ANALYTICAL METHODS USED IN THE PRODUCTION
AND FUEL QUALITY ASSESSMENT OF BIODIESEL
G. Knothe
ABSTRACT. Biodiesel, an alternative diesel fuel derived from vegetable oil, animal fats, or waste vegetable oils, is obtained
by reacting the oil or fat with an alcohol (transesterification) in the presence of a basic catalyst to give the corresponding
mono鈥揳lkyl esters. Two major categories of methods besides other miscellaneous ones have been reported in the literature
for assessing biodiesel fuel quality and/or monitoring the transesterification reaction by which biodiesel is produced. The
two major categories comprise chromatographic and spectroscopic methods. This article considers the various methods in
each category, including advantages and drawbacks, and offers suggestions on selection of appropriate methods.
Keywords. Biodiesel, Chromatographic methods, Fiber鈥搊ptic probe, Fuel quality, Gas chromatography, Gel permeation
chromatography, High鈥損erformance liquid chromatography, Mass spectrometry, Near鈥搃nfrared spectroscopy, Nuclear
magnetic resonance spectroscopy, Physical properties, Spectroscopic methods, Transesterification, Viscometry.
B
iodiesel has significant potential for use as an methanol, in the presence of a basic catalyst such as NaOH
alternative fuel in compression鈥搃gnition (diesel) or KOH, yields the mono鈥揳lkyl esters of the fatty acids,
engines (Knothe et al., 1997; Dunn et al., 1997). It mainly comprising vegetable oils besides glycerol as side
is technically competitive with conventional, product (see figure 1). Methanol, being the least expensive
petroleum鈥揹erived diesel fuel and requires no changes in the alcohol, is the most commonly used alcohol for
fuel distribution infrastructure. While some technical transesterification and accordingly yields methyl esters such
improvements regarding cold鈥揻low properties, reduction of as methyl soyate and rapeseed methyl ester. Recently,
NOx exhaust emissions, and oxidative stability remain, a modifications of the transesterification reaction, such as
major hurdle towards widespread commercialization is the those based on enzymatic catalysis, have been explored
high price of biodiesel. For this reason, in the United States (Nelson et al., 1996).
the commercialization of biodiesel is targeted towards During the transesterification process, intermediate
regulated fleets and mining and marine markets. In these glycerols, mono鈥? and diacylglycerols, are formed, small
markets, environmental and energy security concerns, which amounts of which can remain in the final biodiesel (methyl
are subject to legislation, can override economic aspects. ester) product. Besides these partial glycerols, unreacted
Vegetable oils, such as soybean oil, rapeseed oil (canola triacylglycerols, unseparated glycerol, free fatty acids,
oil), and in countries with more tropical climates, tropical residual alcohol, and catalyst can contaminate the final
oils (palm oil and coconut oil) are the major sources of product. The contaminants can lead to severe operational
biodiesel. However, in recent years, animal fats and problems when using biodiesel, such as engine deposits, filter
especially recycled greases and used vegetable oils have clogging, or fuel deterioration. Therefore, in the United
found increasing attention as sources of biodiesel, the latter States an ASTM (American Society for Testing and
primarily as inexpensive feedstocks (Mittelbach and Materials) standard is under development (Howell, 1997). In
Tritthart, 1988). Regardless of the feedstock, transesterifica- some European countries, such as Austria, the Czech
tion reactions are carried out to produce biodiesel. Early on Republic, France, Germany, and Italy, standards have been
during the renewed interest in vegetable oils as alternative developed that limit the amount of contaminants in biodiesel
diesel fuels, it was observed that the resulting vegetable oil fuel. In these standards, restrictions are placed on the
(or animal fat) esters did not exhibit the operational individual contaminants by inclusion of items such as free
problems, such as engine deposits, coking of injector nozzles, and total glycerol for limiting glycerol and acylglycerols,
etc., associated with neat oils (Bruwer et al., 1980). flash point for limiting residual alcohol, acid value for
The transesterification reaction of triacylglycerols in limiting free fatty acids, and ash value for limiting residual
vegetable oils, usually with a monohydric alcohol such as catalyst. A more detailed discussion of the rationale for each
quality parameter in biodiesel fuel standards is given in the
literature (Mittelbach, 1994; Mittelbach, 1996). Another
paper (Komers et al., 1998) briefly describes some methods
Article was submitted for review in March 2000; approved for used in the analysis of biodiesel, which include procedures
publication by the Power & Machinery Division of ASAE in December 2000.
for determining contaminants such as water and phosphorus
Parts of this article were presented at the 1999 ASAE Annual Meeting as
that will not be dealt with here. The determination of
Paper No. 99鈥?6111.
biodiesel fuel quality is therefore an issue of great importance
Product names are necessary to report factually on available data;
however, the USDA neither guarantees nor warrants the standard of the to the successful commercialization of this fuel.
product, and the use of the name by the USDA implies no approval of the
Continuously high fuel quality with no operational problems
product to the exclusion of others that may also be suitable.
is a prerequisite for market acceptance of biodiesel.
