Eur. J. Lipid Sci. Technol.

107 (2005) 153157

Paolo Bondioli
Laura Della Bella
Stazione Sperimentale
Oli e Grassi,
Milano, Italy

DOI 10.1002/ejlt.200401054

153

An alternative spectrophotometric method for the

determination of free glycerol in biodiesel
The presence of small amounts of free glycerol in fatty acid methyl esters used as diesel fuel or as heating fuel may represent the reason for some failures in engines and
heating systems as well. Several methods are actually available for the determination
of free-glycerol traces in biodiesel, and most on them are based on gas-chromatographic techniques. After a review of the existing methods, a new procedure based on
periodate oxidation of glycerol, leading to the preparation of formaldehyde, on reaction
with acetylacetone and on spectrophotometric measurement at 410 nm is illustrated.
This method is simple, quick and economic and seems to be sufficiently reliable. Data
related to recovery tests and analyses of real samples are shown, also in correlation
with the existing reference method.

1 Introduction
The great development of the renewable fuel biodiesel,
intended as a mixture of fatty acid methyl esters to be
used as diesel fuel alternative, is supported at the
moment by a good regulation system that the European
Community issued by means of CEN Committees, and it
is represented by norms EN 14213:2003 and EN
14214:2003. These two documents set the standard for
biodiesel heating and automotive application, respectively.
In both standards, the specification limit for free glycerol
is set at max. 200 mg/kg, and two alternative analytical
methods are listed for the evaluation, EN 14105:2003 and
EN 14106:2003. The presence of free-glycerol traces in
biodiesel is caused by the reaction from triacylglycerol to
fatty acid methyl ester, giving glycerol as one of the reaction products. It is generally known that glycerol is practically insoluble in oils, fats, and also biodiesel, but a small
amount of glycerol, which can be estimated at around
200 mg/kg, is soluble in biodiesel at temperatures around
0 7C. In addition, glycerol can be easily dispersed in biodiesel in small droplets, and this is the reason why in the
past, it was possible to find biodiesel samples containing
300500 mg/kg of glycerol. Also, the presence of residual

Correspondence: Paolo Bondioli, Technology Department, Stazione Sperimentale Oli e Grassi, Via Giuseppe Colombo 79,
20133 Milano, Italy. Phone: 139 02 7064971, Fax: 139 02
2363953, e-mail: bondioli@ssog.it

2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

soaps may have an influence on final free-glycerol concentration. Finally, the presence of water (we do not have
to forget that biodiesel is a hygroscopic fluid), even at very
low levels (some hundred mg/kg), may cause the
separation of free glycerol, when present in high amounts.
Some distilled biodiesel samples, prepared avoiding preor post-treatments, may contain free glycerol in relatively
high concentration, because free glycerol distils as a head
product during this unit operation. This high content of
free glycerol was one of the main causes in the past for
filter blocking and general failure in diesel systems. After
the adoption of EN regulations about biodiesel, the freeglycerol content is generally below 200 mg/kg in each
sample produced in Europe, and often, we analyze samples having a free-glycerol content close to zero. In this
case, for samples having a free-glycerol concentration
lower than the solubility limit, surely a water washing step
was used during processing. From the beginning of the
biodiesel adventure, it was clear that free-glycerol analysis was a key point for quality control. The first tests were
carried out on a big amount of sample, repeatedly
extracted with water: in the resulting solution, glycerol
was evaluated using the classic periodate method (e.g.
ISO 2879:1975). The first tailor-made method for freeglycerol determination in biodiesel was published by Bailer and de Heuber [1] in 1991. The biodiesel sample was
extracted with water, and on the resulting solution, a biochemical test, mutated from the clinical chemistry, was
carried out. After this method, some GC tests appeared in
the international literature: Bondioli et al. [2] described a
method for free-glycerol determination using a GC
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Research Paper

Keywords: Analytical method, biodiesel, biofuel, renewable material, free glycerol,

quality control.

