A novel method for monitoring the transesterification reaction of oil in biodiesel production by estimation of glycerol

Article abstract of DOI:10.1007/s11746-010-1549-2

A quantitative method is reported for the estimation of glycerol during transesterification of oil to form biodiesel. The reagent used to derivatize glycerol was 9,9-dimethoxyfluorene. Glycerol is estimated by both UV-visible spectrophotometric and high performance liquid chromatography methods. Using the former method, detection limits of 0.05% w/w of glycerol in biodiesel was established. Validation of the developed method was done using the Greenhill method for determination of free glycerol formed during the transesterification reaction.

Full text of DOI:10.1007/s11746-010-1549-2

J Am Oil Chem Soc (2010) 87:747–754
DOI 10.1007/s11746-010-1549-2
ORIGINAL PAPER
A Novel Method for Monitoring the Transesterification Reaction
of Oil in Biodiesel Production by Estimation of Glycerol
•
Sabbasani Rajasekhara Reddy Devamani Titu
•
Anju Chadha
Received: 3 September 2009 / Revised: 11 January 2010 / Accepted: 22 January 2010 / Published online: 16 February 2010
Ó AOCS 2010
Abstract A quantitative method is reported for the esti-
mation of glycerol during transesterification of oil to form
biodiesel. The reagent used to derivatize glycerol was
9,9-dimethoxyfluorene. Glycerol is estimated by both
UV–visible spectrophotometric and high performance
liquid chromatography methods. Using the former method,
detection limits of 0.05% w/w of glycerol in biodiesel was
established. Validation of the developed method was done
using the Greenhill method for determination of free
glycerol formed during the transesterification reaction.
HPLC
HTGC
High performance liquid chromatography
High temperature capillary gas
chromatography
NIR
Near infrared spectroscopy
p-Toluenesulfonic acid
Size exclusion chromatography
Triglycerides
PTSA
SEC
TG
TLC
mp.
Thin layer chromatography
Melting point
NMR
HRMS
RT
Nuclear magnetic resonance
High resolution mass spectra
Retention time
Keywords Non-edible oils ꢀ Transesterification reaction ꢀ
Biodiesel ꢀ Glycerol ꢀ Estimation ꢀ 9,9-Dimethoxyfluorene ꢀ
p-Toluenesulfonic acid ꢀ Jatropha curcas ꢀ
UV–visible spectrophotometric ꢀ HPLC
Introduction
Abbreviations
ASTM
American Society for Testing and Materials
Biodiesel is known for its environmental benefits such as
biodegradability and its non-toxic nature. Moreover, it is
derived from renewable resources [1]. Biodiesel is typi-
cally produced from neat oil or fat by transesterification
with an alcohol, usually methanol, in the presence of an
alkaline catalyst [2]. Alkali metal hydroxides are the most
common catalysts employed in the transesterification
reaction but other metal catalysts [3, 4] as well as bio-
catalysts [5, 6] have also been reported. Successful com-
mercialization of biodiesel depends on a variety of
parameters. One of these parameters is fuel quality as
defined in a provisional standard for biodiesel by the
American Society for Testing and Materials (ASTM) and
existing standards in some European countries [7, 8]. The
fuel standards address quality issues of biodiesel by con-
taminants [glycerol, mono-, di-, and triglycerides (TG) and
alcohol] present in it [7]. Glycerol, a by-product of the
ATR-FTIR Attenuated total reflectance-Fourier
transform infrared
CCDC
CE
Cambridge Crystallographic Data Collection
Capillary electrophoresis
FAME
FID
Fatty acid methyl esters
Flame ionization detection
S. R. Reddy ꢀ D. Titu ꢀ A. Chadha (&)
Laboratory of Bioorganic Chemistry,
Department of Biotechnology,
Indian Institute of Technology Madras,
Chennai 600 036, India
e-mail: anjuc@iitm.ac.in
A. Chadha
National Center for Catalysis Research,
Indian Institute of Technology Madras,
Chennai 600036, India
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J Am Oil Chem Soc (2010) 87:747–754
transesterification reaction, is the major constituent in
determining fuel quality [9].
by UV–visible spectrophotometry, capillary electrophore-
sis (CE) and HPLC [16, 21, 22] have been developed.
