knothe - analytical methods used in biodiesel

8/13/2019 Knothe - Analytical Methods Used in Biodiesel

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Transactions of the ASAE

2001 American Society of Agricultural Engineers 193Vol. 44(2): 193200

ANALYTICALMETHODSUSEDINTHEPRODUCTIONANDFUELQUALITYASSESSMENTOFBIODIESEL

G. Knothe

ABSTRACT.Biodiesel, an alternative diesel fuel derived from vegetable oil, animal fats, or waste vegetable oils, is obtainedby reacting the oil or fat with an alcohol (transesterification) in the presence of a basic catalyst to give the corresponding

monoalkyl esters. Two major categories of methods besides other miscellaneous ones have been reported in the literaturefor assessing biodiesel fuel quality and/or monitoring the transesterification reaction by which biodiesel is produced. Thetwo major categories comprise chromatographic and spectroscopic methods. This article considers the various methods ineach category, including advantages and drawbacks, and offers suggestions on selection of appropriate methods.

Keywords. Biodiesel, Chromatographic methods, Fiberoptic probe, Fuel quality, Gas chromatography, Gel permeationchromatography, Highperformance liquid chromatography, Mass spectrometry, Nearinfrared spectroscopy, Nuclearmagnetic resonance spectroscopy, Physical properties, Spectroscopic methods, Transesterification, Viscometry.

iodiesel has significant potential for use as analternative fuel in compressionignition (diesel)engines (Knothe et al., 1997; Dunn et al., 1997). Itis technically competitive with conventional,

petroleumderived diesel fuel and requires no changes in thefuel distribution infrastructure. While some technicalimprovements regarding coldflow properties, reduction ofNOx exhaust emissions, and oxidative stability remain, amajor hurdle towards widespread commercialization is thehigh price of biodiesel. For this reason, in the United Statesthe commercialization of biodiesel is targeted towardsregulated fleets and mining and marine markets. In thesemarkets, environmental and energy security concerns, whichare subject to legislation, can override economic aspects.

Vegetable oils, such as soybean oil, rapeseed oil (canolaoil), and in countries with more tropical climates, tropicaloils (palm oil and coconut oil) are the major sources ofbiodiesel. However, in recent years, animal fats and

especially recycled greases and used vegetable oils havefound increasing attention as sources of biodiesel, the latterprimarily as inexpensive feedstocks (Mittelbach andTritthart, 1988). Regardless of the feedstock, transesterifica-tion reactions are carried out to produce biodiesel. Early onduring the renewed interest in vegetable oils as alternativediesel fuels, it was observed that the resulting vegetable oil(or animal fat) esters did not exhibit the operationalproblems, such as engine deposits, coking of injector nozzles,etc., associated with neat oils (Bruwer et al., 1980).

The transesterification reaction of triacylglycerols invegetable oils, usually with a monohydric alcohol such as

Article was submitted for review in March 2000; approved forpublication by the Power & Machinery Division of ASAE in December 2000.Parts of this article were presented at the 1999 ASAE Annual Meeting asPaper No. 996111.

Product names are necessary to report factually on available data;however, the USDA neither guarantees nor warrants the standard of theproduct, and the use of the name by the USDA implies no approval of theproduct to the exclusion of others that may also be suitable.

The author is Gerhard Knothe, Research Chemist, USDAARS,National Center for Agricultural Utilization Research, 1815 N. University St.,Peoria, IL 61604; phone: 3096816112; fax: 3096816340; email:[email protected].

methanol, in the presence of a basic catalyst such as NaOHor KOH, yields the monoalkyl esters of the fatty acids,mainly comprising vegetable oils besides glycerol as sideproduct (see figure 1). Methanol, being the least expensive

alcohol, is the most commonly used alcohol fortransesterification and accordingly yields methyl esters suchas methyl soyate and rapeseed methyl ester. Recently,modifications of the transesterification reaction, such asthose based on enzymatic catalysis, have been explored(Nelson et al., 1996).