The author is Gerhard Knothe, Research Chemist, USDA鈥揂RS,
Accordingly, the analysis of biodiesel and the monitoring of
National Center for Agricultural Utilization Research, 1815 N. University St.,
the transesterification reaction have been the subject of
Peoria, IL 61604; phone: 309鈥?681鈥?6112; fax: 309鈥?681鈥?6340; e鈥搈ail:
knothegh@mail.ncaur.usda.gov. numerous recent publications.
Transactions of the ASAE
E 2001 American Society of Agricultural Engineers
Vol. 44(2): 193鈥?200 193
CHROMATOGRAPHIC METHODS
Both GC and HPLC analyses and combinations thereof
have been reported for biodiesel. Gel permeation chromatog-
raphy (GPC) as an analytical tool for analysis of
transesterification products has also been reported. To date,
most chromatographic analyses have been applied to methyl
esters and not higher esters such as ethyl, iso鈥損ropyl, etc. It
is likely that most methods discussed here would have to be
modified to properly analyze the higher esters. For example,
when conducting gas chromatographic analyses, changes in
the temperature programs or other parameters may be
Figure 1. The transesterification reaction of triacylglycerols with
methanol, yielding methyl esters and glycerol. The catalyst is necessary. The original work (Freedman et al., 1986) on GC
usually a base, such as sodium or potassium hydroxide. analysis reported the investigation of methyl and butyl esters
of soybean oil. Apparently, not all individual components
Generally, the major categories of analytical procedures could be separated there in the analysis of butyl soyate, but
for biodiesel comprise chromatographic and spectroscopic classes of compounds could be analyzed. HPLC analysis was
methods, although papers dealing with other methods, applied to some ethyl, iso鈥損ropyl, 2鈥揵utyl, and iso鈥揵utyl
including physical property鈥揵ased ones, have also appeared. esters of soybean oil and tallow (Foglia and Jones, 1997). If
The different categories and procedures will be evaluated an analytical method has been applied to esters higher than
here. As a rule, primarily research with direct reference to methyl, it is noted here accordingly.
biodiesel analysis will be considered. Additional literature is The first report on chromatographic analysis of the
available through the papers cited in the References. transesterification used thin layer chromatography with
flame ionization detection (TLC / FID; Iatroscan instrument)
(Freedman et al., 1984). In another report (Cvengros and
Cvengrosov谩, 1994), TLC / FID was used to correlate bound
GENERAL ASPECTS
glycerol content to acyl conversion determined by GC. It was
The ideal analytical method for a product such as biodiesel
found in this work that if conversion to methyl esters is >
would be able to reliably and inexpensively quantify all
96%, then the amount of bound glycerol is < 0.25 wt.鈥?%.
contaminants even at trace levels with experimental ease in
Although the TLC / FID method is easy to learn and use
a matter of, at most, seconds or even faster for on鈥搇ine
(Freedman et., 1984), it has been largely abandoned because
reaction monitoring. No current analytical method meets
of lower accuracy and material inconsistencies, as well as
these extreme demands. Therefore, compromises are
sensitivity to humidity (Freedman et al., 1984) and the
necessary when selecting methods for analyzing biodiesel or
relatively high cost of the instrument (Cvengros and
monitoring the transesterification reaction.
Cvengrosov谩, 1994).
