1

S.D.COLLEGE OF ENGINEERING & TECHNOLOGY, MUZAFFARNAGAR

(Affiliated to: MAHAMAYA TECHNICAL UNIVERSITY- NOIDA)
A Training Report on

“Biodiesel: Extraction of oil from the seeds of Jatropha curcas, its conversion
to biodiesel & its comparative analysis with commercial diesel”

Submitted for the partial fulfillment of the requirements

For the Award of the degree of
BACHELOR OF TECHNOLOGY
IN
BIOTECHNOLOGY

Submitted by
ABHISHEK PANDEY
(VII SEMESTER; 1008354001)

Submitted To: Under the Supervision of:

Er. NAVEEN DWIVEDI Dr. MANOJ KUMAR UPADHYAY

HEAD, DEPT. OF BIOTECHNOLOGY; SCIENTIST ‘C’, QA & QC DEPT.
S.D. COLLEGE OF ENGG. & TECNOLOGY BIOTECH PARK, LUCKNOW

Mrs. SHUBHA DWIVEDI Er. MANOJ SINGH RAWAT

ASSISTANT PROFESSOR ANALYST, DEPT. OF BIOFUELS
DEPT. OF BIOTECHNOLOGY & BIODIESEL, BIOTECH PARK
S.D. COLLEGE OF ENGG. & TECNOLOGY LUCKNOW
 2

ACKNOWLEDGEMENT

I surpass all the barriers of written words to owe a deep sense of gratitude to reverend Dr. S.N. CHAUHAN (Director
S.D.C.E.T) for providing me an opportunity to present this report at their renowned organization. No words can
express my grateful to him.

I sincerely express my thanks with deep sense of gratitude to Er. NAVEEN DWIVEDI (Head of Department of
Biotechnology; S.D.C.E.T.) for constant encouragement invaluable guidance and all possible help which will be great
inspiration for me throughout my life.

I would like to convey my deep thanks to Mrs. SHUBHA DWIVEDI (Assistant Professor), a teacher, and a guide,
without whose cooperation and help it would not been possible for me to complete this training successfully.

My acknowledgement would not be complete without the mention of all the staff members of the department, who
directly or indirectly helped me a lot during the course of training. I sincerely thankful to Er. ANUPAM SINGH (Asst.
Professor) and Er. ASHWINI SINGH (Asst. Professor), who gave their valuable time and necessary help.

A heartfelt thanks is also extended towards Dr. MANOJ KUMAR UPADHYAY, ( “Scientist C”, Biotech Park ) &
Mr. MANOJ SINGH RAWAT for their guidance, support, and encouragement throughout the course of the project.

I would also like to thank the lab assistant Yogendra Ji for their contribution made in making available all the
requirements.
I can never express feelings to our parents, brother, sister and friends who gave ME help in each task of our work
and giving us strong emotional support. I dedicate whatever I have achieved and attained to the gratefulness of
invisible hand of God.

MEGHA SHARMA

S.D.C.E.T, Muzaffarnagar, U.P.

 3

DECLARATION

I hereby declare that the project work entitled "Biodiesel: Extraction of oil from seeds of
Jatropha curcas, conversion to biodiesel & its comparative analysis with commercial
diesel” submitted to S.D. College of Engineering and Technology, Muzaffarnagar a record of the
work undertaken by me under the guidance of Dr.Manoj Kumar Upadhyay, (Scientist C, QC &
QA department Biotech Park, Lucknow).

The data mentioned in the report was obtained during genuine work done by me. I also declare
that no part of this thesis has previously been submitted to any University or any examining body
for acquiring any diploma or degree.I,therefore,declare that the data & results are true to best of
my knowledge.

MEGHA SHARMA

S.D.COLLEGE OF ENGINEERING AND TECHNOLOGY,

MUZAFFARNAGAR (U.P)

S.N. TOPIC PAGE NO.

 4

1. Overview of biotech park

2. Abstract

3. Aims and objectives

4. Introduction and Plant

Source

5. Review of literature

6. Instruments used

7. Analysis of soil

8. Analysis Of Crude Oil

9. To prepare Biodiesel from

crude oil

10. Analysis of biodiesel

11. Results and Discussions

12. Conclusion

13. Lectures Attended

14. References

DECLARATION

I hereby declare that the project work entitled "Biodiesel: Extraction of oil from seeds of
Jatropha curcas, conversion to biodiesel & its comparative analysis with commercial
diesel” submitted to S.D. College of Engineering and Technology, Muzaffarnagar a record of the
 5

work undertaken by me under the guidance of Dr.Manoj Kumar Upadhyay, (Scientist C, QC &
QA department Biotech Park, Lucknow).

The data mentioned in the report was obtained during genuine work done by me. I also declare
that no part of this thesis has previously been submitted to any University or any examining body
for acquiring any diploma or degree.I,therefore,declare that the data & results are true to best of
my knowledge.

ABHISHEK PANDEY

S.D.COLLEGE OF ENGINEERING AND TECHNOLOGY,

MUZAFFARNAGAR (U.P)

1. OVERVIEW OF BIOTECH PARK

 6

Biotech Park, Lucknow

The State of Uttar Pradesh, a vital hub of scientific activities endowed with rich biological
resources and biodiversity has the unique distinction of having a number of research institutions,
which have expertise and capabilities in biotechnology. In view of this, Lucknow was declared as
the Biotechnology City of India during the 89 th session of the Indian Science Congress on January
3, 2002. These institutions are providing a great impetus and support in the development of
Biotech Park.

The Biotech Park has been set up on 8 acres of land provided by the Department of Science and
Technology, Government of Uttar Pradesh. The thrust areas identified for the initial phase of the
Park are.

 Health Care

 Agriculture

 Environment

Biotech Park is divided into following blocks

BLOCK I: BIO-BUSINESS BLOCK

 7

The Bio-Business Block houses the following:

1. Business Support Facilities
2. Bioinformatics Centre
3. Conference Hall
4. Cafeteria
5. Laboratory Space

BIOINFORMATICS CENTRE

A Bioinformatics Centre has been setup to establish a close network with various institutions and
provide information to industries regarding technological advancements.

BLOCK II: SOLVENT EXTRACTION BLOCK

The extraction unit comprises of solid liquid solvent extraction system with solvent recovery
system for extraction of Photochemical / Lead molecules from high value medicinal plants and
multipurpose reaction cum hydrolysis and solvent recovery unit along with chromatography.

Extraction unit has the following infrastructural facilities:

 8

1. Oil fired steam boiler, evaporation capacity: 500 kg/hr and 150 psi steam pressure for
basic heating requirements to main extraction unit.
2. Hot air tray dryer, High capacity hammer mill and vacuum oven for drying,
grinding/pulverizing of raw materials/finished herbs / medicinal plants.
3. Refrigerated brine chilling circulation unit for carrying out reactions and chilling of
condenser water.
4. Solid liquid extraction unit consisting of two drug holders with agitators of 125 - 150
kg/batch capacity depending on plant bulk density with efficient solvent recovery system
from extract and spent marc.
5. Silica gel chromatography column for chromatographic separation over silica gel with
solvent recycling system, for enriching and isolation of the photochemical in high purity.
Stainless steel reaction vessel with agitator and solvent reflux / recovery system for
production of semi synthetic drug molecules and for chemical transformations of the
photochemical.
6. Heavy-duty spray dryer for drying of herbal / aqueous extracts, capacity 10 – 20 kg/hr for
making powder of herbal extracts.
7. Falling film evaporator for concentrating herbal / aqueous extracts for separation of the
extracts from the plant biomass.
8. Notch type vacuum filtration unit for filtration of aqueous solvent extracts for finer filtration
of the extracts for removal of particulate impurities.
9. Liquid - liquid counter current extraction equipment for the purification of crude extracts by
liquid partitioning.
10. Minor equipments like vacuum pumps, metering pumps, weighing machine, weighing
balance, trolleys etc. for auxiliary infrastructure, process and material handling etc.
11. Capacity: 250 kg biomass/batch
 9

Extraction unit

Block III: Bio-fertilizer, Tissue Culture & Centre Support Block

1. Bio-fertilizer unit – Ground floor

2. Plant Tissue culture unit – First floor

3. Molecular and Analytical laboratory – Second floor

BIOFERTILIZER UNIT

This unit has facilities for perfecting the technology and production of bacterial fertilizers
and pesticides, phosphate solubilizing bacteria (PSB), Azotobacter (with a capacity of up to 240
tones / annum) Trichoderma (with a capacity of up to 500 tones / annum). The trials runs have
been completed in March 2007 and batch production has started.

TISSUE CULTURE AND HARDENING FACILITY

Tissue culture facility at Biotech Park, Lucknow is spread over 2000 sq ft. It has area that
possesses the capacity to raise and multiply banana, potato, jatropha seedlings. The culture
media used in the unit is completely biodegradable and non-toxic.

ADVANTAGES OF PROPAGATION BY TISSUE CULTURE

1. The elimination of diseases and the production of disease free plantlets.

1. The rapid production of large numbers of genetically identical plantlets.
2. Introduction of new varieties and or genotypes.
3. Preservation of germplasm.
4. Production of haploid plants which can be used for plant breeding.
5. Production of plantlets from species in which plant development from seed is difficult.
 10

CAPACITY:
10000 to 100000 plants / batch and it can produce about 2 million plants/ annum

Net house & glass house

ANALYTICAL AND MOLECULAR FACILITY

The facility is equipped with High Performance Liquid Chromatography (HPLC), Gas
chromatography (GC), High Performance Thin Layer Chromatography (HPTLC), Atomic
absorption spectrophotometer (AAS), Nanodrop-spectrophotometer, Gel Electrophoresis, Elisa,
Polarimeter, and other supportive equipments. A wide range of metabolites or molecules can be
quantitatively analyzed using HPLC and HPTLC.

DISTILLATION UNIT

The distillation unit has been set up for obtaining essential oil from aromatic plants such as
Mentha arvensis, Mentha piperata, Mentha cardiaca, Lemongrass, Palmarosa, Citronella, Basil,
Vetiver, and Geranium etc. The recovery of essential oil from different aromatic plants has been
found to be relatively high as compared to conventional distillation unit used by the farmers at
 11

their site. About 1000 kg fresh herbs per batch can be distilled in
the unit. The boiling points of organic compounds can give
important clues to other physical properties. A liquid boils
when its vapour.

VERMICOMPOSTING UNIT

This is the fastest and effective way of recycling of organic waste with the help of earthworms for
the production of useful compost. To utilize the large quantity of agro-waste generated from
distillation and solvent extraction units the vermicomposting unit has been set up at the Park. The
unit will serve as demonstration-cum-training facility for the farmers. Vermicompost  is the product
of composting utilizing various species of worms, usually red wigglers, white worms, and
earthworms to create a heterogeneous mixture of decomposing vegetable or food waste
(excluding meat, dairy, fats, or oils), bedding materials, and vermicast. Vermicast, also known
as worm castings, worm humus or worm manure, is the end-product of the breakdown of organic
matter by species of earthworm. Vermicomposting is
widely used in North America for on-site institutional
processing of food waste, such as in hospitals and
shopping malls. This type of composting is sometimes
suggested as a feasible indoor home composting
method. Vermicomposting has gained popularity in both
these industrial and domestic settings because, as
compared to conventional composting, composting worms most often used are red wigglers
(Eisenia fetida or Eisenia andrei), though European night crawlers (Eisenia
hortensis or Dendrobaena veneta) could also be used.

FACILITIES BEING OPERATED UNDER PUBLIC-PRIVATE PARTNERSHIP MODE

Distillation and vermicomposting unit, biofertilizer unit and tissue culture and hardening facilities
are being run under public-private partnership mode as given below:
 Distillation and vermicomposting units.
 Hindustan Bioenergy Pvt. Ltd.
 Biofertilizer Unit Hindustan Bioenergy Pvt. Ltd.
 Tissue culture and hardening facility.
 12

2. ABSTRACT

Bio-Diesel is an eco-friendly, alternative diesel fuel which is prepared from the domestic
renewable resources i.e. vegetable oils and animal fats. These natural oils and fats are made up
of triglycerides, which on chemical reaction with lower alcohols in presence of fatty acids give
esters. These esters show striking similarity to petroleum derived diesel and are called “Bio-
Diesel". Being renewable and energy effecient, bio diesel can displace petroleun derived diesel
fuels.

In this project crude oil was extracted from the seeds of Jatropha curcas, which was converted
into biodiesel by transesterification reaction. The obtained oil and biodiesel was analysed,
respectively. Also, the soil of the source plant was analysed for porosity, specific density, etc.
Extraction of oil was done in a specially designed apparatus called Soxhlet apparatus using
hexane as a solvent.