During the transesterification process, intermediateglycerols, mono and diacylglycerols, are formed, smallamounts of which can remain in the final biodiesel (methylester) product. Besides these partial glycerols, unreactedtriacylglycerols, unseparated glycerol, free fatty acids,residual alcohol, and catalyst can contaminate the finalproduct. The contaminants can lead to severe operationalproblems when using biodiesel, such as engine deposits, filter

clogging, or fuel deterioration. Therefore, in the UnitedStates an ASTM (American Society for Testing andMaterials) standard is under development (Howell, 1997). Insome European countries, such as Austria, the CzechRepublic, France, Germany, and Italy, standards have beendeveloped that limit the amount of contaminants in biodieselfuel. In these standards, restrictions are placed on theindividual contaminants by inclusion of items such as freeand total glycerol for limiting glycerol and acylglycerols,flash point for limiting residual alcohol, acid value forlimiting free fatty acids, and ash value for limiting residualcatalyst. A more detailed discussion of the rationale for eachquality parameter in biodiesel fuel standards is given in theliterature (Mittelbach, 1994; Mittelbach, 1996). Another

paper (Komers et al., 1998) briefly describes some methodsused in the analysis of biodiesel, which include proceduresfor determining contaminants such as water and phosphorusthat will not be dealt with here. The determination ofbiodiesel fuel quality is therefore an issue of great importanceto the successful commercialization of this fuel.Continuously high fuel quality with no operational problemsis a prerequisite for market acceptance of biodiesel.Accordingly, the analysis of biodiesel and the monitoring ofthe transesterification reaction have been the subject ofnumerous recent publications.

B


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194 TRANSACTIONSOFTHEASAE

Figure 1. The transesterification reaction of triacylglycerols withmethanol, yielding methyl esters and glycerol. The catalyst is

usually a base, such as sodium or potassium hydroxide.

Generally, the major categories of analytical proceduresfor biodiesel comprise chromatographic and spectroscopicmethods, although papers dealing with other methods,including physical propertybased ones, have also appeared.The different categories and procedures will be evaluatedhere. As a rule, primarily research with direct reference tobiodiesel analysis will be considered. Additional literature isavailable through the papers cited in the References.

GENERALASPECTSThe ideal analytical method for a product such as biodiesel

would be able to reliably and inexpensively quantify allcontaminants even at trace levels with experimental ease ina matter of, at most, seconds or even faster for onlinereaction monitoring. No current analytical method meetsthese extreme demands. Therefore, compromises arenecessary when selecting methods for analyzing biodiesel ormonitoring the transesterification reaction.

The categories mentioned above often overlap in organicanalytical chemistry due to the advent of hyphenatedtechniques such as gas chromatography mass spectrometry(GCMS), gas chromatography infrared spectrometry

(GCIR), and liquid chromatography mass spectrometry(LCMS). Few reports exist in the literature on the use ofhyphenated techniques in biodiesel analysis. The mainreasons are likely the higher equipment costs and the higherinvestment in technical skills of personnel needed to interpretthe data. This is the case despite the fact that hyphenatedtechniques could aid in resolving ambiguities remaining afteranalysis by standalone chromatographic methods.

It is important to note that, in order to satisfy therequirements of biodiesel standards, the quantitation ofindividual compounds in biodiesel is not necessary but thequantitation of classes of compoundsis. For example, for thedetermination of total glycerol, it does not matter whichmonoacylglycerol (for example, monolein or monostearin)

the glycerol stems from. The same observation, of course,holds for diand triacylglycerols. It does not even matter forthe determination of total glycerol which type of acylglycerol(mono, di or tri) the glycerol stems from. Thatacylglycerols are quantifiable as classes of compounds byGC is a result of the method.

Furthermore, virtually all methods used in the analysis ofbiodiesel are suitable (if necessary, with appropriatemodifications) for all biodiesel feedstocks, even if theauthors report their methods on one specific feedstock.

CHROMATOGRAPHICMETHODSBoth GC and HPLC analyses and combinations thereof

have been reported for biodiesel. Gel permeation chromatog-raphy (GPC) as an analytical tool for analysis oftransesterification products has also been reported. To date,most chromatographic analyses have been applied to methylesters and not higher esters such as ethyl, isopropyl, etc. Itis likely that most methods discussed here would have to bemodified to properly analyze the higher esters. For example,when conducting gas chromatographic analyses, changes in

the temperature programs or other parameters may benecessary. The original work (Freedman et al., 1986) on GCanalysis reported the investigation of methyl and butyl estersof soybean oil. Apparently, not all individual componentscould be separated there in the analysis of butyl soyate, butclasses of compounds could be analyzed. HPLC analysis wasapplied to some ethyl, isopropyl, 2butyl, and isobutylesters of soybean oil and tallow (Foglia and Jones, 1997). Ifan analytical method has been applied to esters higher thanmethyl, it is noted here accordingly.