Furthermore, Sigma-Aldrich Fine Chemicals developed a
kit (NBB method BQP-02—Greenhill method) for enzy-
matic determination of free and total glycerol in biodiesel
[23]. This method is based on coupled enzyme reactions of
glycerol leading to a spectrophotometric detection (at
540 nm) of quinonimine dye. Although this method is
faster and simpler than chromatographic methods, the
commercially available free glycerol kit is expensive for
routine analysis. As part of an ongoing project in our lab-
oratory on biodiesel from non-edible local oils [5, 24, 25],
herein we report an efficient and novel method for moni-
toring transesterification and assessing biodiesel production
by the estimation of glycerol.
The first method for monitoring the transesterification
reaction was thin layer chromatography with flame ioni-
zation detection (TLC/FID) [10]. It was used to determine
the fatty acid methyl esters (FAME), mono-, di-, and tri-
glycerides. Although its analysis time is quite short, this
method was reported to be sensitive to humidity, showing
material discrepancies and is not as accurate as gas chro-
matography (GC) and high performance liquid chroma-
tography (HPLC) methods developed later. The cost of the
instrument (TLC/FID) is high [7]. TLC on silica gel [11]
developed later is based on the area of TG spot seen on the
TLC in comparison to a standard and is merely qualitative
and does not allow the exact determination of the degree of
conversion. Bansal et al. [12] recently reported a TLC
method and image analysis to detect glycerol in biodiesel.
This method is based on the area of the spot seen on the
TLC in proportion to the amount of glycerol in the sample,
followed by image analysis using the Matlab program.
Subsequently, high temperature capillary gas chromatog-
raphy (HTGC) was developed to monitor esters, tri-, di-,
and monoglycerides in the transesterification reaction in a
single run [13]. A later protocol developed for determining
total glycerol in biodiesel, using Plank’s method (HTGC)
[14] requires the hydroxyl groups of the glycerides and free
glycerol to be derivatized in the presence of expensive
reagents before analysis, limiting its use for routine anal-
ysis. HPLC methods have also been developed as an
alternative to HTGC [15]. For this, derivatization of the
samples is not necessary [16], the analysis time is shorter
and all the reaction mixtures of oils and fats are readily
quantitated [17]. However, both HTGC and HPLC methods
require standards for quantification.
Experimental Procedures
Materials
Melting points of the synthesized compounds were deter-
1
mined using a Toshniwal melting point apparatus. H- and
13C-NMR spectra were recorded on a Bruker 400 instru-
ment at 100 MHz. The chemical shifts are quoted in parts
per million (d) relative to tetramethyl silane for 1H and 13
C
NMR using CDCl3 as solvent and the infrared (IR) spectra
were recorded on a Nicolet 6700 spectrophotometer. Mass
spectra were recorded on a Q TOF micromass spectrome-
ter. Flash chromatography was performed on silica gel
(100–200 mesh) using hexane and ethyl acetate as eluent.
TLC was done using Kieselgel 60 F254 aluminium sheets
(Merck 1.05554). Chemicals, 9-fluorenone, trimethyl
orthoformate, p-toluenesulfonic acid (PTSA) and free
glycerol kit were purchased from Sigma Chemical Co. All
reactions were carried out under an inert atmosphere. The
spectrophotometry analysis was carried out using a UV–
visible spectrophotometer (JASCO, V530). The yield was
determined by HPLC analysis on a JASCO PU-1580 liquid
chromatograph equipped with PDA detector. The column
used to monitor derivatized glycerol was C18 (Qualisil
BDS; 4.6 9 250 mm) and the absorbance was monitored
at 280 nm. Acetonitrile: water (85:15) was used as the
mobile phase at a flow rate of 1 mL/min. Crystallographic
data were collected on a APEX CCD diffractometer
Gelbard et al. [18] described the first work on the uti-
lization of nuclear magnetic resonance (NMR) spectro-
scopy. 1H NMR, for monitoring the yield of the
transesterification reaction and 13C NMR for rapeseed oil
transesterification [19]. The fiber-optic near infrared spec-
troscopy (NIR) method was developed by Knothe [7].