KEYWORDS: Bio-diesel, alternative fuel, Jatropha curcas, hexane, soxhlet apparatus.

 13

3. AIMS AND OBJECTIVES

1. Introduction to Jatropha curcas and its uses.

2. Analysis of soil

3. Study about the extraction process of oil from the seeds using Soxhlet
apparatus and the solvent recovery by rotatory evaporator and its
percentage oil yield.

4. Qualitative analysis of crude oil from seeds of plant.

5. Biodiesel preparation

6. Comparative analysis of biodiesel with commercial diesel

 14

4. INTRODUCTION

4.1 BIODIESEL

The idea of Bio diesel was originated in 1985 (Germany) and the first working Nut-oil engine
was tested in 1986. Due to edible oil demand being higher than its domestic production, there is
no possibility of diverting oil for production of Bio-diesel in India. There are many tree species,
which bear seeds rich in non-edible oil like Jatropha curcas, Pongamia pinnata. Calophyllum
inophyllum, Euphorbia antisyphilitica, E. tithymaloides E. caducifoila, E, royleana, E.neerifolia,
Calotropis igantea and C.procera, they are also recognized as promising petrol-crops for bio
diesel. Biodiesel refers to a vegetable oil- or animal fat-based diesel fuel consisting of long-
chain alkyl (methyl, ethyl, or propyl) esters. Biodiesel is typically made by chemically
reacting lipids (e.g., vegetable oil, animal fat (tallow with an alcohol producing fatty acid esters.

Biodiesel is meant to be used in standard diesel engines and is thus distinct from the vegetable
and waste oils used to fuel converted diesel engines. Biodiesel can be used alone, or blended with
petro diesel. Biodiesel can also be used as a low carbon alternative to heating oil.

The National Biodiesel Board (USA) also has a technical definition of "biodiesel" as a mono-alkyl

ester.

APPLICATIONS:
Biodiesel can be used in pure form (B100) or may be blended with petroleum diesel at any
concentration in most injection pump diesel engines. New extreme high-pressure (29,000
psi) common rail engines have strict factory limits of B5 or B20, depending on manufacturer.
Biodiesel has different solvent properties than petrodiesel, and will degrade
natural rubber gaskets and hoses in vehicles (mostly vehicles manufactured before 1992),
although these tend to wear out naturally and most likely will have already been replaced
with FKM, which is nonreactive to biodiesel. Biodiesel has been known to break down deposits of
residue in the fuel lines where petrodiesel has been used. As a result, fuel filters may become
clogged with particulates if a quick transition to pure biodiesel is made. Therefore, it is
recommended to change the fuel filters on engines and heaters shortly after first switching to a
biodiesel blend.

PRODUCTION LEVEL:
 15

In 2007, biodiesel production capacity was growing rapidly, with an average annual growth rate
from 2002-06 of over 40%.For the year 2006, the latest for which actual production figures could
be obtained; total world biodiesel production was about 5-6 million tonnes, with 4.9 million tonnes
processed in Europe (of which 2.7 million tonnes was from Germany) and most of the rest from
the USA. In 2008 production in Europe alone had risen to 7.8 million tonnes. In July 2009, a duty
was added to American imported biodiesel in the European Union in order to balance the
competition from European, especially German producers. The capacity for 2008 in Europe
totalled 16 million tonnes. This compares with a total demand for diesel in the US and Europe of
approximately 490 million tonnes (147 billion gallons). Total world production of vegetable oil for all
purposes in 2005/06 was about 110 million tonnes, with about 34 million tonnes each of palm oil
and soybean oil.

US biodiesel production in 2011 brought the industry to a new milestone. Under the EPA
Renewable Fuel Standard, targets have been implemented for the biodiesel production plants in
order to monitor and document production levels in comparison to total demand. According to the
year-end data released by the EPA, biodiesel production in 2011 reached more than 1 billion
gallons. This production number far exceeded the 800 million gallon target set by the EPA. The
projected production for 2020 is nearly 12 billion gallons.

PROPERTIES:

Biodiesel has better lubricating properties and much higher cetane ratings than today's lower
sulphur diesel fuels. Biodiesel addition reduces fuel system wear, and in low levels in high
pressure systems increases the life of the fuel injection equipment that relies on the fuel for its
lubrication. Depending on the engine, this might include high pressure injection pumps, pump
injectors (also called unit injectors) and fuel injectors.

The calorific value of biodiesel is about 37.27 MJ/kg.This is 9% lower than regular Number 2 petro
diesel. Variations in biodiesel energy density are more dependent on the feedstock used than the
production process. Still, these variations are less than for petro diesel. It has been claimed
biodiesel gives better lubricate and more complete combustion thus increasing the engine energy
output and partially compensating for the higher energy density of petro diesel.

Biodiesel is a liquid which varies in colour —between golden and dark brown —depending on the
production feedstock. It is immiscible with water, has a high boiling point and low vapour pressure.
The flash point of biodiesel (>130 °C, >266 °F)[ is significantly higher than that of petroleum diesel
(64 °C, 147 °F) or gasoline (−45 °C, -52 °F). Biodiesel has a density of ~ 0.88 g/cm³, higher than
petro diesel (~ 0.85 g/cm³).

Biodiesel has virtually no sulphur content, and it is often used as an additive to Ultra-Low Sulphur
Diesel (ULSD) fuel to aid with lubrication, as the sulphur compounds in petro diesel provide much
of the lubricity.
 16

FEEDSTOCKS:

A variety of oils can be used to produce biodiesel. These include:

 Virgin oil feedstock – rapeseed and soybean oils are most commonly used, soybean oil
accounting for about half of U.S. production. It also can be obtained from Pongamia, field
pennycress and jatropha and other crops such as mustard, jojoba, flax, sunflower, palm oil.
 Waste vegetable oil (WVO);

 Animal fats including tallow, lard, yellow grease, chicken fat,] and the by-products of the

production of Omega-3 fatty acids from fish oil.

 Algae, which can be grown using waste materials such as sewage and without displacing
land currently used for food production.

 Oil from halophytes such as Salicornia bigelovii, which can be grown using saltwater in

coastal areas where conventional crops cannot be grown, with yields equal to the yields of
soybeans and other oilseeds grown using freshwater irrigation .

 Sewage Sludge - The sewage-to-biofuel field is attracting interest from major companies
like Waste Management and startups like InfoSpi.

ADVANTAGES OF BIODIESEL:
1. It is renewable, nontoxic, biodegradable, and suitable for sensitive environments.
2. It is energy efficient that is, it has higher flash point.
3. It displaces petroleum-derived diesel fuel and can be used as a 20% blend in most diesel
equipment with no or only minor modifications.
4. It can reduce global warming gas emissions, tailpipe emissions, including air toxics.
5. It is Biodiesel is an oxygenated fuel so it contributes to a more complete fuel burn and a
greatly improved emission profile.
6. Biodiesel produces fewer particulate, carbon monoxide, greenhouse gases and sulphur
dioxide emissions reducing public health risk.

LIMITATIONS OF BIODIESEL:
1. NOx emissions are higher since biodiesel tends to increase NOx emissions.
 17

2. Engine performance (fuel economy, torque and power) is less than that of diesel by 8% to
15% because of lower energy content of biodiesel (121,000 Btu compared to 135,000 Btu
for diesel fuel)
3. Since its pour point and cloud point is around -10, it solidifies at that temperature during
winter in American and European countries.

4.2 PLANT SOURCES:

 JATROPHA CURCAS (RATANJYOT OR PHYSIC NUT)
 POGMIA PINNATA (KARANJA)
 RICINUS COMMUNICAS (CASTOR OR ARAND)
 MADHUCA INDICA (MAHUA)
 PRUNUS ARMENIACA (KHUBANI OR WILD APRICOT)
 AZADIRACHTA INDICA (NEEM)
 BRASSICA CAMPESTRIS (MUSTARD OR SASRO)
 SIMONDSIA CHINENCIS (JOJOBA)
 DIPLOPNEMA BUTRYRACEAE (CHEURA)

Jatropha curcas
Kingdom: Plantae
Division : Embryophyta
Class : Spermalopsida
Order : Malpaghiales
Family : Euphorbiaceae
Genus : Jatropha
Species:curcas

Jatroph
a curcas plant

Cultivation:
 18

Jatropha curcas is a species native to the American tropics, most likely Mexico and Central
America.

It is cultivated in tropical and subtropical regions around the world, becoming naturalized in some
areas. Jatropha Curcas is a drought resistant perennial, growing well in marginal/poor soil. It is
easy to establish, grows relatively quickly and lives, producing seeds for 50 years.

Climatically Jatropha curcas prefers the warmer regions of tropics and sub-tropics, although it
does well even in slightly cool conditions and can withstand a high frost. It water requirement is
extremely low and withstands long periods of drought by shedding most of its leaves to reduce
transpiration losses. The species is well adapted to arid conditions. Jatropha curcas is also a
suitable species for soil conservation areas and stabilization of shifting sand dunes.

Jatropha produces seeds with an oil content of 27-40% oil (average: 34.4%) that can be
processed to produce a high-quality biodiesel fuel, usable in a standard diesel engine. The
seeds are also a source of the highly poisonous toxalbumin curcin. The oil can be combusted as
fuel without being refined. It barns with clear smoke-free flame, tested successfully as fuel for
simple diesel engine.

BOTANICAL FEATURES:

 Leaves: The leaves have significant variability in their morphology. In general, the leaves
are green to pale green, alternate to sub opposite, and three- to five-lobed with a spiral
phyllotaxis.

 Flowers: male and female flowers are produced on the same inflorescence, averaging 20
male flowers to each female flower, or 10 male flowers to each female flower. The petiole
length ranges from 0.24 to 0.90 inches (6.1–23.1 mm). The inflorescence can be formed in
the leaf axial. Plants are monoecious and also presents hermaphroditic flowers
occasionally.

 Fruits : fruits are produced in winter, or there may

be several crops during the year if soil moisture is
good and temperatures are sufficiently high. Most fruit
production is concentrated from midsummer to late fall
with variations in production peaks where some plants
have two or three harvests and some produce
continuously through the season.
 19

 Seeds: the seeds are mature when the capsule changes from green to yellow. The seeds
contain around 20% saturated fatty acids and 80% unsaturated fatty acids, and they yield
25%–40% oil by weight. In addition, the seeds contain other chemical compounds, such
as saccharose, raffinose, stachyose, glucose, fructose, galactose, and protein. The oil is
largely made up of oleic and linoleic acids.
Furthermore, the plant also contains curcasin,
arachidic, linoleic, myristic, oleic, palmitic,
and stearic acids and curcin.

PROPAGATION:

Jatropha curcas has limited natural vegetative

propagation and is usually propagated by seed. Propagation through seed (sexual propagation)
leads to a lot of genetic variability in terms of growth, biomass, seed yield and oil content. Low
seed viability and the recalcitrant nature of oil seeds also limit seed propagation. However, clonal
techniques can help in overcoming these problems that hinder mass propagation of this tree-
borne oilseed species. Vegetative propagation has been achieved by stem
cuttings, grafting, budding as well as by air layering techniques. The investigation leads to the
recommendation that cuttings should be taken preferably from juvenile plants and treated with
200 micro grams per liter of IBA (rooting hormone) to ensure the highest level of rooting in stem
cuttings. These vegetative methods have potential for commercial propagation of these plants.

UTILIZATION:

Jatropha curcas oil finds wide usage and has high economic potential for large scale industrial
use. However, the cultivation practices of this plant and the economic exploitation of its oil yet
remain to be investigated systematically. Progressive research for systematic cultivation and
detoxification of oil for edible purposes.

 BIOFUEL- When jatropha seeds are crushed, the resulting jatropha oil can be processed
to produce a high-quality biofuel or biodiesel that can be used in a standard diesel car or
further processed into jet fuel, while the residue (press cake) can also be used as
biomass feedstock to power electricity plants, used as fertilizer (it contains nitrogen,
phosphorus and potassium), or as animal fodder. The cake can also be used as feed
in digesters and gasifiers to produce biogas.
 20

 JETFUEL- Aviation fuels may be more widely replaced by biofuels such as jatropha oil
than fuels for other forms of transportation. There are fewer planes than cars or trucks
and far fewer jet fuelling stations to convert than gas stations .

SEED RATE: For one hectare plantation about 5-7.5 kg seeds are required. Fruit starts from
second year if propagated by stem cutting.