The first report on chromatographic analysis of thetransesterification used thin layer chromatography withflame ionization detection (TLC / FID; Iatroscan instrument)(Freedman et al., 1984). In another report (Cvengros and

Cvengrosov, 1994), TLC / FID was used to correlate boundglycerol content to acyl conversion determined by GC. It wasfound in this work that if conversion to methyl esters is >96%, then the amount of bound glycerol is < 0.25 wt.%.Although the TLC / FID method is easy to learn and use(Freedman et., 1984), it has been largely abandoned becauseof lower accuracy and material inconsistencies, as well assensitivity to humidity (Freedman et al., 1984) and therelatively high cost of the instrument (Cvengros andCvengrosov, 1994).

GASCHROMATOGRAPHYGas chromatography has to date been the most widely

used method for the analysis of biodiesel due to its generallyhigher accuracy in quantifying minor components. Note,however, that accuracy of GC analyses can be influenced byfactors such as baseline drift, overlapping signals, etc. It isnot always clear that such factors are compensated for in suchreports on biodiesel analysis. The first report on the use ofcapillary gas chromatography discussed the quantitation ofesters as well as mono, di, and triacylglycerols (Freedmanet al., 1986). The samples were treated withN,Obis(trime-thylsilyl)trifluoracetamide (BSTFA) to give the correspond-ing trimethylsilyl (TMS) derivatives of the hydroxy groups.This kind of derivatization has been carried out in subsequentresearch on GC quantitation of biodiesel. Derivatization toTMS derivatives is important because it improves thechromatographic properties of the hydroxylated materials,and in case of coupling to a mass spectrometer, facilitatesinterpretation of their mass spectra. While the originalresearch (Freedman et al., 1986) used a short (1.8 m) fusedsilica (100% dimethylpolysiloxane) capillary column, otherresearch typically used fusedsilica capillary columns coatedwith a 0.1m film of (5%phenyl)methylpolysiloxane of10 to 15 m length. The analysis of rapeseed ethyl esters wasalso carried out on a GC instrument equipped an FID and witha 1.8 m 4 mm i.d. packed column (Cvengroov et al.,1997).


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Most reports on the use of GC for biodiesel analysisemploy flameionization detectors (FID), although the use ofmass spectrometric detectors (MSD) would eliminate anyambiguities about the nature of the eluting materials sincemass spectra unique to individual compounds would beobtained. Two papers exist in the literature in which the useof mass spectrometric detection is described (Mittelbach,1993; Mittelbach et al., 1996). It can be surmised that theadditional cost of MSDs (and mass spectral interpretation)plays a role in deterring the commercial adoption of this

detection method, although the benefits of mass spectrome-try would likely more than compensate for the costs.The majority of GCrelated papers discuss the

determination of a specific contaminant or class ofcontaminants in the methyl esters. The original report onbiodiesel GC analysis (Freedman et al., 1986) quantifiedmono, di, and triacylglycerols in methyl soyate on a short100% dimethylpolysiloxane column (1.8 m 0.32 mm i.d.).Similar reports on the quantitation of acylglycerols exist(Mariani et al., 1991; Plank and Lorbeer, 1992). The maindifferences are in the specifications of the columns both(5%phenyl)methylpolysiloxane, with differences in pa-rameters such as column length and the temperatureprograms, as well as standards.

Other papers discuss the individual or combineddetermination of other potential contaminants, such as freeglycerol or methanol. A paper describing the use of a massselective detector deals with the determination of glycerol(Mittelbach, 1993) and, in an extension thereof, a secondpaper describes the simultaneous quantitation of glycerol andmethanol (Mittelbach et al., 1996). Other authors have alsoreported the determination of glycerol (Bondioli et al.,1992a) or methanol (Bondioli et al., 1992b). Using the samegas chromatographic equipment as in the previousdetermination of glycerol (Bondioli et al., 1992a) with onlya modification of the oven temperature program, methanolcould be determined. Ethanol was used as a standard forresponse factor determination. The flash point of biodiesel

from palm oil and methanol content were correlated.Underivatized glycerol was detected with 1,4butanediol asa standard on a short 2 m glass column (4 mm i.d.) loadedwith Chromosorb 101 (Bondioli et al., 1992a), while theother method used derivatization and a 60 m 0.25 mm i.d.column with 0.25m film of (5%phenyl)methylpolysi-loxane and was reported to be more sensitive (Mittelbach,1993). The temperature program varied (starting lower whendetermining methanol) (Mittelbach, 1993; Mittelbach et al.,1996), otherwise the column was the same.