The categories mentioned above often overlap in organic
analytical chemistry due to the advent of hyphenated
GAS CHROMATOGRAPHY
techniques such as gas chromatography 鈥? mass spectrometry
Gas chromatography has to date been the most widely
(GC鈥揗S), gas chromatography 鈥? infrared spectrometry
used method for the analysis of biodiesel due to its generally
(GC鈥揑R), and liquid chromatography 鈥? mass spectrometry
higher accuracy in quantifying minor components. Note,
(LC鈥揗S). Few reports exist in the literature on the use of
however, that accuracy of GC analyses can be influenced by
hyphenated techniques in biodiesel analysis. The main
factors such as baseline drift, overlapping signals, etc. It is
reasons are likely the higher equipment costs and the higher
not always clear that such factors are compensated for in such
investment in technical skills of personnel needed to interpret
reports on biodiesel analysis. The first report on the use of
the data. This is the case despite the fact that hyphenated
capillary gas chromatography discussed the quantitation of
techniques could aid in resolving ambiguities remaining after
esters as well as mono鈥?, di鈥?, and triacylglycerols (Freedman
analysis by stand鈥揳lone chromatographic methods.
et al., 1986). The samples were treated with N,O鈥揵is(trime-
It is important to note that, in order to satisfy the
thylsilyl)trifluoracetamide (BSTFA) to give the correspond-
requirements of biodiesel standards, the quantitation of
ing trimethylsilyl (TMS) derivatives of the hydroxy groups.
individual compounds in biodiesel is not necessary but the
This kind of derivatization has been carried out in subsequent
quantitation of classes of compounds is. For example, for the
research on GC quantitation of biodiesel. Derivatization to
determination of total glycerol, it does not matter which
TMS derivatives is important because it improves the
monoacylglycerol (for example, monolein or monostearin)
chromatographic properties of the hydroxylated materials,
the glycerol stems from. The same observation, of course,
and in case of coupling to a mass spectrometer, facilitates
holds for di鈥? and triacylglycerols. It does not even matter for
interpretation of their mass spectra. While the original
the determination of total glycerol which type of acylglycerol
research (Freedman et al., 1986) used a short (1.8 m) fused
(mono鈥?, di鈥? or tri鈥?) the glycerol stems from. That
silica (100% dimethylpolysiloxane) capillary column, other
acylglycerols are quantifiable as classes of compounds by
research typically used fused鈥搒ilica capillary columns coated
GC is a result of the method.
with a 0.1鈥撀祄 film of (5%鈥損henyl)鈥搈ethylpolysiloxane of
Furthermore, virtually all methods used in the analysis of
10 to 15 m length. The analysis of rapeseed ethyl esters was
biodiesel are suitable (if necessary, with appropriate
also carried out on a GC instrument equipped an FID and with
modifications) for all biodiesel feedstocks, even if the
a 1.8 m 脳 4 mm i.d. packed column (Cvengrosov谩 et al.,
authors report their methods on one specific feedstock.
1997).
194 TRANSACTIONS OF THE ASAE
Most reports on the use of GC for biodiesel analysis were monitored, as were peaks at m/z 75 and 103 of the
employ flame鈥搃onization detectors (FID), although the use of additional (silylated) standard ethanol (trimethylethoxysi-
mass spectrometric detectors (MSD) would eliminate any lane). Mass spectrometry in SIM mode has the additional
ambiguities about the nature of the eluting materials since advantage that interfering signals can be avoided, and thus
mass spectra unique to individual compounds would be the use of shorter columns is possible (Mittelbach et al.,
obtained. Two papers exist in the literature in which the use 1996).
of mass spectrometric detection is described (Mittelbach, A further extension of the aforementioned research is the
1993; Mittelbach et al., 1996). It can be surmised that the simultaneous determination of glycerol as well as mono鈥?,
additional cost of MSDs (and mass spectral interpretation) di鈥?, and triacylglycerols simultaneously by GC (Plank and
plays a role in deterring the commercial adoption of this Lorbeer, 1995). Here and in previous work (Plank and
detection method, although the benefits of mass spectrome- Lorbeer, 1992; Plank and Lorbeer, 1995) 10 m (5%鈥損he-
try would likely more than compensate for the costs. nyl)鈥搈ethylpolysiloxane columns with 0.1鈥撀祄 film 鈥?
The majority of GC鈥搑elated papers discuss the 0.25 mm i.d. in Plank and Lorbeer (1992) and 0.32 mm i.d.
determination of a specific contaminant or class of in Plank and Lorbeer (1995) 鈥? were used. Major differences
contaminants in the methyl esters. The original report on were the lower starting temperature of the temperature
biodiesel GC analysis (Freedman et al., 1986) quantified program (Plank and Lorbeer, 1995) and the addition of a
mono鈥?, di鈥?, and triacylglycerols in methyl soyate on a short standard (1,2,4鈥揵utanetriol) for the glycerol analysis. In this
100% dimethylpolysiloxane column (1.8 m 脳 0.32 mm i.d.). work (Plank and Lorbeer, 1992; Plank and Lorbeer, 1995), a
Similar reports on the quantitation of acylglycerols exist cool on鈥揷olumn injector was used instead of the more
(Mariani et al., 1991; Plank and Lorbeer, 1992). The main common split/splitless injector.