OTHER PLANTS WHICH CAN BE USED FOR EXTRACTION OF DIESEL:

21
22
 23

5 .REVIEW OF LITERATURE

Biodiesel is an alternative fuel for diesel engines that is produced by chemically combining
vegetable oils and animal fats with an alcohol to form alkyl esters. Interest in biodiesel has been
expanding recently due to high petroleum prices. It is also possible to make biodiesel from other
oils, fats and recycled oils such as mustard, palm, coconut, peanut, olive, sesame, and safflower
oils, trap greases, and even oils produced from algae, fungi, bacteria, and yeast.
 24

The production of algae to harvest oil for biodiesel has not yet been undertaken on a
commercial scale, but feasibility studies have been conducted to arrive at the above yield
estimate. Prof. Rodrigo E.Teixeira from the University of Alabama in Huntsville
demonstrated the extraction of biodiesel lipids from wet algae using a simple and
economical reaction in ionic liquids.
Biodiesel will blend with petroleum diesel in any proportion. Three specific blends are of special
interest. B2 contains 2% biodiesel in 98% petroleum diesel. Similarly, B20 contains 20% biodiesel
and B100 contains 100% bio-diesel. B2 is of interest because of its lubricity benefits.

METHODS OF BIODIESEL PRODUCTION:

 Direct use and blending
The direct usage of vegetable oils as biodiesel is possible by blending it with conventional diesel
fuels in a suitable ratio and these ester blends are stable for short term usages. But direct use and
blending is not satisfactory and impractical for both direct and indirect diesel engines. The high
viscosity, acid composition, free fatty acid content, as well as gum formation due to oxidation and
polymerization during storage and combustion, carbon deposits and lubricating oil thickening are
obvious problems (Ma and Hanna 1999).

 Micro emulsions
A micro emulsion is defined as a colloidal equilibrium dispersion of optically isotropic fluid
microstructures formed spontaneously from two normally immiscible liquid or more ionic or non-
ionic amphiphiles. Alcohols such as methanol, ethanol and propanol are used as viscosity
lowering additives, higher alcohols are used as surfactants and alkyl nitrates are used as cetane
improvers. Viscosity reduction, increase in cetane number and good spray characters encourage
the usage of micro emulsions but prolonged usage causes problems like injector needle sticking,
carbon deposit formation and incomplete combustion (Ma and Hanna 1999).

 Thermal cracking (pyrolysis)

Pyrolysis is the chemical decomposition or conversion of condensed organic substances into
another by means of heat alone or with the aid of a catalyst. The pyrolyzed material can be
vegetable oils, animal fats, natural fatty acids and methyl esters of fatty acids. Disadvantages of
this process include high equipment cost and need
for separate distillation equipment for separation of various fractions. Also the product obtained
was similar to gasoline containing sulphur which makes it less eco-friendly (Ma and Hanna 1999).
 25

 Transesterification (alcoholysis)
In the transesterification of different types of oils, triacylglycerol react with an alcohol, generally
methanol or ethanol, to produce esters and glycerine. The overall process is normally a sequence
of three consecutive steps, which are reversible reactions. In the first step, from triacylglycerol
diacylglycerol is obtained, from diacylglycerol monoacylglycerol is produced and in the last step,
from monoacylglycerol glycerine is obtained. In all these reactions, esters are produced. Among
the alcohols that can be used in the transesterification process such as methanol, ethanol,
propanol, butanol and amyl alcohol, methanol and ethanol are used most frequently.

Glycerol being denser settles at the bottom and biodiesel can be decanted. The simple
transesterification process discussed above is confronted with two problems, i.e. the process is
relatively time consuming and it needs separation of the catalyst and saponified impurities from
the biodiesel.
The second conventional way of producing biodiesel is using an acid catalyst instead of a base.

 Base-catalysed transesterification mechanism

The transesterification reaction is base catalyzed. Any strong base capable of deprotonating the
alcohol will do (e.g. NaOH, KOH, sodium methoxide, etc.), but the sodium and potassium
 26

hydroxides are often chosen for their cost. The presence of water causes undesirable
base hydrolysis, so the reaction must be kept dry.

In the transesterification mechanism, the carbonyl carbon of the starting ester (RCOOR 1)
undergoes nucleophilic attack by the incoming alkoxides (R 2O−) to give a tetrahedral intermediate,
which either reverts to the starting material, or proceeds to the transesterified product (RCOOR 2).

 Enzyme-catalyzed transesterification
Biocatalysts are becoming increasingly important in biodiesel preparation as it is believed that
these catalysts will eventually have the ability to outperform chemical catalysts (Sulaiman et al.
2007). Biocatalysts are naturally occurring lipases enzymes used to catalyze some reaction such
as hydrolysis of glycerol, alcoholysis and acidolysis, but it has been discovered that they can be
used as catalyst for transesterification and esterification reactions too. The extracellular and the
intracellular lipases are also able to catalyze the transesterification of triacylglycerol effectively.

.
 Lipase mediated transesterification
The nature of a lipolytic reaction catalyzed by lipases is complex as the lipid substrates are water-
insoluble (Sellappan and Akoh 2005). The need for some water to maintain and activate lipases
and the immiscibility of lipids in water make the reaction media heterogeneous by forming a liquid–
liquid interface. The interface activates the enzyme by adsorption, which aids the opening of the lid
on the catalytic site (Sellappan and Akoh 2005). All types of interfaces, such as solid–liquid, liquid,
or liquid–gas, can influence the activity due to the interfacial hydrophobicity. Adsorption of the
enzyme onto the interface initiates a sequence of events before complete catalysis can be
achieved. Adsorption leads to activation and substrate binding followed by catalysis.
 27

 Supercritical process
An alternative, catalyst-free method for transesterification
uses supercritical methanol at high temperatures and pressures in a
continuous process. In the supercritical state, the oil and methanol are in
a single phase, and reaction occurs spontaneously and rapidly. The
process can tolerate water in the feedstock; free fatty acids are
converted to methyl esters instead of soap, so a wide variety of
feedstock’s can be used. Also the catalyst removal step is
eliminated. High temperatures and pressures are required, but energy costs of production are
similar or less than catalytic production routes.

 Ultra- and high-shear in-line and batch reactors

Ultra- and High Shear in-line or batch reactors allow production of biodiesel continuously, semi-
continuously, and in batch-mode. This drastically reduces production time and increases
production volume.

The reaction takes place in the high-energetic shear zone of the Ultra- and High Shear mixer by
reducing the droplet size of the immiscible liquids such as oil or fats and methanol. Therefore, the
smaller the droplet size the larger the surface area the faster the catalyst can react.

 Ultrasonic reactor method

In the ultrasonic reactor method, the ultrasonic waves cause the reaction mixture to produce and
collapse bubbles constantly. This cavitations provides simultaneously the mixing and heating
required to carry out the transesterification process. Thus using an ultrasonic reactor for biodiesel
production drastically reduces the reaction time, reaction temperatures, and energy input. Hence
the process of transesterification can run inline rather than using the time consuming batch
processing. Industrial scale ultrasonic devices allow for the industrial scale processing of several
thousand barrels per day.

6. INSTRUMENTS USED:

6.1.1. ANALYTICAL BALANCE:

Make and Model – DENVER
1. Used for measuring capacity. (Min-50 gram, max-15 gram).
2. It is an electronic device used to weigh chemicals and reagents in very small quantity i.e. up
to 0.001g.
 28

6.1.2. HOT AIR OVEN:

Make and model: JSGW

 Hot air oven is an electrical device used in sterilization.

 The oven uses dry heat to sterilize articles. Generally, they can be operated from 50 to 300 °C
(122 to 572 °F).
 There is a thermostat controlling the temperature. These are digitally controlled to maintain
the temperature.
 Their double walled insulation keeps the heat in and conserves energy, the inner layer being a
poor conductor and outer layer being metallic.
 There is also an air filled space in between to aid insulation.

An air circulating fan helps in uniform distribution of the heat. These are
fitted with the adjustable wire mesh plated trays or aluminium trays and
may have an on/off rocker switch, as well as indicators and controls for
temperature and holding time.

6.1.3. MILLIPORE WATER PURIFICATION SYSTEM:

Make and Model: Millipore- Elix
The production of pure water is indispensable in any laboratory.
However, for most users, the production of pure water also involves high costs and
extensive maintenance due to inefficient or outdates water purification systems.
Furthermore, most water purification systems cannot guarantee the production of
consistently pure water quality. All Millipore Analytical & Laboratory grade water systems
can be fed with tap water. Different standards allow you to make the right choice depending
on your use of purified water.

6.1.4 pH METER:
Make and model: EUTECH 510

 A pH meter is an electronic instrument used for

measuring the pH (acidity or alkalinity) of a liquid.
 A typical pH meter consists of a special measuring
probe (a glass electrode) connected to an electronic
meter that measures and displays the pH reading.
 29

 The pH probe measures pH as the activity of the hydrogen cations surrounding a thin-walled

glass bulb at its tip. The probe produces a small voltage (about 0.06 volt per pH unit) that is
measured and displayed as pH units by the meter.
 The meter circuit is no more than a voltmeter that displays measurements in pH units instead
of volts. The input impedance of the meter must be very high because of high resistance
approximately 20 to 1000M ohm-of the glass electrode probes typically used with pH meters.
 The circuit of a simple pH meter usually consists of operational amplifiers in an inverting
configuration, with a total voltage gain of about 17. The inverting amplifier convert the initial
voltage produced by the probe (+0.059volt per pH) into pH unit, which are then offset by 7 volts
to give a reading on the pH scale.
 At neutral pH 7 the voltage at the probes output is 0volts 0*17+7=7
 At basic pH, the voltage at the probe output ranges from +0 to +0.41 volts
(7*0.059=0.41). So for a sample of pH 10 ie 3 pH units above the neutral,
3*0.059=0.18 volts, the output of meter’s amplifier is 0.18*17+7=10.
 At acidic pH, the voltage at the probe’s output ranges from 0.41 volts to 0. So for a
sample of pH 4 ie 3 pH units below neutral, 3*0.059=0.18 volts, the output of the
meter’s amplifier is 0.18*17+7=4.

CALIBRATION AND USE:

For very precise work the pH meter should be calibrated before and after each measurement. For
normal use calibration should be performed at the beginning of each day. The reason for this is
the glass electrode does not give a reproducible reading over a long period of time. Calibration
should be performed with at least two standard buffer solutions that span a range of pH values to
be measured. For general purpose buffers at pH 4 and pH 10 are acceptable. The pH meter has
one control (calibrate) to set the meter reading equal to the value of the first standard buffer and a
second control (slope) which is used to adjust the meter reading to the value of the second buffer.
A third control allows the temperature to be set.

The calibration process correlates the voltage produced by the probe (approx 0.06 volt per pH
unit) with the pH scale after each single measurement, the probe is rinsed with distilled water or
deionised water to remove traces of the solution being measured, the probe blotted with a clean
tissue to absorb any remaining water which could dilute the sample and thus after the reading and
then quickly immersed in another solution. When not in use, the probe tip must be kept wet at all
times, It is typically immersed in an acidic solution of pH around 3.0. Distilled water or tap water
should never be used for this purpose.
 30

6.1.5. ROTARY EVAPORATOR

Make and model: Roteva

 A simple rotary evaporator system was invented by Lyman C. Craig. It was first
commercialized by the Swiss company Büchi in 1957.
 A rotary evaporator is a device used in chemical laboratories
for the efficient and gentle removal of solvents from samples
by evaporation.
 It consists of the following parts:
- A motor unit that rotates the evaporation flask or vial
containing the user's sample.
- A vapour duct that is the axis for sample rotation, and
is a vacuum-tight conduit for the vapour being drawn
off of the sample.
- A vacuum system, to substantially
reduce the pressure within the
evaporator system.
- A heated fluid bath (generally
water) to heat the sample.
- A condenser with either a coil
passing coolant, or a "cold finger"
into which coolant mixtures such as
dry ice and acetone are placed.
- A condensate-collecting flask at the bottom of the condenser, to catch the distilling
solvent after it re-condenses.
- A mechanical or motorized mechanism to quickly lift the evaporation flask from the
heating bath.

6.1.6. SOXHLET APPARATUS

Make and Model - JSGW
 31

Method was describes by Soxhlet in 1879. It is commonly used example of semi continuous
method applied to extraction of lipids from foods. According to Soxhlet procedure, oil and
fat from solid material are extracted by repeated washing with an organic solvent, usually
hexane or petroleum ether under reflux 5in special glassware. In this method sample is
dried, ground into small particles and placed in a porous cellulose thimble. The thimble is
placed in extraction chamber which is suspended above a flask containing the solvent and
below the condenser. The flask is heated and the solvent evaporates and moves up in the
chamber where it is converted to liquid due to condensation that trickles into the extraction
chamber containing the sample. The extraction chamber is designed such that when the
solvent surrounding the sample exceeds a certain level it overflows and trickles back into
the boiling flask. The solvent collected in the flask is then evaporated and the mass of the
remaining liquid is measured. The percent of the liquid in the initial sample can be
calculated.