Two papers (Mittelbach, 1993; Mittelbach et al., 1996)discuss the use of mass spectrometry as a detection methodbesides flame ionization. In the determination of freeglycerol in biodiesel by GCMS, selected ion monitoring

(SIM) mode was used to track the ions m/z116 and 117 ofbisOtrimethylsilyl1,4butanediol (from silylation of the1,4butanediol standard) and m/z 147 and 205 oftrisOtrimethylsilyl1,2,3propanetriol (from silylation ofglycerol). The detection limit was also improved for rapeseedmethyl ester (RME) when using MS in SIM mode (105%)compared to the FID detector (104%) (Mittelbach, 1993).In straightforward extension of this work, the simultaneousdetection of methanol and glycerol by MS in SIM mode wasreported (Mittelbach et al., 1996). For detection of (silylated)methanol (trimethylmethoxysilane), peaks at m/z59 and 89

were monitored, as were peaks at m/z 75 and 103 of theadditional (silylated) standard ethanol (trimethylethoxysi-lane). Mass spectrometry in SIM mode has the additionaladvantage that interfering signals can be avoided, and thusthe use of shorter columns is possible (Mittelbach et al.,1996).

A further extension of the aforementioned research is thesimultaneous determination of glycerol as well as mono,di, and triacylglycerols simultaneously by GC (Plank andLorbeer, 1995). Here and in previous work (Plank and

Lorbeer, 1992; Plank and Lorbeer, 1995) 10 m (5%phe-nyl)methylpolysiloxane columns with 0.1m film 0.25 mm i.d. in Plank and Lorbeer (1992) and 0.32 mm i.d.in Plank and Lorbeer (1995) were used. Major differenceswere the lower starting temperature of the temperatureprogram (Plank and Lorbeer, 1995) and the addition of astandard (1,2,4butanetriol) for the glycerol analysis. In thiswork (Plank and Lorbeer, 1992; Plank and Lorbeer, 1995), acool oncolumn injector was used instead of the morecommon split/splitless injector.

Nonglyceridic materials that can be present in biodieselalso have been analyzed by GC. Thus, the determination ofsterols and sterol esters in biodiesel (Plank and Lorbeer,1993) has been reported. The stated reason is that the

influence of these compounds, which remain in vegetableoils after processing (and thus in biodiesel after transesteri-fication because they are soluble in methyl esters), onbiodiesel fuel quality has not been determined (Plank andLorbeer, 1993). Detection was carried out with aflameionization detector, as in other GCrelated research,although in this case MS detection would appear especiallydesirable. The method for detection of sterols (Plank andLorbeer, 1993) is virtually identical to the other GC methodreported by the same authors (Plank and Lorbeer, 1992). Theonly differences are the use of sterol standards and a slightmodification of the GC temperature program to spread thesterol peaks under condensation or even overlapping of thepeaks of the other classes of compounds. Derivatization was

carried out again with BSTFA (with 1% trimethylchlorosi-lane), and the column again was a fusedsilica capillarycolumn coated with a 0.1m film of (5%phenyl)methyl-polysiloxane. The total concentration of sterols in rapeseedmethyl ester was reported to be 0.3390.500%, and sterolesters were 0.5880.722%. In another paper on analysis ofsterol content in rapeseed methyl ester (Plank and Lorbeer,1994a), the same authors reported a sterol content of0.700.81%. Other authors (Mariani et al., 1991) also pointedout the presence of sterols and sterol esters in biodiesel, butno analytical data were given.

HIGHPERFORMANCELIQUIDCHROMATOGRAPHYA general advantage of HPLC compared to GC is that

time and reagentconsuming derivatizations are notnecessary, which reduces analyses times. Nevertheless, thereare fewer reports of HPLC applied to biodiesel than GCanalysis. The first report on the use of HPLC (Trathnigg andMittelbach, 1990) described the use of an isocratic solventsystem (chloroform with an ethanol content of 0.6%) on acyanomodified silica column coupled to two gel permeationchromatography (GPC) columns with density detection. Thissystem allowed for the detection of mono, di, andtriacylglycerols as well as methyl esters as classes of


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compounds. The system was useful for quantitating variousdegrees of conversion of the transesterification reaction.

