'"

FTIR spectroscopy-The next generation

of oil analysismethodologies?

This article is by F.R. /Jailde Voorl. aprofessor of food science at McGill

Uniuersity and co-director of theMcGill / R Group, Montreal. Canada,

aud ], Sedman, a doctoral candidate atMcGill University who has been a

major contributor to the developmentof ,.71 R edible oil O1w/ysis methodolc-

gy. She was a recipient of an ADesHonored Student Award during the

AOes Allllllal Meeti11g & Expo earlierthis year in Sa" Diego, California.

Volume II • June 2000 • Inform

Instrumentation

What an IR spectrum tells usInfrared (IR) spectroscopy has had along association with edible oils andtheir analysis, its main application beingthe provision of qualitative structuraland functional group information aboutlipids. Historically, LRspectroscopy hasrarely been used for quantitative analy-sis of fats and oils with the exception ofthe official AOes method for the deter-mination of isolated trans isomers,developed in the 1940s. The amount ofinformation potentially available froman IR spectrum of an oil (Figure I) issubstantial, as every peak and shoulderrepresents, in some way or other, struc-rural or functional group information,either about [he lipid or about impuri-ties associated with it.

At the higher frequency end of thespectrum (3700-3400 cm-l) is a regionwhere compounds containing hydroxylgroups absorb, including water (H-OH)and hydroperoxides (RO-H) and theirbreakdown products. When an oil isclean and dry and has not oxidizedappreciably, these constituents are notpresent, and only the weak first over-tone of the triglyceride ester linkage car-bonyl absorption is discernible in thisregion of the spectrum. At somewhatlower frequencies (3025-2850 em-I) isThe CH stretching region, where threebands are visible: a weak cis doublebond CH absorption (CH=CH) andstrong bands due to the CH2 groups ofthe aliphatic chains of the rriglyceridesas well as the terminal methyl groups.jusr beyond this region, secondary oxi-dation products such as aldehydes andketones absorb, albeit weakly.

Toward the center of the spectrum isa very strong band due to the C=Ostretching absorption of the ester link-age attaching the fatty acids to the glyc-erol backbone of the triglycerides. Nextto it, if lipolysis has occurred, is a band

In an earlier article (ITIR Spectroscopyin Edible Oil Analysis, Inform5:1038-1042, 1994), the potential util-ity of Fourier transform infrared (FTIR)spectroscopy as a routine analytical toolfor the fats and oils industry was out-lined. A reexamination of this potentialappears warranted in light of two recentdevelopments that could provide animpetus for the wider application ofrnR instrumentation in fats and oilsanalysis.

The first of these developments wasthe adoption in 1999 by the AOCS of anew FI1R method for the determinationof isolated trans isomers. By exploitingsome of the inherent advantages ofrnR spectroscopy, this new methodrepresents a substantial improvementover the existing AOCS IR trailsmethod (AOeS Method Cd14d-99).

The second development was theproposal ill late 1999 by the U.S. Foodand Drug Administration (FDA) toamend its nutrition labeling regula-tions to require the inclusion of transfatty acids in the saturated fat compo-nent, together with a foomote report-ing the trans fany acid content. Ifthese amendments are adopted andregulations promulgated, it may beanticipated that the new AOCS fo'TIRmethod will find extensive use, leadingto increased awareness within theindustry of the advantages of FTIRanalysis.

This article will summarize theinformation available from FTIRspectra of fats and oils, its relevanceto routine fats and oils analysis, thestate of development of FTIR analyti-cal methodology, and the issues thatremain to be addressed in order forFTIR spectroscopy to realize irspotential as a practical process andquality--control tool in the edible oilindustry.

0.30

0.25

0.20

(I)oc 0.15ro.0~0sn 0.10.0«

0.05

v(CH)methylene &methyl groups

I

Hydroxylregion

(H20, ROH,

R~OH)

Ester linkage\v(C=O)

Fingerprint region

I

/cisv(C-H)

cis bonds

)0.00 I-----~

v(C=O)alde~ydes

v(C=O),FFAs

Isolatedtrans bonds

C=C-H

Jend /CH2rocking

v(C-H)/ aldehvdes

[I VV J':'v\.!-.. transArans & cisltransconjugated bonds,

3500 3000 2500 2000 1500

Wavenumber (ern")

1000

Figure I. The mid-Infrared spectrum of an edible oil collected on an attenuated total n!!flecance (ATR) c~al. The \;abels indicateabsorption bands or rellont usoc.lllted with triglycerides or other constituents that may be present In an all. FFA, free ratty acid.

due to the carboxyl group (eaOH) offree fatty acids. In this same region, therewould also be carbonyl absorptionbands of aldehydes (R-CHO) andketones (R-CO-R) should the oil be oxi-dized, Continuing to lower frequencies isthe fingerprint region (1500-900 em-l),so called because the pattern of bands inthis region is very characteristic of mol-ecular composition and can be used toidentify substances. At the low-frequen-cy end of the fingerprint region, a banddue to the CH=CH bending absorptionof isolated trans double bonds would beobserved should trans-containinguiglycerides be present in the oil. Thecorresponding absorptions of conjugar-

ed dieues containing trolls/trails andcis/trails double bonds, such as are pre-sent in polyunsaturated oils that haveundergone oxidation, appear at slightlyhigher frequencies. Beyond the isolatedtrails band is another group of CHabsorptions, in this case bending vibra-tions, including a very Strong cisabsorption.