6.1.7 UV-VISIBLE SPECTROPHOTOMETER:

Make and model: Labtronics

 Ultraviolet-visible spectrophotometry refers to absorption spectroscopy or reflectance

spectroscopy in the ultraviolet-visible spectral region.
 This means it uses light in the visible and adjacent (near-UV and near-infrared (NIR)) ranges.
The absorption or reflectance in the visible range directly affects the perceived colour of the
chemicals involved.
 It works on the principle of Beer- Lambert law.
 UV/Vis spectroscopy is routinely used in analytical chemistry for the quantitative determination
of different analytes, such as transition
metal ions, highly conjugated organic
compounds, and biological macromolecules.

 Spectroscopic analysis is commonly carried out in

solutions but solids and gases may also be
studied.
 A UV/Vis spectrophotometer may be used as a
detector for HPLC.
 The presence of an analyte gives a response assumed to be proportional to the concentration.
 32

 For accurate results, the instrument's response to the analyte in the unknown should be
compared with the response to a standard; this is very similar to the use of calibration curves.
 The response (e.g., peak height) for a particular concentration is known as the response factor.

6.1.8 KJELDAHL UNIT:

Make and model: KEL plus (D, 06L, VAC)

Johan Kjeldahl first introduced the Kjeldahl nitrogen method in 1883 at a meeting of the Danish
Chemical Society. Kjeldahl nitrogen determinations are performed on food and beverages,
meat, feed, grain, waste water, soil and many other samples. By this method NITROGEN of
both organic & inorganic samples can be analysed.

The method comprises of 3 steps:

1. Digestion - the decomposition of nitrogen in organic samples utilizing

a concentrated acid solution. This is accomplished by boiling a homogeneous sample in
concentrated sulphuric acid. The end result is an ammonium sulphate solution.
2. Distillation - adding excess base to the acid digestion mixture to
convert NH4+ to NH3, followed by boiling and condensation of the NH 3 gas in a receiving
solution.
3. Titration - to quantify the amount of ammonia in the
receiving solution.

Digestion system principle:

Sulphuric acid has been used to digest the organic samples.
Heating with sulphuric acid decomposes the organic
substance by oxidation so as to liberate the free nitrogen as ammonium sulphate.
Organic N + H2SO4 → (NH4)2SO4 + H2O + CO2 + other sample matrix by products
The acid digestion mixture is diluted and made strongly alkaline with NaOH.
(NH4)2SO4 + 2NaOH → 2NH3+ Na2SO4 + 2H2O

The Kjeldahl flask is attached to a water condenser and is heated to boil off the NH 3 gas from
the digest. The tip of the condenser is submerged in a flask of acidic receiving solution, either
standard acid or boric acid solution, to again trap the distilled NH 3 in receiving solution. The
majority of the NH3 is distilled and trapped in the receiving acid solution within the first 5 or 10
minutes of boiling.
 33

Titration process principle:

There are 2 types of titration:
o back titration
o direct titration
Both indicate ammonia present in the distillate with a
colour change & allow for calculation of unknown
concentrations.
If boric acid is used as the receiving solution then:
NH3 + H3BO3 → NH4+: H2BO3 - + H3BO3
The boric acid captures the ammonia gas, forming an
ammonium-borate complex and the initial pink colour discharges.

6.1.9. DESSICATOR:

 Desiccators are sealable enclosures containing desiccants used for preserving moisture
sensitive items such as cobalt chloride.
 A common use for desiccators is to protect chemicals which are hygroscopic or which react
with water from humidity.
 Desiccators are sometimes used to remove traces of water from almost dry samples.
 In order to weight a substance, watch glass or weighing bottles or crucibles are used.

6.1.10 FLAME SPECTROPHOTOMETER:

Make and model: Systronics

 Flame photometry concerns to the measurement of the EM radiation in UV-VIS range emitted
by the vaporised atoms after they are electronically excited by the thermal energy of the flame.
 Flame photometry is a method used for the
determination of elements which can be easily excited
for example, alkali and alkaline earth metals.

 This method is based upon the measurement of

intensity of radiation emitted, in the visible region,
when a metal atom is introduced into a flame. The
 34

wavelength of the radiation (or the colour), emitted tells us what the element is, and the
intensity of the radiation tells us how much of the element is present.
 The processes occur in the flame are Desolvation, Vaporisation, Atomisation, Excitation,
Emission of radiation.

6.1.11 FUME HOOD:

Make and model: NEWTON

A fume hood or fume cupboard is a type of

local ventilation device that is designed to limit exposure to
hazardous or toxic fumes, vapours or dusts. A fume hood is
typically a large piece of equipment enclosing five sides of a
work area, the bottom of which is most commonly located at a
standing work height.

Other related types of local ventilation devices include: clean

benches, biosafety cabinets, glove boxes and snorkel exhausts.
All these devices address the need to control airborne
hazards or irritants that are typically generated or released within the local ventilation device. All
local ventilation devices are designed to address one or more of three primary goals:

 to protect the user from inhaling toxic gases(fume hoods, biosafety cabinets, glove boxes);
 to protect the product or experiment (biosafety cabinets, glove boxes);

 to protect the environment (recirculating fume hoods, certain biosafety cabinets, and any
other type when fitted with appropriate filters in the exhaust airstream).

Secondary functions of these devices may include explosion

protection, spill containment, and other functions necessary to the
work being done within the device.

6.1.12 HOT PLATE WITH MAGNETIC STIRRER:

Make & model –REMI 5MLH PLUS
It is a laboratory device that employs a rotating magnetic field to
cause a stir bar (also called "flea") immersed in a liquid to spin
very quickly, thus stirring it. The rotating field may be created either by a rotating magnet or a set
of stationary electromagnets, placed beneath the vessel with the liquid. Since glass does not affect
a magnetic field appreciably (it is transparent to magnetism), and most chemical reactions take
place in glass vessels (i.e. see beaker (glassware) or laboratory flasks), magnetic stir bars work
well in glass vessels. On the other hand, the limited size of the bar means that magnetic stirrers
 35

can only be used for relatively small (under 4 liters) experiments. They also have difficulty dealing
with viscous liquids or thick suspensions. For larger volumes or more viscous liquids, some sort of
mechanical stirring is typically needed.

7. ANALYSIS OF SOIL

EXPERIMENT-1

Objective: To Determine the moisture content of soil.

Principle:
Water content or moisture content is the quantity of water contained in a material, such as soil
(called soil moisture), rock, ceramics, or wood on a volumetric or gravimetric basis. The property is
used in a wide range of scientific and technical areas.

Materials:
Petri plate, Hot air oven, Dessicator, Analytical balance, Distilled water.

2Method:
 36

1. Wash the Petri plate with distilled water and keep it in hot air oven for drying. Weigh the
empty Petri.
2. Take 5g of soil and weigh it in analytical balance. Then keep the soil in hot air oven at
1100C for 1 hour, followed by keeping in dessicator for 15 minutes.
3. Measure the weight of Petri and record the values.
4. Keep the Petri plate in hot air oven again for 30 minutes and record the weight after
keeping it in dessicator for 10 minutes. Repeat the step twice.
5. At last keep the Petri in hot air oven for 15 minutes and in dessicator for 5 minutes. Repeat
the process till the weight becomes constant up to three decimal places.

Formula:
% Moisture content = Initial weight of the soil – Final weight of the soil X 100
Initial weight of the soil

Observations:
Weight of Petri plate with soil (initial) = 42.29469g
Weight of Petri plate with soil (final) = 42.28169g

Calculations:
%Moisture Content = 42.29469-42.28169 * 100
5.0022

= 0.2598%

Result: The moisture content of soil is 0.2598%

Precautions:
1. All glass wares must be cleaned
2. Weighing should be done properly.
 37

EXPERIMENT-2

Objective: To Determine the pH of Soil.

Principle:
Soil supports a number of organic and inorganic chemical reactions. Many of these reactions
depend on pH of the soil, which is controlled primarily by concentration of free H + ions. If
substance is acidic then its pH will be less than 7 and if it is basic then it will have pH more than 7.
Alkaline soils have relatively low concentration of hydrogen ions. Hydrogen ions are made
available to the soil matrix by the dissociation of water by the activity of plant roots and many other
chemical weathering reactions.

Materials:
Soil sample, Glass vial, Analytical balance, 250ml beaker, pH meter, Buffer of pH 4 and pH 7,
Distilled water
 38

Method:
1. Take 30 gm of soil in a 250ml beaker and add 75 ml distilled water. Mix the contents
properly.
2. Allow the soil particles to settle down for 1 hour.
3. Calibrate the pH meter using buffers of ph 4 and pH 7.
4. Once the soil particles are settled, measure the pH. Take the mean of three readings.

Observation:
Buffer calibration:
pH 7.0 = 7.02
pH 4.0 = 4.03

Result: The pH of the soil of Jatropha curcas is 7.2.

Precaution:
1. All glass ware must be cleaned,
2. Probe of pH meter should be cleaned with distilled water,
3. pH meter should be calibrated with both buffer (pH 4 & pH 7) solutions.

EXPERIMENT-3

Objective: To Determine the Water Holding Capacity of Soil.

Principle:
Soil water retention is essential to life. It provides an ongoing supply of water to plants between
periods of replenishment (infiltration) so as to allow their continued growth and survival. Different
soils have different water holding capacity. The spaces that exist between soil particles, called
pores, provide for the passage and/or retention of gasses and moisture within the soil profile. The
soil’s ability to retain water is strongly related to its particle size, amount of organic component, soil
composition, etc.

Materials:
Beaker, Funnel, Non absorbent cotton, Soil sample, measuring cylinder

Method:
 39

1. Take 2g of soil and place it in a funnel (base plugged with non absorbent cotton).
2. Pour 10 ml of distilled water into the funnel.
3. Leave it undisturbed for 1 hour.
4. Measure the amount of water left.

Formula:
Water holding capacity = Initial volume of water – amount of water left × 100
Initial amount of water
Observations:
Total quantity of water = 10ml
Amount of water left = 7.6ml

Calculation:
Water holding capacity = 10 – 7.6 ×100 = 24%
10
Result: The water holding capacity of jatropha soil is 24%
Precaution:
1. Beaker & measuring cylinder should be cleaned.
2. Non-absorbent cotton should be fixed in the funnel carefully.
EXPERIMENT-4

Objective: To Determine the bulk density of soil.

Principle:
Bulk density is a property of powders, granules and other "divided" solids, especially used in
reference to soil. It is defined as the mass of many particles of the material divided by the total
volume they occupy. The total volume includes particle volume, inter-particle void volume and
internal pore volume. Bulk density is of greater importance than particle density in understanding
the physical nature of the soil.

Materials:
Pycnometer, Analytical balance, Soil sample

Method:
1. Empty pycnometer was weighed.
2. Pycnometer was filled with soil up to the neck.
 40

3. The pycnometer filled with soil was weighed.

Formula:
Bulk density = (wt. of pycnometer + soil) – (wt. of empty pycnometer)
Volume of the pycnometer
Observations:
Volume of the pycnometer = 25ml.
Weight of empty pycnometer = 20.92545g.
Weight of pycnometer + Soil =51.63514g.

Calculations:
Bulk density= 51.63514-20.92545=1.228 g/cc
25
Result: The bulk density of jatropha soil is 1.228 g/cc

Precautions:
1. Pycnometer must be cleaned, and dried.
2. Analytical balance should be calibrated.
EXPERIMENT-5

Objective: To Determine the particle density of Soil.

Principle:
Soil particle density is a measure of the mass per unit volume of the soil solids only. Texture and
structure do not affect particle density. However, organic matter, which is a soil solid, readily
influences particle density. Organic matter weighs much less per unit volume than soil minerals.
Soils high in organic matter have lower particle densities than soils similar in texture that are low in
organic matter. Soil particle density generally increases with soil depth because of the concurrent
decrease in organic matter.

Materials:
Pycnometer, Soil Sample, Analytical balance, distilled water, Hot air oven

Method:
1. Take the weight of empty pycnometer on an analytical balance.
2. Fill the pycnometer with distilled water till neck and weigh it again.
 41

3. Empty and dry the pycnometer and add 5 g of soil in the pycnometer.
4. Fill the pycnometer with distilled water till neck.
5. Take the weight of pycnometer + soil + water.

