

biodiesel does not have the light fractions. The flash point of QUSO reduced after

transesterification into methyl ester. This observation is due to the increase in volatile

81

components in the fuel. This reduction in flash point of biodiesel complies with the report of

Sivaramakrishnan and Ravikumar (2012) for the methyl esters from common oils such as palm,

peanut, rapeseed and sunflower. The flash point of QUSO biodiesel is higher than the petroleum

diesel fuel which makes it safer. It is a very important parameter to be determined considering

handling, storage and safety of the fuel (Dias et al., 2008).

Cloud point is one of the key flow properties for winter fuel specifications. All biodiesel exhibit

poor cold flow properties with cloud points higher than those of petroleum diesel fuel

(Ewerenmadu, 2011) and the same applies to QUSO biodiesel. The cloud point of QUSO (23°C)

was significantly reduced (from 23-13°C) through transesterification to biodiesel fuel. This is in

agreement with biodiesels derived from common vegetable oils such as sunflower (7.2-12), palm

(31-13), and babassu oil (20-4) as reported by Singh and Singh, (2010). According to ASTM

standards, no limit is specified for cloud point. The reason is that the climatic conditions in the

world vary considerably, thus affecting the needs of biodiesel users in a specific region.

The fatty acid compositions of QUSO are presented in table 10. A total of eight fatty acids were

identified, ranging from C16 – C21. The major fatty acids that occurred in the QUSO are

Octadecanoic acid (33.44%), hexadecanoic acid (10.45%), 9-Octadecenoic acid (46.36%), and

11-Nonadecenoic acid (5.70%). The remaining components occurred in small percentages as

shown in table 9.The saturated fatty acids were 46.44%, monounsaturated acids were 52.06%

while a polyunsaturated fatty acid, 9, 12-Octadecenoic acid was found as a minor component

(2.17%). The result of the GC analysis of QUSO has been reported elsewhere (Louppe et al.,

2008). Octadecanoic, 9-Octadecenoic and 9,12-Octadecenoic acids were the most abundant

saturated, monounsaturated and polyunsaturated fatty acids from the report of Louppe and

colleagues, which corresponds to the findings from this research. The composition and nature of

fatty acids present in triglycerides are transferred to the biodiesel made from them and so are

other properties. The physicochemical characteristics of oils rich in long chain and low

unsaturation level of fatty acids make them better resources for biodiesel production (Pinto et al.,

2005). The fatty acid profile of QUSO has significant impact on the positive properties and

potential application of QUSO for biodiesel production.

The table 11 indicates that the FAME from QUSO contained mainly 9-Octadecenoic acid methyl

ester and Octadecanoic acid methyl ester, which are comparable to fatty acid composition in

QUSO feedstock. A total of ten fatty acids were identified in the FAME ranging from C16 – C22.

82

The saturated FAME (47.82%) were mainly Hexadecanoic acid methyl ester (14.46%), followed

by Octadecanoic acid methyl ester (24.13%) and Aracidic acid methyl ester (5.56%) and a few

minor ones. In the group of monoene families (50.36%), the two major fatty acids are 9-

Octadecenoic acid methyl ester (49.09%) and 11-Eicosenoic acid methyl ester (1.29%). Besides

this, two fatty acid derivatives, 9-Hexadecenal and 13-Octadecenal, also occurred in minor

amounts. The degree of saturation and chain length determines many important properties of

fatty acid methyl esters (biodiesel) including resistance to oxidation, viscosity, cloud and flash

points. Monounsaturated fatty acid methyl esters have been reported to strike the best balance

between cold flow properties and oxidative stability to enhance biodiesel test results and produce

a better burning fuel. In this connection, methyl ester has been suggested by many as a

compound of enrichment in biodiesel fuel for improving fuel properties (Knothe, 2008). Analysis

of oils to determine the type and relative abundance of its fatty acid content would provide a

useful guide in their selective applications for biodiesel production.

4.4 CONCLUSION

QUSO occupies a special place among the oil plants suitable for biodiesel production because it

is less useful for nutritional purposes and less competitive for other applications.