differences are in the specifications of the columns 鈥? both Non鈥揼lyceridic materials that can be present in biodiesel
(5%鈥損henyl)鈥搈ethylpolysiloxane, with differences in pa- also have been analyzed by GC. Thus, the determination of
rameters such as column length 鈥? and the temperature sterols and sterol esters in biodiesel (Plank and Lorbeer,
programs, as well as standards. 1993) has been reported. The stated reason is that the
Other papers discuss the individual or combined influence of these compounds, which remain in vegetable
determination of other potential contaminants, such as free oils after processing (and thus in biodiesel after transesteri-
glycerol or methanol. A paper describing the use of a mass fication because they are soluble in methyl esters), on
selective detector deals with the determination of glycerol biodiesel fuel quality has not been determined (Plank and
(Mittelbach, 1993) and, in an extension thereof, a second Lorbeer, 1993). Detection was carried out with a
paper describes the simultaneous quantitation of glycerol and flame鈥搃onization detector, as in other GC鈥搑elated research,
methanol (Mittelbach et al., 1996). Other authors have also although in this case MS detection would appear especially
reported the determination of glycerol (Bondioli et al., desirable. The method for detection of sterols (Plank and
1992a) or methanol (Bondioli et al., 1992b). Using the same Lorbeer, 1993) is virtually identical to the other GC method
gas chromatographic equipment as in the previous reported by the same authors (Plank and Lorbeer, 1992). The
determination of glycerol (Bondioli et al., 1992a) with only only differences are the use of sterol standards and a slight
a modification of the oven temperature program, methanol modification of the GC temperature program to spread the
could be determined. Ethanol was used as a standard for sterol peaks under condensation or even overlapping of the
response factor determination. The flash point of biodiesel peaks of the other classes of compounds. Derivatization was
from palm oil and methanol content were correlated. carried out again with BSTFA (with 1% trimethylchlorosi-
Underivatized glycerol was detected with 1,4鈥揵utanediol as lane), and the column again was a fused鈥搒ilica capillary
a standard on a short 2 m glass column (4 mm i.d.) loaded column coated with a 0.1鈥撀祄 film of (5%鈥損henyl)鈥搈ethyl-
with Chromosorb 101 (Bondioli et al., 1992a), while the polysiloxane. The total concentration of sterols in rapeseed
other method used derivatization and a 60 m 脳 0.25 mm i.d. methyl ester was reported to be 0.339鈥?0.500%, and sterol
column with 0.25鈥撀祄 film of (5%鈥損henyl)鈥搈ethylpolysi- esters were 0.588鈥?0.722%. In another paper on analysis of
loxane and was reported to be more sensitive (Mittelbach, sterol content in rapeseed methyl ester (Plank and Lorbeer,
1993). The temperature program varied (starting lower when 1994a), the same authors reported a sterol content of
determining methanol) (Mittelbach, 1993; Mittelbach et al., 0.70鈥?0.81%. Other authors (Mariani et al., 1991) also pointed
1996), otherwise the column was the same. out the presence of sterols and sterol esters in biodiesel, but
Two papers (Mittelbach, 1993; Mittelbach et al., 1996) no analytical data were given.
discuss the use of mass spectrometry as a detection method
besides flame ionization. In the determination of free HIGH鈥揚ERFORMANCE LIQUID CHROMATOGRAPHY
glycerol in biodiesel by GC鈥揗S, selected ion monitoring A general advantage of HPLC compared to GC is that
(SIM) mode was used to track the ions m/z 116 and 117 of time鈥? and reagent鈥揷onsuming derivatizations are not
bis鈥揙鈥搕rimethylsilyl鈥?1,4鈥揵utanediol (from silylation of the necessary, which reduces analyses times. Nevertheless, there
1,4鈥揵utanediol standard) and m/z 147 and 205 of are fewer reports of HPLC applied to biodiesel than GC
tris鈥揙鈥搕rimethylsilyl鈥?1,2,3鈥損ropanetriol (from silylation of analysis. The first report on the use of HPLC (Trathnigg and
glycerol). The detection limit was also improved for rapeseed Mittelbach, 1990) described the use of an isocratic solvent
methyl ester (RME) when using MS in SIM mode (10鈥?5 %) system (chloroform with an ethanol content of 0.6%) on a
compared to the FID detector (10鈥?4 %) (Mittelbach, 1993). cyano鈥搈odified silica column coupled to two gel permeation
In straightforward extension of this work, the simultaneous chromatography (GPC) columns with density detection. This
detection of methanol and glycerol by MS in SIM mode was system allowed for the detection of mono鈥?, di鈥?, and
reported (Mittelbach et al., 1996). For detection of (silylated) triacylglycerols as well as methyl esters as classes of
methanol (trimethylmethoxysilane), peaks at m/z 59 and 89
Vol. 44(2): 193鈥?200 195
compounds. The system was useful for quantitating various the complexity of the gas chromatograms and to obtain more
degrees of conversion of the transesterification reaction. reliable peak assignments (Lechner et al., 1997).
