Fuel 87 (2008) 3369–3372

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Fuel
journal homepage: www.elsevier.com/locate/fuel

Thin layer chromatography and image analysis to detect glycerol in biodiesel

Karan Bansal, Jonathan McCrady, Alan Hansen, Kaustubh Bhalerao *
Department of Agricultural and Biological Engineering, University of Illinois – Urbana Champaign, 1304 W. Pennsylvania Avenue, Urbana, IL 61801, USA

a r t i c l e i n f o a b s t r a c t

Article history: Glycerol is a by-product of biodiesel production and is also considered a contaminant in the final product.
Received 11 October 2007 Current US and EU standards place limits on the permissible levels of free glycerol and bound (uncon-
Received in revised form 12 February 2008 verted) mono, di and triglycerides in biodiesel, which can be quantified using gas or high pressure liquid
Accepted 28 April 2008
chromatography.
Available online 2 June 2008
We report a method based on thin layer chromatography that can be effectively used to determine the
presence of glycerol in biodiesel. Visual and machine (image analysis) detection limits of 0.1% and 0.2% v/v
Keywords:
of free glycerol, respectively, were established using this procedure. In conjunction with a suitable sam-
Biodiesel
Thin layer chromatography
ple enrichment procedure, this procedure could provide a cost effective alternative to glycerol detection.
Glycerol Ó 2008 Elsevier Ltd. All rights reserved.
Image analysis

1. Introduction 1.1. Glycerol standards

The method widely used to produce biodiesel consists of a
transesterification reaction of a vegetable oil with methanol in
the presence of a base catalyst. This reaction yields mono-alkyl es-
ters of the fatty acids in the vegetable oil and glycerol as a by-prod-
uct. While purification procedures exist, the final fuel product can
be contaminated by partial glycerols, unreacted triacylglycerols,
unseparated glycerol, free fatty acids, residual alcohol, and catalyst
[1]. These contaminants can create severe problems in the engine,
such as engine deposits, corrosion, and failures. The unconverted
mono, di, and triglycerides increase the fuel viscosity, causing an
increase in pressure within the fuel system and poor fuel atomiza-
tion in the combustion chamber. Glycerol also settles in the fuel
tank and can clog fuel filters, accelerating the wear and tear of
the entire combustion system [2].
Other contaminants, not within the scope of this study, also
pose serious concerns in its utilization in combustion engines:
The presence of free fatty acids in the finished fuel indicates the
fuel is being oxidized and is decomposing; alcohol lowers the flash
point of the fuel and can pose a serious safety risk when handling
the fuel and can also break down rubber components in the fuel
system; and catalyst leads to corrosion in the fuel system [2].
The hygroscopic nature of glycerol may contribute synergistically
to processes such as corrosion and fuel oxidation as well.
Biodiesel fuel quality is of great importance as production and
consumption levels rapidly increase around the world. Fuel quality
and quality standards were the second most important research
priority identified in Annual Biodiesel Technical Workshop in Jan-
uary 2005 organized by the National Biodiesel Board. Continued
research in biodiesel quality is especially necessary, as the variabil-
ity in final fuel quality tracks the variability in the lipid composi-
tion of the various feedstocks, of both plant and animal origin [3].
The ASTM standard D6751-07a applied in the US sets forth the
specifications that must be met for a fatty acid ester product to carry
the designation ‘‘biodiesel fuel” or ‘‘B100” [4]. Products that meet
the specification, by implication, will perform properly as a com-
pression ignition fuel either as B100 or in blends with any petro-
leum-derived diesel fuel defined by ASTM specification D 975
Grades 1-D, 2-D, and low sulfur 1-D and 2-D [5]. The European stan-
dard EN 14214 includes an additional specification, the oxidative
stability test, over the ASTM standard [6]. Both standards specify
limits for contaminants, including glycerol at 0.2 wt% and 0.02wt%
for total and free glycerol, respectively. Total glycerol includes free
and unconverted glycerol in the form of mono, di and triglycerides.
1.2. Present methods for measuring glycerol

The measurement of glycerol concentrations in various solvents

* Corresponding author. Tel.: +1 217 244 6569; fax: +1 217 244 0323.
E-mail address: bhalerao@uiuc.edu (K. Bhalerao).
is not a new problem. It is an indicator in several metabolic
processes and beverages, and has been measured using multiple
approaches. High pressure liquid chromatography, gas chromatog-
raphy and enzymatic methods coupled with amperometric mea-
surements constitute the main methods for determining glycerol