The QUSO produced in this research was analyzed for the physical and chemical properties.

Most of the properties evaluated conformed to the ASTM and EN standard values. Due to the

high FFA content of QUSO, a two step reaction was preferred to process the oil. The

pretreatment process using a strong acid catalyst has shown to provide good conversion yields

and high final product. Therefore, the experimental result confirms the effectiveness of the 2 step

acid-base transesterification as a promising area of research for the production of biodiesel in

large scale.

The produced biodiesel is within the recommended standards of biodiesel fuel, good in quality

and with satisfactory yield. Biodiesel produced from QUSO can be considered a suitable

alternative fuel for use in Nigeria.

The GC MS analysis of QUSO revealed the component fatty acids and fatty methyl esters

profiles. Hexadecanoic acid (stearic) and Octadecenoic acid (oleic) were found to be the

predominant acids in the oil and biodiesel. From the fatty methyl ester profile, it could be

concluded that the biodiesel is an Oleic-Stearic acid biodiesel. In addition, the overall results

83

showed that it was effective to produce good quality biodiesel from QUSO judging by the

physicochemical properties and the unique fatty acid profile.

84

REFERENCES

Abayeh, O. J., Onuoha, E. C., and Ugah, I. A. (2007). Transesterified Thevitia nerifolia seed

oil as biodiesel fuel. Global Journal of Environmental Research. 1(3): 124-127

Adebayo, G. B, Ameen, O. M., and Abass, L. T (2011). Physicochemical properties of

biodiesel produced from Jatropha curcas oil and fossil diesel. Journal of

Microbiology and Biotechnology Resources 1:12 – 16.

Adesanwo, J. K., Aiyelaagbe, O. O. and Moronkola, D. O. (2009). Antimicrobial and

cytotoxic activity of the stem and root bark extracts of Quassia undulata and

isolated constituents. Toxicological and Environmental Chemistry. 91(5): 999-

1003

Afolabi, I. S. (2008). Chemical qualities of oils from some fresh and market vegetable crops

within Kwara state of Nigeria. Biokemistri. 20(2): 71-75

Aini, N. M., Yusoff, M. M. and Azhari, H. A. (2009). Chromatographic methods to analyze

geometrical and positional isomers of fatty acids: A review. National Conference

On Postgraduate Research (NCON-PGR), Universiti Malaysia Pahang. Pp 165-

175

Ajaiyeoba, E. O., Abalogu, U. I. and Oduola, A. M. (1999). Invivo antimalarial activities of

Quassia amara and Quassia undulata plant extracts in mice. Journal of

Ethnopharmarcy. 67: 321-325

Akbar, E., Yaakob, Z., Kamarudin, S. K., Ismail, M. and Salimon, J. (2009). Characterization

and composition of Jatropha curcas oil seed from Malaysia and its potential as

biodiesel feedstock. European Journal of Scientific Research. 29: 396-403

Alamu, O. J., T. A. Akintola, Enweremadu, and Adeleke, A. E. (2008). Characterization of

palm-kernel oil biodiesel through NaOH catalysed transesterification process.

Scientific Research and Essay 3:308-311

Al-Zuhair, S. (2008). Enzymatic production of biodiesel from waste cooking oil using

lipase. The Open Chemical Engineering Journal. 2: 84-88

Archana, J., Pankaj, S. and Bachheti, R. K. (2011). Physicochemical characterization of seed

oil of Jatropha curcas collected from Dehradun (Uttarakhand) India.

International Journal Of Applied Biology and Pharmaceutical Technology. 2:

127-130

ASTM international (2002). Standard Test Methods for Biodiesel (B 100) Analysis. Annual

book of standards, D 6751. ASTM international, West Conshohoncken. Pp 734-

738

Asuquo, J. E and Anusiem, A. C. I. (2010). Extraction and Characterisation of shea butter oil.

World Journal of Applied Science and Technology 2. 282–288.