Formula:
Particle density = Weight of soil taken
Weight of soil displaced
Observations:
Weight of empty pycnometer (a) = 20.92545g
Weight of soil = 5.11111g
Weight of pycnometer + water + soil (b) = 48.59875g
Weight of pycnometer + water (c) = 45.41121g
Weight of water(y) = c – a = 24.48576g
Weight of soil + water(x) = b – a = 27.6733g
Weight of soil displaced (x-y) = 27.6733g- 24.48576g
= 3.18754g

Calculation:
Particle Density = weight of soil
x-y

= 5.11111 = 1.6035 g/cc

3.18754
Result:
The particle density of the soil sample is 1.6035g/cc.

Precautions:
1. Pycnometer must be cleaned,
2. Analytical balance should be calibrated.
 42

EXPERIMENT-6

Objective: To Determine Porosity Of Soil.

Principle:
Porosity is the measure of the void space in a material is measured as a fraction between 0 to 1 or
as a percentage between 0 to 100%. The term is used in multiple fields including ceramics,
manufacturing metallurgy earth science & concentration.

Formula:
Porosity = 1 – Bulk density ×100
Particle density
Observations:
Bulk density of the soil sample = 1.228g/cc
Particle density of the soil sample = 1.6034g/cc

Calculations:
Porosity =1-1.228×100 = 23.42%
1.6034
Result:
 43

The porosity of soil sample is 23.42%

Precautions:
1. Pycnometer must be cleaned, and properly dried.

2. Analytical balance should be calibrated.

EXPERIMENT-7

Objective: To Determine the total Organic Content of soil.

Principle:
The second major component of soils is organic matter produced by organisms. The total organic
matter in soil, except for materials identifiable as undecomposed or partially decomposed biomass
is called humus. This solid, dark-coloured component of soil plays a significant role in the control
of soil acidity, in the cycling of nutrients, and in the detoxification of hazardous compounds. Humus
consists of biological molecules such as proteins and carbohydrates as well as the humic
substances (polymeric compounds produced through microbial action that differ from metabolically
active compounds).

Materials:
Conical flask, pipette, burette, measuring cylinder, 0.5M ferrous ammonium sulfate, 1 N potassium
dichromate, diphenyl amine, sulphuric acid, orthophosphoric acid.

Method:
1. Take 0.5 g soil in a conical flask.
2. Add 5 ml of 1 N potassium dichromate to it.
 44

3. Add 10 ml conc. Sulphuric acid to the conical flask, and cover it with aluminium foil. Leave it
for half an hour.
4. Add 100 ml distilled water and 5 ml orthophosphoric acid to it.
5. Add a few drops of diphenylamine indicator and titrate it with 0.5 M ferrous ammonium
sulphate.
6. Prepare a blank sample similarly and titrate it.

Formula:
Total organic content (%) = 10 (B-S) ×0.39 × MCF
B ×Weight of soil

Observation:
Reading of blank (B) = 10.4 ml
Reading of sample (S) = 6.9 ml
Weight of soil sample = 0.50814 g

Moisture content (%) = 0.030736

Moisture correction factor (MCF) = 100 + 0.030736= 1.00030736

100

Calculations:
Total organic content = 10(10.4-6.9)×0.39×1.00030736 = 2.584%
10.4 × 0.50814
Result:

The Total Organic content in soil is found to be 2.584%

Precautions:

1. Burette & conical flask must be cleaned with distilled water.

2. H2SO4 should be handled very carefully.
 45

EXPERIMENT-8

Objective: To Determine the Available Nitrogen in the soil.

Principle:
A known weight of soil is mixed with excess of alkaline KMnO 4 (to oxidize the organic nitrogen
present in the soil). Ammonia gas thus formed is absorbed in a known volume of standard acid
(Boric acid), excess of which is titrated against standard alkali using methyl red as indicator.
Alk.KMnO4 has been used as an extracting reagent for the characteristics of the nature of nitrogen
in organic manures.

Materials:
Three volumetric flask of 100ml, Conical flask, Soil sample, Electrical balance, 0.32% KMnO 4,
2.5% NaOH solution, 2.5% Boric acid, Methyl Red indicator, 0.02N H 2SO4

Method:
1. Take 5g of soil in the DTL tube and add 10 ml of distilled water.
2. Add 25ml of 0.32% KMnO 4 solution and 25ml of 2.5% NaOH solution to the sample and
attach it to the distillation unit.
3. Pipette out 25 ml of 2.5% Boric acid into a conical flask and add 3-4 drops of methyl red
indicator. Attach the set up to the receiving end of the distillation unit.
4. Run the process for 9 minutes and titrate it with 0.02N H 2SO4.
Formula:
 46

Available Nitrogen = (14 x Titre value x Normality of H2SO4 x 2.24x106) Kg/hec.

Weight of soil x 1000
Observation:
Weight of soil = 5.000 g
Initial Reading = 0.0 ml
Final Reading = 1.2 ml

Calculations:
Available nitrogen = 14×1.2×0.02×2.24×106 =150.528 kg/hec
5.000×1000

Result: The available nitrogen in soil sample is 150.528 kg/hec

Precautions:
1. The Glassware used must be properly cleaned and dried.
2. Burette should be properly cleaned.
3. The pink color after adding phenolphthalein should be carefully observed.
 47

EXPERIMENT-9

Objective: To determine of available phosphorus in the soil by Olson’s method using UV-
Vis spectrophotometer.

Principle: The available phosphorus is extracted by treating the soil with 0.5N sodium bicarbonate
solution (pH 8.5), which reacts with ammonium molybdate to form hetropholy acid,
molybdophosphoric acid which is reduced by stannous chloride (tin chloride) to intensely coloured
molybdenum blue.

Materials: Sodium bicarbonate, sodium hydroxide, DarcoG60 Charcoal, ammonium molybdate,

stannous chloride, potassium dihydrogen phosphate, conc. H 2SO4, Whatmann filter paper, vortex
mixer, glycerol, UV-Vis spectrophotometer. Molybdenum blue.

Method:
Preparation of sample

 Extraction of phosphate:

 Take 2.5 g of soil sample;

 add 50 ml of 0.5N sodium bicarbonate solution and maintain the pH to 8.5

 Add a pinch of DarcoG60 Charcoal. Shake the mixture for 10 minutes over vortex mixture.

 Filter through what Mann filter paper. Collect the filtrate in a clean and dry Erlenmeyer flask
or beaker.
 48

 Color development: Add with thorough mixing after each addition, 400µl molybdate reagent
and 0.5 ml stannous chloride reagent.
Rate of color development and intensity of the color depends on the temperature of the final
solution.

 Color measurement: After 10 minutes but before 12 minutes using the same specific
interval for all determinations measure colour at 690 nm, spectrophotometerically.

 Preparation of calibration curve: Prepare a calibration curve of absorbance v/s phosphate

concentration, by running working standards in the same manner as the sample.

 Always run a blank on reagent and distilled water because the color first develops and then
fades. Maintain equal timing condition for sample and standards.

Observation and calculation:

S.No. Concentration of phosphorous Absorbance at 690 nm

(ppm)

Standard

1. 0.2500 0.2030

2. 0.5000 0.4721

3. 1.0000 0.8896

Sample

1. 1.9204 1.7902

Result:

The concentration of Phosphorus in the soil sample is 1.9204ppm

Precautions:
 49

1. Glassware should be cleaned properly.

2. Prepare the standards accurately.

Phosphorus in ppm
 50

EXPERIMENT-10

Objective: Estimation of Sodium and Potassium in soil by Flame Photometer

Principle:
When a solution containing salts of Sodium or Potassium is atomized into a gas flame, light
characteristics of this element are emitted, the intensity being a function concentration. Salts of
many other elements cause similar emission. The flame photometer consists of apparatus for
giving a reproducible amount of emitted light for a give concentration of element in the test
solution, and for determining the intensity of such emission as a function of concentration of the
element without excessive interference from other emitted light.

Materials and Equipments:

 Soil Sample
 Flame Photometer
 Hot air Oven

Procedure:
1. Preparation of standard sodium solution
Weigh accurately 2.5422g of sodium chloride previously dried at 120º C to constant mass
and cooled to room temperature in a dessicator over silica gel. Transfer the solution
quantitatively to 1 Liter volumetric flask and dilute to mark. Mix well and store in a polythene
bottle. This solution contains 1mg/ml of sodium.
2. Preparation of working standard solution
Into a 100ml volumetric flask take 10ml of the standard solution, add 2ml of the standard
solution and 2ml conc.HCl and make up the volume upto the mark. This solution will contain
100µg/ml of sodium. Dilute 1, 2, 3, 4 and 5 ml of this solution to 100ml volumetric flasks.
Number these flasks as 1, 2,3,4,5 respectively.
3. Preparation of Standard Potassium solution
Weigh accurately 1.9068g of KCl dried at 400º C and cool in a dessicator. Transfer to a
250ml beaker with washing.
Dissolve in 100ml of water. Add 10ml of conc. HCl. Transfer to 1Litre volumetric flask and
dilute to mark. Now mix well and store in a polythene bottle. This solution contains 1mg/ml
of potassium.
 51

4. Preparation of Test Solution

Weigh accurately 0.2 g of sample and transfer to a 100 ml beaker. Add 5ml of conc. HCl,
5ml Conc. Nitric acid and 10ml of water. Boil for 30 – 40 minutes till reaction is complete.

Evaporate to syrupy consistency, extract the residue with 2% Hydrochloric Acid, filter if
necessary, then transfer to a 100 ml volumetric flask and make the volume to the mark.
Switch on the flame photometer in advance to allow sufficient time for stabilization and
calibrate with standard matching solutions (Na / K) and then take the reading of the test
solution.

Calculation:
Concentration of alkali metal, percent by mass = A × D / 10,000

Where, A = Conc. in µg/ml of alkali metal in sample solution.

D = Dilution Factor of the sample.

Observations:
Standards
Concentration of Na and K Observed Concentration of Observed Concentration of
in standard solution (ppm) Sodium (ppm) Potassium (ppm)
1 1.04 1.00
3 3.11 3.05
5 5.24 5.33
10 9.69 9.69
15 15.16 15.01
Sample
1 0.02 0.27

Result:
The concentration of Sodium in the soil sample is 0.02 ppm and the concentration of Potassium is
0.27 ppm.

Precautions:
1. Glassware should be properly cleaned.
2. Standards should be prepared accurately.
8. ANALYSIS OF OIL

EXPERIMENT-1
 52

Objective: To determine the average weight of the seeds of the Jatropha curcas.

Materials:
Sample, Electronic analytical balance, Petri plate

Method:

1. Take 25 seeds of Jatropha curcas and divided them into 5 batches (5 seeds/batch).
2. Weigh each batch of 5 whole seeds on an analytical balance and note their readings.
3. Break the seeds to expose their kernels and weigh them.
4. Weigh the seed coats of each batch and note their readings.
5. Calculate the average weight of whole seeds, kernels and seed coats, and average weight
of all the entities of one seed. This is the average weight with respect to one seed.

Observations:

Batch Whole seed weight Kernel weight Seed coat weight

(in g) (in g) ( in g )
1. 3.317 2.072 1.230
2. 3.57 2.242 1.324
3. 3.615 2.339 1.275
4. 3.060 1.784 1.263
5. 3.429 2.126 1.289
Average 3.3982 2.1126 1.2762
weight

Calculations:
1. Average weight of whole seed (w.r.t batch) = 16.9602 / 5 = 3.39204 g
Average weight of whole seed (w.r.t. seed) = 16.9602 / 25 = 0.6784 g
2. Average weight of kernel (w.r.t batch) = 10.563 / 5 = 2.1126 g
Average weight of kernel (w.r.t seed) = 10.563 / 25 = 0.42252g
3. Average weight of seed coat (w.r.t batch) = 6.375 / 5 =1.2762 g
Average weight of seed coat (w.r.t seed) =6.375 / 25 = 0.255 g

Precautions:
1. In each Petri dish seeds should be of same variety and equal in number.
2. Good quality of seeds should be taken.
 53

EXPERIMENT-2

Objective: To determine the percentage Oil yield from seeds of Jatropha curcas.

Principle:
 54

Solvent extraction is a selective extraction procedure for isolating

and concentrating a valuable substance from aqueous solution
with the aid of an organic solvent. In the process the aqueous
Solution containing the substance of interest, often at low
concentration together with other dissolved substances is mixed
with an organic solvent containing reagent. The substance of
interest reacts with the reagent to form a chemical compound
which is more soluble in the organic than in the aqueous solution
and thus gets transferred to the organic solvent and is then
recovered.

