MOI UNIVERSITY

SCHOOL OF SCIENCE AND AEROSPACE STUDIES

DEPARTMENT OF CHEMISTRY AND BIOCHEMISTRY

ESTERFICATION AND PHYSICO-CHEMICAL ANALYSIS OF BIODIESEL FROM

CROTON SEEDS

BY GROUP 2

1.FREDRICK KANU SC/33/19

2.AKISA MIRELLE SC/02/19

3.DENNIS GITONGA SC/21/19

i
DECLARATION

We declare that this thesis my original work and has not been submitted elsewhere for

examination, award of a degree or publication.

Submission Date…………………

ii
ACKNOWLEDGEMENT

We wish to articulate our gratitude to all staff in the Chemistry laboratory at Moi University for

the assistance offered during our research work; for offering valuable assistance on the use of

various instruments and permission to use various equipment. Our sincere appreciation goes to

our supervisors, Dr.Cherutoi for assistance, guidance and thought provoking discussions that

enabled us to come up with ideas that contributed to the success of this project. We also wish to

thank Dr Florence Opondo for immense support during laboratory sessions.

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ABSTRACT

Croton oil was extracted from powdered Croton megalocarpus seeds using Rotary Evaporation

method with n-hexane as the solvent. The optimum conditions for preparation of biodiesel from

the oil through a two-stage chemical process in reactor were determined by varying parameters

such as temperature, oil: alcohol mole ratio and a mount of catalysts. The optimum conditions

established were temperatures of 50 and 60 oC for esterification and transesterification,

respectively, methanol: oil mole ratio of 3:1 and 6:1 for esterification and transesterification,

correspondingly and a base catalyst mass of 1% (w/w) of the Croton oil for transesterification.

The optimum reaction times established were 1 and 2 hours for transesterification and

esterification correspondingly. A maximum yield of 88% biodiesel which had an acid value of

0.336 mg KOH/g, a density of 0.8858 g/cm3.

iv
TABLE OF CONTENTS
DECLARATION.............................................................................................................................ii

ACKNOWLEDGEMENT..............................................................................................................iii

ABSTRACT...................................................................................................................................iv

TABLE OF CONTENTS................................................................................................................v

LIST OF FIGURES.......................................................................................................................vii

LIST OF REACTION EQUATIONS..........................................................................................viii

LIST OF ABBREVIATIONS, SYMBOLS AND ACRONYMS..................................................ix

CHAPTER ONE..............................................................................................................................1

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

1.1 Background information............................................................................................................1

1.2 Biodiesel....................................................................................................................................2

1.3 Glycerol.....................................................................................................................................4

1.4 Fatty acids..................................................................................................................................5

1.5 Statement of the problem...........................................................................................................5

1.6 Objectives of the study..............................................................................................................5

1.6.1 General Objective...............................................................................................................5

1.6.2 Specific Objectives.............................................................................................................5

1.7 Justification and significance of the study.................................................................................6

1.7.1 Justification of study...........................................................................................................6

1.7.2 Significance of study...........................................................................................................6

CHAPTER TWO.............................................................................................................................8

LITERATURE REVIEW................................................................................................................8

2.1 Croton megalocarpus.................................................................................................................8

2.3 Transesterification.....................................................................................................................9

v
 2.3.1 Kinetics and mechanism of transesterification.................................................................10

2.3.2 Alkali-catalysed transesterification...................................................................................11

2.4 Characteristics of diesel fuels..................................................................................................11

2.5. Physico-chemical analysis of Biodiesel..................................................................................12

2.5.1 Saponification and acid values..........................................................................................12

2.5.2 Flash point.........................................................................................................................12

2.5.3 Density..............................................................................................................................13

2.5.4 Cloud and pour point.........................................................................................................14

vi
LIST OF FIGURES

vii
LIST OF REACTION EQUATIONS

Reaction equation 2.1 Balanced General equation for transesterification.…………...………...13

Reaction equation 2.2 Mechanism of alkali-catalysed transesterification ……………….…...15

Reaction equation 4.1 General esterification equation.…………………….…………………..71

xvi

viii
LIST OF ABBREVIATIONS, SYMBOLS AND ACRONYMS

ASAL Arid and semi-arid lands

ASTM American Society for Testing and Materials

ATP Adenosine triphosphate

B5 5% biodiesel, 95% diesel blend

B10 10% biodiesel, 90% diesel blend

B15 15% biodiesel, 85% diesel blend

B20 20% biodiesel, 80% diesel blend

B50 50% biodiesel, 50% diesel blend

B100 100% biodiesel

BGM Bacteria growth media

BHA Butylhydroxyanisole

BHT Butylated hydroxytoluene

CFPP Cold filter plug point

CI Compression ignition

CO Carbon monoxide (Carbon (II) Oxide)