Relevance to the U1a1ysisof edible oilsClearly, mid-lR spectroscopy can pro-vide substantive qualitative informationabout fats and oils and, in principle,since IR band intensities are linearlyrelated to the concentration of the

absorbing species, a correspondingamount of quantitative information canalso be made available. However,extracting valid and unambiguousquantitative data is generally nor a sim-ple matter owing to band overlap, theeffects of intermolecular interactions,and crher complicating factors dis-cussed in Computerized QllontitativeInfrared Analysis, ASTM STP 934, edit-ed by C.L McClure, American Societyfor Testing and Materials, Philadelphia,1987. The advances in chemomerricsthat have paralleled the development offTlR spectroscopy have, to a largedegree, alleviated many of these prob-lems and thereby greatly enhanced the

Volume II • Jun.. 2000 • Inwrm

'"

".

In principle,the spectralinformation

provided by FTIRspectroscopy can be

related directly toimportant quality

parameters associatedwith fats and oils.

Volume I I • June 2000 • Inform

Instrumentation

magnetic resonance (AOeS MethodCd 16-81). The full spectrum couldpossibly also be used to evaluate therelative amounts of saturated,monounsaturated, and polyunsaturat-ed fatty acids in a far or oil, presentlydetermined by gas chromatography(e.g., AOeS Method Ce 1c-89).

On the basis of the outline above, itis apparent that quantitative IR spec-troscopy. beyond its established utilityfor [he determination of trans isomers,has the potential to provide an alterna-tive means of determining a range ofphysical and chemical propertiespresently determined by standardAOes methods. This potential couldnot be realized with traditional disper-sive IR spectrometers because of instru-mental, sample handling, and computa-tional limirarions. This situationchanged with the advent of l-TLR spec-trometers, which became a viable alter-native to dispersive instruments forquality control laboratories when theCOSt of FrIR instruments started todrop in the late 1980s. The very sub-stantial advantages of Ff1R instrurnen-ration (wavelength accuracy, scanspeed, acquisition of digitized spectra,and computational capabilities) relativeto conventional dispersive insrrumenra-rion provided an instrumental platformfrom which high-quality, reliable, andreproducible spectral data could beobtained and accurate quantitativeinformation extracted.

quantitative analysis capabilities of IRspectroscopy.

In principle, the spectral informa-tion provided by ITI R spectroscopycan be related directly to importantquality parameters associated withfats and oils. From the DH stretchingregion, one could determine the pres-ence of moisture or hydrcperoxides,the latter traditionally measured bythe chemical peroxide value (PV) test(ADCS Method Cd 8-53 or ca 8b-90).The intensities of the bands in the eHregion and of the ester linkage absorp-tion are related to the average molecu-lar weight of an oil, commonly evalu-ated by saponification number (SN)determinations (ADCS Method Cd 3-25). The CDDH absorption is indica-rive of lipolysis and related to the freefatty acid (FFA) content of an oil(ADeS Method Ca 5a-40). The pres-ence of spectral features related toaldehydes and ketones as well as con-jugated dienes is indicative of theaccumulation of secondary oxidationproducts. These are commonly mea-sured via the tbiobarbiruric acid(TBA) test (ADCS Method Cd 19-90)or anisid.ine value (AV) test (AOeSMethod Cd 18-90). The isolated trails

band provides a direct measure of iso-lated trans isomers (AOCS Method Cd14-95 or 14d-96I, while combinationsof cis and trans absorptions provide ameasure of rota! unsaturation oriodine value (IV), commonly deter-mined iodometrically (AOCS MethodCd 1-25 or Cd Ib-87) or by gas chro-matography (AOCS Method Cd lc-85). The complete spectrum of arefined, melted fat characterizes itsoverall triglyceride composition,which in turn can be related to itssolid fat index or conrenr (SFUSFC),usually determined by dilatomerry(ADeS Method Cd 10-57) or nuclear

Analytical methodology researchand developmentThe McGill JR Group was formed in1990 to advance the application offTlR spectroscopy to food and edibleoil analysis based on the potential utili-ty of this analytical technique to theindustry. Some of the advantages thatfTlR analysis has been shown to confer

in this regard include the following:• Time-consuming separation tech-

nologies [gas chromatography (GC),high-performance liquid chromatogra-phy (HPLc)1 and solvent-based chemi-cal analyses are avoided.