However, in this method, the presence of low levels of
contaminants in biodiesel cannot be completely quantified.
The insolubility of some contaminants (e.g., glycerol) in
methyl esters contribute to this problem. Later, overcoming
some of these limitations ATR-FTIR methods (attenuated
total reflectance) were reported and results by these
methods were validated with size exclusion chromatogra-
phy (SEC) [7]. In addition, a SEC method was developed
for monitoring the transesterification reaction of oil in
biodiesel production [20].
˚
equipped with Mo–Ka (l = 0.7107 A) radiation. GC
analysis was performed with Clarus 500 Gas Chromato-
graph Perkin Elmer-USA. Sample analysis was carried out
on a Zebron ZB-5HT fused silica capillary column
(15 m 9 0.32 mm 9 0.10 lm). Samples (5 lL) were
injected by an auto sampler injector at an oven temperature
of 50 °C. After an isothermal period of 1 min at 50 °C, the
GC oven was heated at 15 °C min-1 to 170 °C, then at
Recently, methods for the determination of small
amounts of free glycerol present in commercial biodiesel
samples, using the periodate oxidation of glycerol followed
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J Am Oil Chem Soc (2010) 87:747–754
749
3 °C min-1 to 200 °C and at 20 °C min-1 to 350 °C (hold
for 10 min for a total run time of 36.5 min). Flow rates of
the gases: Nitrogen—1.5 mL/min, Hydrogen—45.0 mL/
min, Air—450.0 mL/min. The injector and detector tem-
peratures were 360 and 360 °C respectively. Biodiesel was
prepared from Jatropha curcas oil and TG was separated
from Jatropha curcas oil by column chromatography prior
to use (Ravikumar et al., unpublished data).
Scheme 1 Estimation of glycerol in biodiesel
Methods
1H), 4.06 (dd, J = 6.8, 8.4 Hz, 1H), 2.02 (t, J = 6.0 Hz,
1H).
Synthesis of 9,9-Dimethoxyfluorene 1
13C NMR (CDCl3, 100 MHz) dC (ppm) : 144.3, 143.3,
140.0, 139.3, 131.0, 130.2, 128.4, 128.3, 124.0, 123.5,
120.0, 120.0, 113.1, 77.3, 67.0, 62.3.
A mixture of 9-fluorenone 3 (9.0 g, 50 mmol), p-toluene-
sulfonic acid (PTSA, 0.38 g, 1.95 mmol), trimethyl
orthoformate (12 mL) and CH3OH (60 mL) was stirred for
36 h at room temperature. The clear yellow solution was
made neutral with triethylamine and the suspension was
filtered to remove the tosylate salt. The filtrate was evap-
orated to dryness, and the residue was recrystallized from
CH3OH to give 8.8 g (78%) of colorless needles of com-
pound 1.
1H NMR (CDCl3, 400 MHz) dH (ppm) : 7.2–7.7 (m, 8 H,
ArH) and 3.3 (s, 6 H).
13C NMR (CDCl3, 100 MHz) dC (ppm) : 141.5, 130.0,
127.8, 124.5, 120.1, 107.9, 52.0
IR (cm-1): 3408, 3063, 2926, 1611, 1482, 1450, 1254,
1209, 1115, 1057, 993, 936, 732.
HRMS (M ? Na?) : Calc. 277.0841, Obs. 277.0846
(C16H14O3 Na), mp. 72–75 °C.