CP Cloud Point

DG Diglycerides

DHA Docosahexaenoic acid

DI Direct injection

EGR Exhaust gas recirculation

EN European standard

EPA Eicosapentaenoic acid

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EU European Union xvii

FAME Fatty acid methyl ester

FAAE Fatty acid alkyl ester

FFA Free fatty acid

FID Flame ionization detector

GC Gas chromatography

HC Hydrocarbons

H2SO4 Sulphuric acid

IDI Indirect injection

IF Inorganic fraction

IUPAC International Union of Pure and Applied Chemistry

KOH Potassium hydroxide

LA Linolenic acid

LB Lysogeny broth

MAG Monoacylglyceride

MG Monoglycerides

MS Mass spectrometer

MSS Minimum salt solution

NACOSTI National Council of Science, Technology and Innovation

NaOH Sodium hydroxide

NOx Nitrogen oxides

OSI Oxidation stability index

PAH Polycyclic/Polynuclear aromatic hydrocarbons

x
PM Particulate matter

PP Pour Point xviii

ppm Parts per million

PRG Propyl gallate

PUFA Polyunsaturated fatty acids

PYG Pyrogallol

rpm revolutions per minute

RSC Royal Society of Chemistry

SI Spark ignition

SmF Submerged Fermentation

SOF Soluble organic fraction

SSF Solid state Fermentation

SVO Straight vegetable oil

TBARS Thiobarbiturate acid reagents

TBHQ tert-butylhydroquinone

TAG Triacylglyceride

TDG Transportation of dangerous goods

xi
CHAPTER ONE

INTRODUCTION

1.1 Background information

There is an increasing demand for alternative and renewable sources of fuel due to the depletion

of fossil fuel reserves and the need to reduce pollution from internal combustion engines.

Biodiesel, made from animal fats and plant or restaurant waste oils, is viewed as a promising

substitute for Petro diesel. The use of non-edible and waste oils as sources of environment-

friendly renewable substitutes has created interest in biodiesel research. There are challenges

associated with producing biodiesel, including the adverse effects of free fatty acids and water on

the base-catalyzed transesterification reactions used for commercial biodiesel production. The

project investigates the optimum conditions for preparing biodiesel from Croton oil using two-

stage chemical processes. Diesel engines are preferably used due to their efficiency and cost, this

is because high compression within the engine which produces heat, causing ignition of the fuel.

Diesel-powered engines are commonly used in cars, trucks, buses, locomotives, ships,

agricultural machinery and electrical power generators [Robert, 2001].

Strict world treaties on managing amount of exhaust emissions such as the Copenhagen Summit

of December, 2009 agreed to keep the global rise in temperature at a maximum of 2 oC this 21st

century. This has made many countries to come up with specific measures, aimed at reducing the

levels of certain contaminants from internal combustion engines. The European Union (EU) has

1
given each member state a target of at least 10% of transport fuel to be obtained from renewable

sources by 2020

Base-catalysed transesterification reactions employing potassium hydroxide (KOH) or sodium

hydroxide (NaOH) are extensively employed for commercial preparation of biodiesel because of

their fast rates. These reactions are however, adversely affected by free fatty acids (FFAs) and

presence of water [Van Gerpen et al., 2004]. The fatty acid methyl esters (FAME), which are the

widely utilized biodiesel, are normally produced by transesterification of oils or fats using

methanol and alkaline catalyst [Upadhyay and Sharma, 2013].

Despite ready availability of large quantities of non-edible plant and waste oils such as Croton,

kitchen waste and Nile perch viscera oil that can be used as sources of biodiesel feedstocks in

many developing countries such as Kenya, no intensive effort has been made to prepare and use

biodiesel on large scale. However, some of oil feedstocks possess high FFA content which

makes the widely employed alkali-catalyzed transesterification reactions inappropriate. Optimum

conditions for conversion of each specific non-edible oil feedstock with high FFA should

therefore be established using the two-stage chemical and enzymatic processes that may be

employed in saleable production of biodiesel. In this study, optimum conditions for preparing

biodiesel from Croton oil using two-stage chemical and enzymatic processes were investigated.

The physicochemical properties and performance characteristics of Croton biodiesel were also

tested and compared with recommended ASTM values and those of petrodiesel.

2
1.2 Biodiesel

Biodiesel is an unconventional fuel that can be prepared from plant or animal feedstocks.

Biodiesel is attracting much consideration globally either for blending purposes or direct

substitute for conventional diesel in compression ignition (CI) engines [Dermibas, 2009]. The

American standard specification (ASTM 67-02) describes biodiesel as a fuel composed of

methyl or ethyl esters of fatty acids derived from renewable plant oil or animal fat feedstocks.