HPLC with pulsed amperometric detection (the detection In a study on analytical methods for determining biodiesel
limit is usually 10 to 100 times lower than for amperometric in mixtures with conventional diesel fuel, silica cartridge
detection, and the detection limit is 1 碌g/g) was used to (Sep鈥揚ak) chromatography with hexane / diethyl ether as
determine the amount of free glycerol in vegetable oil esters solvents was employed to separate biodiesel from
(Lozano et al., 1996). The major advantage of this detection conventional diesel fuel (Bondioli et al., 1994).
method was its high sensitivity. The simultaneous detection A fully automated LC鈥揋C instrument was employed in
of residual alcohol is also possible with this technique. the determination of acylglycerols in vegetable oil methyl
Reaction mixtures obtained from lipase鈥揷atalyzed esters (Lechner et al., 1997). Hydroxy groups were
transesterification were analyzed by HPLC using an acetylated, and then the methyl esters (sterols and esterified
evaporative light scattering detector (ELSD) (Foglia and sterols elute with methyl esters) and acylglycerols were
Jones, 1997). This method is able to quantitate product esters, pre鈥搒eparated by LC (variable wavelength detector). The
free fatty acids, and the various forms of acylglycerols. A solvent system for LC was hexane / methylene chloride /
solvent system consisting of hexane and methyl tert.鈥揵utyl acetonitrile 79.97:20:0.05 and GC (flame鈥搃onization
ether (each with 0.4% acetic acid) with a gradient elution detector) was performed on a 10 m (5%鈥損henyl)鈥搈ethylpo-
profile was used. It can be applied to esters higher than lysiloxane column. One LC鈥揋C run required 52 minutes.
methyl, as discussed above. LC鈥揋C was also applied to the analysis of sterols in
In an extensive study (Holcapek et al., 1999), biodiesel derived from rapeseed oil (Plank and Lorbeer,
reversed鈥損hase HPLC was used with different detection 1994a; Plank and Lorbeer, 1994b). On鈥搇ine LC鈥揋C was
methods: UV detection at 205 nm, evaporative light applied to the analysis of sterols from five different types of
scattering detection, and atmospheric pressure chemical methyl esters (Plank and Lorbeer, 1994b). The vegetable oil
ionization mass spectrometry (APCI鈥揗S) in positive鈥搃on methyl esters were those of rapeseed oil, soybean oil,
mode. Two gradient solvent systems were used: one sunflower oil, high鈥搊leic sunflower oil, and used frying oil.
consisting of mixing methanol (A) with 5:4 2鈥損ropanol / The sterols were silylated prior to analysis with N鈥搈ethyl鈥?
hexane (B) from 100% A to 50:50 A:B 鈥? a non鈥揳queous N鈥搕rimethylsilyltrifluoracetamide (MSTFA). No saponifica-
reversed phase (NARP) solvent system 鈥? and the other tion and off鈥搇ine pre鈥搒eparation was required. The methyl
consisting of mixing water (A), acetonitrile (B), and 5:4 esters were separated from the sterols by LC with a hexane
2鈥損ropanol / hexane (C) in two linear gradient steps (30:70 / methylene chloride / acetonitrile 79.9:20:0.1 solvent
A:B at 0 min, 100% B in 10 min, 50:50 B:C in 20 min, and system. Gas chromatography was again carried out with a
last isocratic 50:50 B:C for 5 min). The first solvent system 12 m (5%鈥損henyl)鈥搈ethylpolysiloxane column and FID
was developed for rapid quantitation of the transesterifica- detection. Total concentrations of free sterols were 0.20鈥?0.35
tion of rapeseed oil with methanol by comparing the peak weight鈥?% for the five samples, while sterol esters displayed
areas of methyl esters and triglycerols. The contents of a range of 0.15鈥?0.73 weight鈥?%. Soybean oil methyl ester was
individual acids (using normalized peak areas) were subject at the lower end (0.20% and 0.15%, respectively), while
to error, and the results differed for the various detection rapeseed oil methyl ester was the higher end (0.33% and
methods. The sensitivity and linearity of each detection 0.73%, respectively). In a comparison of two methods,
method varied with the individual triacylglycerols. APCI鈥? saponification and isolation of the sterol fraction with
MS and ELSD had decreased sensitivity with increasing subsequent GC analysis and LC鈥揋C analysis of sterols in
number of double bonds in the fatty acid methyl esters, while rapeseed oil methyl ester (Plank and Lorbeer, 1994b), despite
UV will not quantify the saturates. APCI鈥揗S was stated to the sophisticated instrumentation required, LC鈥揋C was
be the most suitable detection method for the analysis of recommended because of additional information, short
rapeseed oil and biodiesel. analysis time, and reproducibility. The total sterol content in
rapeseed methyl ester was found to be 0.70鈥?0.81 weight鈥?%.