85

Ayugi, D. A., Ayorinde, F. O., Gugssa, A., Allen, A., Izevbigie, B. E., Eribo B. and

Anderson W. A. (2011). Biological activity and mass spectrometric analysis of

Vernonia amygdalina fractions. Journal of Biosciences and Technology. 2(3):

287-304

Bagali, S., Shridhar K. V., Beena M. V., and Paramjeet K. B. (2010). Optimization and

characterization of castor seed oil. Leonardo Journal of sciences 17: 59-70.

Belewu, M. A, F. Adekola, Adebajo G. B, Ameen, O. M., Muhammed, N. O, Olaniyan, A.

M., Adekola, O. F. and Musa, A. K. (2010). Physico chemical characterization of

biodiesel from Nigerian and Indian Jatropha Curcas seeds. International Journal

of Biological and Chemical sciences 4 (2) 524 – 529.

Bello, E. I., and Makanju, A. (2011). Production, characterization and evaluation of castor oil

biodiesel as alternative fuel for diesel engines. Journal of Emerging Trends

Engineering and Applied Sciences. 2 (3): 525-530

Berchmanns, H. J. and Hirata, S. (2008). Biodiesel production from Jatropha curcas seed oil

with a high content of fatty acids. Bioresources Technology. 99: 1716-1721

Bhagdeo, M. (2005). Determination of extractables from cranberry seeds using supercritical

CO2. Postgraduate thesis, Virginia polytechnic and state university. USA p 17

Canacki, M., and Van Gerpen, J. (2001). Biodiesel production from oils and fats with high

free fatty acids. American Society Of Agricultural Engineering. 44: 1429-1436

Da Silva, N. D., Batistella, B. C., Filho, M. R., and Maciel, W. R. (2006). Optimization of

biodiesel production from castor oil. Applied Biochemistry and Biotechnology.

13: 405-414

Demirbas, A. (2002). Biodiesel production from vegetable oils by supercritical methanol.

Journal Of Scientific and Industrial Research. 64: 858-865

Demirbas, A. (2008). Comparison of transesterification methods for production of biodiesel

from vegetable oils and fats. Energy Conversion and Management. 49: 125-130

Demirbas, A. (2009). Political, economic and environmental impacts of biofuels: A review.

Applied Energy. 86: 108-117

Demirbas, A. and Karslioglu, S. (2007) Biodiesel production facilities from vegetable oils

and Animal fats. Energy Sources. 29: 133 – 141.

Dias, J. M., Alvim-Farraz, M. C. M. and Almeida, M. F. (2008). Comparism of the

performance of different homogenous alkali catalysts during transesterification of

waste and virgin oils and evaluation of biodiesel quality. Fuel. 85: 3572-3578

Drapcho, C. M., Nhuan, P. N., Walker, T. H. (2008). Biofuels Engineering Process

Technology. The McGraw-Hill Companies, New York, P 220.

86

Enweremadu, C. C., Rutto, H. L., and Oladeji, J. T. (2011). Investigation of the relationship

between some basic flow properties of shea butter biodiesel and their blends with

diesel fuel. International Journal of The Physical Sciences. 6 (4): 758-767

Eromosele, I. C., Eromosele, C. O., Akintoye and Komolafe I. O (1993) Characterization of

oils and chemical analysis of the seeds of wild plants. Plant foods for Human

Nutrition 46: 361 – 365.

Eze, S. O. O. (2012). Physicochemical properties of oil from some selected underutilized oil

seeds available for biodiesel preparation. African Journal of Biotechnology.

11(42): 10003-10007

Fagerquest, C. K., Neese, R. A. and Hellerstein, M. K. (1999). Molecular ion fragmentation

and its effects on mass isotopomer abundances of fatty acid methyl esters ionized

by electron impact. American Society for Mass Spectrometry. 10: 430-439

Fan, X., Burton, R. and Austic, G. (2009). Preparation and characterization of biodiesel

produced from recycled canola oil. The Open Fuels and Energy Science Journal.

