Advances in Bioscience and Biotechnology, 2013, 4, 900-907 ABB Published Online September 2013 (
Production of mono-, di-, and triglycerides from waste
fatty acids through esterification with glycerol
N. A. Mostafa1, Ashraf Maher2, Wael Abdelmoez2*
1Faculty of Applied Medical Science, Taif University, Taif, KSA
2Chemical Engineering Department, Faculty of Engineering, Minia University, Minia, Egypt
Email: *
Received 10 July 2013; revised 10 August 2013; accepted 25 August 2013
Copyright © 2013 N. A. Mostafa et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Currently monoglyceride and diglyceride are repre-
senting important products, as they have numerous
applications such as modifying agents in food and
pharmaceutical industries. In this work, the produc-
tion of these economically value added compounds by
estrifying the fatty acids with the glycerol is presented.
Effects of various reaction parameters were optimized
to obtain high yield of mono, di- and triglycerids. The
effects of temperature (180˚C to 260˚C), ZnCl2 cata-
lyst concentration (0.1%, 0.2%, 0.3%), glycerol to
fatty acids molar ratio (1:1, 1:2, 1:3, 3:1), agitation
speeds (200, 500, 1000 rpm), type of reaction system
(opened and closed) and type of fatty acids including
oleic and palmatic acids on esterification efficiency of
fatty acids were investigated. The optimum conditions
of esterification reaction were at temperature 195˚C,
molar ratio 1:1, amount of catalyst 0.3% Zncl2, and
agitation 500 rpm. Analysis of yield showed that at
the optimum conditions mondi and triglycerids were
produced in high purity, up to 99%. Infrared spec-
troscopy IR and thin layer chromatograph TLC
proved that the final product contains mono, di- and
Keywords: Esterification; Fatty Acids; Glycerol;
Monoglycerides; Diglycerides; Zinc chloride;
Soapstock is a by product of the caustic refining of oils
and fats. Fatty acids are the main component in the soap-
stock in the form of sodium salts. These fatty acids could
be separated by soapstock acidulation to give waste oils.
Distilled fatty acids could be obtained from waste oils by
two processes namely splitting and distillation. These fr ee
fatty acids could be used as a starting material for the
production of very valuable products such as mono-, di-
and triglycerides by esterification reaction with glycerol
[1]. As consumers become more aware and concerned
about the impact of the food they eat and the substances
they commonly use on their health and general well-
being, there is a growing interest in designer lipids. Among
the lipid classes, surfactants, such as monoacylglycerols
(monoglycerides, MAG), are desired by the food, cos-
metic, pharmaceutical and chemical industries [2-5]. Pro-
duction of tailor-made designer MAG with targeted fatty
acids therefore offers promising industrial opportunities.
The commercial synthesis of fatty acid esters of glycerol
is carried out by two different routes: direct esterification
of the fatty acid with the glycerol (Glycerolysis), cata-
lyzed by a homogeneous acid, such as sulphuric or sul-
fonic acids, or by transesterification of triglycerides and
polyalcohol catalyzed by alkaline hydroxides like NaOH,
KOH or Ca(OH)2 and sodium salts of low molecular
weight a lco hols , su ch as meth an ol . The e steri cation me-
thod is best suited for the production of designer MAG
because the desired free fate acid (FFA) can easily be
selected prior to MAG formation [6]. It could be esti-
mated that approximately 80% of manufacturing proc-
esses use acid catalysts [7]. The complete steps for es-
terification reaction are demonstrated in Figure 1. The
major factors affecting the conversion efficiency of the
esterification process are molar ratio of alcohol/oil,
amount of catalyst, reaction temperature, catalyst type
and stirring speed according t o reaction duration [8,9].
Esterification of glycerol and fatty acids was studied
under reduced pressure with and without the assistance
of various metal chlorides and oxides as catalysts. Also
super acids like sulphated zirconia and niobium acid wer e
used as catalyst, for the reason that they could prevent
*Corresponding a uthor.