The European Union Specification (EN14214) also accepts this definition [Minodora et. al.,

2010]. Biodiesel may be prepared from edible, non-edible and waste plant or animal oils or fats.

Owing to diminishing world's crude oil deposits, and food security issues, non-edible plant and

waste oils have attracted increased interest as possible sources of raw materials for production of

alternative fuel. Some of the processes that have been developed for biodiesel production involve

reaction of the oils with alcohol under supercritical temperatures or in the presence of chemical

or enzyme catalysts [Kumari et al., 2009].

Biodiesel is mainly produced on a large scale by reacting animal fats or vegetable oils with

alcohols, mainly methanol, to provide fatty acid methyl esters (FAME). A catalyst like potassium

or sodium hydroxide is usually used and glycerol is produced as a by-product [Van Gerpen et al.,

2004]. Biodiesel can work in any internal CI engine either in its pure form or blended in various

proportions with petrodiesel since they have similar characteristics [Dermibas, 2009]. Biodiesel

has become quite important owing to great demand on the current fossil fuel reserves and its

lower emission of hazardous gases as compared to petrodiesel fuelled engines [Park et al., 2008].

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Biodiesel made from non-edible oils, restaurant waste and animal fats can therefore be used as an

alternative low-cost fuel which is globally acceptable and economically competitive.

Biodiesel is at the moment regarded as an important substitute fuel and is being seen as a suitable

alternative to petrodiesel. Some of the advantages of biodiesel in comparison to petrodiesel

includes non-toxicity, biodegradability, renewable, non-contribution to global warming, has

negligible content of aromatic compounds and sulfur, higher cetane number and flash point

[Taher et al., 2011]. Additional profits of biodiesel include improved lubricating abilities and

reduced hazardous exhaust emissions in comparison to petrodiesel [Dermibas, 2008].

The use of agricultural crops such as sunflower oil, safflower oil, corn oil, coconut oil palm oil,

and soyabean oil as sources of biodiesel feedstocks, however, contends with their use as food

and can therefore not effectively fulfil the world's requirement for biodiesel. The direct use of

vegetable or plant oils also has some negative effects, such as increased viscosity and low

volatility, which may result in partial ignition in CI engines, and hence deposition of carbon.

Extensive work has been done to come up with derivatives of biodiesel that have properties

resembling those of petrodiesel [Taher et al., 2011]. This project targeted the use of non-edible

plant seed oil, Croton megalocarpus for biodiesel production using enzyme and two-stage

process.

Schumacher et al., [2001] stated that the use of biodiesel in automobile engines leads to 4, 47

and 66% decline in total hydrocarbon (HC), CO and smoke or particulate matter (PM) emissions

respectively, while Dermibas, [2008] reported a reduction of 42 and 55% in CO and PM

emissions correspondingly, in comparison to petrodiesel. The observations were explained by the

4
presence of oxygen in biodiesel molecules and their higher cetane number. Although biodiesel

environmental considerations with regards to emissions are very positive, its use normally leads

to higher emission of nitrogen oxides. Reports have, however, shown that the levels of NOx

emissions for biodiesel fuelled engines can be reduced by modifying injection timing and

combustion temperatures

1.3 Glycerol

Glycerol is an odorless, colourless, viscous and simple polyol liquid compound that is

extensively employed in pharmaceutical formulations. Glycerol contains three -OH groups that

are accountable for its ability in to dissolve in water and absorb moisture.

1.4 Fatty acids

Fatty acids or fats are carboxylic esters derived from the alcohol, glycerol and are commonly

referred to as triglycerides (TG). Since each fat is composed of glycerides from different

carboxylic acids with proportions varying from one fat to the other, fats from different sources,

have their characteristic composition. The even numbers are a result of the usual biosynthesis of

fats from acetate units, in stages that nearly resemble malonic ester synthesis. Both unsaturated

and saturated fatty acids, having one or more double bonds per molecule occur naturally.

1.5 Statement of the problem

The negative impact of fossil fuels on the environment, including hazardous fumes and global

warming. There are problems associated with limited fossil fuel supplies and increased demand.

Biodiesel is presented as a potential alternative, which can mitigate pollution and stabilize fuel

5
prices. The study focuses on the production of biodiesel from Croton megalocarpus oil using

enzymatic and two-stage chemical processes.

1.6 Objectives of the study

1.6.1 General Objective

The general objective of this study was to establish optimum conditions for production of

biodiesel from Croton megalocarpus oil using two-stage chemical process and to determine

characteristics of the biodiesel.

1.6.2 Specific Objectives

The specific objectives of this study were:

1. To determine optimum conditions for two-stage transesterification of Croton

megalocarpus seed oil.

2. To determine various organic compounds, present in croton oil through phytochemical

screening

3. To determine physico-chemical properties of Croton biodiesel

1.7 Justification and significance of the study

1.7.1 Justification of study

The increased industrialization and world population has resulted in extra energy demands.

