BKC2732 Chemical Engineering Laboratory II

SEMESTER 2 SESSION 2022/2023

LAB TECHNICAL REPORT

THE ENZYMATIC SYNTHESIS OF ETHYL ETHANOATE BY USING
IMMOBILISED LIPASE

Nur Hidayah binti Kamarudin, Noorain Fatihah binti Kamal, Muhammad Afiq Ikmal Bin Afandi,
Shazlina binti Suhaimi, Ammar Hanif bin Mohd Zaidi, Asyrafulkhair bin Aseb
Section 01, K01
Dr Nurul Aini Binti Mohd Azman
Faculty of Chemical and Process Engineering Technology, College of Engineering
Universiti Malaysia Pahang, 26300 Gambang, Pahang, MALAYSIA.

ABSTRACT

The enzymatic synthesis of ethyl ethanoate utilizing immobilized lipase was investigated in this
study. The use of immobilization enzymes is one method for improving enzyme stability and, in
particular, reducing the difficulty of separating the substrate from the catalyst. The effect of the
lipase enzyme in the reaction as well as the effect of temperature on the conversion to ethyl
ethanoate were analyzed. Sequentially, phenolphthalein, ethanol, acetic acid solution, and NaOH
solution were prepared. 1g of phenolphthalein powder was mixed with 10 mL of 99.8% ethanol
and was ready when all of the phenolphthalein was completely dissolved in the ethanol after
water was added until the solution reached a volume of 50 mL for phenolphthalein preparation.
In the beaker, 3.51 mL of ethanol with up to 50 mL of distilled water were mixed well, and 2.86
mL of acetic acid and up to 50 mL of distilled water were mixed well to prepare the ethanol and
acetic acid solution. To prepare the NaOH solution, 500 mL of 0.1 M NaOH solution mixed with
2g of NaOH was transferred into the burette for titration. The reaction and titration processes
were continued in the experiment. At 27°C, 50 mL of each ethanol and acetic acid solution was
measured and combined with 0.3 g of lipase before being poured into a beaker. Every two
minutes, 1.0 mL of solution from the beaker was pipetted into a conical flask using a 1 mL
pipette. 2-3 drops of phenolphthalein indicator were added to 1.0 mL of extracted solution in a
conical flask. The mixture was titrated with NaOH solution until a faint pink solution appeared.
As the temperature rises, the rate of esterification increases due to the kinetic energy of the
molecule. The conversion rate at 37°C is higher than at 27°C because as the temperature rises, so
does the process's conversion rate since molecules react rapidly. The higher the temperature, the
faster each molecule travels, increasing collisions and, as a result, increasing the kinetics of the
reaction. As the temperature rises, the production of ethyl ethanoate from acetic acid and ethanol
treated with lipase accelerates.

Keywords: Ethyl ethanoate; immobilized lipase; enzymatic synthesis; conversion rate;

esterification

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1. Introduction

Ethyl ethanoate is one of the most regularly utilized small esters. It is a flammable,
colorless liquid with a pleasant odor that is used in fragrances, flavorings and medications. Ethyl
ethanoate is a common ingredient in nail polish removers and lacquer thinners because it serves
as an organic solvent. Ethyl ethanoate, often known as ethyl acetate, is a solvent and diluent that
is popular due to its inexpensive cost, low toxicity and pleasant odor. Esters, which are taste and
aroma molecules generated from short-chain carboxylic acids like acetic acid, are widely
employed in the food, beverage, cosmetic, and pharmaceutical sectors. Since the immobilized
enzyme has several appealing properties, such as reusability and accessibility, it has been
extensively researched and used in a variety of biocatalysis processes. It is now produced via
ecologically friendly chemical processes utilizing acetic acid as a feedstock. This project requires
students to investigate the ester development potential using various feedstocks and catalysts.
The enzymatic synthesis of ethyl ethanoate is promising due to its mild reaction
conditions, good catalysis efficiency, and intrinsic selectivity. The use of immobilized lipase
makes it considerably easier to separate the substrate from the catalyst. Ethanol is less costly than
acetic acid and is widely accessible from the petroleum industry's downstream. On the other
hand, acetic acid is freely available. The esterification process is shown in Eq.1.
CH3CH2OH + CH3COOH ↔ CH3COOCH2CH3 + H2O (Eq.1)
As a result, the goal of this study is to see how temperature affects the enzymatic
production of ethanol through the esterification of ethanol and acetic acid utilizing immobilized
lipase. The experiment was carried out using an acid-base titration between sodium hydroxide
and the acid content of ethanol and acetic acid mixture to determine the amount of sodium
hydroxide used, which corresponds to the degree of completion of esterification and the
conversion of the reactant at each interval of 2 minutes, ranging from 0 to 20 minutes at two
different temperatures, 27 °C and 37 °C.