N. A. Mostafa et al. / Advances in Bioscience and Biotechnology 4 (2013) 900-907 901
Figure 1. Estrification reaction.
corrosion, their separation is easy, and also high FFA
conversions could be achieved [10].
Recently, heterogeneous acid catalysts have been more
widely favored over homogeneous ones since they are
more separable and thus easier to recover. There have
been only a few studies on non-catalytic esterification
and/or transesterification reactions which lead to much
simpler purification and environmentally friendly proc-
esses [11]. To avoid separation required in homogenous
catalytic system, researches have explored the use of
heterogeneous catalyst, such as glycerol esterification
with lauric and oleic acid using solid cationic resins and
zeolitic materials. More recently, mesoporous catalysts
containing SO3H groups have been reported to be effi-
cient catalysts in the esterification of glycerol with fatty
acids, where high yields of mono derivative could be
obtained [7,12]. The synthesis of triglycerides by enzy-
matic esterification of polyunsaturated fatty acids (PUFA)
with glycerol is well known by using Novozym 435 [13].
In the current work we demonstrate the feasibility of
producing mono-, di- and triglycerides from waste fatty
acids by direct esterification reaction with glycerol in a
solvent free system.
2.1. Materials
Mixture of fatty acids Saturated and unsaturated were
supplied from Al Manaar Company, Alexandria, Egypt.
Palmitic acid, ethanol, methanol chloroform, sodium Hy-
droxide, potassium iodide, sodium thiosulfate and acetic
acid were obtained from El-Nasr Company for chemicals,
Cairo, Egypt. Sulfuric acid and phosphoric acid were
provided by united company for chemicals, Cairo, Egypt.
Distilled water was prepared in our department. Thin-
Layer Chromatography paper (TLC), Kieselgel 60 f254
precoated plates, E. Merck, Dermastadt, Germany.
2.2. Equipment
Esterification reactions were carried out in a labora-
tory-built apparatus. An apparatus consists of laboratory
conical flask 250 ml with 30 ml work ing volume. Esteri-
fication reaction was under atmospheric pressure (opened
system), temperature of the reactor was controlled using
hot plate (controlled with internal thermostat). All the
reactants (fatty acids, glycerol and catalyst) were weighted
and charged into the reactor. Then the temperature was
increased through adjustin g the thermostat. Th e magnetic
stirrer was allowed to operate after 2 - 3 min (to heat up
the mixture). After passing the desired reaction time, the
reactor was removed from the hot plate. Samples were
withdrawn from the reaction mixture for analysis. The
reaction mixture was cooled to the ambient temperature
by immersing it into a water bath. Also the esterification
process in closed system was investigated, where all the
reactors were isolated.
2.3. Optimum Conditions
Several reaction conditions were optimized including tem-
perature, reaction time, type of catalyst, concentration of
catalyst and molar ratio of glycerol to free fatty acids.
The studied reaction temperatures were from 180˚C to
260˚C. The molar ratios of glycerol to fatty acids were
1:3, 1:2, 1:1 and 3:1. Concentrations of catalyst were
0.0%, 0.1%, 0.2%, and 0.3%. Effect of agitation speeds
were in the range of 100 rpm to 1000 rpm.
2.4. Reversible Reaction
Three runs were conducted to study the effect of the re-
verse reaction of esterification. Reverse reaction experi-
ments were run at 195˚C for 60 min using 1:1 molar ra-
tion without using a catalyst. To find out the best way of
demolishing the effect of the reverse reaction on the
overall yield of the esterification, the reaction mixture
was rapidly cooled down by three different mechanism
using refrigeration, water cooling, or air cooling. Then
the yield of the esterification was measured in each case
to investigate the conversion in each sample.
2.5. Analytical Methods
2.5.1. Determination of the Fatty Acids Conversion
Titration method was selected for the determination of
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N. A. Mostafa et al. / Advances in Bioscience and Biotechnology 4 (2013) 900-907
the fatty acids conversion during the esterification reac-
tion because it was simple and efficient analytical me tho d
that could be performed without the need for highly spe-
cialized equipment [14].