Crystallographic data for derivatized glycerol 2: CCDC
# 738893. Intensity data were collected on an APEX CCD
˚
diffractometer equipped with Mo–Ka (l = 0.7107 A)
radiation. The intensity data were corrected for Lorentzian,
polarization and absorption effects. C16H14O3, M =
˚
254.10, tetragonal, a = 22.6156(9) A, b = 22.6156(9),
3
˚
˚
˚
(3) A, c = 11.6576 A, a = b = c = 90°, V = 5962.5(5) A ,
space group P4nc, Z = 2, dcalc. = 1.226 g/cm3, 20874/3928
reflections collected/unique, final R indices [I [ 2s(I)]
R1 = 0.0835, wR2 = 0.2214, R indices (all data) R1 =
0.1063,
Melting point (mp.): Observed 85–86 °C (reported mp.
86–88 °C) [26].
Estimation of Glycerol in Biodiesel
wR2 = 0.2483. The crystallographic data for deriva-
tized glycerol 2 have been deposited with the Cambridge
Crystallographic Data Centre. Copies of this information
may be obtained free of charge from the Director, CCDC,
12 Union Road, Cambridge, CB2 1EZ, UK [Fax: ?44-
(1223)336033, E-mail: deposit@ccdc.cam.ac.uk or http://
www.ccdc.cam.ac.uk].
A mixture of 0.9 g of biodiesel (from Jatropha curcas oil),
glycerol (100 mg, 1.01 mmol), 9,9-dimethoxyfluorene
(339 mg, 1.5 mmol) and dry toluene (3 mL) were placed in
a 10 mL single neck round bottom flask under an inert
atmosphere. PTSA (30 mg, 0.157 mmol) was added and
stirred for 2.5 h at 80 °C (Scheme 1). The reaction was
monitored by TLC using hexane/ethyl acetate (7:3) as
eluent. Then, the reaction mixture was diluted with ethyl
acetate and treated with 2 mL of saturated aq. NaHCO3.
The separated organic phases were washed with brine,
dried over Na2SO4, and concentrated. The product was
then purified by column chromatography on silica gel,
using hexane/ethyl acetate (7:3) as the solvent to give a
white colorless solid 2 in 0.265 mg (96%) of isolated yield.
The structure of the derivatized glycerol 2 was confirmed
from their spectral data from IR, NMR, HRMS and single
crystal XRD. The reaction mass was analyzed by UV–
visible spectrophotometry and HPLC methods.
Control Experiment with TG and Biodiesel
in the Absence of Glycerol
A mixture of TG (500 mg), biodiesel (500 mg) and
9,9-dimethoxyfluorene (339 mg, 1.5 mmol) and dry tolu-
ene (3 ml) was placed in a 10 mL single neck round
bottom flask under an inert atmosphere. PTSA (30 mg,
0.157 mmol) was added and stirred for 2.5 h at 80 °C
(Scheme 2). Then, the reaction mixture was diluted with
ethyl acetate and treated with 2 mL of saturated aq.
NaHCO3. The separated organic phase was washed with
brine, dried over Na2SO4 and concentrated under reduced
pressure. Samples were analyzed by using UV–visible
spectrophotometry and HPLC.
1H NMR (CDCl3, 400 MHz) dH (ppm) : 7.44–7.14 (m,
8H), 4.72 (m, 1H), 4.45 (dd, J = 6.4, 8.4 Hz, 1H), 4.32
(dd, J = 4.4, 11.6 Hz, 1H), 4.24 (dd, J = 5.6, 11.6 Hz,
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J Am Oil Chem Soc (2010) 87:747–754
anhydrous Na2SO4 and concentrated under reduced pres-
sure. The residue was purified by column chromatography
on silica gel, using hexane/ethyl acetate (99:1) as the sol-
vent to give a colorless solid 4 in 354 mg (72%) of isolated
yield. The structure of the derivatized monoglyceride 4 was
confirmed based on the spectral data from IR, NMR and
HRMS.