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2. Methodology
2.1. Materials
99.8% Ethyl Alcohol, Sodium Hydroxide, Acetic Acid, Phenolphthalein and Enzyme
Lipase

2.2. Experimental
2.2.1 Preparation of Phenolphthalein Indicator
1g of phenolphthalein powder will be weighted. The phenolphthalein powder will be
mixed with 10 mL of 99.8% ethanol. Deionized water will then be added into the volumetric
flask until the solution reaches the volume of 50 mL and is ready when all phenolphthalein is
completely dissolved in ethanol.

2.2.2 Reaction of Ethanol and Acetic Acid

3.51 mL of Ethanol was measured and diluted with distilled water up to 50 mL to produce
1.2 M of Ethanol solution. 2.86 mL of Acetic Acid was measured and diluted with distilled water
up to 50 mL to produce 1.0M of Acetic Acid solution. Both solutions will be mixed well in the
beaker using a magnetic stirrer. Next, the thermometer is used to measure the temperature and set
it to 27°C. Once 0.4g lipase enzyme is added, the reaction time is recorded. Samples are taken
every 2 minutes for titration process purposes. The steps are repeated with temperature at 37°C.

2.2.3 Preparation of NaOH solution

2g of NaOH will be weighed and then dissolved in 500mL of distilled water to produce
0.1 M NaOH solution. 500 mL of 0.1 M NaOH solution will be poured into the burette for the
titration process.

2.2.4 Titration Process

1 mL of sample from ethanol and acetic acid reaction is taken and 2-3 drops of
phenolphthalein indicator will be added to the conical flask. The mixture will be titrated with
NaOH solution until a faint pink solution appears. The amount of NaOH titrated will be
measured. The titration procedure continues until the titrated volume of NaOH becomes constant.
The procedure was repeated for different samples taken every 2 minutes. The experiment will be
repeated at a mixed solution temperature of 37°C.

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3. Result and discussion

3.1. Preparation of solutions

Preparation of 50 ml of 1.0M Ethanol Preparation of 50 ml of 1.0M Acetic Acid

MW of Ethanol = 46.07 g/mol MW of Acetic Acid = 60 g/mol
Density = 789 g/L Density = 1050 g/L
Purity = 95% Purity = 99.8%
Molarity = 0.95(789) / 46.07 Molarity = 0.998(1050) / 60
= 16.26 mol/L = 17.48mo/L
M1V1 = M2V2 M1V1 = M2V2
16.26V1 = 1.2(50ml) 17.48V1 = 1(50ml)
V1 = 3.7 ml V1 = 2.86 ml

Preparation of 500 ml of 0.1M Sodium Hydroxide

MW of NaOH = 40 g/mol
Mole = 0.1 mol/L x 0.5 L = 0.05 mol
Mass of NaOH needed= 0.05 mol x 40 g/mol = 2.0 g

For every 1 ml of Ethanol-acetic mixture solution, it consists of:

0.1𝐿 𝑥 1.2𝑀
0.1𝐿+0.1𝐿
𝑥 0. 001 = 0.6 x 10-3 mol acetic acid

CH3COOH (aq) + NaOH (aq) 🡪 H2O (l) + CH3COONa+ (aq)