However, due to the limited discovery of new fossil fuel reserves, the increased demands has led

to an unpredictable increase in oil price as well as inflation in the price of basic commodities.

6
The rate at which fossil fuels are currently being used is more rapid than they can be renewed

and this has led to fears of their depletion and unsustainability. The emergence of newly

industrialized nations and increased world population has also led to excessive emission of

pollutants into the environment that cannot be readily assimilated. Therefore, it is important to

come up with alternative renewable environment-friendly sources that can be sustainable on a

long term basis. The increased hazardous emissions from internal combustion engines and

corresponding environmental problems can be mitigated by substituting fossil fuels with green

renewable energy sources. Production of biodiesel from non-edible sources such as Croton does

not pose food security issues as compared to food sources such as soy bean and vegetable oils.

1.7.2 Significance of study

Petroleum-derived diesel is the most commonly used source of energy in the transport sector as

well as domestic and commercial engine driven appliances such as generators. The attainment of

Kenya's vision 2030 of becoming a newly industrialized, middle-income nation will result in

increased demand and consumption of fuel. The establishment of large-scale production and use

of biodiesel from locally available non-edible feedstocks can lead to huge savings in foreign

exchange, reduce emissions of certain harmful gases, improve rural income through

diversification of rural economies, contribute to conservation of ecosystem, improve biodiversity

conservation efforts and ensure environmental sustainability in increased fuel supply, provide

energy security and avert fuel crises that might disrupt economic growth targets and increase

inflation.

7
CHAPTER TWO

LITERATURE REVIEW

2.1 Croton megalocarpus

Croton megalocarpus Hutch. belongs to the family Euphorbiaceae. Since the plant grows readily

in many parts of East Africa at altitude ranges of 4,000 to 6,700 feet, it is commonly used as a

shade tree in coffee plantations and homes. It may reach a height of 120 feet, with a clear

cylindrical pole measuring between 40 to 60 feet in length and trunk diameters of 2 to 4 feet. The

plant may start bearing fruits at 3 years and matures at about 11 years (Bolza and Keating, 1972).

Croton oil can be easily extracted from its dry nuts using a rotary evaporator or mechanical

pressing machine to obtain straight vegetable oil (SVO) which can be used for biodiesel

preparation while .In Kenya, the plant is widely distributed in many forests, farms and homes

especially rift valley around Moi university.

2.2. Esterification

The effect of concentrated sulphuric acid catalyst on esterification of free fatty acid (FFA) was

determined by adding different volumes of acid to a mixture of croton oil and methanol in a

beaker. Since the esterification reaction is a reversible reaction, a FFA:methanol ratio of 1:3 was

used to shift the equilibrium to the right and improve the yield of ester .Exactly 2 mL of 99%

pure methanol was added to the croton oil in a beaker and stirred for 5 minutes. A graduated

pipette was used to add 98% concentrated sulfuric to the mixture. The mixture was continuously

stirred in a fume chamber

8
2.3 Transesterification

Transesterification is the conversion of large triglycerides (TGs) into short chain alkyl ester

molecules whose size and physical properties are similar to those of petrodiesel (Sinha et al.,

2008). Although every molecule of a triglyceride stoichiometrically needs three molecules of

alcohol for complete reaction, surplus alcohol is normally added to alter the equilibrium of the

reversible reaction and improve the yield of biodiesel. The mole ratio employed depends on the

9
oil feedstock, catalyst used, reaction time and temperature. Some of the most widely used

alcohols for transesterification include methanol, ethanol and propanol. The yield of biodiesel

does not depend on the nature of alcohol used and the choice is usually based on the cost.

Excessive amount of alcohol causes increased emulsification and makes separation of glycerol

quite challenging. The most suitable oil: alcohol ratio must therefore, be determined. During

transesterification, the ester bonds are first broken then followed by cleavage of hydroxyl bond.

However, in esterification process, hydroxyl bonds are broken before the ester bonds resulting in

the formation of glycerol as a co-product in transesterification and water in esterification

Transesterification can be carried out using different catalysts such as alkali, acid or enzymes

(lipases) which promote hydrolysis of triglycerides to fatty acid alkyl esters (FAAE) and

glycerol. The biodiesel is then purified from glycerol through a separation step that involves

washing with warm water a number of times.