Scheme 2 Compound 1 with TG and biodiesel in the absence of
glycerol
1H NMR (CDCl3, 400 MHz) dH (ppm): 7.55–7.25
(m, 8H), 4.85 (m, 1H), 4.56 (m, 1H), 4.40 (m, 2H), 4.19
(m, 1H), 2.38 (t, 2H, J = 7.48), 1.25–1.65 (m, 26H), 0.88
(t, 3H, J = 6.52)
Estimation of Glycerol in Biodiesel with One Pot Real
System Experiments
One gram of Jatropha curcas oil, KOH (1%, 10 mg) and
dry methanol (1:10 M ratio 0.45 mL) was placed in a
10 mL single neck round bottom flask under a nitrogen
atmosphere. The reaction mass was stirred for 30 min at
60 °C for complete conversion (Scheme 3). The pH of the
reaction mixture was adjusted between six and seven using
glacial acetic acid and 1 mL of toluene was added (to
remove trace amounts of moisture and methanol) and
concentrated under reduced pressure. To this reaction
mass, 9,9-dimethoxyfluorene (339 mg, 1.5 mmol), 3 mL of
dry toluene and PTSA (30 mg, 0.157 mmol) were added
under an inert atmosphere. The reaction mass was stirred at
80 °C for 2.5 h (Scheme 3). The reaction was monitored
by TLC using hexane/ethyl acetate (7:3) as eluent. Then,
the reaction mixture was diluted with ethyl acetate and
treated with 2 mL of saturated aq. NaHCO3. The separated
organic phase was washed with brine, dried over Na2SO4,
and concentrated under reduced pressure. Samples were
analyzed by using UV–visible spectrophotometry and
HPLC methods.
13C NMR (CDCl3, 100 MHz) dC (ppm): 173.7, 144.3,
142.9, 140.0, 139.4, 130.6, 130.2, 128.3, 128.2, 124.1,
123.5, 120.1, 120.0, 113.6, 74.8, 67.7, 64.2, 34.1, 32.0,
29.7, 29.6, 29.5, 29.4, 29.2, 29.1, 24.9, 22.7, 14.1
IR (cm-1): 2922, 2852, 1738, 1450, 1302, 1253, 1210,
1172, 1172, 1115, 1057, 994, 834, 760.
HRMS (M ? H?): Calc. 493.3319, Obs. 493.3318
(C32H45O4), mp. 50–55 °C.
Detection of Various Concentrations of Glycerol
in Biodiesel
To each of seven 10 mL single neck round bottom flasks,
was added different amounts of glycerol (5, 10, 20, 30, 40,
50 and 100 mg) and biodiesel (995, 990, 980, 970, 960,
940, 950 and 900 mg) respectively, to make a final volume
of reaction mass of one gram. To each flask 9,9-dimeth-
oxyfluorene (339 mg, 1.5 mmol) and dry toluene (3 mL)
was added under inert atmosphere. PTSA (30 mg,
0.157 mmol) was added to the reaction mass and stirred for
2.5 h at 80 °C. Then, the reaction mixture was diluted with
ethyl acetate and treated with 2 mL of saturated aq.
NaHCO3. The separated organic phase was washed with
brine, dried over Na2SO4 and concentrated under reduced
pressure. Samples were analyzed by using UV–visible
spectrophotometry.
Synthesis of Spiro[1,3]dioxolane-2,90-fluorene]-4-ylmethyl
Palmitate 4
A mixture of derivatized glycerol 2 (254 mg, 1 mmol),
palmitic acid (256 mg, 1 mmol) and dry dichloromethane
(10 mL) was placed in a 50 mL single neck round bottom
flask under a nitrogen atmosphere. Dicyclohexylcarbodi-
imide (DCC) (226.6 mg, 1.1 mmol) and 4-(dimethyl-
amino)-pyridine (DMAP) (6 mg, 0.04 mmol) was added to
the reaction mass at 0 °C for 5 min. After the addition, the
reaction mass was stirred for 4 h at room temperature
under an inert atmosphere (Scheme 4). The suspension was
filtered, and the filtrate was treated with 5 mL of saturated
aq. NaHCO3 and extracted with dichloromethane. The
separated organic phase was washed with brine, dried over
Progress of Transesterification as a Function of Time
Progress of transesterification reaction of Jatropha curcas
oil to biodiesel formation by estimation of the glycerol in
one pot real system experiments was carried out for 5, 10,
15, 20 and 30 min. Five 10 mL single neck round bottom
flasks were taken and to each flask 1 g of oil, KOH (1%,
10 mg) and dry methanol (1:10 M ratio 0.45 mL) was
Scheme 3 One pot real system
experiment for the monitoring
transesterification reaction
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751
used as substrate analogues for nucleoside glycosylase
enzymes [26, 27]. Another derivative of compound 1 i.e.