GEL PERMEATION CHROMATOGRAPHY
One report exists which describes the use of GPC (which
is very similar to HPLC in instrumentation except for the SPECTROSCOPIC METHODS
nature of the column and the underlying separation principle, Spectroscopic methods also have been reported for the
namely molecular weight of the analytes for GPC) for the analysis of biodiesel and/or monitoring of the transesterifica-
analysis of transesterification products (Darnoko et al., tion reaction. These methods are 1H nuclear as well as 13C
2000). Using a refractive index detector and tetrahydrofuran magnetic resonance (NMR) spectroscopy and near鈥搃nfrared
as mobile phase, mono鈥?, di鈥?, and triacylglycerols as well as (NIR) spectroscopy.
the methyl esters and glycerol could be analyzed. The
method was tailored for palm oil, and standards were selected
NUCLEAR MAGNETIC RESONANCE
accordingly. Reproducibility was good, with the standard
The first report on spectroscopic determination of the
deviation at different rates of conversion being 0.27鈥?3.87%.
yield of a transesterification reaction utilized 1H鈥揘MR
(Gelbard et al., 1995). Figure 2 depicts the 1H鈥揘MR
LIQUID CHROMATOGRAPHY WITH GAS CHROMATOGRAPHY
spectrum of a progressing transesterification reaction. These
The combination of liquid chromatography (LC) with gas authors used the protons of the methylene group adjacent to
chromatography (GC) has also been reported. The purpose of the ester moiety in triglycerols and the protons in the alcohol
the combination of the two separation methods is to reduce moiety of the product methyl esters to monitor the yield. A
196 TRANSACTIONS OF THE ASAE
Figure 2. 1H鈥揘MR spectrum of a progressing transesterification reaction. The signals at 4.1鈥?4.3 ppm are caused by the protons attached to the
glycerol moiety of mono鈥?, di鈥?, or triacylglycerols. The strong singlet at 3.6 ppm indicates methyl ester (鈥揅O2CH3) formation. The signals
at 2.3 ppm result from the protons on the CH2 groups adjacent to the methyl or glyceryl ester moieties
(鈥揅H2CO2CH3 for methyl esters). These signals can be used for quantitation.
simple equation is given by the authors (their terminology is allowed the determination of transesterification kinetics,
slightly modified here): which showed that the formation of partial acylglycerols
from the triglycerols is the slower, rate鈥揹etermining step.
铮? 2 AME 铮?
C = 100 脳 铮? 铮? (1)
铮? 3Aa鈭扖H 铮?
铮? 铮? NEAR鈥揑NFRARED SPECTROSCOPY
2
More recently, NIR spectroscopy has been used to monitor
where
the transesterification reaction (Knothe, 1999). The basis for
C = conversion of triacylglycerol feedstock (vegeta鈥?
quantitation of the turnover from triacylglycerol feedstock to
ble oil) to the corresponding methyl ester.
methyl ester product are differences in the NIR spectra of
AME = integration value of the protons of the methyl
these classes of compounds. At 6005 cm鈥?1 and at
esters (the strong singlet peak).
4425鈥?4430 cm鈥?1, the methyl esters display peaks, while
= integration value of the methylene protons.