2: 113-118

Fjerbaek, L., Christensen, K. V. and Norddahl, B. (2008). A review of the current state of

biodiesel production using enzymatic transesterification. Biotechnology and

Bioengineering. 102(5): 1298-1315

Freitas, S. V. D., Pratas, M. J., Ceriani, R., Lima, S. A. and Coutino, A. P. (2010). Evaluation

of predictive models for viscosity of biodiesel. Energy Fuels. 25: 352-358

Fukuda, H., Kondo, A., and Noda, H. (2001). Biodiesel fuel production by transesterification

of oils. Journal of Bioscience and Engineering. 92: 405-416

Goering, E., Schwab W., Daugherty, J. , Pryde, H. and Heakin, J. (1982). Fuel properties of

eleven vegetable oils. Trans American Society of Agricultural Engineering

25:1472–1483

Gunstone, F. D. (1999). Fatty Acid and Lipid Chemistry. Aspen publishers Gaithersburg,

Maryland P 12.

Hamilton, R. J and Hamilton, S. (1992). Lipid Analysis A Practical Approach. Oxford

University press, Walton street, Oxford Pp 36 – 49.

Harbourne, J. B. (1973). Phytochemical methods: A guide to modern techniques of plant

analysis. Chapman and Hall, London. P 127

Hartig, C., Loffhagen, N. and Harms, H. (2005). Formation of trans fatty acids is not

involved in growth-linked membrane adaptation of pseudomonas putida. Applied

and Environmental Microbiology. 71(4): 1915-1922

Hoffman, G. (1986). Edible Oil and Fats in Hershedoerfer, S. M. (eds). Quality Control in

Food Industry. 2nd

edition. Academic press, London. Pp 407-504

87

Hossain, A. B. M., Nasrulhaq, B. A., Salleh, A., and Chandran, S. (2010). Biodiesel

production from waste soybean oil biomass as renewable energy and

environmental recycled process. African Journal of Biotechnology. 9 (27): 4233-

4240

Igwenyi, I. O., Offor, C. E., Aloh, P. M., Orji, G. S., and Ugwu, O. O. (2011). Potentials of

Afzelia Africana vegetable oil in biodiesel production. Progress in Renewable

Energies. 1: 8-14

Ivanoiu, A., Schmidt A., Peter F., Rusnac, L. M. and Ungurean M. (2011). Comparative

study on biodiesel synthesis from different vegetable oils. Chemical Bulletin of

“Poletechnica” Universityh of Timisoara. 56(70): 94-98

Johnston, M. (2006). Evaluating the potential of large-scale biodiesel deployments in a

global context. Masters dissertation, University of Wisconsin-Madison. P 18

Kai, Y. and Pryde, E. H. (1982). Production of branched-chaihhn fatty acids from sterculia

oil. Journal of American oil Chemical society. 57(7): 300-305

Kale, S. S., Darade, V. and Thakur, H. A. (2011). Analysis of fixed oil from Sterculia foetita

linn. International Journal of Pharmaceutical Sciences and Research. 2(11):

2908-2914

Kinast, J. A. (2003) Production of biodiesel from multiple feedstocks and properties of

biodiesels and biodiesel/biodiesel blends. National renewable energy laboratory.

Cole Boutevard Colorado. P 29

Knothe, G. (2001). Analytical methods used in the production and fuel quality assessment of

biodiesel. American Society of Agricultural Engineers. 44(2): 193-200

Knothe, G. (2002a). Current perspectives on biodiesel. Inform. 13: 900-9003

Knothe, G. (2002b). Structure indices in fatty acid chemistry. How relevant is the iodine

value? Journal of American Oil Chemical Society. 79(9): 847-854

Knothe, G. (2005). Dependence of biodiesel fuel properties on the structure of fatty acid

alkyl esters. Fuel Processing Technology. 86: 1059-1070

Knothe, G. (2008). “Designer biodiesel”: Optimiziing fatty ester composition to improve fuel

properties. Energy and Fuels. 22: 1358-1364

Knothe, G. Van Gerpen, J. and Krahl, J. (2005). The Biodiesel Handbook. AOCS press,

Champaign, USA. P 56

Leca, M., Tcacenco, L., Micutz, M., and Staicu, T. (2010). Optimization of biodiesel

production by transesterification of vegetable oils using lipases. Romanian

Biotechnology Letters. 15: 5 – 7

Leiner, I. (1980). Toxic Constituents of Plant Foodstuffs. Academic press, Newyork. P 380.