Materials:
Seeds (Jatropha curcas whole seed), Mortar pestle, Weighing Balance, Soxhlet Apparatus,
Cotton, Porcelain piece, Rotary Evaporator, Hexane

Method:
a) Extraction of oil using Soxhlet:
1. Take 100g of seed samples and crush with the help of mortar pestle.
2. Place cotton at the end of thimble and fill it with crushed
seeds.
3. Take 600ml Hexane in the round bottom flask and add a
piece of porcelain.
4. Fix the apparatus, open the cooling water (condenser) and
set the temperature of the mantle to 40ºC for few minutes
till the hexane boils than decrease the temperature below
10ºC and leave the apparatus for 8 hrs.

b) Solvent recovery (to remove hexane from the mixture)

1. Put the mixture of oil and hexane (from Soxhlet) to rotatory evaporator with following set up:
1. Heating water bath temp.: 45º C

2. Rotations per minute: 300 rpm

3. Cooling Water bath temp: 4-5º C

 55

4. Vacuum: 280mbr (Hexane gets collected in another flask, leaving behind the oil)

2. Keep the oil on water bath to remove the little amount of hexane left in the oil.
3. Calculate the yield.

Formula Used:
% Yield (w/w) = Weight of oil extracted × 100
Weight of seed

% Yield (v/w) = Volume of oil extracted × 100

Weight of seed

Observations and Result:

Jatropha Weight of Volume of Weight of Oil yield Volume of Oil yield
seeds
seeds (g) solvent oil (W/w %) oil (V/w %)
Batch (ml) extracted extracted
(g) (ml)
1. 102 600 31.32 30.70 34 33.33
2. 105 600 32.82 31.25 35 33.33
3. 100 600 28.41 28.41 30 30.00
4. 102 600 33.23 32.57 35 34.31
5. 109 600 32.87 30.15 35 32.11

Precautions :-

1. Glasswares must be cleaned.

2. Weight of seeds must be taken accurately.
3. Jatropha seeds should be crushed properly.
4. Water supply should be continuous in soxhlet apparatus.
EXPERIMENT-3

Objective: To Determine the Moisture Content of oil from seeds of Jatropha curcas.

Principle:
Moisture content is the quantity of water content in a material, such as soil, rock, ceramics or wood
on a volumetric or gravimetric basis. The property is used in a wide range of scientific and
 56

technical areas and is expressed as a ratio which can range from 0(completely dry) to the value of
the materials porosity at saturation.

Materials:
Oil samples (Jatropha curcas whole seed and kernel), Beaker (5ml), Hot air oven, Analytical
Balance, Micropipette, Desiccator

Method:
1. Weigh the empty beaker.
2. Pour 1ml crude oil in the beaker and weigh the beaker (initial weight).
3. Put the beaker in hot air oven at 50ºC for 1 hr.
4. Put the beaker in desiccator for 15 minutes and allow it to cool at room temperature.
5. Weigh the beaker + sample.
6. Again put the beaker in oven for 30mins at 50ºC and then desiccator for 10 minutes
7. Repeat the procedure till the weight becomes constant (final weight).

Formula:
Moisture content %= Initial weight – Final weight X 100
Initial weight

Observations:
Weight of empty beaker = 4.94595 g
Weight of beaker + 1ml oil = 5.81846 g
Final weight of beaker + 1ml oil = 5.79002 g
Constant weight = 5.79002 g

Calculations:

% moisture content = 5.81846 -5.79002 ×100

0.8214

= 3.46%

Result:
The moisture content of crude oil is 3.46 %
 57

Precautions:-
1. Glassware must be clean.
2. Weight of oil must be measured accurately.
3. Time for desiccation at every interval should be same.

EXPERIMENT-4

Objective: To Determine the specific gravity of Oil from seeds of Jatropha curcas.

Principle:
Specific gravity is defined as the ratio of the density of the given substance to the density of water
when both are at the same temperature. Substances with specific gravity >1 are denser than
water and thus sink in it while the substances with specific gravity <1 are less dense than water
and thus float in it. Specific gravity is a special case of Relative Density.
 58

Specific gravity is mathematically expressed as: P1 / P2

Where P1 is the density of the substance and P2 is the density of water.

Material:
Oil Samples, Distilled water, Pycnometer, Analytical Balance, Micropipette

Method:
1. Weigh the empty pycnometer (X).
2. Pour 1ml distilled water in pycnometer and again weigh the pycnometer (Y).
3. Wash the pycnometer and dry it.
4. Pour 1ml crude oil sample in pycnometer and weigh it (Z).

Formula:
Specific gravity= Weight of oil = Z–X
Weight of Water Y–X

Observation:
Weight of empty pycnometer (X) = 20.8730 g
Weight of pycnometer + 1ml oil (Z) = 21.8550 g
Weight of pycnometer + 1ml distilled water (Y) = 21.8740 g

Calculations:

Specific gravity = 21.8550-20.6334

21.8740- 20.8730

= 0.8790

Result: The specific gravity of crude oil is 0.8790.

EXPERIMENT-5

Objective: To Determine the Acid Value of Crude oil from the seeds Jatropha curcas.

Principle:
Acid value (or "neutralization number" or "acid number" or "acidity") is the mass of potassium
hydroxide (KOH) in milligrams that is required to neutralize the free fatty acid present in one gram
of chemical substance. The acid number is a measure of the amount of carboxylic acid groups in a
chemical compound. In a typical procedure, a known amount of sample dissolved in organic
 59

solvent is titrated with a solution of potassium hydroxide with known concentration and with
phenolphthalein as a color indicator. The acid number is used to quantify the amount of acid
present in the sample. It is the quantity of base, expressed in milligrams of potassium hydroxide
that is required to neutralize the acidic constituents in 1 g of sample.

Materials:
Oil samples (Jatropha curcas), Ethanol (neutralized – 95%), Phenolphthalein indicator, 0.1N KOH,
Burette, Conical flask, Funnel, Hot Water Bath

Method:
1. Weigh 2g of oil sample in conical flask, add 5 ml 95% neutralized ethanol, and cover it with
foil.
2. Add 3-4 drops of phenolphthalein indicator in it.
3. Clean the burette with hot water and fill it with 0.1N KOH solution.
4. Titrate the sample with KOH and note the readings.
5. Calculate the acid value.

Formula:
Acid Value (A.V.) = 56.1 X Titer Value X Normality
Weight of Sample taken
Observation:
Initial value = 12.0 ml
Final value = 14.0 ml
Titre value= 2.0 ml

Calculations:
Acid value = 56.1×2.0×0.1= 5.60221 meq/g
2.00278

Result: The acid value of crude oil is 5.60221meq/g.

Precautions:
1. Conical flask & burette must be cleaned.
2. Working platform should be clean.
 60

EXPERIMENT-6

Objective: To Determine the Saponification Value of Crude oil from seeds of Jatropha
curcas.

Principle:
Saponification value is a measure of the free acid and saponifiable ester groups. It is
expressed as the number of milligrams of potassium hydroxide required to neutralize the free
acids and saponify the esters contained in one gram of the material. Following reactions are
involved in the calculation of saponification value.
 61

R COOH + KOH → R COOK + H2O

KOH + HCl → KCl + H2O
Materials:
Oil samples, 0.5N methanolic KOH, 0.5M HCl, Phenolphthalein indicator, Burette, Conical
flask, Micropipette, Funnel
Method:
1. Take 5g of oil in a conical flask.
2. Add 50ml of 0.5N methanolic KOH in it.
3. Keep the flask on boiling water bath to saponify fats completely. (30 minutes approx.)
4. Cool and titrate it with 0.5M HCl using phenolphthalein indicator.
5. Conduct the blank determination along with the sample, using same period for blank.

Formula:
Saponification Value= Concentration conversion factor (28.05) X (Blank – Sample)
Weight of Sample taken
Observation:

Sample Weight of Initial Reading of Final reading of burette Titre Value

oil burette (ml) (ml)
(g) (ml)

Blank 0.0 0 47.7 47.7

Sample
5.0447 5 22 17

Calculations:
Saponification value of crude oil = 28.05×(47.7-17) = 172.044
5.0053

Result:

The saponification value for crude oil is 172.044

Precautions:
1. The glassware used must be properly cleaned and dried.
2. The titration step should be carried out carefully.
 62

EXPERIMENT-7

Objective: To Determine the peroxide value of Crude Oil.

Principle:
Peroxide value is defined as the mill equivalents of peroxides per kilo gram of sample. It is
titrimetric determination. The Peroxide value of an oil or fat is used as a measurement of the
extent to which rancidity reactions have occurred during storage. The double bonds found in fats
and oils play a role in autoxidation. The best test for autoxidation (oxidative rancidity) is
determination of the peroxide value. Peroxides are intermediates in the autoxidation reaction.
Autoxidation is a free radical reaction involving oxygen that leads to deterioration of fats and oils
which form off-flavors and off-odors. Peroxide value, concentration of peroxide in an oil or fat, is
 63

useful for assessing the extent to which spoilage has advanced. The peroxide value is determined
by measuring the amount of iodine which is formed by the reaction of peroxides (formed in fat or
oil) with iodide ion.
2 I- + H2O + ROOH -> ROH + 20H- + I2

Materials:
Oil Samples, Saturated KI Solution, 0.1N Sodium thiosulphate, Acetic acid, Chloroform, Starch
indicator, Distilled water, Burette, Conical flasks, Funnel

Method:
1. Weigh 5g of oil in a flask.
2. Add 30ml of chloroform and acetic acid mixture (2:3).
3. Add 0.5ml saturated KI solution and add 30 ml of distilled water after 1 min.
4. Titrate the sample with 0.1N Na2S2O3. With vigorous shaking until yellow colour disappears.
5. Add 0.5ml 1% starch solution and continue titration, shaking vigorously to release all iodine
from chloroform layer until brownish blue colour just disappears.
6. Conduct the same with blank.

Formula:
Peroxide Value= (Sample – Blank) × N × 1000
Weight Of the sample taken

Observation:
Weight of oil = 5.159 g

For Blank:
Initial reading = 6.5ml
Final reading = 13ml
Titre value = 6.5ml

For sample:
Initial reading = 13.0
Final reading = 19.5
Titre value = 6.5
 64

Calculations and Result:

For Jatropha seeds:
Per oxide Value of crude oil = (6.5-6.5) × 1.006×100
5.159

= 0.00 meq/kg

Precautions:
1. Conical flask & burette must be cleaned.

2. Readings of burette should be taken very carefully

EXPERIMENT-8

Objective: To Determine the solubility of crude oil in various solvents.

Materials:
Centrifuge tubes, Micropipette, Ethyl Acetate, Methanol, Hexane, Oil samples

Method:
1. Add 200µl of oil sample in three different centrifuge tubes.
2. Add 200 µl of methanol, ethyl acetate, hexane in these tubes respectively.
3. Shake the tubes vigorously.

Observations and Result:

 65

Solvent
Sample Methanol Ethyl Acetate Hexane

Jatropha curcas Insoluble soluble Soluble

(whole seed)

Precautions:

1. All eppendorf tubes should be cleaned.

2. Ratio of oil & solvent should be equal.
3. The contents of the eppendorf tubes should be properly missed before taking down the results.

9. To Prepare Biodiesel from Crude Oil

Bio-diesel is the name of a fuel alternative of the conventional, petroleum based diesel engine fuel,
which is manufactured from vegetable oils or animal fats by catalytic reacting these with a short-
chain aliphatic alcohol (methanol or ethanol), typically using a process called trans esterification,
or alcoholysis.
 66

Transesterification chemistry:
Animal and plant fats and oils are typically made of triglycerides which are caters of free fatty acids
with the trihydric alcohol, glycerol. In the transesterification process, the alcohol is deprotonated
with a base to make it a stronger nucleophile. Commonly, ethanol or methanol’s are alcohol. A
reaction scheme for trans esterification is as follows:

R1, R2, and R3 in this diagram represent long carbon chains that are too lengthy to include in the
diagram. Animal and plant fats and oils are typically made of triglycerides which are esters of free
fatty acids with the trihydric alcohol, glycerol. In the transesterification process, the alcohol is
deprotonated with a base to make it a stronger nucleophile. Commonly, ethanol or methanol is
used. As can be seen, the reaction has no other inputs than the triglyceride and the alcohol. Heat,
as well as an acid or base are used to help the reaction proceed more quickly. Almost all biodiesel
is produced from virgin vegetable oils using the base-catalyzed technique as it is the most
economical process for treating virgin vegetable oils, requiring only low temperatures and
pressures and producing over 98% conversion yield.