Aa鈭扖H2 triacylglycerols only exhibit shoulders (see fig. 3). Ethyl
The factors 2 and 3 derive from the fact that the methylene esters could be distinguished in a similar fashion. (Knothe,
carbon possesses two protons and the alcohol (methanol鈥揹e- 1999). Using quantitation software, it is possible to develop
rived) carbon has three attached protons. a method (using partial least squares regression) for
Turnover and reaction kinetics of the transesterification of quantifying the turnover of triacylglycerols to methyl esters.
rapeseed oil with methanol were studied by 13C鈥揘MR The absorption at 6005 cm鈥?1 gave better results than the one
(Dimmig et al., 1999) with benzene鈥揹6 as solvent. The at 4425 cm鈥?1. Note that the mid鈥搑ange IR spectra of
signals at approximately 14.5 ppm of the terminal methyl triacylglycerols and methyl esters of fatty acids are almost
groups unaffected by the transesterification were used as identical and offer no possibility for distinguishing. It
internal quantitation standard. The methyl signal of the appears that ethyl esters, and perhaps even higher ester, may
product methyl esters registered at around 51 ppm, and the be distinguished similarly by NIR from triacylglycerols, but
glyceridic carbons of the mono鈥?, di鈥?, and triacylglycerols no results have been reported yet.
registered at 62鈥?71 ppm. Analysis of the latter peak range
Vol. 44(2): 193鈥?200 197
spectroscopic methods, which can be correlated by simple
equations, were in good agreement. Two NMR approaches
were used, one being the use of the methyl ester protons (peak
at 3.6 ppm in figure 2) and the protons on the carbons next to
the glyceryl moiety (a鈥揅H2; peaks at 2.3 ppm in figure 2)
(Knothe, 2000), for which the equation for determining
conversion (in %) is:
5 脳 I ME
CME = 100 脳 (2)
5脳 I ME + 9 脳 ITAG
where I refers to integration values, and the subscripts ME
and TAG refer to methyl esters and triacylglycerol. The
second approach was the use of the methyl ester protons and
the protons of the glyceryl moiety (peaks at 4.1鈥?4.3 ppm in
figure 2) in the triacylglycerols (Knothe, 2000).
In connection with the spectroscopic assessment of
biodiesel fuel quality and monitoring of the transesterifica-
tion reaction, a paper that discusses determining the amount
of biodiesel in lubricating oil (Sadeghi鈥揓orabchi et al., 1994)
is noteworthy. The investigated problem is significant
because biodiesel can cause dilution of the lubricant, which
can ultimately result in engine failure. The dilution was
attributed to the higher boiling range of biodiesel
Figure 3. NIR spectra of soybean oil (SBO), methyl soyate (SME), and
(Sadeghi鈥揓orabchi et al., 1994; Siekmann et al., 1982)
SME containing significant amounts of methanol. The inscribed wave
compared to conventional diesel fuel, whose more volatile
numbers highlight the possibilities for distinguishing the spectra
components have less chance to dilute the lubricant. These
and thus quantifying the components.
authors used mid鈥搑ange IR spectroscopy with a fiber鈥搊ptic
probe to determine the amount of biodiesel in lubricating oil.
NIR spectra were obtained with the aid of a fiber鈥搊ptic
The range used was 1820鈥?1680 cm鈥?1, which is typical for
probe coupled to the spectrometer, which renders their
carbonyl absorption and which is not observed in
acquisition particularly easy and time鈥揺fficient.
conventional diesel fuel nor in the lubricating oil (note that
Contaminants of biodiesel cannot be fully quantified by
this range is not suitable for distinguishing vegetable oils and
NIR at the low levels called for in biodiesel standards. The
their methyl esters because they have nearly identical
accuracy of the NIR method in distinguishing triacylglycer-
carbonyl absorptions in the mid鈥揑R range). Previous to this
ols and methyl esters is in the range of 1鈥?1.5%, although in
work, other authors had used IR spectroscopy (without the
most cases better results are achieved. To circumvent this
aid of a fiber鈥搊ptic probe) in the range 1850鈥?1700 cm鈥?1 to
difficulty, an inductive method can be applied. The inductive
analyze biodiesel in lubricating oil (Siekmann et al., 1982).
method consists of verifying 鈥? by, say, gas chromatography
The carbonyl absorption at 1750 cm鈥?1 was not disturbed by
鈥? that a biodiesel sample meets prescribed biodiesel
the absorption of oxidation products at 1710 cm鈥?1.