154

P. Bondioli and L. Della Bella

packed column in which a water/alcohol extract containing an internal standard was injected. Three years later,
Mittelbach et al. [3] published a method allowing the
contemporary determination of methanol and free glycerol by injecting a TMS-derivatized biodiesel sample into
a capillary GC. While the first GC method was further
developed, adapted for capillary GC and used as a basic
paper for method EN 14106:2003, the second one was
abandoned. In the same years, a method allowing the
contemporary determination of free and bonded glycerol
(mono-, di- and triacylglycerols) was published by Plank
and Lorbeer [4], and this method, developed and standardized under the CEN activity, represents the actual solution for this problem as method EN 14105:2003. This
procedure, very accurate, easy to handle and satisfactory, is in common use in all biodiesel laboratories, and it
is generally known as Christina Planks method, from
the name of the so nice lady who immediately afterwards
left the biodiesel sector for other businesses. Very
recently, Sigma Aldrich Co. presented the BQP-02 Kit
(glycerin determination by enzymatic analysis) allowing
the determination of both free and total glycerol in a very
easy and precise way. This method is actually under
evaluation of the National Biodiesel Accreditation Commission (NBAC, USA), and it can be considered as a valid
alternative of the ASTM 6584 procedure (the American
version of Christina Planks method). The necessity for the
availability of a second free-glycerol evaluation method
was underlined and accepted by the CEN TC 307/WG1
Committee, because in some samples, the presence of
traces of volatile products (solvents used as a carrier for
additives, traces of volatile hydrocarbons from tanks,
carrier or pipeline) may cause interference in free-glycerol
determination according to EN 14105:2003. The use of an
alternative method is sometimes necessary for an efficient control of biodiesel. Unfortunately, the standardized
method EN 14106:2003 does not guarantee the necessary performance in terms of repeatability (r) and reproducibility (R) in comparison with the set specification limit,
and for this reason, it needs to be substituted by a better
one. From these starting considerations, we began our
tests with few but clear guidelines in our mind: the new
method might use techniques other than GC or enzyme,
because in these two fields, probably the other authors
approached 100% of the available potential.
After a wide examination of available possibilities in the
literature, several solutions were identified using the periodate oxidation of free glycerol to give formaldehyde and
in the successive quantification of formaldehyde after
derivatization. Starting from a paper published by Benassi
et al. [5], describing the evaluation of formaldehyde traces
in cosmetic products after the preparation of the corresponding dinitrophenyl hydrazone (DNPH), we did some

2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Eur. J. Lipid Sci. Technol. 107 (2005) 153157

tests, but the procedure consisting of extraction, periodate reaction, DNPH preparation and RP-HPLC analysis
was too complicated, and the obtained performances
were not satisfactory. Also, the detection limit, after dilution and sample manipulation, was not as low as necessary for our evaluation purposes. Our attention was finally
turned to the reaction between formaldehyde and two
molecules of acetylacetone in the presence of ammonium
acetate, leading to the formation of 3,5-diacetyl-1,4dihydrolutidine (Fig. 1), generally known as Hantzschs
reaction.
Although the precise details of this multicomponent condensation are not known, a reasonable mechanism can
be found in well-known organic chemistry texts [6, 7].
This derivative has a very high specific absorption at
410 nm, and this property greatly improves the potential
to get a very low detection limit for glycerol estimation.
The use of the acetylacetone reaction is a widely used
technique for the evaluation of formaldehyde (or formaldehyde-producing substances) in different application fields. Wu et al. [8] recently published a study where
an HPLC method and an acetylacetone method were
compared in performance for the determination of formaldehyde traces in cosmetic products. An interesting
example of the use of this reaction is reported by Kondoh
and Takano [9], who used a post-column reactor located
at the outlet of an HPLC equipment for the selective
detection of triacylglycerols according to the sequence
triacylglycerol ? saponification ? free glycerol ? periodate reaction ? formaldehyde ? Hantzschs reaction?
spectrophotometric detection. The authors carried out a
very comprehensive study about the reaction conditions,
and this work represented the starting point of our research. Also, in the field of environmental analysis, we can

Fig. 1. Structure of 3,5-diacetyl-1,4-dihydrolutidine.

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Eur. J. Lipid Sci. Technol. 107 (2005) 153157

find examples of formaldehyde evaluation by means of
the acetylacetone reaction, as reported by Kormann et al.
[10] during their studies about the photochemical degradation of methane and non-methane volatile organic
compounds.

2 Materials and methods

2.1 Materials
Biodiesel samples from different origins were collected
and previously analyzed using the EN official procedures.
A glycerol-free biodiesel sample was prepared by
washing several times with distilled water, in a separator
funnel, a current product. After washing, the residual
moisture was removed by heating at 80 7C and stirring the
sample under a residual pressure of 0.1 kPa for 30 min.
This sample was then additivated as previously described
[2] with known amounts of free glycerol dissolved in
ethanol, in order to obtain different concentrations of free
glycerol in biodiesel for analysis. An ethanolic mother solution containing 0.1 mg/mL of 99% reference glycerol
(Carlo Erba, Milano, Italy) was prepared in a 100-mL calibrated flask. Using a precision microsyringe having a capacity of 200 mL, five 100-g samples of glycerol-free biodiesel were spiked with appropriate amounts of solution
in order to cover the range between 0 and 200 mg/kg of
free glycerol. Immediately after additivation, the samples
were stopped and carefully homogenized before analysis.
All used reagents were of analytical grade. The spectrophotometric analyses were carried out using a CARY
mod. 1E UV-Vis spectrophotometer (Varian, Lake Forest,
CA, USA).