From the chemical equation above, 1 mole of Acetic acid required 1 mole of NaOH to neutralize.
Therefore, 0.6 x 10-3 mole of Acetic acid required 0.6 x 10-3 mole of NaOH to be neutralized. The
theoretical amount of 0.1 M NaOH is required to neutralize 1 ml of Ethanol-Acetic acid mixture
solution.
VNaOH = NNaOH / MNaOH
= 0.6 x 10-3 mol / 0.1 mol
= 0.006 ml

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3.2. Results of experiment

Table 1: Volume of NaOH required to neutralize 1 ml of Ethanol-Acetic acid mixture solution

extracted from distillation flask every 2 minutes interval throughout the esterification process at
different temperature of 27oC and 37oC
Time (min) 27oC 37oC
Volume of Conversion, 𝑋 Volume of Conversion, 𝑋
1−𝑋 1−𝑋
NaOH X NaOH X
required required
(mL) (mL)
0 4.1 0.8038 4.0980 2.9 0.8840 7.6207
2 4.1 0.8038 4.0980 2.8 0.8880 7.9286
4 4.0 0.8077 4.2000 2.8 0.8880 7.9286
6 3.9 0.8115 4.3061 2.7 0.8920 8.2593
8 3.7 0.8192 4.5319 2.7 0.8920 8.2593
10 3.7 0.8192 4.5319 2.6 0.8960 8.6154
12 3.6 0.8231 4.6522 2.5 0.9000 9.0000
14 3.6 0.8231 4.6522 2.5 0.9000 9.0000
16 3.5 0.8269 4.7778 2.3 0.9080 9.8696
18 3.4 0.8308 4.9091 2.3 0.9080 9.8696
20 3.4 0.8308 4.9091 2.3 0.9080 9.8696

𝑇ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑁𝑎𝑂𝐻 𝑟𝑒𝑞𝑢𝑖𝑟𝑒𝑑−𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑁𝑎𝑂𝐻 𝑟𝑒𝑞𝑢𝑖𝑟𝑒𝑑 𝑝𝑟𝑎𝑐𝑡𝑖𝑐𝑎𝑙𝑙𝑦 𝑎𝑡 𝑡

𝐶𝑜𝑛𝑣𝑒𝑟𝑠𝑖𝑜𝑛 (%) = 𝑇ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑁𝑎𝑂𝐻 𝑟𝑒𝑞𝑢𝑖𝑟𝑒𝑑
𝑥100%

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Figure 1: Volume of NaOH required vs Time

When Ethanol reacts with Acetic Acid it is esterification process:

CH3COOH+CH3CH2OH🡪CH3COOCH2CH3+H2O
Can be simplified into:
A+B🡨🡪 C+D
Assumption:
1. The reason is elementary and irreversible reaction
2. It is isothermal reaction
3. Amount of lipase extracted during extraction of mixture solution is neglected as the
extracted amount is relatively small
4. Acetic acid is chosen as limiting reactant
5. The change in concentration of extracted 5 ml solution is very small
6. There is no pressure drop in this reaction, where y=1

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𝑑𝑋
Design equation: CA0 𝑑𝑡
= -rA

Rate Law: -rA = kCACB

Stoichiometry: CA = CA0 ((1−𝑋)𝑇0
1−ε𝑋)𝑇
𝑦, ε = 0

CA = CA0 (1-X)
CB = CA0 (1-X)
Combine: CA0 𝑑𝑋𝑑𝑡 = k CA02 (1-X)2
𝑥
1
∫ 𝑑𝑋
0 (1−𝑋)
2
= kCA0t

The problem can be solved by using the linear equation: Y = mX+ C

From the integral: Y = X/(1-X)
m = k CA0
X=t
k= rate constant
Cao= initial concentration of acetic acid

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Figure 2: Conversion vs Time at varied temperature

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Figure 3: X/(1-X) vs Time

From the graph, the value of the reaction rate constant, k can be determined from the formula
below:
k = m / CA0
where, m is a slope of the graph and CA0 is the initial concentration of Acetic acid with 1.0 mol/L
According to Figure 1, at 27oC the value of m is 0.0773, so
k1 = 0.0773 / 1.0 = 0.0773 L/mol.min
At 35oC, the value of m is 0.2459, so
k2 = 0.2459 / 1.0 = 0.2459 L/mol.min

From the calculation above, the conversion at 37oC is higher than the conversion at 27oC with
the value of k2 being larger than k1. In this case, Acetic acid is a weak acid while Sodium
Hydroxide is a strong base as a result it will undergo a neutralization reaction that produces salt
and water. Besides, based on Arrhenius, molecules move faster at higher temperatures since it
will increase its kinetic energy, which will increase the collision of molecules. Therefore, it will

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result in an increase in the esterification.Not to mention that the optimum temperature for lipase
activity is about 35°C.