HPLC with pulsed amperometric detection (the detectionlimit is usually 10 to 100 times lower than for amperometricdetection, and the detection limit is 1 g/g) was used todetermine the amount of free glycerol in vegetable oil esters(Lozano et al., 1996). The major advantage of this detectionmethod was its high sensitivity. The simultaneous detectionof residual alcohol is also possible with this technique.

Reaction mixtures obtained from lipasecatalyzed

transesterification were analyzed by HPLC using anevaporative light scattering detector (ELSD) (Foglia andJones, 1997). This method is able to quantitate product esters,free fatty acids, and the various forms of acylglycerols. Asolvent system consisting of hexane and methyl tert.butylether (each with 0.4% acetic acid) with a gradient elutionprofile was used. It can be applied to esters higher thanmethyl, as discussed above.

In an extensive study (Holcapek et al., 1999),reversedphase HPLC was used with different detectionmethods: UV detection at 205 nm, evaporative lightscattering detection, and atmospheric pressure chemicalionization mass spectrometry (APCIMS) in positiveionmode. Two gradient solvent systems were used: one

consisting of mixing methanol (A) with 5:4 2propanol /hexane (B) from 100% A to 50:50 A:B a nonaqueousreversed phase (NARP) solvent system and the otherconsisting of mixing water (A), acetonitrile (B), and 5:42propanol / hexane (C) in two linear gradient steps (30:70A:B at 0 min, 100% B in 10 min, 50:50 B:C in 20 min, andlast isocratic 50:50 B:C for 5 min). The first solvent systemwas developed for rapid quantitation of the transesterifica-tion of rapeseed oil with methanol by comparing the peakareas of methyl esters and triglycerols. The contents ofindividual acids (using normalized peak areas) were subjectto error, and the results differed for the various detectionmethods. The sensitivity and linearity of each detectionmethod varied with the individual triacylglycerols. APCI

MS and ELSD had decreased sensitivity with increasingnumber of double bonds in the fatty acid methyl esters, whileUV will not quantify the saturates. APCIMS was stated tobe the most suitable detection method for the analysis ofrapeseed oil and biodiesel.

GELPERMEATIONCHROMATOGRAPHYOne report exists which describes the use of GPC (which

is very similar to HPLC in instrumentation except for thenature of the column and the underlying separation principle,namely molecular weight of the analytes for GPC) for theanalysis of transesterification products (Darnoko et al.,2000). Using a refractive index detector and tetrahydrofuranas mobile phase, mono, di, and triacylglycerols as well as

the methyl esters and glycerol could be analyzed. Themethod was tailored for palm oil, and standards were selectedaccordingly. Reproducibility was good, with the standarddeviation at different rates of conversion being 0.273.87%.

LIQUIDCHROMATOGRAPHYWITHGASCHROMATOGRAPHY

The combination of liquid chromatography (LC) with gaschromatography (GC) has also been reported. The purpose ofthe combination of the two separation methods is to reduce

the complexity of the gas chromatograms and to obtain morereliable peak assignments (Lechner et al., 1997).

In a study on analytical methods for determining biodieselin mixtures with conventional diesel fuel, silica cartridge(SepPak) chromatography with hexane / diethyl ether assolvents was employed to separate biodiesel fromconventional diesel fuel (Bondioli et al., 1994).

A fully automated LCGC instrument was employed inthe determination of acylglycerols in vegetable oil methylesters (Lechner et al., 1997). Hydroxy groups were

acetylated, and then the methyl esters (sterols and esterifiedsterols elute with methyl esters) and acylglycerols werepreseparated by LC (variable wavelength detector). Thesolvent system for LC was hexane / methylene chloride /acetonitrile 79.97:20:0.05 and GC (flameionizationdetector) was performed on a 10 m (5%phenyl)methylpo-lysiloxane column. One LCGC run required 52 minutes.

LCGC was also applied to the analysis of sterols inbiodiesel derived from rapeseed oil (Plank and Lorbeer,1994a; Plank and Lorbeer, 1994b). Online LCGC wasapplied to the analysis of sterols from five different types ofmethyl esters (Plank and Lorbeer, 1994b). The vegetable oilmethyl esters were those of rapeseed oil, soybean oil,sunflower oil, higholeic sunflower oil, and used frying oil.