(4S, 6S)-4-(2-piperidyl)spiro[1,3-dioxolane-2,90-[9H]-fluo-
rene] which is an analogue of the dioxolanes dexoxadrol
and etoxadrol was used as potential phencyclidine-like
agent [28]. In addition, recently compound 1 was coupled
with 1,2-, syn-, anti-1,3-diols and 1,3-sulfanyl alcohols to
produce flexible and optically transparent molecules [in
UV–visible molecules] which shows remarkable changes
in the optical rotation values [29].
Scheme 4
added and heated to 60 °C. Reactions were stopped at 5,
10, 15, 20 and 30 min. The pH of the reaction mixture was
adjusted between 6 and 7 using glacial acetic acid and
1 mL of toluene was added (to remove trace amounts of
moisture and methanol) and the reaction mass was con-
centrated under reduced pressure. To each flask 9,9-di-
methoxyfluorene (339 mg, 1.5 mmol) and dry toluene
(3 mL) were added under an inert atmosphere. PTSA
(30 mg, 0.157 mmol) was added to the reaction mass and
stirred for 2.5 h at 80 °C. Then, the reaction mixture was
diluted with ethyl acetate and treated with 2 mL of satu-
rated aq. NaHCO3. The separated organic phase was
washed with brine, dried over Na2SO4 and concentrated
under reduced pressure. Samples were analyzed by using
UV–visible spectrophotometry.
In this study, compound 1 was coupled with glycerol in
the presence of biodiesel using a catalytic amount of PTSA
and dry toluene (solvent) at 80 °C for 2.5 h to give the
corresponding derivatized glycerol
2 in 96% yield
(Scheme 1). This is the first ever report on synthesis of
derivatized glycerol 2 and the crystal structure of deriva-
tized glycerol 2 is shown in Fig. 1. The yield of derivatized
glycerol 2 was calculated using UV–visible spectropho-
tometry and HPLC methods from the crude reaction mix-
ture. In the UV–visible spectrophotometric method, the
yield was calculated based on formation of 3 at 380 nm
(Fig. 2), since the product 2 and the byproduct 3 had a
common kmax at 290 nm. In the HPLC method, the yield
was calculated based on formation of derivatized glycerol 2
using the standard graph (Fig. 3). The real system experi-
ment was performed from one gram of Jatropha curcas oil.
Biodiesel formation in this was followed by glycerol
estimation (Scheme 3). The derivatized glycerol 2 (RT
3.8 min) and the byproduct fluorenone (RT 4.8 min) were
monitored at 280 nm using acetonitrile/water (85:15)
(Fig. 4). The monoglyceride derivative 4, was synthesized
using the reported procedure [30] (Scheme 4). The com-
pound 4 on HPLC analysis had a retention time of 144 min.
Sample Analysis by UV–Visible Spectrophotometry
Five milligrams of reaction mixture was dissolved in
10 mL of ethanol and the absorbance was measured for
compound 3 at 380 nm using 1 mL quartz cuvettes. The
yield of derivatized glycerol 2 was calculated based on the
formation of 3.
Sample Analysis by the HPLC Method
Five milligrams of reaction mixture was dissolved in
10 mL of acetonitrile, from this 20 lL was taken for the
analysis. The derivatized glycerol 2 was monitored at
280 nm and the yield of derivatized glycerol 2 was cal-
culated from the standard graph.