3.3 What is the offset to eliminate the mass transfer effect of the immobilized lipase in the
reaction kinetic study?

There several suggestions can be addressed regarding reducing mass transfer effect. Mass transfer
effect can be eliminated by minimizing diffusion path length which is by reducing the thickness
of the immobilization matrix or layer where the lipase is immobilized. The reduction can be
achieved by using thin films or membranes with high surface area-to-volume ratios.

Next, the mass transfer effect can be reduced by increasing the surface area by utilizing
immobilization techniques which provide a higher surface area for the lipase. For instance, high
transfer area can be achieved by using porous materials, beads, or membranes that offer a large
interfacial area between the enzyme and the reaction mixture.

Last but not least, the mass transfer effect can be reduced by optimizing enzyme loading whereby
the optimal amount of lipase to immobilize needs to be determined. Not to mention that, several
factors need to be considered such as reaction conditions, substrate concentration, and specific
activity of the lipase. Overloading the immobilization matrix can lead to diffusion limitations.

3.4 How the limiting reactant measured?

The limiting reactant can be determined by comparing the stoichiometry of the reaction
and the initial amounts of the reactants. The stoichiometry of the esterification reaction between
ethanol and acetic acid is 1:1, meaning that one molecule of ethanol reacts with one molecule of
acetic acid to produce one molecule of ethyl ethanoate. The reactant that is completely consumed
first will be the limiting reactant. The other reactant, which is still present in excess after the
reaction is complete, is called the excess reactant. In this experiment, 50 mL of 1.2 M ethanol and
50 mL of 1.0 M acetic acid were used. By calculating the number of moles for each reactant, it
was found that there were 0.06 moles of ethanol and 0.05 moles of acetic acid. Since acetic acid
had fewer moles compared to ethanol, it was identified as the limiting reactant.

3.5 What rate law is used to elaborate the kinetics of the esterification reaction?
The rate law for the enzymatic synthesis of ethyl ethanoate using immobilized lipase can
be expressed as -rA = kCACB, where k is the rate constant, CA represents the concentration of
acetic acid, and CB represents the concentration of ethanol. Since the stoichiometric coefficients
for acetic acid and ethanol are the same in the balanced equation, the rate law can be simplified to
-rA = kCA2, indicating that the reaction is a second-order reversible reaction. The rate law
indicates that the rate of the reaction is directly proportional to the square of the concentration of

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acetic acid. It's important to note that the rate law may differ if other factors, such as the presence
of inhibitors or activators, temperature, or pH, significantly affect the reaction kinetics.
Experimental determination of the rate law and its parameters is necessary to accurately describe
the kinetics of the enzymatic synthesis of ethyl ethanoate in this specific system.

4.0 Conclusion and recommendations

In conclusion, the effect of temperature on the enzymatic esterification of acetic acid and
ethanol to produce ethyl ethanoate using immobilized lipase had been examined at 27°C and
37°C. The rate of enzymatic synthesis of ethyl ethanoate from the esterification reaction process
of acetic acid and ethanol can be determined by identifying the amount of sodium hydroxide
(NaOH) solution titrated every 2 minutes at different temperatures between 27°C and 37°C. As a
result, the equilibrium conversion of the chemical process can be calculated. According to the
results, the amount of NaOH required to neutralize the ethyl ethanoate solution at 27°C is more
than the amount of NaOH required to neutralize and synthesize the ethyl ethanoate solution at
37°C. As an outcome, at 27°C, the equilibrium conversion of this exothermic reversible reaction
is greater than at 37°C. The higher the temperature of the reaction process, the higher the rate of
reaction as the kinetic energy increases due to ion collisions with one another more frequently
and faster when compared to the lower reaction temperature, which shortens the reaction time,
representing a positive influence on the ester yield. It is additionally shown when the estimated
reaction rate constant is 0.077 Lmol -1 min -1 at 27°C and 0.246 L mol -1 min -1 at 37°C.
However, due to the fact that this is an exothermic reversible reaction, the estimated equilibrium
conversion value for 27°C is 0.8303 and 0.9080 for 37°C. This is considering increases in
temperature cause the equilibrium to shift to the left to supply some energy, but decreases in
temperature cause the equilibrium to shift to the right. As a result, we can conclude that the
greater the temperature, the lower the reaction's equilibrium conversion.