10
2.3.1 Kinetics and mechanism of transesterification

Transesterification of triglycerides involves two-step mechanism that proceeds through

hydrolysis of the ester units to liberate glycerol and fatty acids, followed by esterification process

using an alcohol such as methanol. The mechanism for transesterification of triglycerides that is

widely accepted ,where each product is formed upon the addition of reagents. However, none of

the models used explicitly explains the formation of the mono- and diglycerides, the effects of

temperature on deactivation of enzymes or equilibrium limitation for the conversion

2.3.2 Alkali-catalysed transesterification

In alkali catalysed transesterification of croton oil, the alkali catalyst is dissolved in alcohol and

the solution mixed with oil in a beaker with vigorous stirring for 30 minutes at about 60 0c. A

positive transesterification reaction yields two separate layers, ester and crude glycerol. The first

stage in base catalysed reaction involves formation of an alkoxide and a protonated catalyst as a

result of the reaction between the base and alcohol. This is followed by the nucleophilic attack of

the alkoxide at the carbonyl group of the triglyceride to produce an intermediate. Subsequently,

the alkyl ester and the respective anion of the diglyceride are formed from the intermediate. The

diglyceride and monoglyceride intermediates are eventually transformed through a similar

process into a combination of FAAE's and glycerol. Alkaline metal alkoxides like CH3O-K+ are

quite effective in improving the rates of transesterification reactions and normally give excellent

yields (>98%) within a short duration of less than 1 hour even at marginal quantities (0.5 mol%).

11
2.4 Characteristics of diesel fuels
Diesel fuels produce power when atomized, mixed with air in the cylinder and burned at a high

pressure in a compression ignition (CI) engine. For the fuel to burn effectively, it should undergo

ignition at an appropriate time, release adequate energy on combustion and provide a large

amount of energy output per unit volume. The diesel fuel should flow freely and burn readily

even at low temperatures, be non-corrosive to the engine and contain no sediments that can plug

filters or block orifices. The fuel should also not cause excessive pollution on burning, be

relatively safe to handle in terms of flammability and toxicity and its viscosity should be

specified within a fairly narrow range. Some of the essential characteristics of diesel fuels are

briefly described in the preceding sections

2.5. Physico-chemical analysis of Biodiesel

2.5.1 Saponification and acid values
The saponification value or number of a fatty acid or ester is the amount of KOH needed to

neutralize one gram of the oil or fat. The saponification value is thus inversely proportionate to

the relative formula masses of the fatty acids obtained from the esters. The knowledge of

saponification value can be employed to determine the amount of alcohol required to react with

the TG during transesterification reactions [Vogel, 1989]. The acid value is the amount of KOH

12
needed to react completely with free fatty acids available in a plant or animal oil/fat. The acid

value is normally expressed as mg KOH/g of the oil/fat or % FFA present in the TG. The acidity

of biodiesel can cause poor fuel stability, corrosion of fuel tanks, fuel transmission pipes and

engine cylinders. The acidity can also result in production of sediments in some types of fuel

injection systems.

2.5.2 Flash point

The flash point is the temperature at which the fuel forms an inflammable vapour combination

with air at normal pressure. Since the flash point of diesel fuel is at least 55 oC, it is much safer

to transport and store than petrol or kerosene whose flash points are -40 and 30 oC respectively

[Stone, 1999]. Flash point can be established by heating up the fuel in equipment with a sealed

chamber such as a Pensky cup apparatus until it produces a vapour that readily catches fire when

a flame is placed over the liquid fuel. The flash point of a fuel does not have any effect on its

operation in an engine or auto combustion abilities. However, it can provide a valuable indicator

on the presence of pollutants, since the presence of small quantities of contaminants greatly

affects its value. [http://www.ufa.com/petroleum/resources/fueel/diesel_fuel_resources.html].

Flash point is a very important measure in determining the safety requirements for transportation

and storage of fuels. The value of flash point is used in the classification of fuels and other

liquids during transport such as the Transportation of Dangerous Goods (TDG)) regulations. It is

also used in setting safety measures needed in fuel handling and storage and requirement for

insurance and fire regulations.

13
2.5.3 Density
Density of a fuel is the ratio of its mass to volume at a particular temperature. Densities of fuels

are usually determined at a standard temperature of 15 °C using a density meter. Knowledge of

density can be used for determination of quantity calculations and evaluating ignition quality.

The density of plant oils is contingent on their source and constitution. Generally, the density of

plant oils is normally greater than that of their corresponding biodiesel and petrodiesel fuel

[Lujaji et al., 2010]. Density is a very important property of fuel, since the quantity of fuel that

should be delivered into engine cylinders by injection systems such as pumps and injectors for

proper combustion in the engines is determined by their densities. Density values of biodiesel are

also very essential in designing biodiesel production systems such as reactors, distillation units,

separation process, storage tanks and process piping.

Suitable fuels should have adequate amounts of less dense and volatile components for easy

starting of vehicles under cold environments and fast warm-up. The fuels should also possess

denser constituents that provide power and fuel economy once the engine attains maximum

operating temperature. The fuel density requirement depends on the size of engine, design,

nature of speed, load variations, starting and atmospheric situations. Engines used in fast

changing load and speed operations like buses and trucks may require less dense and more

volatile fuels for improved operation, especially in relation to smoke emission. However, the use

of denser fuels such as diesel usually results in better fuel economy since they have higher

energy content than the less dense fuels.