88

Loupe, D. O., Amoako, A. A. and Brink, M. (2008). Plant Resources of Tropical Africa Vol.

7 (1). Backhuys publishers, Leiden, Netherlands Pp 487 – 501.

Ma, F. and Hanna, A. M. (1999). Biodiesel production: A review. Bioresources Technology.

70: 1-15

Mehdi, A. and Hamid, R. K. (2011). Characterisation and transesterification Iranian bitter

almond oil for biodiesel production. Applied Energy, 2011. 88(7): 23 - 77.

Moser R. B. (2009). Biodiesel production, properties and feedstocks. Invitro Cell

Development Biology. 45: 229-266

National standard for biodiesel (2003). Setting national fuel quality standards – paper 6.

Department of environment and heritage, Australia. P 25

Nayak B. S. and Patel, K. N. (2010). Physicochemical characterization of seed and seed oil

of Jatropha curcas collected from Bardoli (South Gujarat). Sains Malaysiana.

39(6): 951-955

Olatunji, O. M., Akor, A. J., Aboroei, J. F. N. and Akintayo, C. O. (2011). Transesterification

for the preparation of biodiesel from crude oil of milk bush. Research Journal of

Environmental and Earth sciences. 3(4): 358-363

Osai, E.C (2011). Production of biodiesel from fluted pumpkin (Telfaira occidentalis hook

F.) seed oil. Masters dissertation. Department of Biochemistry, University of

Nigeria Nsukka.

Oyugi, D. A., Ayorinde, F. O., Gugssa, A., Allen, A., Izevbigie, E. B., Eribo, B. and

Anderson, A. W. (2011). Biological activity and mass spectrometric analysis of

Vernomia amygdalina fractions. Journal of Biosciences and Technology. 2(3):

287-304

Ozcimen, D. and Yucel, S. (2010). Novel methods in biodiesel production. Biofuels

Engineering Process Technology. 7: 353-383

Palligarnai, T. V. and Briggs, M. (2008). Biodiesel Production – current state of the art and

challenges. Journal of Industrial Microbiology and Biotechnology 35: 421 - 430.

Parthiban, K. T., Selvan, P., Paramathma, M., Kanna, S. U., Kumar, P., Subbulakshmi, V.

and Vennila, S. (2011). Physicochemical characterization of seed oil from

jatropha curcas genetic resources. Journal of Ecological and Natural

Environment. 3(5): 163-167

Pinto, A. C., Guarieiro, L. L. N., Rezende, M. J., Ribeiro, N. M., Torres, E. A., Lopes, W. A.,

Pereira P. A. and Andrade J. B. (2005). Biodiesel: An overview. Journal of

Brazilian Chemical Society. 16: 1313-1330

Popovich, M. and Hering, C. (1959). Fuels and Lubricants. John Wiley and sons, Inc.

newyork. P 187.

89

Raja, S. A., Smarth ,D. S. R. and Lee, R. L. C. (2011). Biodiesel production from jatropha

oil and its characterization. Research Journal of Chemical Sciences. 1: 81-87

Ramamurthi, S., Manohar, V. and Mani, V. V. S. (1998). Characterization of fatty acid

isomers in dehydrated castor ion by gas chromatography and gas chromatography

mass spectrometry techniques. Journal American Oil Chemical Society. 75:

1297-1303

Rao, P. V. (2011). Experimental investigations on the influence of the properties of jatropha

biodiesel on the influence, combustion and emission characterization of a DI – CI

engine. World Academy of Science Engineering and Technology. 75: 835-868

Refaat, A. A., Attia, N. K., Sibak, H. A., El-Sheltawy, S. T. and El-Diwani, G. J. H. (2008).