Next, for the recommendations, to avoid contamination of the solution, it is recommended

that the equipment used as a beaker, measuring cylinder, dropper, conical flask, magnetic stirrer,
volumetric flask, and burette be thoroughly cleaned and rinsed with distilled water before
conducting the experiment. Second, add a suitable amount of phenolphthalein to the solution to
ensure that it is thoroughly mixed in. Next, ensure that the titration procedure of NaOH and
ethanol with acetic acid is sufficient based on drop by drop until the entire combined solution
transforms from clear to light pink for high precision in measuring the volume of NaOH used.
Finally, increase the amount of immobilized lipase enzyme used to accelerate the reaction.

References
[1] Cooler A. S. (1999) Binary Flow Systems. J. Fluid Mech 999:999-996.
[2] Icer D.F., Adams J.A. (1977) Mathematical Elements for Computer Simulation. McGraw Hill, NY.

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[3] Nygus, G. (1983) Numerical Analysis Using Finite Element Method. PhD Thesis, NTU Mech. Eng. Dept., Lagos.
[4] Cancer Research UK (2003). Cancer statistics reports for the UK.
http://www.cancerresearchuk.org/aboutcancer/statistics/cancerstatsreport/ (accessed 13.03.03).

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Appendix

Preparation of Ethanol
The molarity of ethanol, M, can be calculated using the equation below

M= (purity(%) x density)/MW

MW=molecular weight

M= 0.95(789) / 46.07
= 16.26 mol/L

Volume at M1 can be calculated using the equation below:

𝑀1𝑉1 = 𝑀2𝑉2

Where

𝑀1 =concentration in molarity (moles/liters) of the concentrated solution

𝑉1 =-volume of the concentrated solution
𝑀2 = concentration in molarity of the dilute solution
𝑉2 = volume of dilute solution

Therefore, V1= (1.2 X 50) / 16.26 = 3.7 mL of concentrated Ethanol

Preparation of Acetic Acid

The molarity of Acetic Acid, M, can be calculated using the equation below

M= (purity(%) x density)/MW

MW=molecular weight

M= 0.998(1050) / 60
= 17.48 mol/L

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Volume at M1 can be calculated using the equation below:

𝑀1𝑉1 = 𝑀2𝑉2

Where
Therefore, V1= (1.0 X 50) / 17.48 = 2.86 mL of concentrated Acetic Acid

Preparation of 50 ml of 0.1M Sodium Hydroxide

MW of NaOH = 40 g/mol
Mole = 0.1 mol/L x 0.5 L = 0.05 mol
Mass of NaOH needed= 0.05 mol x 40 g/mol = 2.0 g

Raw data obtained from experiments are as follows:

Time (min) 27oC 37oC
Volume of Volume of
NaOH NaOH
required required
(mL) (mL)
0 4.1 2.9
2 4.1 2.8
4 4.0 2.8
6 3.9 2.7
8 3.7 2.7
10 3.7 2.6
12 3.6 2.5
14 3.6 2.5
16 3.5 2.3
18 3.4 2.3
20 3.4 2.3

Table 1: Data of Volume of NaOH required within 20 minutes

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Figure 1: Volume of NaOH required versus Time at 27 degree celsius and 37 degree celsius

Figure 2: X/(1-X) versus time at varied temperature

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Figure 3: Conversion vs Time at varied temperature

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