Results and Discussion
Estimation of glycerol formed during the transesterification
of oil using 9,9-dimethoxyfluorene 1 as a reagent and
PTSA as a catalyst by UV–visible spectrophotometry and
HPLC was carried out. The compound 1 was synthesized
from commercially available inexpensive materials [26] in
78% yield. Most importantly, compound 1 and catalyst
(PTSA) were stable, solid and easy to use at room tem-
perature. Earlier compound 1 was used for the synthesis of
p-nitrophenyl b-D-ribofuranoside derivatives, which were
Fig. 1 Crystal structure of spiro[[1, 3]dioxolane-2,90-fluorene]-4-
ylmethanol 2
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J Am Oil Chem Soc (2010) 87:747–754
Fig. 4 HPLC chromatogram of the crude reaction mixture (transe-
sterification followed by derivatization) of real system experiment
from one gram of Jatropha curcas oil for biodiesel formation in
30 min, monitored by estimation of the derivatized glycerol 2
Fig. 2 Standard graph for 9-fluorenone (e380 = 281 M-1 cm-1) by
UV–visible spectrophotometry
Standard Graph for 9-Fluorenone Using a UV–Visible
Spectrophotometer
The standard graph for compound 3 is shown in Fig. 2. The
correlation between concentration and recorded absorbance
is linear up to 0.76 units, absorbance measured at 380 nm
and the calculated R2 is 0.99. The molar extinction
coefficient for compound 3 was determined to be
e380 = 281 M-1 cm-1
.
Standard Graph for Derivatized Glycerol 2 by HPLC
Figure 3 shows the standard graph for derivatized glycerol
2 in acetonitrile, at varying concentrations. The correlation
between concentration and recorded area is linear up to
7 9 106 lAU], the calculated R2 is 0.99. The solvent used
was acetonitrile: water (85:15) at a flow rate of 1 mL/min.
The derivatised glycerol 2 was monitored at 280 nm using
a PDA detector.
Fig. 3 Standard graph for derivatized glycerol 2 by HPLC
Control Experiment with TG and Biodiesel
in the Absence of Glycerol
A mixture of 2, 3 and 4 were subjected to HPLC using
acetonitrile/water (85:15) and this was used as a standard
reference for further analysis.
The control reaction was examined in the absence of
glycerol using the 500 mg of TG and 500 mg of biodiesel
in the presence of compound 1 as described in Scheme 2.
From this experiment it was observed that compound 1
was completely converted into compound 3 without
forming any derivatized glycerol. It was concluded that
compound 1 has no effect on TG and biodiesel and the
reaction is between glycerol and 1 (Scheme 2). This was
confirmed by both UV–visible spectrophotometry and
HPLC methods.
The sample from the real system experiment (Scheme 3)
did not show 4 (derivative of monoglyceride). Furthermore,
a known amount of glycerol (90 mg) and monoglyceride
2,3-dihydroxypropyl palmitate (10 mg) in one gram of
biodiesel containing compound 1, under conditions shown
in Scheme 1 did not show the formation of 4. All this
indicates the absence of derivatized monoglyceride.
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753
One-Pot Real System Experiments
for the Estimation of Glycerol
Table 1 Results on various concentrations of glycerol detection in
biodiesel
Entry
Glycerol
(mg)
2 (mg)
Reacted
glycerol (mg)a
Yield (%)
The parameters of the glycerol estimation were opti-
mized and used for a one pot real system experiment
starting from the Jatropha curcas oil to biodiesel for-
mation followed by glycerol estimation (Scheme 3).
Biodiesel was prepared by transesterifying Jatropha
curcas oil using the reported procedure [31]. The total
content of biodiesel in the final product was 99%, as
confirmed by GC and 1H-NMR analysis. The proposed
method using UV–visible spectrophotometry and HPLC,
revealed that the amount of glycerol formed from one
gram of Jatropha curcas oil on complete conversion to
biodiesel gave 110 2.0 and 109 1.5 mg respectively,
whereas the expected yield for 100% conversion was
115 2.0 and 114 1.5 mg.