0016-2361/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fuel.2008.04.033
3370 K. Bansal et al. / Fuel 87 (2008) 3369–3372

concentrations in aqueous solutions. These methods, especially Table 1

chromatographic methods have also been adapted to measure glyc- Biodiesel sample specifications

erol in biodiesel successfully, and may be reviewed in [7–9]. How- Property B100 from Growmark FS
ever, these methods require expensive equipment and trained Carbon (% by mass) 77.42
professionals. Compared to the ease with which biodiesel lends it- Hydrogen (% by mass) 12.29
self to small scale manufacture, the availability of simple, effective, Oxygen (% by mass) 10.29
and inexpensive quality testing methods is severely lacking. This Sulfur (ppmm) <50
Nitrogen (ppmm) 10
creates a barrier to entry for the local, small scale production of bio- Ash (ppmm) 20
diesel where the capability to produce biodiesel may not be suffi- Density (kg/l at 21 °C) 0.8796
ciently complemented with the availability of analytical testing Specific gravity (ppmm) 0.8805
facilities. Gross calorific value (MJ/kg) 39.78
Net calorific value (MJ/kg) 37.17

1.3. Objectives

The ninth most important research priority listed at the Annual

Table 2
Biodiesel Technical Workshop in 2005 was developing faster and
Standard and sample preparation
simpler test methods. In cognizance of this need our main objec-
tive in developing this assay was cost effectiveness. Thin layer Sample composition Glycerol percentage

chromatography (TLC) has been frequently used as a simple, inex- 0 100 5 4 3 2 1

pensive method for analyzing or visually separating components of 1–5% v/v
different heterogeneous solutions. TLC, and its even simpler coun- Biodiesel (ml) 2 – 1.9 1.92 1.94 1.96 1.98
terpart, paper chromatography, has been used to determine the Glycerol (ll) – 100 100 80 60 40 20
Water (ml) – 1.9 – – – – –
glycerol concentration in several different solutions as well. How-
ever, it has never been tested for feasibility in the measurement of 0.1–0.8% v/v
0 100 0.8 0.6 0.4 0.2 0.1
glycerol in biodiesel.
Biodiesel (ml) 2 – 1.984 1.988 1.992 1.996 1.998
Our secondary objective was to develop a framework for quan- Glycerol (ll) – 100 16 12 8 4 2
tifying the amount of glycerol in a given solution. We therefore Water (ml) – 1.9 – – – – –
developed an image analysis procedure involving acquiring the im-
0% and 100% indicate pure biodiesel and glycerol, respectively.
age of the TLC plate using an easily available document scanner
and a computer. An image analysis algorithm serves to analyze
and quantify the size of the glycerol spots with respect to the
amount of glycerol in the biodiesel. second marked points respectively from the left on the TLC plate.
Similarly, for each of the two series, the sample solutions were
2. Materials and methods spotted from points 3 to 7, respectively, as shown in Fig. 1a. The
spotting procedure was repeated three times, allowing the spots
The following reagents were obtained: Potassium permanga- to dry between spotting, consuming 6 ll of sample in all cases.
nate and 1-butanol from Sigma Inc. (St. Louis, MO). Glycerol, so- After the spots dried, the TLC plates were run in 30 ml of buta-
dium hydroxide, and acetone from Fisher Scientific Inc. nol:water (1:1) v/v for 25 min in a 1000 ml beaker. The size of the
(Hampton, NH), and TLC plastic sheets (Silica Gel 60 F254) from beaker and volume of the mobile phase ensure that the line of
Merck Inc. (Whitehouse Station, NJ). spots remained above the liquid level. The plate was removed
The biodiesel sample was obtained from Growmark FS (Bloom- and allowed to dry horizontally. The dried plate was dipped in
ington, IL). Its properties as measured by Phoenix Chemical Labora- freshly prepared potassium permanganate solution (0.5% KMnO4
tory Inc. (Chicago, IL) are as shown in Table 1. The free and total in 1 N NaOH). The plate was removed immediately and scanned
glycerol contents were within ASTM specification (<0.2% total gly- at 600 dpi in the uncompressed JPEG format using a Hewlett Pack-
cerol by weight). ard desktop scanner model ScanJet 4370 (Palo Alto, CA). Each glyc-
erol concentration series was spotted on at least five TLC plates and
2.1. Preparation of standard solutions and testing procedure run independently, to get an estimate of the operator and pipette
variability in spotting the samples on the ceramic plate. Examples
The biodiesel standard was prepared by adding 2 ml of pure of the scanned images are shown in Fig. 1.
biodiesel (B100) to 8 ml of acetone. For the glycerol standard
100 ll of pure glycerol was added in 1.9 ml of distilled water and
8 ml of acetone. Two sets of samples prepared for quantification 3. Quantification of glycerol contamination
contained of 1–5% glycerol in one series and 0.1–0.8% glycerol for
the other. The sample solutions consisting of biodiesel with vary- 3.1. Image analysis
ing amounts of glycerol is shown in Table 2. As an example, to pre-
pare 0.4% glycerol sample, 1 ml of biodiesel was added to 8 ml of Fig. 1 clearly shows that the area of the spot varies in proportion
acetone followed by the addition of 900 ll of biodiesel and then to the amount of glycerol in the sample. We explored the possibil-
92 ll making a total of 1.992 ml of biodiesel in the sample. To this ity of performing image analysis on the scanned images to quantify
8 ll of pure glycerol was added. This procedure allowed us to deal the amount of glycerol seen on the TLC plates. The image analysis
with the problem of viscosity of biodiesel and minimize the error was performed using MatlabÒ (MathWorks Inc., Natick, MA).
in pipetting. The algorithm for image analysis used was as follows: the
A TLC plate of size 10 cm  10 cm was used for all experiments. images were first cropped to include only the glycerol spots to re-
A line was drawn 1 cm from the bottom using a pencil. Seven move irrelevant image features. They were converted from the
points were marked on this line at equal intervals. Two microliters Red–Green–Blue (RGB) color space to the Luminosity–Red/
of the biodiesel and glycerol standards were spotted at the first and Green–Yellow/Blue (L*a*b*) color space, which attempts to capture
human-like perceptional differences between colors [10]. This is
 K. Bansal et al. / Fuel 87 (2008) 3369–3372 3371

