Chapter three

3. Material and method

The experimental work has been done in laboratory of Addis Ababa science and technology university
department of chemical Engineering

3.1. Materials
3.1.1. Equipment

The equipment used in the laboratory experiment are:

 Soxhlet apparatus
 Oven drier
 Weight balance
 Ball mill
 Sieve (screener)
 Rotary device
 Magnetic stirrer
 Heater
 Filter
 Stop watch
 Flask
 Beaker
 Volumetric cylinder
 pH meter
 Conductometer
 Refractometer
3.1.2 Chemicals

The chemicals used in the laboratory experiment are:

 Hexane
 Ethanol
 Acetone
 Starch
 Acetic acid
 Hydrochloric acid
 Sodium hydroxide
 Potassium hydroxide
 Sodium carbonate
  Sodium thiosulphate
 Phenolphthalein indicator
 Chloroform
 Potassium iodide
 Distilled water
3.2. Method
3.2.1. Raw material Preparation

This is the initial stage of sample preparation and it includes the following:

Mango seed collection: mango seed was collected from juice houses of AASTU.

Cleaning: the outer part of the seed was separated from the chaffs and other impurities. This
Preparation is very important since any impurity in the seed will eventually reflect on the oil
extracted.

Drying: After the mango seed has been cleaned thoroughly; the mango kernel part was separated
and dried in an electric oven at 1070C for 10 hours to reduce the moisture content of the seed and
the moisture has been measured using electronic weighing balance.

Size Reduction: Mango seed kernel was milled using electronic miller to provide a greater
surface area using sieve analyses.

A. Determination of moisture content of the seeds

A sample of the kernel was randomly weighed and dried in an oven at 107 0C and the weight was
measured every two hours. T he procedure was repeated until a constant weight was obtained. The
Percentage moisture in the kernel was calculated using the following: ‘M’ initial mass and ‘m’
final mass

( M −m )
Moisture content ( % )= ∗100 %
(M)

B. Size reduction and sieve analysis of the seed

The mango kernel seed was crushed by the miller and the sample has been vibrated using the
sieve until the particle size becomes 0.5mm since we have not used the size as a factor of our
experiment.
 3.2.2. Oil Extraction Process

 200ml of hexane was measured in a measuring cylinder and then poured into three
necked round bottom flasks.
 The round bottom flask was placed on a heating mantle.
 It was then uncorked and the extractor of the Soxhlet was placed on it.
 A condenser was placed on the extractor and properly connected to a tap.
 A piece of filter paper was rolled and used to make a conical cylinder.
 30g of the kernel powder was weighed and poured into the conical cylinder.
 The heating mantle was switched on and set at temperatures of 55, 60 and 70 C. The
extraction was left to continue for 2, 4 and 6 hours.
 After such hours, the heating mantle was switched off and the apparatus was allowed to
cool.
 The product was taken to a rotary evaporator for further separation of the hexane and oil.
 A condenser was then connected to the round bottom flask which still sat on the heating
mantle and the conical flask was attached to the other end of the condenser to collect the
hexane that vaporizes from the mixture in the round bottom flask.
 The heating mantle was switched on and set at temperature of 690C until the hexane was
vaporized.
 The total yield of oil was expressed in percentage

Determination of percentage of oil extracted: The percentage yield was calculated using the
formula:

mass of the oil

percentage yield ( % )= ∗100 %
mass of the sample
 3.3. Characterization of the Extracted Oil
The acidity, iodine, peroxide and saponification values are the major characterization parameters
for oil quality. Physical properties like moisture content, impurity (gum content), specific
gravity, viscosity, PH value and conductivity and chemical properties like saponification value,
free fatty acid, acid value and peroxide value were determined for the oil which was extracted
using optimum operating parameters

3.3.1. physical characteristic of the oil

3.3.1.1. Determination of Moisture of oil
5gm of oil was weighted and putted in a beaker and then dried in an oven at 110 0C for 1 hour.
The beaker was removed from the oven and cooled and weighed. The process was repeated until
a constant weight was observed and the moisture and volatile matter of the oil was determined.

3.3.1.2. Specific Gravity

The specific gravity – SG - is a dimensionless unit defined as the ratio of density of the mango
oil to the density of water at a specified temperature. This was done by measuring the density of
mango oil in reference to the density of distilled water at 200C using a specific gravity bottle.

Procedure:
I. The mass of the bottle was weighed while empty. It was then filled with mango oil
and weighed again
II. The difference in weights was divided by the mass of equal volume of water to obtain
the specific gravity of the mango oil.
3.3.1.3. Determination of PH
2 gm of the sample was taken and putted in to a clean dry 25 ml beaker. The PH electrode was
standardized with a buffer solution first and then immersed in to the sample and the PH was read.
 3.3.1.4. Determination of conductivity
2 gm of the sample was taken and putted in to a clean dry 25 ml beaker. The conductor meter
was standardized with a buffer solution first and then immersed in to the sample and the
conductivity was read.