14
2.5.4 Cloud and pour point
The cloud point (CP) is the temperature at which visible wax in diesel or biowax in biodiesel

begins to form, giving the fuel a hazy look. Large crystals combine form clusters that finally

become widespread enough to inhibit free flow of the fluid at lower temperatures than CP. If a

fuel is used at temperatures below its CP, the crystals formed may plug filters or form sediments

at the bottom of a storage tank. Cloud point thus provides an indicator of the tendency of the

fuel.

CHAPTER 3

15
 METHODOLOGY

3.1 SAMPLE COLLECTION AND PREPARATION

Matured Croton megalocarpus fruits were collected at Moi University environment around
Guest houses near F houses. The fruits were dried for seven days then decorticated manually
to remove the outer shell after which the seeds were further dried for two days

3.2 OIL EXTRACTION

In preparation for oil extraction the dried croton megalocarpus seeds were crushed or grinded
into powder using mortar and pestle and weighed.

Picture 1: crushed croton seeds

Solvent extraction method, using n-hexane as the solvent, was employed in extracting the oil
from the grounded seed meal. The choice of n-hexane as the extraction solvent was owing to the
fact that it is non-poisonous and volatile with high affinity for oil. Besides, it can be easily
recovered. 450ml of n-hexane was added to 150g of grounded croton seeds in a conical flask and
waited for 72 hours. At the end of oil extraction, the extract was filtered and the solvent was
recovered using a rotary evaporator.

16
 Picture 2: rotary evaporator

The oil was placed in a sample beaker left out to dry at room temperature.

Picture 3: croton oil

The percentage yield of oil is calculated by equation below

%Yield = weight of oil × 100%

Weight of sample

17
3.3 Determination of the Characteristics of the Oil
The oil extracted from the croton seeds was characterized and its physicochemical
parameters were determined to be sure that it would be suitable for biodiesel production.
3.3.1 Saponification value determination

The saponification value of Croton oil was determined using the method described by Vogel,

[1989]. Approximately 0.5M solution of KOH was prepared by dissolving 14 g of KOH in 10 ml

of distilled water and the solution made to the mark with 95% ethanol in a 500 ml volumetric

flask. The solution was left to decant for 24 hours after which it was filtered. 2.0g of the oil was

placed in a 250ml conical flask and 25 ml of alcoholic KOH was added. A reflux condenser was

attached and the flask content refluxed for 30 min on a water bath with continuous swirling until

it simmered. The excess potassium hydroxide was titrated against 0.5M hydrochloric acid using

phenolphthalein indicator while still hot, and a mean titre value (titration value=a ml) of 20.18

ml was obtained. A blank determination was carried out under the same condition (titration value

=b ml). The saponification value was then calculated using Equation (2).

Saponification Value = (b-a) × 28.05

Weight of oil sample

Where b and a are the volumes hydrochloric acid used for blank and sample titrations

respectively .

3.3.2 Acid value and free fatty acids

A 25 ml of diethyl ether was mixed with 25 ml methanol and 1 ml phenolphthalein

solution and carefully neutralized with 0.1 M sodium hydroxide. 5 grams of croton oil was
dissolved in the mixed neutral solvent and titrated with aqueous 0.1 M sodium hydroxide shaking

18
constantly until a pink colour which persisted for 15 seconds was obtained. The titration was
carried out in triplicate in the experiments and the mean titre used to determine the and acid
value.

The following equation was used to calculate the acid value (Robinson and Stockes, 1959).

Acid value = titration (ml) x 5.61

weight of sample used

The FFA figure is usually calculated as oleic acid (1 ml 0.1 M sodium hydroxide =

0.0282 g oleic acid), in which case the acid value = 2 x FFA.

3.3.3 Density

An empty measuring cylinder was weighed before being filled with croton oil and reweighed to
obtain the mass of oil . The volume was enough to ensure that the oil fully covers the bottom of
the cylinder. The density of oil was calculated by the equation below

Density = (Mass of oil ÷ Volume of oil)

3.4 Qualitative Phytochemical screening of croton oil

Phytochemicals to Test procedure Inference( if positive)

be screened

STEROIDS -2 mL of the oil extract was dissolved red color at the upper layer

19
 in 10 mL of chloroform and yellow on the sulphuric

-10 mL of concentrated H2SO4 was acid layer with green

added by the side of the test tube fluorescence

TANNINS -5 drops of 0.1% FeCl3was added to 2 ml Brownish green color

of the oil extract in a test tube observed.