Fig. 1. (a) The position of the pencil line and the sample spots with respect to the bottom edge of the TLC plate. After developing the TLC plates in KMnO4, the glycerol and
biodiesel bands show up as yellow spots on a purple/pink background. The green streaks are due to shaking off of excess KMnO4. The biodiesel being hydrophobic, moves with
the butanol phase, while hydrophilic glycerol remains in the slower moving water phase. The 0.1% glycerol in biodiesel spot (far right in b) is distinctly visible. High resolution
color scans have been uploaded as Supplemental documents A and B. (For interpretation of the references to color in this figure legend, the reader is referred to the web
version of this article.)

Fig. 2. The image analysis algorithm identifies the glycerol spots correctly over the background, detects their edge and computes the area of the spot size. As seen in Fig. 1b, it
fails to identify the spot corresponding to 0.1% glycerol which is visible on the scan. It also suffers from an error due to the green streak on the 0.2% glycerol spot size.
However, it does not fail to identify the presence of 0.2% glycerol in the sample. (For interpretation of the references to color in this figure legend, the reader is referred to the
web version of this article.)

especially beneficial, since we seek to distinguish between glycerol other of the same TLC plate within the span of approximately
spots, which are characteristically bright, yellow, and not red, one minute immediately after removing the plates from KMnO4.
against the reddish background. In the L*a*b* space, this translates
to subtracting the second channel (red–green) from the first (lumi-
nosity) and adding the third channel (yellow–blue) to the result. Glycerol spot size
0.8

This operation increases the contrast of the spots over the back-
ground and is followed by an automated thresholding process that 1–5% Glycerol
● 0.1–0.8% Glycerol
produces a 2-bit color depth image, with the spots in white and
everything else in black. Fig. 2 shows the black and white image
produced at this stage of the analysis corresponding to the scans
0.6

in Fig. 1. The boundaries for the identified spots were identified

Relative spot size

using edge detection and their areas were calculated. Any spot
smaller than 100 pixels in area was considered noise and dis-
carded. The remaining spot sizes were highlighted, and their areas
0.4

were denoted adjacent to the spots on the image. The MatlabÒ

source code for this procedure is available upon request.

