Characterization of fatty acid methyl esters in biodiesel using high-performance liquid chromatography

Characterization

of

fatty

acid

methyl

esters

in

biodiesel

using

high-performance

liquid

chromatography

Neng-Chou

Shang

,

Rong-Zhen

Liu

,

Yi-Hung

Chen

*

,

Ching-Yuan

Chang

,

Rong-Hsien

Lin

a_{DharmaDrumCollegeofHumanitiesandSocialScience,Taipei100,Taiwan} b

DepartmentofChemicalEngineeringandBiotechnology,NationalTaipeiUniversityofTechnology,Taipei106,Taiwan

c

GraduateInstituteofEnvironmentalEngineering,NationalTaiwanUniversity,Taipei106,Taiwan

d

DepartmentofChemicalandMaterialEngineering,NationalKaohsiungUniversityofAppliedSciences,Kaohsiung807,Taiwan

1. Introduction

Biodiesel composedof fattyacidmethylesters (FAMEs)is a

productofthetransesterificationoftriglycerideswithmethanol.

Biodieselhas received a great deal of attention because of its

potential advantages in energy conservation and air pollution

reduction compared to fossil diesel fuel [1,2]. Recently, some

studiesattemptedtoinvestigatetheapplicationofheterogeneous

catalystsforthesynthesisofbiodiesels[3,4].Thepropertiesofa

biodieselarestronglydependentontheFAMEcompositionofthe

vegetable oils. The iodine value and oxidation stability of the

biodieselincludedinthespecificationsofbiodieselstandards,such

asASTMD6751andEN14214,arerelatedtothenumberofthe

unsaturatedcarbon–carbondoublebonds[5,6].Areliable

analyti-caltechniqueisessentialtoquantifytheindividualFAMEsofthe

biodieselproductfromthetransesterificationofvegetableoils.

Gaschromatography(GC)isthemostwidelyusedmethodfor

the quantification of FAMEs; however, gas chromatography is

oftenconsideredtobelaborintensivetoperform[7,8].

Compre-hensive two-dimensional gas chromatography (GCGC) has

recently been developed to efficiently quantify FAMEs [9–11].

Reversed-phasehigh-performanceliquidchromatography(HPLC)

equipped with various detectors has also been employed to

determinethecompoundsthataregeneratedduringthe

produc-tionofbiodiesel.OnegeneraladvantageofHPLCcomparedtoGCis

toreduceanalysistimebecausethetime-andreagent-consuming

derivatization is not necessary [12]. The detection techniques

associated with the HPLC system include refractive index (RI),

ultraviolet (UV), fluorescence, evaporative light scattering, and

massspectrometricdetection.TheapplicationofRIorUVdetection

is relatively common and lower in cost than other detector

systems.SomepreviousstudiesusingtheHPLCsystemwithRIor

UVdetentionforthequantificationofFAMEsaresummarizedin

Table1.

OnefrequentproblemthatoccursduringHPLCanalysisofthe

quantification of FAMEs is poor chromatographic separation

between methyl palmitate (MP) and methyl oleate (MO)

[8,14,16–18,21,23]. MPandMOchromatographic peaksoverlap

inHPLCanalysisandcausedifficultiesinquantification.According

to previous literatures as well as our experiments using the

gradient elution methodto separateMP and MO in HPLC, the

separation and quantification of MO and MP has been very

challenging. Palmitic and oleic acids are the main fatty acid

components of many oils and fats, including canola, corn,

cottonseed, rapeseed,safflower,soybean, sunflower,tallow oils,

andotheroils.Areliablemethodisstillrequiredfortheproper

JournaloftheTaiwanInstituteofChemicalEngineers43(2012)354–359

ARTICLE INFO

Articlehistory:

Received24August2011

Receivedinrevisedform10November2011 Accepted12November2011

Availableonline14December2011 Keywords:

Biodiesel Methylpalmitate Methyloleate

Refractiveindexdetector Ultravioletdetector

High-performanceliquidchromatography

ABSTRACT

Thisstudypresentsanewmethodforcharacterizingthecompositionofabiodieselthatiscomposedof fatty acid methyl esters (FAMEs) with high-performance liquid chromatography (HPLC). The chromatographicpeaksofmethylpalmitate(MP)andmethyloleate(MO),whicharethemainFAMEs, usually overlap chromatographically in the HPLC analysis. HPLC shows poorperformance for the separationofMPandMO,evenbyapplyingagradientelutionprogram,andisdifficulttouseforthe accuratequantificationofMPandMO.Amathematicalmethodhasbeendevelopedtoestimatethe individualmassesofMPandMOfromtheiroverlappingpeaksintheHPLCchromatogramwithrefractive index(RI)andultraviolet(UV)detectorsinseries.Asaresult,theindividualmassesofMPandMOinthe artificialmixturescanbequantifiedaccurately.Furthermore,FAMEcompositionandtheyieldofthe biodieselthatwasobtainedfromthetransesterificationofsoybeanoilwerequantifiedforverificationin thisstudy.Therefore,thepresentapproachcanprovideusefulinformationtopreciselyestimatethe FAMEcompositioninbiodieselswiththeemploymentofHPLC.