Determination of free glycerol in biodiesel

155

sample extraction, reaction and mother glycerol reference

solutions. Glycerol reference stock solution: weigh
approx. 150 mg (accuracy 6 0.1 mg) of glycerol into a 50mL calibrated flask. Dissolve in the working solvent and
fill up to the mark. This solution contains approx. 3 mg/mL
of glycerol. Glycerol reference working solution: using a
precision pipette, transfer 1 mL of glycerol reference
stock solution to a 100-mL calibrated flask. Dilute and fill
up to the mark using the same solvent. This solution
contains approx. 0.03 mg/mL of glycerol. Both solutions
are stable for some weeks.

2.3 Methods
2.3.1 Preparation of the calibration curve
Into a series of ten 10-mL test tubes, transfer 0.00, 0.25,
0.50, 0.75, 1.00, 1.25, 1.50, 1.75 and 2.00 mL of a
0.036 mg/mL glycerol solution. Dilute with the working solvent in such a way as to get a final volume of 2 mL in each
tube. Add 1.2 mL of a 10 mM sodium periodate solution
and shake for 30 s. After that, add 1.2 mL of a 0.2 M acetylacetone solution and put in a water bath thermostated at
70 7C for 1 min, stirring manually. After the reaction time, the
sample must be immediately cooled by immersing the tube
in a beaker containing tap water. The water must be periodically changed to maintain a nearly constant temperature (2025 7C). The samples are finally read in a spectrophotometer set in double beam mode at 410 nm.

2.3.2 Sample analysis

Acetic acid stock solution: a 1.6 M (9.6 g/100 mL) aqueous solution was prepared. Ammonium acetate stock
solution: a 4.0 M (30.8 g/100 mL) aqueous solution was
prepared. Both solutions are stable over time. Mixed in
equal volumes, these solutions result in a buffer solution
at pH 5.5. Acetylacetone solution, 0.2 M: dissolve in a test
tube approx. 200 mL (195 mg) of acetylacetone in 5 mL of
acetic acid stock solution and 5 mL of ammonium acetate
stock solution. This reagent must be prepared daily.
Sodium periodate solution, 10 mM: weigh into a test tube
approx. 21 mg of sodium meta periodate, add 5 mL of
acetic acid stock solution, swirl to dissolve the periodate,
and after periodate is completely dissolved, add 5 mL
ammonium acetate stock solution. This reagent must be
prepared daily. Working solvent: mix equal volumes of
distilled water and 95% ethanol. This solvent is used for

Into a 10-mL test tube, weigh 1 g of biodiesel sample

(accuracy 6 0.1 mg), dissolve in 4 mL of hexane and add
4 mL of extraction solvent. Tightly stop the tube and
shake the sample vigorously (using a Vortex mixer if
available) for 5 min. Centrifuge for 15 min at 2000 rpm.
After centrifugation, remove the main part of the upper
layer using a Pasteur pipette. Transfer exactly 0.5 mL of
the lower layer into a 10-mL test tube. Add 1.5 mL of
working solvent. Add 1.2 mL of a 10 mM sodium periodate solution and shake for 30 s. After that, add 1.2 mL of a
0.2 M acetylacetone solution and put in a water bath
thermostated at 70 7C for 1 min, stirring manually. After
the reaction time, the sample must be immediately cooled
by immersing the tube in a beaker containing tap water.
The water must be periodically changed to maintain a
nearly constant temperature. The samples are finally read
in a spectrophotometer set in double beam mode at
410 nm, against a blank sample prepared in the same way
as the samples, after addition of 2 mL of working solvent
to the test tube.

2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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2.2 Working reagents

156

P. Bondioli and L. Della Bella

Eur. J. Lipid Sci. Technol. 107 (2005) 153157

3 Results and discussion

The calibration curve obtained from the procedure
described above is shown in Fig. 2. The correlation between concentration and recorded absorbance is linear
up to 1.5 absorbance units, and the calculated R2 is .0.99.
After calibration, several recovery tests were carried out in
order to understand if the proposed method can be considered as suitable for a further panel evaluation. For this
purpose, five samples containing different added
amounts of free glycerol were analyzed. On each sample,
six independent tests (weighing, extraction, color development, spectrophotometric analysis, etc.) were carried
out.
The results of the recovery tests are reported in Tab. 1.
Tab. 1. Recovery tests.