The sterols were silylated prior to analysis with NmethylNtrimethylsilyltrifluoracetamide (MSTFA). No saponifica-tion and offline preseparation was required. The methylesters were separated from the sterols by LC with a hexane/ methylene chloride / acetonitrile 79.9:20:0.1 solventsystem. Gas chromatography was again carried out with a12 m (5%phenyl)methylpolysiloxane column and FIDdetection. Total concentrations of free sterols were 0.200.35weight% for the five samples, while sterol esters displayeda range of 0.150.73 weight%. Soybean oil methyl ester wasat the lower end (0.20% and 0.15%, respectively), whilerapeseed oil methyl ester was the higher end (0.33% and0.73%, respectively). In a comparison of two methods,saponification and isolation of the sterol fraction with

subsequent GC analysis and LCGC analysis of sterols inrapeseed oil methyl ester (Plank and Lorbeer, 1994b), despitethe sophisticated instrumentation required, LCGC wasrecommended because of additional information, shortanalysis time, and reproducibility. The total sterol content inrapeseed methyl ester was found to be 0.700.81 weight%.

SPECTROSCOPICMETHODSSpectroscopic methods also have been reported for the

analysis of biodiesel and/or monitoring of the transesterifica-tion reaction. These methods are 1H nuclear as well as 13Cmagnetic resonance (NMR) spectroscopy and nearinfrared(NIR) spectroscopy.

NUCLEARMAGNETICRESONANCEThe first report on spectroscopic determination of the

yield of a transesterification reaction utilized 1HNMR(Gelbard et al., 1995). Figure 2 depicts the 1HNMRspectrum of a progressing transesterification reaction. Theseauthors used the protons of the methylene group adjacent tothe ester moiety in triglycerols and the protons in the alcoholmoiety of the product methyl esters to monitor the yield. A


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Figure 2. 1HNMR spectrum of a progressing transesterification reaction. The signals at 4.14.3 ppm are caused by the protons attached to the

glycerol moiety of mono, di, or triacylglycerols. The strong singlet at 3.6 ppm indicates methyl ester (CO2CH3) formation. The signals

at 2.3 ppm result from the protons on the CH2groups adjacent to the methyl or glyceryl ester moieties

(CH2CO2CH3for methyl esters). These signals can be used for quantitation.

simple equation is given by the authors (their terminology isslightly modified here):

=

23

2100

CH

ME

A

AC

(1)

whereC = conversion of triacylglycerol feedstock (vegeta

ble oil) to the corresponding methyl ester.AME = integration value of the protons of the methyl

esters (the strong singlet peak).

2CHA = integration value of the methylene protons.

The factors 2 and 3 derive from the fact that the methylene

carbon possesses two protons and the alcohol (methanolde-rived) carbon has three attached protons.

Turnover and reaction kinetics of the transesterification ofrapeseed oil with methanol were studied by 13CNMR(Dimmig et al., 1999) with benzened6 as solvent. Thesignals at approximately 14.5 ppm of the terminal methylgroups unaffected by the transesterification were used asinternal quantitation standard. The methyl signal of theproduct methyl esters registered at around 51 ppm, and theglyceridic carbons of the mono, di, and triacylglycerolsregistered at 6271 ppm. Analysis of the latter peak range

allowed the determination of transesterification kinetics,which showed that the formation of partial acylglycerolsfrom the triglycerols is the slower, ratedetermining step.

NEARINFRAREDSPECTROSCOPY

More recently, NIR spectroscopy has been used to monitorthe transesterification reaction (Knothe, 1999). The basis forquantitation of the turnover from triacylglycerol feedstock tomethyl ester product are differences in the NIR spectra ofthese classes of compounds. At 6005 cm1 and at44254430 cm1, the methyl esters display peaks, whiletriacylglycerols only exhibit shoulders (see fig. 3). Ethylesters could be distinguished in a similar fashion. (Knothe,

1999). Using quantitation software, it is possible to developa method (using partial least squares regression) forquantifying the turnover of triacylglycerols to methyl esters.The absorption at 6005 cm1gave better results than the oneat 4425 cm1. Note that the midrange IR spectra oftriacylglycerols and methyl esters of fatty acids are almostidentical and offer no possibility for distinguishing. Itappears that ethyl esters, and perhaps even higher ester, maybe distinguished similarly by NIR from triacylglycerols, butno results have been reported yet.