Five grams of the sample at time intervals was dis-
solved in ethanol and ph enolphthalein indicato r was used
to determine the pH ch ange during esterification reaction .
The titration was carried out against aqueous solution of
0.1 N KOH. Acid value (A.V) was determined according
to the equation given below:
.mg KOHg oil
N: normality of KOH solution
V: the volume of solution employed for titration, ml
M: weight of fatty acids sample, g
The conversion of FFA was calculated by the follow-
ing equation:
... conversion%100
Where Ai is the initial acidity of the mixture and At
acidity at any time, t.
2.5.2. Thin-Layer Chromatography for the Produced
TLC was used to identify the types of glycrides produced
during the esterification reaction (i.e mono, di or triglyc-
erides). TLC experiments were carried out by dissolving
one drop of the esterification products into 0.5 ml mix-
ture of chloroform and methanol (9:1). Then, a drop from
the final mixture was applied over the TLC paper im-
mersed into a 100 ml-beaker containing 10 ml of a mix-
ture of chloroform and methanol (9:1) which allows a
contact between the edges of the TLC paper with the
solvent mixture. The TLC paper was allowed to stand
until the solvent reach a level which must be just below
the end line. Then, the TLC paper was dried and the
bands of the different produced glycerides were detected
under UV lamp. In some cases, the bands were not cl ea r l y
appearing under the UV lamp. Accordingly, the TLC
paper was exposed to an iodine vapor which allows a
direct visualizing of the band by naked eye.
Monoglyceride, diglyceride and triglyceride were the ma-
jor products obtained in this study. First, it was necessary
to optimize the esterification conditions. To find out the
optimum esterification conditions, the esterification proc-
ess was carried out under different operating conditions
at any time only one parameter was changing while the
other parameters were kept con st a n t .
3.1. Effect of Type of the System
To investigate the effect of type of the system on the
esterification yield, the same procedures described above
were followed by applying both closed and opened sys-
tems (isolated and atmospheric systems). The esterifica-
tion reactions were carried out using molar ratio of 3:1
(glycerol to fatty acids) at 180˚C for 510 min. Figure 2
shows the effect of both systems on the obtained estrifi-
cation yield. The results indicated that the closed system
and the opened syste m were come to equilibriu m point at
nearly the same time. However in the opened system the
conversion (91%) was higher than that obtained by
closed system (72%). Consequently it could be concluded
that the opened system based process is more efficient
than the closed one. This finding maybe due to the pres-
ence of the side reactions products in the estrification
reactor inhibit the reaction to be proceed, therefore when
these by products allowed to get out from the reactor,
favorabl e p roducts formation c ou ld be obtained.
3.2. Effect of Temperature and Time
Both temperature and time are having major effects on
the conversion of the esterification process. Accordingly,
they were studied and optimized together. The obtained
results showd that that by increasing the reaction tem-
perature, the reaction conversion increases rapidly. Fig-
ure 3 shows that after 20 min, the conversion reached
99%. However, the energy consumed was very high (260˚C)
which is not practical from the economic point of view.
Therefore another esterification reaction was carried out
within a temperature range of 180 ˚C - 210˚C as sh own in
Figure 4. The results revealed that by increasing the
Figure 2. Effect of the type of esterification system (opened
system & closed system) at 180˚C, 3:1 molar ratio and 510 min
of the unsaturated fatty acid.
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N. A. Mostafa et al. / Advances in Bioscience and Biotechnology 4 (2013) 900-907 903
Figure 3. Effect of different temperatures on the esterification
yiled of saturated fatty acid after 40 min and 3:1 molar raio
Figure 4. Effect of temperature at 3:1 gly:fat molar ratio on the
esterification of unsaturated fatty acid.
esterification time, the esterification yield increased up to
a maximum conversation. To determine the optimum tem-
perature of esterification process, not only the maximum
yield of esterification should be considered but also the
time required to reach the reaction temperature was taken
into account. Heating time to reaction temperature was
certainly longer and energy consumption was surely greater
for higher reaction temperature. Consequently, a faster
reaction at a lower temperature is desirable. From this
economical point of view, the optimum temperature for
the esterification was found to be 195˚C at which the
conversion reached up to 99%.