standards. The NIR spectrum of this sample would then be
recorded. The NIR spectrum of the feedstock would also be
recorded, as well as the spectra of intermediate samples at
OTHER METHODS
conversions of, say, 25%, 50%, and 75%. With these samples,
a quantitative NIR evaluation method could be established. VISCOMETRY
When another transesterification reaction is subsequently The viscosity difference between component triacylglyc-
conducted, the NIR spectrum would indicate that the reaction erols of vegetable oils and their corresponding methyl esters
(within the prescribed parameters such as time and resulting from transesterification is approximately one order
temperature) has attained conversion to a product that (within of magnitude (Knothe et al., 1997; Dunn et al., 1997). For
experimental error of NIR) conforms to standards. It can be example, the viscosity of soybean oil is 32.6 mm2/s (38掳C)
safely assumed that this result is correct, even if not all and that of methyl soyate is 4.41 mm2/s (40掳C) (Knothe et al.,
potential contaminants have been fully analyzed. Only if a 1997, and references therein). The high viscosity of the
significant deviation is indicated by NIR (beyond vegetable oils was the cause of severe operational problems,
experimental error) would a detailed investigation by a more such as engine deposits (Bruwer et al., 1980; Knothe et al.,
complex method such as GC be necessary. The NIR 1997; Dunn et al., 1997). This is a major reason why neat
procedure is considerably less labor鈥搃ntensive, faster, and vegetable oils largely have been abandoned as alternative
easier to perform than GC. diesel fuels in favor of mono鈥揳lkyl esters such as methyl
While the first NIR paper used a model system to describe esters.
monitoring of transesterification and for developing The viscosity difference forms the basis of an analytical
quantitation methods, a second paper applied the method to method, viscometry, applied to determining the conversion
a transesterification reaction in progress on a 6 L scale. Here, of vegetable oil to methyl ester (De Filippis et al., 1995).
spectroscopic results were obtained not only by NIR but also Viscosities determined at 20掳C and 37.8掳C were in good
by 1H鈥揘MR and NIR (Knothe, 2000). The results of both agreement with GC analyses conducted for verification
198 TRANSACTIONS OF THE ASAE
purposes. The viscometric method, especially results SUMMARY AND OUTLOOK
obtained at 20掳C, is reported to be suitable for Several analytical methods have been investigated for fuel
process鈥揷ontrol purposes due to its rapidity (De Filippis et quality assessment and production monitoring of biodiesel.
al., 1995). Similar results were obtained from density The most intensively studied method is GC, while HPLC,
measurements (De Filippis et al., 1995). NMR, and NIR have also been studied. GC is also the method
used for verification that biodiesel meets prescribed
TITRATION FOR DETERMINING FREE FATTY ACIDS standards due to its ability to detect low鈥搇evel contaminants,
Titration methods for determining the neutralization although improvements to this method are possible. Physical
number (NN) of biodiesel were described (Komers et al., properties鈥揵ased methods have been explored less, and it
1997). Two methods for determining strong acids and free appears that this may be an area for further study. However,
fatty acids in one measurement were developed. One method, no method can simultaneously satisfy all criteria of
of particular interest, used potentiometry, while the other simultaneously determining all trace contaminants with
used two acid鈥揵ase indicators (neutral red, phenolphthalein). minimal investment of time, cost, and labor. The following
The potentiometric method is more reliable, and even with approach therefore appears to be a reasonable compromise.
the use of two indicators, the NN values derived from the A fast and easy鈥搕o鈥搖se method that may be adaptable to
titration method are 10鈥?20 rel.鈥?% greater than the real acidity production monitoring, such as NIR (or viscometry), can be
of the sample. used for routine analyses. For example, if measurements by
NIR (or viscometry) at several turnover ratios indicate that
ENZYMATIC METHODS the transesterification reaction is progressing as desired and
An enzymatic method for analyzing glycerol in biodiesel that the NIR spectrum (viscosity) of the biodiesel product
was described to test for completeness of the transesterifica- agrees with that of one known to meet biodiesel standards,
tion reaction (Bailer and de Hueber, 1991). Solid鈥損hase then further, more complex, analyses would be unnecessary.
extraction of the reaction mixture with subsequent enzymatic Only if NIR (or viscometry) analyses indicate that there is a
analysis was applied. This method was originally intended as potential problem with the product would more complex and
a simple method for glycerol determination, but reproduc- time鈥揷onsuming analyses, for example by GC, be warranted
ibility and complexity concerns exist (Bondioli et al., 1992a; to determine the exact cause of the problem.
Lozano et al., 1996). Determining the blend levels of biodiesel in blends with
conventional diesel fuel is also an area that can be expanded.
BIODIESEL BLENDS
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200 TRANSACTIONS OF THE ASAE
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