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Figure 3. NIR spectra of soybean oil (SBO), methyl soyate (SME), and

SME containing significant amounts of methanol. The inscribed wavenumbers highlight the possibilities for distinguishing the spectra

and thus quantifying the components.

NIR spectra were obtained with the aid of a fiberopticprobe coupled to the spectrometer, which renders theiracquisition particularly easy and timeefficient.

Contaminants of biodiesel cannot be fully quantified byNIR at the low levels called for in biodiesel standards. Theaccuracy of the NIR method in distinguishing triacylglycer-ols and methyl esters is in the range of 11.5%, although inmost cases better results are achieved. To circumvent thisdifficulty, an inductive method can be applied. The inductivemethod consists of verifying by, say, gas chromatography

that a biodiesel sample meets prescribed biodieselstandards. The NIR spectrum of this sample would then berecorded. The NIR spectrum of the feedstock would also berecorded, as well as the spectra of intermediate samples atconversions of, say, 25%, 50%, and 75%. With these samples,a quantitative NIR evaluation method could be established.When another transesterification reaction is subsequentlyconducted, the NIR spectrum would indicate that the reaction(within the prescribed parameters such as time andtemperature) has attained conversion to a product that (withinexperimental error of NIR) conforms to standards. It can besafely assumed that this result is correct, even if not allpotential contaminants have been fully analyzed. Only if asignificant deviation is indicated by NIR (beyond

experimental error) would a detailed investigation by a morecomplex method such as GC be necessary. The NIRprocedure is considerably less laborintensive, faster, andeasier to perform than GC.

While the first NIR paper used a model system to describemonitoring of transesterification and for developingquantitation methods, a second paper applied the method toa transesterification reaction in progress on a 6 L scale. Here,spectroscopic results were obtained not only by NIR but alsoby 1HNMR and NIR (Knothe, 2000). The results of both

spectroscopic methods, which can be correlated by simpleequations, were in good agreement. Two NMR approacheswere used, one being the use of the methyl ester protons (peakat 3.6 ppm in figure 2) and the protons on the carbons next tothe glyceryl moiety (CH2; peaks at 2.3 ppm in figure 2)(Knothe, 2000), for which the equation for determiningconversion (in %) is:

TAGME

MEME II

IC

+

=95

5100 (2)

where Irefers to integration values, and the subscripts MEand TAG refer to methyl esters and triacylglycerol. Thesecond approach was the use of the methyl ester protons andthe protons of the glyceryl moiety (peaks at 4.14.3 ppm infigure 2) in the triacylglycerols (Knothe, 2000).

In connection with the spectroscopic assessment ofbiodiesel fuel quality and monitoring of the transesterifica-tion reaction, a paper that discusses determining the amountof biodiesel in lubricating oil (SadeghiJorabchi et al., 1994)is noteworthy. The investigated problem is significantbecause biodiesel can cause dilution of the lubricant, whichcan ultimately result in engine failure. The dilution wasattributed to the higher boiling range of biodiesel

(SadeghiJorabchi et al., 1994; Siekmann et al., 1982)compared to conventional diesel fuel, whose more volatilecomponents have less chance to dilute the lubricant. Theseauthors used midrange IR spectroscopy with a fiberopticprobe to determine the amount of biodiesel in lubricating oil.The range used was 18201680 cm1, which is typical forcarbonyl absorption and which is not observed inconventional diesel fuel nor in the lubricating oil (note thatthis range is not suitable for distinguishing vegetable oils andtheir methyl esters because they have nearly identicalcarbonyl absorptions in the midIR range). Previous to thiswork, other authors had used IR spectroscopy (without theaid of a fiberoptic probe) in the range 18501700 cm1toanalyze biodiesel in lubricating oil (Siekmann et al., 1982).

The carbonyl absorption at 1750 cm1

was not disturbed bythe absorption of oxidation products at 1710 cm1.

OTHERMETHODSVISCOMETRY

The viscosity difference between component triacylglyc-erols of vegetable oils and their corresponding methyl estersresulting from transesterification is approximately one orderof magnitude (Knothe et al., 1997; Dunn et al., 1997). Forexample, the viscosity of soybean oil is 32.6 mm2/s (38C)and that of methyl soyate is 4.41 mm2/s (40C) (Knothe et al.,1997, and references therein). The high viscosity of thevegetable oils was the cause of severe operational problems,

such as engine deposits (Bruwer et al., 1980; Knothe et al.,1997; Dunn et al., 1997). This is a major reason why neatvegetable oils largely have been abandoned as alternativediesel fuels in favor of monoalkyl esters such as methylesters.