3.2. Statistical analysis

0.2

●
●
●
The examples in Figs. 1 and 2 show the limits of our analytical
methodology. There are a few technical difficulties in the whole ●

procedure. After developing the chromatogram in KMnO4, two fac- Error bar = 1 SD
●
0.0

tors change rapidly. Firstly, the glycerol spots continue to spread
and become larger and more easily distinguishable. We observed
this as a difference between the computed areas of two scans of
the same TLC plate taken one after the other approximately a min-
ute apart (data not shown). Secondly, the KMnO4 background rap-
idly changes color, starting from purple, through pink and finally
yellow, thereby reducing the contrast between the yellow spot
sizes and the background. These two effects are counter to each
other from an image analysis perspective.
The delay between scans also adds distortion in the image anal-
ysis procedure. In all cases we took two scans one following the
Lowess curves
0.1 0.2 0.5 1.0 2.0 5.0
Glycerol percentage
Fig. 3. The glycerol spot area computed from the image analysis algorithm varies
along a continuum as a function of the amount of glycerol in the biodiesel sample.
Error bars represent the variability in the spot size over five replicates and up to
three consecutive scans per TLC plate relative to that of the pure glycerol spot. The
points are connected with a lowess curve. It does not appear that the spot size
varies in a linear (or log-linear) manner. The error bars for 0.1% and 0.2% include 0 in
their bounds since the image analysis algorithm failed to properly identify the
glycerol spots in some cases, although they were always visible to the eye.
3372 K. Bansal et al. / Fuel 87 (2008) 3369–3372
In some cases, especially for the lower concentrations of glycerol,
we scanned the image for a third time as well. In these cases, typ-
ically the first scan revealed a spot which was visible to the naked
eye but was below the noise threshold of 100 pixels to be visible to
the image analysis algorithm. The spot size was normalized with
respect to the area of the pure glycerol sample in each image. This
removed the problem of increasing spot size with time, as the rel-
ative ratios of the areas were seen to be more or less constant. This
allowed us to pool the data from all the scans. The resulting statis-
tical analysis is presented in Fig. 3. The error bars represent one
standard deviation on either side of the data point. The error com-
prises pipetting error, error in application of sample to the TLC
plate and error in image analysis due to various image artifacts
such as the KMnO4 streaks on the chromatogram.
4. Discussion
Thin layer chromatography provides a simple, cost effective
way of rapidly determining glycerol contamination in a biodiesel
sample. Using a few easily available materials, a commercial scan-
ner and some image analysis software, we have demonstrated the
ability to detect concentrations of free glycerol as low as 0.1% by
volume. While the prescribed maximum limit on glycerol in bio-
diesel is placed at 0.02%, it should be noted that this procedure
does not incorporate any method for enriching or preprocessing
the biodiesel sample. It is conceivable that such a process would
increase the sensitivity by an order of magnitude or more. Even
as it stands, the method is capable of detecting 0.1% glycerol in a
6 ll sample, which is equivalent to an absolute volumetric detec-
tion limit of 6 ml.
While proven methods such as gas and high pressure liquid
chromatography exist, and can provide detection capabilities that
are 100 times lower than the prescribed limits, these methods
are considerably more expensive. The TLC procedure is much bet-
ter suited for smaller operations or portable, rapid, in-field tests on
unknown biodiesel samples.
Using immiscible butanol and water as the mobile phase confers
a significant advantage. The clear separation of biodiesel and glyc-
erol suggests that other constituents of biodiesel do not produce
false positives in the assay. The procedure is also amenable to quan-
tification through image analysis. The size of the spot changes
monotonically as a function of the percentage of glycerol in the
sample. From the results in Fig. 3, it is clear that a calibration curve
can be constructed from standard biodiesel–glycerol mixtures.
We used a very simple image analysis procedure based only on
the color information in the image. We are aware that there exist
far more sophisticated approaches to this problem, including shape
information, travel distance of the spot size and better approaches
to eliminating noise. The focus of these experiments was to devel-
op a simple assay procedure for detecting free glycerol, and the
threshold at which the assay is reliable. The fact that we can also
quantify it comes as an added benefit. With suitable sample prep-
aration, this procedure may become viable as an inline alternative
to other (GC and HPLC) methods.
5. Conclusion
Thin layer chromatography provides a feasible, economical
route to detecting glycerol contamination levels in biodiesel. The
procedure to detect glycerol described here is robust and easily
executed. The ability to quantify detection levels also allows the
computation of assay reliability. To be truly useful however, the
assay needs to gain an order of magnitude in sensitivity to detect
the prescribed 0.02% free glycerol contamination in biodiesel. We
believe that a suitable sample preparation procedure to enrich
the amount of glycerol in a measurement sample will provide that
increase in gain. If such a procedure is correspondingly simple to
use, the TLC-based glycerol detection kit could become an extre-
mely valuable tool for the producers and consumers of biodiesel
alike.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.fuel.2008.04.033.
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