Production, optimization and quality assessment of biodiesel from waste

vegetable oil. International JHournal of Environmental Science Technology. 5(1):

75-82

Retledge, C. and Cohen, Z. (2008). Microbial and algae oils: Do they have a feature for

biodiesel or as commodity oils? Lipid Technology. 20(7): 155-160

Rodriquez-Acosta, M., Sandoval-Ramirez, J. and Zeferino-Diaz, R. (2010). Journal of

Mexican Chemical Society. 54(2): 88-91

Schuchardt, U., Sercheli, R. and Vargas, M. R. (1998). Transesterification of vegetable oils:

A review. Journal of Brazilian Chemical Society. 9: 199-210

Scragg, A. (2003). Environmental Biotechnology. Oxford university press, Oxford. P 301

Shalaby, E. A. and El-Gendy N. (2012). Two steps alkaline transesterification of waste

cooking oil and quality assessment of produced biodiesel. International Journal

of Chemical and Biochemical Sciences. 1: 30-35

Shereena, K. M., and Thangaraj, T. (2009). Biodiesel: An alternative fuel produced from

vegetable oils by transesterification. Electronic Journal of Biology. 5: 67-74

Shridhar, B. S., Beena, K. V. and Paramjeet, K. B. (2010). Optimization and characterization

of castor seed oil. Leonardo Journal of Sciences. 17: 59-70

Singh, R. K., and Pahdi, K. S. (2009). Characterization of jatropha oil for the production of

biodiesel. Natural Product Rediance. 8: 127-132

Singh, S. P. and Singh, D. (2010). Biodiesel production through the use of different sources

and characterization of oils and their esters as the substitute of biodiesel: A

review. Renewable and Sustainable Energy Reviews. 14: 200-216

Sivaramakrishnan, K. and Ravikumar, P. (2012). Determination of cetane number of

biodiesel and it’s influence on physical properties. ARPN Journal of Engineering

and Applied Sciences. 7: 205-211

90

Stamis, H., Xenakis, A., Menge, U. and Kolisis, N. K. (1993). Kinetic study of lipase

catalyzed esterification reaction in water-in-oil microemulsions. Biotechnology

and Bioengineering. 42: 931-937

Tekade, P. V., Mahodaya, O. A., Khandeshwar, G. R. and Joshi, B. D. (2012). Green

synhthesis of biodiesel from various vegetable oil and characterization by FT-IR

spectroscopy. Scientific Reviews and Chemical Communications. 2(3): 208-211

Thiruvengadaravi, K. V., Nandagopal, J., Bala, S. S. V., Kirupha, D. S., Vijayalakshmi, P.

and Sivanesan, S. (2009). Kinetic study of the esterification of free fatty acids in

non-edible Pongamia pinnata oil using acid catalyst. Indian Journal of Science

and Technology. 2: 20-26

Turkan, A. and Kalay, S. (2008). Study of the mechanism of lipase catalysed methanolysis of

sunflower oil in tert-butanol and heptanes. Turkish Journal of Biochemistry. 33

(2): 45-49

Van Gerpen, J. (2004). Biodiesel processing and production. Fuel Processing Technology.

86: 1097-1107

Van Gerpen, J., Shanks,. B., and Pruszko, R. (2004). Biodiesel Production Technology.

National Renewable Energy Laboratory,Springfield. P 90.

Vasudevan, P. T. and Briggs, M. (2008). Biodiesel production – Current state of the art and

challenges. Journal of Industrial Microbiology and Technology. 35: 421-430

Xu, Y. and Hanna, M. (2009). Synthesis and characterization of hazelnut oil based biodiesel.

Industrial Crops and Products 29: 473–479.

Yoon, H. S., Park, S. H. and Lee, S. C. (2008). Experimental investigation on the fuel

properties of biodiesel and its blends at various temperatures. Energy and Fuels.

22: 652-656

Zhang, Y., Dube, M. A., McLean, D. D. and Kates, M. (2003). Biodiesel production from

waste cooking oil: 1. Process design and technological assessment. Bioresource

Technology. 89: 1-16