3.3.2. Characterization of the Chemical Property of Oil

3.3.2.1. Determination of Saponification value
2 gm of the sample was taken and added in to 250 ml flask. 25 ml of alcoholic potassium
hydroxide solution was added in to the flask. Then few drops of phenolphthalein indictor were
added and the excess potassium hydroxide was titrated with 0.5 N hydrochloric acid to the end
point, until the pink color of the indictor just disappears. The same procedure was conducted for
the blank and the saponification value (SV) expressed as the number of milligrams of KOH
required to saponify 2 gm of fat was calculated.

3.3.2.2. Determination of acid value

1.9 gm of the sample of oil was weighed and putted in to 250 ml conical flask and 50 ml of hot
ethyl alcohol was added to the flask. After few drops of phenolphthalein was added, the mixture
was boiled for about five minutes and while it was hot titrated with 0.5N sodium hydroxide
solution and then the acid value was determined. Since the acidity is frequently expressed as free
fatty acid from acid value free fatty acid was calculated.
3.3.2.3. determination of peroxide value
30 ml of a mixture of acetic acid and chloroform in addition to 1 ml of potassium iodide and
0.5ml of starch solution was added to 5 gm of oil and titrated with a solution of sodium
thiosulphate and sodium carbonate.
 3.3.2.4. Ester Value
Ester value was obtained by subtracting the acid value from the saponification value. Ester value
represents the number of milligrams of potassium hydroxide required to saponify the esters
present in 1g of the oil.

3.4. Design of the Experiment

Factorial design is used to investigate the effect of each factor. In a factorial experiment all the
possible combinations factor levels would be tested and it would be possible to determine the
effect of individual factors and to assess the effect of change of two or more variable at a time.
This method of experiment design helps to differentiate the significance of the main and the
interaction factors. 2 factors were investigated as mentioned earlier; these were: time of
extraction and Extraction temperature.
Factors Levels: For each factor, three levels were considered.
Replicates: Each independent experiment was repeated two times.
Number of runs: For “m” levels, “n” factors and “k” replicate, the number of experimental runs
that need to be performed is equal to (k*mn)
In this research m=3, n=2 and k=2 thus, 2*32 = 18 experimental runs were performed.
The levels that were selected for each factor are:
 Extraction time (hr.): 2, 4 and 6.
 Extraction temperature: 55, 60 and 70
 Chapter Four
4. Results and Discussion
4.1. Results
4.1.1. Moisture content of the seed kernel: three samples are taken (19.6, 19.8 and 11.7
gram) of the mango seed kernel, the moisture content of the samples was determined.

Sample weight in gram

Sample 1 Sample 2 Sample 3

Initial mass 19.6 19.8 11.7
Mass after 2 hrs 17.92 18.1 9.84
Mass after 4 hrs 16.47 15.64 8.76
Mass after 6 hrs 13.31 14.01 7.43
Mass after 8 hrs 11.2 11.1 6.35
Mass after 10hrs 11.2 11.1 6.35
final moisture 42.85 43.94 45.72

4.1.2. Oil Extraction

Percentage Yield of Extraction: The percentage extraction yield was calculated by using yield equation

Number of Factors Results (g) Oil yield (%)

experiments
Mea
Temperature, Contact Replicate Replicate Replicate Replicate
n
C time(hr.) 1 2 1 2

1 55 2 2.6 2.62 8.67 8.73 8.7

2 60 2 2.5 2.49 8.33 8.3 8.315

3 70 2 1.6 2.1 5.33 7.0 6.165

4 55 4 2.3 2.13 7.67 7.1 7.385

5 60 4 1.7 1.94 5.67 6.46 6.065

6 70 4 2.8 2.76 9.33 9.2 9.265

7 55 6 2.4 2.45 8.0 8.16 8.08

 8 60 6 2.19 2.18 7.3 7.26 7.28

9 70 6 1.6 1.84 5.33 6.13 5.73

4.1.3. Characterization of the extracted oil
Using process parameter that gave a maximum oil yield (extraction time = 4hr and extraction
temperature=70 0C) oil was extracted then the physical and chemical properties were studied.
4.1.3.1. physical properties
i. Moisture of extracted oil
The moisture and volatile matter of the oil was determined by oven method. 5 gm of oil was
taken and put in oven and the weight was recorded at 1 hour and 2 hours. The result obtained is
summarized in the table below:

Time
(hr.) 0 1 2

Weight
(gm) 5 4.88 4.88

(M – m) is loss in gm in material on drying = (5.0gm – 4.88gm) = 0.12gm

M is weight in gm of oil taken for the test = 5.0gm
( M −m )
MoistureMoisture content ( % )= ∗100 %
(M)
=2.4%

ii. Specific gravity

m= the mass of the bottle while empty.
M= mass of the bottle with mango oil
W= mass of equal volume of water
M at 300C = 35.55
m at 300C = 13.60
W at 300C = 37.692
Substituting the above values in the equation
( 35.55−13.6 )
specific gravity = =¿0.911
( 37.692−13.6 )
Hence the density of oil can be determined using:
( ρ oil )
specific gravity =
( ρwater )
Where:
ρoil = density of mango seed kernel oil
ρwater = density of water = 1000 kg/m3
Therefore, density of oil was 911 kg/m3
iii. pH value of oil
The pH value of oil was determined by pH electrode as measuring experimental procedures were
stated the pH value of kernel oil was triplicated and the results obtained are summarized in the
table:

Run pH value mean

1 5.79

2 5.80 =5.783

3 5.76
Table:
iv. conductivity value
2 gm of the sample was taken and putted in to a clean dry 25 ml beaker. The conductor meter
was standardized with a buffer solution first and then immersed in to the sample and the
conductivity was read.
Table:
conductivity
Run mean
value @25 0c
C(w/mk)

1 0.168

2 0.166 =0.1653

3 0.162
 4.1.3.2. Chemical characteristic

i. Saponification value
Saponification number was determined by using titration. Saponification is a number that
expresses in milligram of quantity of potassium hydroxide required to saponify 1 gram of oil/fat.
The required solutions were prepared with the required concentration.
Steps:
( 5.6 )
 Preparation of 0.5N alcoholic potassium hydroxide solution: =6.59g of
( 0.85 )
85 percent pure KOH was dissolved in 200ml 80 percent ethanol.
 Preparation of 0.5N hydrochloric acid solution:
A stock solution of 13.7ml of hydrochloric acid was poured in to 500ml distilled water.
 After 2 gm of fat was dissolved in alcoholic KOH and heated gently for 1 hour, it was
titrated with HCl to the end point.
 Similar titration was done for the blank.
 In both case the value of HCl was recorded.
Saponification value was calculated using equation
B is volume of HCl required for the blank = 37.56ml
S is volume of HCl required for the sample = 23.99ml
N is normality of HCl = 0.5N
W is weight of fat taken for the test = 2 gm
M= molar mass of potassium hydroxide=56.1
Substituting the above values in equation:
M∗ ( B−S )∗N
saponification value =
W
56.1∗( 37.56−23.99 )∗0.5 N
saponification value=
2g
=190.32mgKOH/gm
 ii. Acid value
Acid value is the measure of total acidity of the lipid involving contributions from all the
constituent fatty acids that make up the glyceride molecule. Titration method was used to
determine the acid value.
The required solutions were prepared with the required concentration as follows.
 Preparation of 80 percent ethyl alcohol:
19.6ml distilled water was added in to 80.4ml 99.5 percent absolute ethanol.
 Preparation of 0.5N sodium hydroxide solution:
10.1gm of 99 percent NaOH was dissolved in 500ml distilled water.

V is volume of NaOH used by the sample during titration = 0.25ml

N is normality of NaOH = 0.5N
W is weight of oil taken for test = 3.005gm
Substituting the above values in equation
M∗Δ V ∗N
acid value=
W
 iii. free fatty acid
Free fatty acids are those acids that are not present as triglycerides (fats) in the biodiesel. In the
saponification reaction, the triglycerides hydrolyze to yield fatty acids that react with the alkali to
form soap whereas the free fatty acids directly react with the base while conducting the
experiments to find out the acid value.
acid value
free fatty acid=
2

iv. Ester Value

Ester value was obtained by subtracting the acid value from the saponification value. Ester value
represents the number of milligrams of potassium hydroxide required to saponify the esters
present in 1g of the oil.
Ester Value=( saponification value−acid value )

v. Peroxide value
The amount of peroxide value of oils indicates the degree of primary oxidation and therefore its
likeliness of becoming rancid. A lower number of peroxide value indicates a good quality of oil
and a good preservation status. Results are expressed as meqO2/Kg.

The required solutions were prepared with the required concentration as follows:

 Preparation of a solution of acetic acid and chloroform

90ml acetic acid was mixed with 60ml chloroform
 Preparation of 1% starch solution
50ml of distilled water was measured and boiled. Then, 0.5gm starch was added to the
boiled water and was stirred. 30 minutes later the solution was filtered using filter paper.
 Preparation of 0.01N sodium thiosulphate
0.25gm of sodium thiosulphate is dissolved in 80ml of distilled water. After the solution
is heated 0.02gm sodium carbonate was added to it and it was shaken for a minute and
20ml of distilled water was added.
 Preparation of potassium Iodide solution.
1 spoon of potassium iodide was dissolved in 2ml of distilled water.
 5gm of oil was heated at 600C and 30ml of the acetic acid-chloroform solution was
added to it. Then, 1ml KI solution was added to the mixture followed by an addition of
30ml of distilled water and after shaking the solution 0.5ml of 1% starch solution was
added.
 Finally, the solution was titrated with sodium thiosulphate until the black color was
removed.
ΔV is change in volume of the titrate=1.9ml
N is Normality of the titrate= 0.01N
W is weight of the sample
Δ V ∗N∗1000
Per oxide value=
W
= 3.8 meq/Kg