ß2011TaiwanInstituteofChemicalEngineers.PublishedbyElsevierB.V.Allrightsreserved.

*Correspondingauthor.Tel.:+886227712171x2539;fax:+886287724328. E-mailaddress:[email protected](Y.-H.Chen).

ContentslistsavailableatSciVerseScienceDirect

Journal

of

the

Taiwan

Institute

of

Chemical

Engineers

j o urn a lhom e pa g e :ww w . e l se v i e r. c om / l oca t e / j t i ce

1876-1070/$–seefrontmatterß2011TaiwanInstituteofChemicalEngineers.PublishedbyElsevierB.V.Allrightsreserved. doi:10.1016/j.jtice.2011.11.005

separationofMPandMOinthebiodieselsampleswhenHPLCis applied.

Themainobjectiveofthisstudyistoproposeamathematical

method for HPLC analysis to precisely quantify the individual

amounts of FAMEs, particularly MP and MO. The method was

developed based on the distinct characteristics of RI and UV

detection and the additivity of the integrated areas for the

overlappingpeaks.TheapplicabilityoftheUVwavelengthselected

at210nmisconfirmedbasedonthefeaturesoftheUVspectraand

thecalibrationcurvesoftheFAMEs.Asaresult,HPLCanalysisthat

adoptedRIdetectionincooperationwithUVdetectionissuggested

forobtainingaccurateinjectedmassesofbothMPandMO.The

FAMEcompositionandthetransesterificationyield(YFAME)during

themethanolysisofsoybeanoilinabatchreactoraredetermined

accordingtotheproposedmethod. Theaccuracyof thepresent

methodisfurther confirmedby comparisonwiththeanalytical

resultsthat wereobtainedfrom1_{Hnuclearmagneticresonance}

(NMR)spectroscopy.

2. Experimental

2.1. Materials

RefinedsoybeanoilwaspurchasedfromUni-President(Tainan,

Taiwan)withamolecularweightof875g/moland adensityof

0.917g/ml.ThemethanolobtainedfromMallinckrodtChemicals

(Phillipsburg,NJ,USA)wasACScertifiedwiththechemicalformula

of CH3OH, 32g/mol molecular weight, 0.79g/ml density, and

64.78Cboilingpoint.Potassiumhydroxide(KOH)withapurityof

85%waspurchasedfromRiedel-deHae¨n(St.Gallen,Switzerland).

Thestandardsof allFAMEs,includingMP, MO,methyl stearate

(MS),methyllinoleate(ML),andmethyllinolenate(MLn), were

purchased from Fluka (St. Gallen, Switzerland). These FAME

standards were prepared at the predetermined concentrations

(MP:2.60–30mg/ml,MS:5.06–6.06mg/ml,MO:2.58–30mg/ml,

ML: 60.7–61.3mg/ml, and MLn: 7.14–7.34mg/ml) by dilution

withacetonitrile(99.8%inHPLCgrade,MallinckrodtChemicals)for

HPLCanalysis.

2.2. Methanolysisofsoybeanoil

The methanolysis of the soybean oil was performed in an

airtightreactormadeof Pyrexglassandequippedwithawater

jackettomaintainaconstantsolutiontemperatureat608C.The

designofthereactorwasbasedonthecriteriafortheshapefactors

ofastandardsix-bladeturbine.Themolarratioofmethanoltooil

and the homogeneous catalyst (KOH) dosage based on the oil

weight were 6 and 1% (w/w), respectively, for conducting the

methanolysisreactionbasedon theoptimum transesterification

conditionsforvegetableoils,assuggestedbyFreedmanetal.[24].