The viscosity difference forms the basis of an analyticalmethod, viscometry, applied to determining the conversionof vegetable oil to methyl ester (De Filippis et al., 1995).Viscosities determined at 20C and 37.8C were in goodagreement with GC analyses conducted for verification


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purposes. The viscometric method, especially resultsobtained at 20C, is reported to be suitable forprocesscontrol purposes due to its rapidity (De Filippis etal., 1995). Similar results were obtained from densitymeasurements (De Filippis et al., 1995).

TITRATIONFORDETERMININGFREEFATTYACIDSTitration methods for determining the neutralization

number (NN) of biodiesel were described (Komers et al.,1997). Two methods for determining strong acids and free

fatty acids in one measurement were developed. One method,of particular interest, used potentiometry, while the otherused two acidbase indicators (neutral red, phenolphthalein).The potentiometric method is more reliable, and even withthe use of two indicators, the NN values derived from thetitration method are 1020 rel.% greater than the real acidityof the sample.

ENZYMATICMETHODS

An enzymatic method for analyzing glycerol in biodieselwas described to test for completeness of the transesterifica-tion reaction (Bailer and de Hueber, 1991). Solidphaseextraction of the reaction mixture with subsequent enzymaticanalysis was applied. This method was originally intended as

a simple method for glycerol determination, but reproduc-ibility and complexity concerns exist (Bondioli et al., 1992a;Lozano et al., 1996).

BIODIESELBLENDSBlends of biodiesel with conventional diesel fuel

represent a common utilization of biodiesel. In the UnitedStates, B20 (a blend of 20% biodiesel with 80%conventional diesel fuel) is recognized as an alternativediesel fuel under criteria of the Energy Policy Act (EPACT).In France, biodiesel is utilized as a lowerlevel blend withconventional diesel fuel. In this connection, a problem that

has found little attention to date in the literature isdetermining or verifying the blend level of biodiesel inconventional diesel fuel in such blends. NIR spectroscopyusing the same peaks for quantitation as those for monitoringtransesterification and fuel quality appears to be suitable forsuch purposes (Knothe, manuscript submitted for publica-tion). MidIR spectroscopy as used for the determination ofbiodiesel in lubricating oil (SadeghiJorabchi et al., 1994)should also be suitable. The use of the NIR range, however,would permit using a spectrometer without any changes ininstrument settings for reaction and fuel quality monitoringas well as for determining blend levels. Generally, it appearsthat spectroscopic methods may be more suitable to addressthe problem of determining / verifying blend levels.

Especially GC would appear to yield very complexchromatograms due to the numerous components ofconventional diesel fuel. In HPLC, as done previously, theclasses of compounds may elute and be analyzable, thusreducing complexity. Methods based on physical propertiesmay also be suitable for determining biodiesel blend levels.

SUMMARYANDOUTLOOKSeveral analytical methods have been investigated for fuel

quality assessment and production monitoring of biodiesel.The most intensively studied method is GC, while HPLC,NMR, and NIR have also been studied. GC is also the methodused for verification that biodiesel meets prescribedstandards due to its ability to detect lowlevel contaminants,although improvements to this method are possible. Physicalpropertiesbased methods have been explored less, and itappears that this may be an area for further study. However,

no method can simultaneously satisfy all criteria ofsimultaneously determining all trace contaminants withminimal investment of time, cost, and labor. The followingapproach therefore appears to be a reasonable compromise.A fast and easytouse method that may be adaptable toproduction monitoring, such as NIR (or viscometry), can beused for routine analyses. For example, if measurements byNIR (or viscometry) at several turnover ratios indicate thatthe transesterification reaction is progressing as desired andthat the NIR spectrum (viscosity) of the biodiesel productagrees with that of one known to meet biodiesel standards,then further, more complex, analyses would be unnecessary.Only if NIR (or viscometry) analyses indicate that there is apotential problem with the product would more complex and

timeconsuming analyses, for example by GC, be warrantedto determine the exact cause of the problem.

Determining the blend levels of biodiesel in blends withconventional diesel fuel is also an area that can be expanded.

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knothe - analytical methods used in biodiesel

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