FLAVONOIDS -The 2 ml of 10% NaOH was added to An intense yellow color

2 ml of the oil extract in a test tube. was formed which turned

colorless upon addition of

2 mL of dilute HCl

SAPONINS

-The 2 mL of the oil extract was diluted About 0.1 cm layer of foam

with 2 mL of distilled water and agitated in was formed

a test tube for 5 min.

TERPENOIDS -The 2 mL of the oil extract was A clear upper and

mixed with 2 mL of chloroform and 1 lower layer with a

mL of concentrated H2SO4 was reddish-brown

carefully added to form a layer. interphase

Table 1 Phytochemical screening procedures.

20
 1 3.5 BIODIESEL PRODUCTION

Materials. Materials and apparatus used in the production of the biodiesel are as follows:
thermometer, retort stand, pipette, measuring cylinder, separating funnel, magnetic stirrer,
conical flask, digital weighing balance, stop watch, heat source ,distilled water, ethanol, and
croton oil.

two steps are used in the production of the biodiesel

I. Esterification reaction which involves reduction of the fatty acid contained in the croton
oil
II. transesterication process

ESTERIFICATION
As obtained in the test carried out on the croton oil, it was discovered that the free fatty acid
(FFA) contents of oil are high (21.6 ). Hence, it became necessary to reduce it. %

Procedure. Croton oil was poured into a conical ask and heated to a temperature of 60○C. A
mixture of concentrated sulfuric acid with ethanol was heated separately at 50○C then added to
the heated oil in the conical flask.The mixture was stirred for 1 hour and allowed to settle for 2
hours.

Trans-esterification process

A solution of potassium ethoxide was prepared in a 250 mL beaker using 0.25 g of potassium
hydroxide pellet and 63 ml of ethanol. The solution was properly stirred until potassium
hydroxide pellet was completely dissolved and the solution was heated to 60○C. 50 g of oil was
measured and poured into 250 ml conical flask and heated to a temperature of 50○C. The
potassium ethoxide solution was then poured into the warm croton oil and stirred vigorously for

21
one hour using a magnetic stirrer. The mixture was then allowed to settle for 24 hours in a
separating funnel. The biodiesel was then poured into a separate beaker, while the lower
layer(which comprises of glycerol and soap) was collected from the bottom of the separating
funnel. Warm water was then used to wash the biodiesel to remove any excess glycerol and soap
that remain in the funnel.This was done until the clear water was seen below the biodiesel in the
separating funnel.The washed sample was dried by placing it on a heat source and excess water
still in the biodiesel was removed. The quantity of biodiesel collected was measured and
recorded.

6 Physicochemical analysis of biodiesel

The biodiesel produced after the transesterification process was carried out was
characterized to obtain its physicochemical parameters as outlined below.
Flash point
150ml conical flask was filled to a certain level with the biodiesel and heated at slow constant
rate on aheat source. The flash point was taken as the lowest temperature when the application
of the test flame caused the vapour above the biodiesel sample to ignite.
Cloud point
A cylindrical tube was filled with the biodiesel to a certain level and clamped with a wooden
clamp bearing thermometer. The sample was allowed to cool below 0oC in a fridge until its
colour appearance turned white/cloudy. The temperature at which this cloudy appearance
colour occurred was recorded as the cloud point of the biodiesel.
Viscosity
The produced biodiesel sample was poured into a long necked funnel with a finger placed at its
bottom. A timer was set and started as the finger was released. When the sample ran off
completely, the timer was stopped. The analysis was repeated four times and the average time
was taken. The viscosity was thereby calculated using the readings taken.
6Determination of Density

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 A conical flask was weighed and filled to a volume of 15ml with the biodiesel.The weight of
the glass and the biodiesel was measured also.The weight of the biodiesel was determined, and
the density was calculated as mass per unit volume.

Determination of saponification value (SV)

2 g of each sample was added to excess alcoholic KOH and the solution was heated for two
minutes to saponify the oil. The unreacted KOH was back - titrated with standardized 0.1 M
HClusing phenolphthalein as indicator. The SV was calculated from equationas follows;

SV = (V-B) x M x 56.1
Sample weight (g)

Where; V = Sample titre value; B = Blank titre value; M = Molarity of the HCL; 56.1 =
Molecular weight of KOH

Determination of Acid value

2 g of the sample was dissolved in 30 mL ethyl alcohol and the mixture was boiled in a
water bath for 2 min and then titrated with a 0.1N KOH solution in the presence of
phenolphthalein as an indicator. Acid value is expressed as mgKOH/g of oil.