Thestirringspeedwas800rpmtoensurethepropermixingoftwo

immisciblephases.Thesamplesweretakenfromthereactoratthe

desiredtimeintervalsduringthetransesterificationprocessofthe

oil,andthetotalsamplingvolumewaswithin5%ofthesolution

volume.Thesampleswereimmediatelyquenchedbyanicebathto

terminate thechemical reactions and then stored overnight to

producethephaseseparation[16,25].Thestabilizedvolumesof

theupperesterandlowerglycerolphaseswererecordedpriorto

the following analyses. The ester phase was washed with a

saturatedsaltsolutionatthreetimesitsvolumetoremovepossible

residualsofmethanol,KOH,andglycerol[26].

2.3. HPLCand1_HNMRanalyses

TheconcentrationsoftheFAMEswereanalyzedintheHPLC

systemmodelL-2000series(Hitachi,Tokyo,Japan)equippedwith

the silica-based column model STR ODS-II (5

m

250mm4.6mmI.D.)(ShinwaChemicalIndustries,Ltd.,Tokyo,

Japan). The HPLC system has a pump (model L-2130)with an

onlinevacuumdegasser,anautosampler(modelL-2200)witha

variableinjectioncapacityfrom0.5to500

m

(modelL-2490)inserieswithadiodearraydetector(UVdetector)

(modelL-2455).ThisUVdetectorcansupplythefullUVspectrum

foreverycomponentintheHPLCseparation.Theacquisitionand

processing of data were performed using the EZChrom Elite

softwareprovided withtheHPLCinstrument. Thecolumnoven

modelSuperco-150(Enshine,Taipei,Taiwan)wasusedtokeepthe

columntemperatureconstantat408C.Themobilephaseinthe

HPLCanalysiswaspureacetonitrileataflowrateof1.0ml/min.

TheinjectionvolumeofthesamplesintheHPLCanalyseswasset

at 1–100

m

sampleswereperformedinduplicatetoconfirmtheobservations.

TheNMRspectroscopyofthesoybeanbiodieselwasperformed

onaVarianUnityINOVA500NMRspectrometer(VarianInc.,Palo

Alto,CA,USA)operatingat500MHzusingCDCl3asthesolvent.The

YFAME value was determined from the signal area ratio of the

methoxyprotonsofthemethylestersat3.7ppmtothemethylene

protons adjacenttotheester groupat 2.3ppm in the1_HNMR

spectrum[16,27–29].

3. Resultsanddiscussion

3.1. HPLCpatternsofsoybeanoilbiodiesel

TheHPLCchromatogramsusingtheRIdetectorforthesoybean

oil biodiesel is illustrated in Fig. 1(a), and the chromatogram

Table1

PreviousliteraturesusingtheHPLCsystemwithUVorRIdetectiontoquantifyFAMEs.

Authors Detectora

Mobilephaseconditions Noureddinietal.[13] RI Acetone:acetonitrileat50:50 SakaandKusdiana[14] Methanol

Coluccietal.[15] Tetrahydrofuran

Meheretal.[16] Methanol

Holcˇapeketal.[17] UV205 Gradientelution:(1)2-propanolandhexane.(2)Acetonitrile,2-propanol,hexane,andwater

Komersetal.[18] Gradientelution:(1)acetonitrile:water(80:20).(2)Acetonitrileandhexane:2-propanol(40:50) Jeongetal.[19] Acetonitrile:acetone:water(48:48:4)

Shibasaki-Kitakawaetal.[20] Gradientelutionofacetonitrile,2-propanol,andultra-purewater Juane´daandSe´be´dio[21] UV210 Hexane:acetonitrile(100:2)

Talukderetal.[22] Gradientelutionofmethanol,isopropanol,andhexane Tu¨rkanandKalay[8] Gradientelutionofacetonitrileandacetone

Nightingaleetal.[23] UV220 Hexane:isopropanolof98:2 a

Thesubscriptsdenotethewavelength(nm)ofUVdetection.

fatty acid methyl esters (FAMEs); in contrast, UV detection is

remarkably sensitive to the FAMEs that contain unsaturated

carbon–carbon double bonds. The artificial mixtures with the

weightratioofMPtoMOfrom3:1to1:45havebeenquantified

successfullytodemonstratetheapplicabilityofthepresentmethod.

The FAME composition and transesterification yield during the

methanolysisofthesoybeanoilthatwereestimatedbythepresent

methodareconsistentwiththoseobtainedintheliteraturesandby

1_{HNMRanalysis,respectively.Thisstudypresentsareliableand}

practicalapproachforthequantificationoftheindividualmassesof

MPand MOfromthe overlappingpeaks intheHPLCanalysisof

biodiesels. Acknowledgement

ThisstudywasfinanciallysupportedbytheNationalScience

CouncilofTaiwan.

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