3.3. Effect of Molar Ratio on the Esterification
The second studied parameter was the optimum esterifi-
cation molar ratio (glycerol:fatty acids). In this part, all
experiments were carried out at the optimum obtained
temperature (195˚C). The esterification process was car-
ried out at 1:1, 1:2, 1:3 and 3:1 (glycerol to fatty acids).
As shown in the Figure 5, it could be easily concluded
that 1:1 molar ratio gave the maximum conversion under
the required lower amount of raw material which in turn
reduces the running cost of the process.
3.4. Effect of the Concentration of Catalyst on
the Esterification Yield
After investigating the effect of temperature, time and
molar ratio without catalyst, the effect of concentration
of the catalyst (0.1%, 0.2%, and 0.3%) on the esterifica-
tion yield under optimum temperature of 195˚C and 1:1.
molar ratio was investigated. Figure 6 shows the effect
of catalyst percentage on the esterification yield. As s ho wn
from the figure, by increasing the catalyst concentration,
the time required to reach the maximum conversion de-
creases. So that 0.3% (by weight) ZnCl2 was considered
to be the optimum dose, where the maximum yield of
Figure 5. Effect of glycerol:fatty acid molar ratio on the esteri-
fication of unsaturated fatty acid with glycerol at 195˚C.
Figure 6. Effect of the concentration of catalyst on the esteri-
fication yield at optimum temperature (195˚C).
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N. A. Mostafa et al. / Advances in Bioscience and Biotechnology 4 (2013) 900-907
99% could be obtained after 100 min. Further increasing
in the catalyst concentration will make the downstream
processes including catalyst separation more difficult.
3.5. Effect of Degree of Agitation (rpm)
To investigate the effect of the degree of agitation (rpm),
the esterification process was carried out at different agi-
tating speeds (1 00 , 500 and 1000 rp m) at 195 ˚C, 1:1 fatty
acids to glycerol and without catalyst. The results are
presented in Figure 7. As shown from the figure the
maximum conversion of 99% w as obtained at 500 rpm.
3.6. Effect of the Reverse Reaction
Reverse reaction effect was stopped using three different
cooling mechanisms as described in the method part. The
results were presented in Figure 8. As shown in the fig-
ure, the yield of esterification were 92%, 90%, and 88%
for reactions cooled down by refrigerator, cooling water
and air cooling, respectively. The rapid cooling of the
reaction mixture prevents (through low temperature) the
reversible reaction, which in turn keeping the conversion.
Accordingly, by decreasing the cooling temperature the
effect of reversible reaction is minimized.
3.7. Effect of the Type of Fatty Acids on the
Esterification Yield
In this part of the study, all experiments were carried out
at the optimum obtained conditions including tempera-
ture (195˚C), molar ratio (1:1) and different types of fatty
Figutr 7. Effect of the degree of agitation at 195˚C, 1:1 (gly:fa t)
molar ratio.
Figure 8. Effect of reversible reaction at optimum temperature.
acids (oleic acid, stearic acid and palmitic acid Figure 9
shows the effect of the type of fatty acids on th e esterifi-
cation yield. The results showed that the yield of esteri-
fication recorded a maximum conversion at time of 360,
240, 210 min in case of oleic acid, stearic acid and
palmitic acid, respectively. From these results it co uld be
concluded that the fatty acid chain length and the degree
of unsaturation are having a considerable effect on the
esterification reaction. Since long chain and unsaturated
fatty acids need higher reaction time compared with the
smaller chai n and saturat ed fatty acids.