Materials:
 67

Crude oil samples (Jatropha curcas whole seed, Kernel), Conical Flasks, Volumetric Flasks,
Beakers, Separating Funnels, Micropipette, pH meter, Magnetic Stirrer, Burette, KOH, Isopropyl
alcohol, Methanol

Method:
A) Titration Step:
Titration is done to estimate the amount of KOH that will be required to produce
methanolic/alcoholic KOH which acts as a catalyst in the transesterification reaction of oil to
produce a fatty acid methyl ester (FAME) and glycerol.

1. Dissolve 0.1g KOH in 100ml distilled water (in volumetric flask).

2. Clean the burrette distilled water.

3. Take 1ml of oil in a conical flask and add 10ml isopropyl alcohol to it.

4. Add 4-5 drops of phenolphthalein indicator.

5. Titrate the sample with KOH solution till light pink colour
appears. Also check the pH of the solution. The pH of the
sample should lie between 8-9.

7. Repeat the process and take the average of three readings.

B) Transesterification Step:

1. Take 25ml of oil in a conical flask.

2 .Place it on a magnetic stirrer and maintain the temperature

between 45- 50oC.

3. Add methanol as per the titrated value in another conical flask and add the required
amount of KOH (determined from titration step).

4. This mixture of methanol and KOH was added in the oil in three phases (in ratio of 2:1:1)
and temperature was maintained till the time as mentioned in table.

5. Continue the process for 45 minutes and then pour the mixture in a separating funnel.
Leave the set up for overnight separation.
 68

C) Washing:
1. After removing glycerin from the biodiesel pour warm water into the separating funnel and
leave it for few minutes. Remove the water carefully.
2. Repeat this step till the water becomes clear.
3. Take out the biodiesel and heat it to remove the methanol.
4. Amount of biodiesel formed was observed and yield was calculated.

10. Comparative Analysis of biodiesel and commercial diesel

Experiment no.-1

Objective- To determine the moisture content of biodiesel and commercial diesel.

The same principle and procedure was followed as that for the determination of moisture content
of crude oil.

Formula:
Moisture content %= Initial weight – Final weight X 100
 69

Initial weight of sample

Observations and calculations:

 Weight of empty beaker = 4.68966 g
 Weight of beaker + 1 ml biodiesel = 5.7130 g
Weight of sample = 1.0234 g
 Weight of beaker + 1 ml commercial diesel = 5.6937g
Weight of sample = 1.0121g

S.No. Sample Initial wt. Final wt. Constant

(g) (g) wt.

1. Jatropha 5.7130 5.7123 5.7123

curcas
(whole seed)
biodiesel

2. Commercial 5.6937 5.6931 5.6931

diesel

Calculation:

 For Biodiesel

Moisture content % = 5.7130 – 5.7123 X 100

1.0234
= 0.068%
 For Commercial diesel

Moisture content % = 5.6937– 5.6201 X 100

1.0121
=0.16%
 70

Result:

The moisture content of Biodiesel is 0.068%

The moisture content of Commercial Biodiesel is 0.016%

Experiment no.-2

Objective: To Determine the solubility of Biodiesel and commercial biodiesel in various

solvents.

Observations:

Solvent
Sample Methanol Ethyl Acetate Hexane Acetone

Jatropha curcas Insoluble Soluble Soluble soluble

(whole seed)
 71

Biodiesel

Solvent
Sample Methanol Ethyl Acetate Hexane Acetone

Commercial Insoluble Soluble Soluble soluble

Biodiesel

Result:
Both Biodiesel and commercial diesel samples are soluble in hexane, ethyl Acetate and acetone
but insoluble in Methanol.

Precautions:

1. All eppendorf tubes should be cleaned.

2. Ratio of oil & solvent should be equal.
3. The contents of the eppendorf tubes should be properly missed before taking down the results.

Experiment no. 3

Objective: To determine the specific gravity of biodiesel and commercial diesel.

The same principle and procedure was followed as that for the determination of specific gravity of
crude oil.

Observations:

 FOR BIODIESEL

Weight of pycnometer (X) = 20.87898 g

 72

Weight of pycnometer + 1 ml water (Y) = 21.89159 g

Weight of pycnometer + 1 ml biodiesel (Z) = 21.62807 g

 FOR COMMERCIAL DIESEL

Weight of pycnometer (X) =20.97731 g

Weight of pycnometer + 1 ml water (Y) = 21.97882 g

Weight of pycnometer + 1 ml Commercial biodiesel (Z) = 21.78324 g

Calculations & Results:

 FOR BIODIESEL
Specific gravity= Weight of biodiesel = Z–X
Weight of Water Y–X

= 21.62807-20.87898 /21.89157-20.87898

=0.84936

 FOR COMMERCIAL DIESEL

Specific gravity= Weight of commercial diesel = Z – X

Weight of Water Y–X

=21.78324-20.97731/21.97882-20.97731

= 0.804714

S.no. Sample X Y Z Specific

gravity
(g) (g) (g)

1. Jatropha 20.87898 21.89157 21.62807 0.849360

curcas
(whole seed)
biodiesel

2. Commercial 20.97731 21.97882 21.78324 0.804714

diesel
 73

Result:

The specific gravity of Biodiesel is 0.849360.

The specific gravity of commercial Biodiesel is 0.804714.

Experiment no.4

Objective- To determine the acid value of biodiesel and commercial diesel.

The same principle and procedure was followed as that for the determination of acid value of
crude oil.

Observations & calculations:

S.no. Sample weight Initial Final Titre

of sample reading reading value
of burette of burette
(ml
(g) (ml) (ml) )
 74

1. Jatropha 2.06114 7 7.6 0.6

curcas
(whole
seed)

2. Commercial 2.06109 7 7.2 0.2

diesel

 FOR BIODIESEL
Acid Value (A.V.) = 56.1 X Titer Value X Normality
Weight of Sample taken
=56.1 X 0.6X 0.1 =1.63307
2.06114

 FOR COMMERCIAL DIESEL

= 56.1 X 0.2X 0.1 =0.54437
2.06109

Result:

The acid value for biodiesel is 1.63307 meq/g.

The acid value for Commercial biodiesel is 0.54437 meq/g

Experiment no.5

Objective-To determine saponification value of biodiesel and commercial diesel.

The same principle and procedure was followed as that for the determination of saponification
value of crude oil.

Formula:
Saponification Value= Concentration conversion factor (28.05) X (Blank – Sample)
Weight of Sample taken

Observations:
 75

Sample Wt. of oil Initial Final Titre

reading of reading of value(ml)
(g)
burette(ml) burette(ml)

a). BLANK:

Biodiesel - 0.0 49.7 49.7

Commercia -
l diesel 0.0 54.0 54.0

b).SAMPLE:

Biodiesel 5.0014 0.0 28.6 28.6

Commercial 5.1027 0.0 50.0 50.0
diesel

Calculations:

 FOR BIODIESEL

Saponification Value= (28.05) X (49.7-28.6) = 118.33

5.0014

 FOR COMMERCIAL DIESEL

Saponification Value= (28.05) X (54-50) =21.988

5.1027

Result:

Saponification value for biodiesel is 188.33

Saponification value for commercial biodiesel is 21.988

 76

Experiment no.6

Objective: To determine the peroxide value of biodiesel and commercial diesel.

The same principle and procedure was followed as that for the determination of peroxide value of
crude oil.

Observations:
 77

Sample Wt. of oil Initial reading of Final Titre

burette(ml) reading of value(ml)
(g)
burette(ml)

a). BLANK:

Jatropha - 0.0 4.0 4.0

curcas(whol
e seed)
Biodiesel

Commercial - 0.0 22.0 22.0

diesel

b).SAMPLE:

Jatropha 5.00245 4.0 8.0 4.0

curcas
(whole seed)
Biodiesel

Commercial 5.1872 22.0 44.0 22.0.

diesel

Calculations

 FOR BIODIESEL

Peroxide Value= (4.0 – 4.0) × 0.1× 1000 = 0.0 meq/g

5.00254
 FOR COMMERCIAL DIESEL
Peroxide Value= (22.0 – 22.0) × 0.1× 1000= 0.0 meq/g
5.00254

Result: The peroxide value of both and commercial biodiesel is 0.0 meq/g
 78

11. Results and Discussions

Jatropha curcas due to its potential in adapting to extreme weather conditions and its ability to
grow in fallow lands, waste lands and its cost effectiveness makes it a good source for the
production of biofuel on a large scale. As a country and around the world we should make
changes for our future and our planet. Biodiesel is a beneficial alternative fuel source that has both
economical and environmental benefits. Diesel-powered cars generally have a better fuel
economy and produce less greenhouse gas pollution than equivalent gasoline engines . This
greater fuel economy is due to the higher energy per-liter content of diesel fuel and the intrinsic
 79

efficiency of the diesel engine. While diesel's 15% higher density results in 15% higher
greenhouse gas emissions per liter compared to gasoline modern diesel-engine automobiles
offset this by having 20-40% better fuel economy. Gases. This results in significantly lower carbon
dioxide emissions per kilometer.

 ANALYSIS OF SOIL

S.NO PARAMETERS RESULT

1. Moisture content 0.2598%

2. ph 7.2

3. Water holding capacity 24%

4. Bulk density 1.228 g/cc

5. Particle density 1.6035 g/cc

6. Porosity 23.42%

7. Total organic content 2.584%

8. Available nitrogen 150.528 kg/hec

9. Available phosphorus 1.9204ppm

10. Available sodium 0.02ppm

11. Available potassium 0.27

 ANALYSIS OF JATROPHA CRUDE OIL,BIODIESEL & COMMERCIAL DIESEL

S.NO PARAMETERS JATROPHA JATROPHA COMMERCIAL

CRUDE OIL BIODIESEL DIESEL

1. Moisture content 3.46% 0.068% 0.016%

2. Specific gravity 0.8790 0.8493 0.8047

3. Acid value 5.6022 meq/g 1.6307 meq/g 0.54431 meq/g

4. SAP value 172.44 118.33 21.988

5. Peroxide value 0.00 meq/g 0.00 meq/g 0.00 meq/g

 80

MOISTURE CONTENT
1
0.8
0.6
0.4
0.2
MOISTURE
0 CONTENT
 81

SPECIFIC GRAVITY
1
0.95
0.9
0.85
0.8
0.75 SPECIGIC
0.7 GRAVITY
 82

ACID VALUE
2.5
2
1.5
1
0.5
0 ACID VALUE
 83

PEROXIDE VALUE VALUE

1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1 PEROXIDE VALUE
0 VALUE

Conclusion
 84

With surging energy needs and depleting resources, the need for a renewable energy source is
ever increasing. Disputes will arise if the current use of edible oils is prolonged, this brought about
the need of extracting crude oil from a non-edible source.

My project aimed at extracting crude oil from the seeds of non-edible plant, Jatropha curcas. The
crude oil was then converted to its alkyl methyl ester and was analyzed for its physiochemical
properties. The properties of the alkyl methyl ester were compared to commercial diesel and an
analysis was made. Jatropha curcas due to its potential in adapting to extreme weather conditions
and its ability to grow in fallow lands, waste lands and its cost effectiveness makes it a better good
source for the production of biofuel on a large scale. It was noticed during the course of the project
that a temperature of 45-50°C, a reaction time of about 45 minutes was ideal for the
transesterification reaction. Alkaline catalysts in the form of KOH proved to be ideal for the
reaction and will also prove successful at an industrial scale due to their cost effectiveness.

Diesel fuels also contain high quantities of sulfur leading to a negative impact on the environment.
They prevent the use of catalytic diesel particulate filters to work properly. These filters reduce
emissions by using particulate and nitrogen oxide emissions due to diesel. Laws to reduce the
sulfur content have been enforced by many refineries but the result is a less lubricating fuel.
Biodiesel is low sulfur containg fuel.