1
Free glycerol [mg/kg], theoretical 45
Free glycerol [mg/kg] found,
45
average value of six
independent measurements
Recovery [%]
100
Standard deviation
2.8
% RSD
6.2
r (repeatability)
9.6

2
70
67

Test no.
3
4
87
77

96 89
4.8 4.7
7.2 6.1
16.8 16.2

128 211
116 198

91 94
2.1 4.6
1.8 2.3
7.3 15.9

The sample indicated in the first column (Test no. 1) was in

reality the sample regarded as blank sample, glycerol
free. After obtaining this unexpected result, we analyzed
the sample according to methods EN 14105:2003 and
EN 14106:2003, in order to understand whether a residual
glycerol amount was still present in the sample.
While using method EN 14106:2003, we obtained a GLC
path with no peaks in the glycerol region; using
EN 14105:2003, a small peak having the glycerol RT was
present. After integration, and regardless of the rules for
the expression of results using only two decimals, we
calculated a residual glycerol content in agreement with
the value found for the sample.
For this reason, for samples 2, 3, 4 and 5, the theoretical
values shown in Tab. 1 represent the sum of the endogenous glycerol content plus the glycerol added to the
sample in ethanolic solution.
From the results shown in Tab. 1, we can see that the
recovery test gave us very encouraging results, even if the
average found values are always lower than the theoretical ones. This fact is probably due to the easy solubility of
ethanol (and by consequence of glycerol) in the hexane/
biodiesel phase, leading to glycerol losses from the
water/alcohol phase. Some tests were carried out by
changing the phase volume ratio or the composition of
the extraction solvent, but the best results can be
obtained under the above-described conditions.

Fig. 2. Calibration curve: free-glycerol

concentration in biodiesel reported
assuming a sample of 1000 g biodiesel.

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Eur. J. Lipid Sci. Technol. 107 (2005) 153157

Determination of free glycerol in biodiesel

In order to complete our work and to better understand

the potential of the suggested procedure, we analyzed
three real samples, previously analyzed in duplicate
according to the reference procedure EN 14105:2003.
From the results reported in Tab. 2, we can see that there
is a good correlation between the reference GC method
and the new spectrophotometric one. The % RSD is
practically constant in the range of the examined concentrations, and this is a further confirmation of the suitability of this method for the scope.
Tab. 2. Free glycerol determination in three real biodiesel
samples
Sample no.

Free glycerol [wt-%] EN 14105:2003

0.01 1 0.02 0 0.02 5
Free glycerol [mg/kg] found, average value 118
205
230
of eight independent measurements
Standard deviation
3.7
8.6
9.4
% RSD
3.2
4.2
4.1
r (repeatability)
12.5 28.7 31.3

Lower-case digits not allowed by the test method,

expression of results.

At the end of our work, we also analyzed two samples

consisting of a diesel fuel/biodiesel mixture, containing 5
and 20% (wt-%) of biodiesel. For the analysis of these
samples, we used the same procedure as described
above, with the only exception of the sample filtration
after color development and immediately before spectrophotometer readings. The second change was in the
sampling procedure after solvent extraction. Instead of
the volume of 0.5 mL of the lower layer, as obtained after
centrifugation, an amount of 2.0 mL was taken, in order to
increase the quantity of glycerol submitted to periodate
oxidation and color development.
More tests are necessary in this case in order to evaluate
the robustness and the suitability of the proposed procedure on this substrate.

4 Conclusions
At the end of our experiments, we are now able to suggest
an original procedure that can be regarded as a potential
alternative for the actually available methods for the
evaluation of free-glycerol content in biodiesel. The pro-

2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

157

cedure seems to be reliable, easy to handle and cheap for

the daily use in quality control. During the experiments,
we also demonstrated the feasibility of this test applied to
a diesel fuel/biodiesel blends. This new method is now
available for consideration by the standardization committees.

Acknowledgments
Many thanks to Prof. Dr. Peter Swiderky (Technical University of Luebeck, Germany), Dr. Nicoletta Ravasio
(CNR, Milano, Italy) and to Dr. Federica Zaccheria (University of Milano, Italy), who kindly provided the necessary advice to understand the mechanism of Hantzschs
reaction.

References
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[5] C. A. Benassi, A. Semenzato, A. Bettero: High performance
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[6] D. T. Davies: Aromatic Heterocyclic Chemistry. Oxford Science Publications, Oxford (UK) 1992, p. 3637.
[7] K. Peter, C. Vollhardt: Chimica Organica. Zanichelli, Bologna
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[Received: July 28, 2004; accepted: January 5, 2005]

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