1
2
3
4
5
6
7
a
5
10
13
26
4.79
9.50
19.2
29.5
38.4
47.0
96.0
96.0
95.0
96.0
98.3
96.0
94.0
96.0
20
53
30
82
40
106
130
265
50
100
Amount of reacted glycerol was measured by the UV–visible
spectrophotometric method
Table 2 Progress of transesterification reaction as a function of time
Entry
Time (min)
Glycerol (mg)a
Biodiesel (%)b
1
2
3
4
5
a
5
10
15
20
30
68
93
61
Detection of Various Concentrations
of Glycerol Biodiesel
84 (83)
86
95
105
110
95 (94)
[99 ([99)
The efficiency of the proposed method was determined by
varying concentrations of glycerol ranging from 5 to
100 mg/g of biodiesel. Furthermore, the detection limits of
glycerol as low as 0.05% w/w (5 mg/10 g) of glycerol in
biodiesel was established. The correlation between actual
glycerol taken in the reaction and the reacted glycerol
starting from 5 mg to 100 mg is shown in Fig. 5 (R2 is
0.99). The yields (Table 1) of these reactions were ana-
lyzed based on UV–visible spectrophotometry since the
standard deviation between both methods (UV–visible
spectrophotometry and HPLC) was less than one. In
addition to this, UV–visible spectrophotometry was faster,
easy to use and all further experimental analysis were
carried out by this method.
The amount of glycerol formed was measured by the UV–visible
spectrophotometric method
b
The yield of biodiesel formed was determined based on glycerol
formed as a function of time
c
The yield of biodiesel as determined by GC
Progress of the Transesterification Reaction
as a Function of Time
Progress of the transesterification reaction was monitored
for biodiesel formation from the Jatropha curcas oil using
the developed method. The glycerol formed was estimated
at different time intervals of 5–30 min (Table 2). It was
found that the concentration of glycerol and biodiesel
increased with time as expected. These results were vali-
dated by GC estimation of biodiesel at different time
intervals of 10, 20 and 30 min (Table 2).
Validation of the Proposed Method
by the Greenhill Method
Validation of the proposed method was carried out using
the Greenhill method for determination of free glycerol
[23]. One gram of oil on complete conversion to biodiesel,
yields 115 mg of glycerol (based on 800 as the MW of
Jatropha curcas oil) [32]. It must be mentioned here that
the molecular weight of Jatropha curcas oil varies from
region to region i.e. 800 and 887.7 [33]. The Greenhill
method gave 117 1.5 mg of glycerol while the devel-
oped method gave 115 2.0 mg of glycerol.
Fig. 5 Actual glycerol (mg) versus reacted glycerol
123

754
J Am Oil Chem Soc (2010) 87:747–754
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Conclusions
The method developed in this work is novel, efficient and
uses stable solid reagents which are easy to handle at room
temperature (9,9-Dimethoxyfluorene and PTSA). Glycerol
was estimated both by UV–visible spectrophotometry and
HPLC. Using this method quantitative estimation of glyc-
erol and therefore biodiesel was carried out up to a
detection limit of 0.05% w/w of glycerol in biodiesel.
Progress of transesterification reaction as a function of time
based on glycerol estimation, revealed that the concentra-
tion of glycerol and biodiesel increased with time as
expected. Validation of the developed method was done
using the Greenhill method.
Acknowledgments Funding from the Department of Science and
Technology (DST), Government of India is gratefully acknowledged.
The authors thank the Sophisticated Analytical Instrumentation
Facility (SAIF), Indian Institute of Technology Madras and Dr. Babu
Varghese for the X-ray data collection. The authors also acknowledge
valuable suggestions received during the drafting stage from Profes-
sor R. Ravi and G. Ravikumar.
21. Bondioli P, Della Bella L (2005) An alternative spectrophoto-
metric method for the determination of free glycerol in biodiesel.
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