Appendix
 85

Molarity = wt.of sampleX 1000

molecular wt. X volume (ml)

Normality = wt.of sampleX 1000

equivalent wt. X volume (ml)

10 ml 1N Potassium Dichromate (eq. wt. = 49.03)

K2Cr2O7 (g) Water (ml)

0.49 100

50 ml 0.5M Ferrous Ammonium Sulphate (mol. wt. = 392.13)

Fe(NH4)2(SO4)2.6H2O (g) Water (ml)

9.8032 50

10 ml 95% Ethanol

C2H5OH (g) Water (ml)

9.5 0.5

100 ml 0.1N Potassium Hydroxide (eq. wt. = 56.11)

KOH (g) Water (ml)

0.56 100

100 ml 0.5N Methanolic Potassium Hydroxide

KOH (g) Methanol (ml)

0.2805 100

100 ml 0.5M Hydrochloric acid (density = 1.180 g/ml, mol. wt. = 36.46)

HCl (ml) Water (ml)

4.47 (for 34.5% pure HCl) 93.53

60 ml 3:2 Acetic acid-Chloroform solution

CH3COOH (ml) CH3CL (ml)

 86

36 24

100 ml 0.1N Sodium thiosulphate (eq. wt. = 124.09)

Na2SO3 (g) Water(ml)

1.2409 100

100 ml 0.32% Potassium Permanganate solution (mol. wt. = 158.032)

KMno4 (g) Water(ml)

0.32 100

100 ml 2.5% Sodium Hydroxide solution (mol. wt. = 39.997)

NaOH (g) Water(ml)

2.5 100

100 ml 2.5% Boric acid solution (mol. wt. = 61.83 mg)

H3BO3(g) Water(ml)
2.5 100

Saturated KI (Potassium Iodide) solution- 1ml water was taken in a test tube & KI is
added till precipitates formed.

1% Starch solution- Dissolve a pinch of starch in 1ml water and heat till solution become
clear.

12. LECTURES ATTENDED

   ENZYME IMMOBILIZATION
 87

As enzymes are biological catalysts that promote the rate of reactions but are not themselves
consumed in the reactions; they may be used repeatedly for as long as they remain active.
However, in most of the processes, enzymes are mixed in a solution with substrates and cannot
be economically recovered after the reaction and are generally wasted. Thus, there is an incentive
to use enzymes in an immobilized or insolubilized form so that they may be retained in a
biochemical reactor for further catalysis. This is done by Enzyme immobilization which may be
defined as-
                   “The process whereby the movement of enzymes, cells, organelles, etc. in space is
completely or severely restricted usually resulting in a water-insoluble form of the enzyme.”
Immobilized enzymes are also sometimes referred to as sound, insolubilized, supported or matrix-
linked enzymes.

SALIENT FEATURES OF ENZYME IMMOBILIZATION:-

1. The enzyme phase is called as carrier phase which is water insoluble but hydrophilic
porous polymeric matrix, e.g. agarose, cellulose, etc.
2. The enzyme phase may be in the form of fine particulate, membranous, or
microcapsule.
3. The enzyme in turn may be bound to another enzyme via cross linking.
4. A special module is produced employing immobilization techniques through which
fluid can pass easily, transforming substrate into product and at the same time facilitating
the easy removal of catalyst from the product as it leaves the reactor.
5. The support or carrier utilized in immobilization technique is not stable at particular
pH, ionic strength, or solvent conditions. Hence, may be disrupted or dissolved releasing
the enzyme component after the reaction.

Advantages of enzyme immobilization:-

 Multiple or repetitive use of a single batch of enzymes.
 Immobilized enzymes are usually more stable.
 Ability to stop the reaction rapidly by removing the enzyme from the reaction solution.
 Product is not contaminated with the enzyme.
 Easy separation of the enzyme from the product.
 Allows development of a multienzyme reaction system.
 Reduces effluent disposal problems.
 88

Disadvantages of enzyme immobilization:-

 It gives rise to an additional bearing on cost.
 It invariably affects the stability and activity of enzymes.
 The technique may not prove to be of any advantage when one of the substrate is found to
be insoluble.
 Certain immobilization protocols offer serious problems with respect to the diffusion of the
substrate to have an access to the enzyme.

TECHNIQUE OF ENZYME IMMOBILIZATION:-

1.     Carrier binding.
· Physical adsorption.
· Covalent bonding.
· Ionic bonding.
2.     Cross linking.
3.     Entrapment.
· Occlusion within a cross linked gel.
· Microencapsulation.

PHYSICAL ADSORPTION:-
This method is based on the physical adsorption of enzyme protein on the surface of water-
insoluble carriers. Examples of suitable adsorbents are ion-exchange matrices, porous carbon,
clay, hydrous metal oxides, glasses and polymeric aromatic resins.

The bond between the enzyme and carrier molecule may be ionic, covalent, hydrogen,
coordinated covalent or even combination of any of these.
Immobilization can be brought about by coupling an enzyme either to external or internal surface
of the carrier.

Advantages of adsorption:-
·       Little or no confirmation change of the enzyme.
·       Simple and cheap.
·       No reagents are required.

·       Wide applicability and capable of high enzyme loading.

 89

Disadvantages of adsorption:-
·       Desorption of the enzyme protein resulting from changes in temperature, pH, and ionic
strength & slow

Covalent bonding:-
Covalent binding is the most widely used method for immobilizing enzymes. The covalent bond
between enzyme and a support matrix forms a stable complex. The functional group present on
enzyme, through which a covalent bond with support could be established, should be non
essential for enzymatic activity.
The most common technique is to activate a cellulose-based support with cyanogen bromide,
which is then mixed with the enzyme.
The protein functional groups which could be utilized in covalent coupling include:
 Amino group
 Carboxylic group
 Phenol ring
 Indole group
 Imidazole group
On the other hand examples of the polymeric supports include:
    ·       Amino and related groups of polysaccharides and silica gel etc.
·       Carboxylic acid and related groups of polyglutamic acid, carboxy methyl cellulose.
·       Aldehyde and acetal groups of polymers.
·       Amide gr. Of polypeptide.

The polymers may be engaged in direct coupling as well as could be modified by other
coupling groups or activating groups. The most commonly used polymers are polysaccharides,
polyvinyl alcohol, silica and porous glasses.

Advantages of covalent coupling:-

 The strength of binding is very strong, so, leakage of enzyme from the support is absent or
very little.
 This is a simple, mild and often successful method of wide applicability
Disadvantages of covalent coupling:-
·       Enzymes are chemically modified and so many are denatured during immobilization.
·       Only small amounts of enzymes may be immobilized (about 0.02 grams per gram of matrix).
 90

Cross linking:-
This method is based on the formation of covalent bonds between the enzyme molecules, by
means of multifunctional reagents, leading to three dimensional cross linked aggregates.
The most common reagent used for cross-linking is glutaraldehyde.
Advantages of cross linking:-
·       Very little desorption(enzyme strongly bound)
·       Best used in conjunction with other methods.
Disadvantages of cross linking:-
·       Cross linking may cause significant changes in the active site.
 
Entrapment:-
In entrapment, the enzymes or cells are not directly attached to the support surface, but simply
trapped inside the polymer matrix. Entrapment is carried out by mixing the biocatalyst into a
monomer solution, followed by polymerization initiated by a change in temperature or by a
chemical reaction.
Polymers like polyacrylamide, collagen, cellulose acetate, calcium alginate or carrageenan etc are
used as the matrices.
Advantages of entrapment:-
 Loss of enzyme activity upon immobilization is minimized.
Disadvantages of entrapment:-
 The enzyme can leak into the surrounding medium.
 Another problem is the mass transfer resistance to substrates and products.
 Substrate cannot diffuse deep into the gel matrix.

1. Occlusion within a cross linked gel:- 

In this entrapment method, a highly cross-linked gel is formed as a result of the polymerization
which has a fine "wire mesh" structure and can more effectively hold smaller enzymes in its cages.
Amounts in excess of 1 g of enzyme per gram of gel or fibre may be entrapped.
Some synthetic polymers such as polyarylamide, polyvinylalcohol, etc... and natural polymer
(starch) have been used to immobilize enzymes using this technique.

2. Microencapsulation:-
This entrapment involves the formation of spherical particle called as “microcapsule” in which a
liquid or suspension of biocatalyst is enclosed within a semi permeable polymeric membrane. 
 91

2. Biotechnology in India:

Biotechnology is the offshoot of science in which living beings are used for making products. Flora
and fauna and microorganisms like bacteria are used for this purpose. In the field of medicine and
agriculture, bio technology has been used to produce food, test for diseases and to remove waste.
Biotechnology is divided into three sub field-red, white and green biotechnology.

Red biotechnology deals with genetically changed microorganisms being used for manufacturing
products like insulin and vaccine for medical use. It is due to research in red biotechnology that
antibiotics for various infections have been developed and vaccines to bolster the bodies’
resistance to various diseases were developed. It has also been used in reproductive technologies
like invitro fertilization, DNA profiling, forensics and transplantation technologies.

White biotechnology deals with creating useful chemicals for the industrial sector through
organisms like moulds or yeast. This type of bio technology is also referred to as grey
biotechnology. White biotechnology has proven to be of immense benefit environmentally in
cleaning oil spills and in storing DNA samples of endangered species for future research. It is also
useful for removing excess nutrients in soil and water and for detection of landmines.

Green biotechnology also called agricultural biotechnology is to do with factors pertaining to

agriculture. Green biotechnology is concerned with the genetic modification of plants and animals
to produce environmentally friendly species.

10.2. Importance of Biotechnology:

In today's era, when people are exposed to so many physical disorders, biotechnology plays a
vital role in developing medicines, vaccines, energy production, and conservation. To keep pace
with the competitive world, India has launched a comprehensive programme in biotechnology to
make use of the resources available. In India the Department of Biotechnology (DBT) was
established in the year 1986 under the ministry of Science and Technology.

It is imperative that India has to keep up with the increasing demand for food from the ever
expanding population. Agricultural land is also shrinking. Genetic engineering of plants to increase
their yield is the way to go in future.
 92

Biotechnology can be used in a wide range of economic activity ranging from environment, animal
husbandry, medicinal and aromatic plants, bio fuels, aquaculture and products like silk and
leather.

Future of the Bio Technology Sector According to reports bio technology industry in India has
become the fourth largest adopter of biotech crop in the world, replacing Canada. The Indian
biotechnology industry is slated to become a US$ 5 billion industry by 2010. India is gaining
recognition in the field of clinical trial. A large number of companies are providing research and
development expertise to global pharmaceutical companies.

13. RERENCES

 Kondu, H. Fukuda and H Noda, Biodiesel fuel production by transesterification of oils,

Journal of Bioscience and Bioengineering, Vol 92(5), 2001, pp. 405-416.

 Macor, A. Mirandola, A. Stoppato, C. Carraretto and S. Tonon, Biodiesel as alternative fuel:

Experimental analysis and energetic evaluations, Energy,Vol 29, 2006, pp. 2195-2211.
 93

 C.L. Lee, D.S. Robinson and R.S. Antony, Biodiesel production from jatropha oil and its
characterization; Research Journal of Chemical Sciences, Vol. 1(1), 2011, pp. 215-220.

 E. Akbar; Characteristic and Composition of Jatropha Curcas Oil Seed from Malaysia and
its Potential as Biodiesel Feedstock; European Journal of Scientific Research, Vol 29(3),
2009, pp 396-403.

 H.J. Berchmans and S. Hirata, Biodiesel production from crude Jatropha curcas seed oil
with a high content of free fatty acids, Science Direct, Bioresource technology, Vol 99,
2008, pp. 1716-1721.

 H. Masjuki, Biofuel as diesel fuel alternative: An overview, Journal of Energy, Heat and
Mass Transfer, Vol 15, 1993, pp. 293-304.

 J.H. Van Gerpen and M. Canackci, Biodiesel production via acid catalysis, Transactions of
the ASAE, 2009, Vol 42, pp. 1203-1210.

 M.A. and M.A. Hanna, Biodiesel production a review, Bioresource technology, Vol 70, 2001,
pp. 1-15.

 M. Mittleback and W. Haas, Detoxification experiments with seed oil from Jatropha curcas,
Industrial Crops Produce, Vol 12(2), 2000, pp. 111-118.

 P.K. Barua, Biodiesel from Seeds of Jatropha Found in Assam, India, International Journal
of Energy, Information and Communications, Vol 2(1), 2011, pp 197-206.

 P. Nakpong and S Wootthikanokkhan, Optimization of biodiesel production from Jatropha

curcas L. Oil via alkali-catalysed methanolysis, Journal of sustainable Energy and
Environment, Vol 1(1), 2010, pp. 105-109.

 P.S. Rao and V.K. Gopalakrishnan, Vegetable oils and their methyl esters as fuels for
diesel engines, Indian Journal of Technology, Vol 29, 1991,pp. 292-297.

 R.K. Singh and S.K. Padhi; Characterisation of the Jatropha oil for the preparation of
biodiesel; Natural Product Radiance, Vol 8(2), 2009, pp. 127-132.

 Tyson et al, Biodiesel handling and use guidelines” National Renewable Energy Laboratory
(NREL), Golden Co. NREL/TP-580-30004.

 Y. Takeda, Development Study on Jatropha curcas (Sabu dum) oil as a substitute for diesel
engine oil in Thailand, Journal of Agricultural Associates, China, Vol 20, 1982, pp. 1-9.
94