3.8. Esterification Reaction Using the Optimized
In this part, the esterification of free fatty acids was per-
formed under the obtained optimum conditions. The time
course of the esterification was measured and shown in
Figure 10. The data showed that the maximum conver-
sion (99%) was obtained after 100 min (instead of 180
min in the temperature effect experiments). Accordingly
the esterification processes was achieved in a considera-
bly short time and lower temperature. A sample was
taken for IR and TLC analysis to prove the composition
of the obtained esterification products. Figure 11 shows
Figure 9. The esterification yield under the optimum condition
(195˚C, 100 min, 0.3% ZnCl2, 500 rpm).
Figure 10. Effect of degree of saturation and number of atoms
of the fatty acids on the esterification reaction at optimum
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N. A. Mostafa et al. / Advances in Bioscience and Biotechnology 4 (2013) 900-907
Copyright © 2013 SciRes.
the IR analysis, the results proved the existance of the
carbonyl group (at wavenumber 1738.96 of the ester for-
mation [15].
plant with a capacity of 100 ton of fatty acids/day using
ASPEN HYSYS 2006 as shown in Figure 13. As dem-
onstrated in the figure the process starts with a feeding
pump (P-100) which was used for transferring the free
fatty acids to the esterification reactor (convertor type
reactor, CRV-100) through a heat exchanger (E-100)
equipped with control valve (VLV-102). Glycerol was
transferred to mixing tank through control valve (VLV-
100) and zinc chloride was mixed with glycerol in the
same mixing tank (V-100). Then the mixture was pumped
to reactor (CRV-100) through a transfer pump (P-101)
equipped with a flow control (VLV-101). The product
w as cooled by the exchang er (E101). Th e resulting mixture
was transferred to a washing tank (V-101). The washing
was carried out using fresh water. Then, the washed mix-
ture was transferred to a settling tank (V-102) using a
control valve (VLV-103). The mixtu re inside the settling
tank is divided into two phases. The first phase is com-
posed mainly of esterified fatty acids and the second
phase is composed mainly of catalyst, glycerol and water.
The first phase was pumped through control (VLV-104)
to a fractionation tower (T-100) equipped with ten stages
fractionating the feed into glycerol from the top and es-
terified fatty acids from the bottom. The results of the
simulation were summarized in Table 1. As shown in
Table 1 the yield of the final product was found to be
Figure 12 shows the TLC paper plate of both estrfied
and free fatty acid. The results showd the existence of the
three main products of monoglyceride, diglyceride, and
Now let us compare the results obtained in this study
with others. Lilis Hermida [16] used a mesoporous cata-
lyst in order to catalyzing the estrification reaction, the
optimum conditions obtained were, 5% catalyst, 2:1 glyc -
erol:fatty acid and 750 rpm which were higher than that
obtained in this study. Furthermore the conversion ob-
tained was 95% which was less than the 99% obtained in
this study. Such a result indicated the effectiv eness of the
proposed route for production of mono-, di- and triglyc-
Consequently, the esterification performed in the pre-
sent work will make two major benefits; firstly, it will
make the production of biodiesel more competitive with
the existing diesel fuel market. Secondly, it will produce
which is the most important emulsifier used in food in-
The last step of this article was building an estrification
Figure 11. IR analysis of esterification unsaturated fatty acids.
N. A. Mostafa et al. / Advances in Bioscience and Biotechnology 4 (2013) 900-907
Figure 12. TLC analysis of esterified and non esterified fatty acids.
Figure 13. Ester production plant from fatty acids and glycerol.
Table 1. Results of the simulation.
M3/hr 4.9
Free fatty acid Mass% 99%
m3/hr 1.05
Glycerol make up Mass% 99
kg/hr 16.5
ZnCl2 Mass% 99
m3/hr 1
Washing water Mass% 100
m3/hr 5.09
Fatty acid ester Mass% 96
m3/hr 0.4267
glycerol Mass% 72.7
96% ester and 4% glycerol at the bottom of the distilla-
tion tower. On the other hand, at the top of the tower it
was recovered for about 73% glycerol.
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