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 CHAPTER FOUR

RESULTS AND DISCUSSION

Oil extraction and properties of Croton oil

The solvent extraction method produced 47.84 g of Croton oil from 150 g of crushed croton

seeds which represented a yield of 31.89% (v/w). The results of the physicochemical properties

of Croton megalorcapus oil are presented in Table 1 together with the corresponding limits

(ASTMD6751)

Properties oil ASTM6751(recommended value)

Acid value (mg/KOH/g) 3.927 2.52

Saponification value(mg/KOH/g) 192.14 188

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Density (g/cm3) 0.9043 0.92

Free fatty acids (mg/KOH/g) 1.9635 -

Visual Color yellow -

Table 2

The saponification value is slightly higher than the recommended values ASTM standards of the

oil. The saponification value is used for measuring the average molecular weight of oil. A high

saponification value in oil indicates a higher proportion of low molecular weight fatty acids in

the oil or vice versa.

Compared to the maximum amount recommended for oils to be used for biodiesel production,

the values of acid value and free fatty acids are higher. Ideally the oil’s acid value should be less

than 1 mgKOH/g with all raw materials being anhydrous (water content < 0.3%) (Freedman et

al., 1984).

The standard for diesel engine fuel states that the fuel should have a density between 0.86 and

0.90 g/cm3,thus crude croton megalocarpus oil is slightly suitable for direct usage as a substitute

for diesel fuel since its density value is slightly similar to the recommended ASTM values.

The visual colour of the croton oil was yellow

Phytochemical constituents in C. megalorcapus seed oil

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Phytochemicals screened Observation inference

STEROIDS red color apperared at the upper layer and present

yellow color on the sulphuric acid layer

with green fluorescence

TANNINS Brownish green color was observed. present

FLAVONOIDS No observation color change absent

SAPONINS About 0.1 cm layer of foam was formed present

TERPENOIDS A clear upper and lower layer with a present

reddish-brown interphase

Table 3

Tannin has antibacterial activity, so its presence in the seed oil shows that the oil possesses some

anti-bacterial effects. Terpenoids and Steroids are both present in very high concentration which

may be due to their non-polar nature which obviously favors their increased concentration in the

oil. Steroidal compounds are of importance and interest in pharmacy due to their relationship

with sex hormones. The presence of steroids in the oil is an indication that it contains compounds

that are related to sex hormones improvement. The absence of flavonoids in the oil indicates that

26
C. megalocarpusseed oil not will have pharmacological properties because flavonoids are

biological antioxidants. Also,Saponin serves as anticancer as well as an antioxidant.

Physicochemical properties of the C. megalorcapuss biodiesel

The two stage process resulted in a high biodiesel yield of 90.7% (v/v) .The results of the

physicochemical properties of the Croton megalorcapus biodiesel are presented

in Table 4 below together with the corresponding limits (ASTMD6751 and EN14214) set for

biodiesel standard.

Property Biodiesel ASTM6751 EN14214

Acid value (mg/KOH/g) 0.58 0.8 maximum 0.5 maximum

Kinematic viscosity@40○C (mm2/s) 6.8 1.9-6.0 3.5-5.0

Density (g/cm3) 0.92 0.85-0.9 0.86-0.9

Cloud point (○C) 8 -2 to -12 -

Flash point (○C) 122 130 maximum >101

Visual Color brown - -

Table 4

Cloud point is a criterion used for low temperature performance of fuel and a higher cloud point

can affect the engine performance and emission adversely under cold climatic conditions. The

cloud point of the biodiesel produced in this study(8○C) is higher than the specifications for

27
biodiesel as seen in Table 4The presence of high cloud point may limit the use of the biodiesel

under low temperature.

One of the most important characteristics of any fuel is the flash point which is defined as the

lowest temperature at which it can vaporize to form an ignitable mixture in air . It is a property

that must be considered when assessing the overall flammability hazard of a material. The flash

point is between the specifications range for biodiesel. This makes the produced biodiesel in this

study safe for use and storage

Another very important property of fuel is the viscosity. It is a measure of resistance of a liquid

to flow due to internal friction of one part of a fluid moving over another. Fuels with high

viscosity has higher tendency to cause problems in the engine . The viscosity of the biodiesel

produced (6.8mm2/s) is above the standards (1.9-6.0) as given by ASTM for biodiesel.Again,

temperature affects the viscosity of biodiesel such that an increase in temperature reduces

viscosity and causes the fuel to flow more easily which is favorable to fuel injection efficiency

and atomization.

The density of a fuel is also an important factor for good engine performance. The higher the

density, the more difficult it becomes to pump the fuel. The density of the biodiesel produced

from Croton megalorcapus seed oil is 0.92g/cm3which is slightly within the acceptable range

according to ASTM and EN14214. Density of biodiesel depends upon the raw materials used for

biodiesel production and the methyl ester profile .

28
Considering that the presence of free fatty acids influences fuel aging, the ASTM specifies a

maximum acid value of 0.8mg/KOH/g of sample. The acid value in the biodiesel produced is

within the specified values.

29