• A single instrument can be used tocarry out a wide range of analyses.

• A variety of sample types can beanalyzed owing to the substantial sam-ple-handling flexibility of FnR spec'rroscopy,

• Powerful chemomemc software isavailable for calibranon developmentand discriminate analysis.

10 Calibration transfer betweeninstruments is feasible owing to theexcellent wavelength accuracy of FTIRspectrometers.

Quantitative FrIR analysis hasevolved steadily and there is now ampleevidence that it is poised to become animportant Technique for the analysis offats and oils. Its most obvious rolewould be as a quality or process controltool for refining, deodorization, hydro-genation, and inreresrerificarionprocesses, where parameters such asmoisture, FFA, PV, IV, trans content,and SFI are of consequence.

The McGill IR Group has demon-strated [he basic concepts, principles,and lirnirarions of a variety of !-TIRanalytical methods, with most of thisresearch having been published in theJournal of the American Oil Chemists'Society. Most of the methods proposedby the McGill IR Group and othersinvolve concepts and complexities thatare nOT familiar to many analysts.Accordingly, although interest in thisanalytical technology has beenexpressed by various edible oil proces-sors and users, these methods are not assrraighrforward 10 implement as theAOCS isolated tram FrIR method. Toovercome this impediment, the optimal

approach is the development of analyti-cal packages or systems that allow pre-calibrated methods to be implementeddirectly.

Packaging analytical methodsAnalytical packages developed to facili-tate the application of !-TIR methodswithin the fats and oils industry mayconsist of some or all of the followingelements:

10 Built-in calibrationts] for a particu-lar analysis;

10 Operating software that converts ageneral-purpose FrIR spectrometer into

'17

an edible oil analyzer dedicated to 3

particular analysis;• An analytical protocol defining the

sample handling accessory, data acquisi-tion parameters, operating conditions(temperature, rime, pathlengtb, erc.);

• System check routines to ensureanalytical performance is not compro-mised; and

• Automatic data processing, Out-put/storage and data management capa-bilities.

Some instrument manufacturers arealready moving ahead, packaging andmarketing selected oil analysis methods.

ACO)11plele

:>olulio" for

Edible Oil AnalysisIt's everything youneed for AOCSapproved FTIRanalysis of edible0115,Includingrapid and accuratedetermination of

• Trans fatty acid content, and• Isolated trans geometric isomers.

The new MIDAC M2000 EOA (Edible OilAnalyzer) is a complete solution to your

needs, Including spectrometer. sampleInterface, custom calibrations and economical

price. Contact us today and be on your way tothe answers you need.

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Volume I I • June 2000 • Inform

6"

Even thoughsubstantial research

has gone intodeveloping FTIR fats

and oils analysismethodology,

its practicalimplementation

as a process controltool is minimal

at this point in time.

Volume I I • June 2000 • Inform

Instrumentation

(e.g., grating, filter, Fl'lg., mid- or near-IR) that meets the analytical specifica-tions of the official method in terms ofaccuracy and reproducibility in a col-laborarive srudy is sanctioned for use asa milk analyzer.

Although the AOes has not takenthis approach to date, it has in placethe basic elements of an excellent per-formance-based validation mechanismin the form of the AOeS LaboratoryProficiency (Smalley) Program.Although the focus of this program islaboratory certification, samples ana-lyzed within it are presently availableas secondary reference materials. Thisprogram could also readily serve a val-idation role, simply by producingmore samples and retaining a portionof them to serve as validation samples.The analysis of the Smalley samples innumerous laboratories yields excellentreference values together with stan-dard deviations which provide an indi-cation of the performance characteris-tics of the AOCS official methodswhich are the norm for some of thebest laboratories in the world. It thusprovides realistic analytical specifica-tions rhat can be used as criteria forapproval of new FfIR methods. Byusing this rype of validation approach,FTIR-based oil analysis packagescould all be compared on a uniformbasis. Thus, the approval of themethod would be based solely on irsperformance, rather than the analyti-cal approach, the insrrumenr rype, orthe amenabiliry to fit within the con-fines of a "prescriptive" method.

Bomem (St.-Jean Baptiste. Quebec) andPerkinElmer (Wellesley, Massachusetts)are marketing proprietary Ff-mid-IRand Ff-near-IR oil analysis systems,respectively, using software packagesthat address some of the elements listedabove. Thermal-Lube (Point-Claire,Quebec, Canada) has taken anotherapproach, developing a UniversalMethod Platform for InfraredEvaluation (UMPIRE) software pack-age that would allow a particular mRmethod to be run on a variety of instru-ments and produce standardized results.

Validation of new methodologiesNo matter what the approach, commer-cial mR oil analysis packages willneed to be validated to ensure that theyperform as advertised. This is a role thatwould be besr fulfilled by third-parryprofessional organizations such as theAOCS and AOAC International, whichhave a long history of analyticalmethodology development and evalua-rion.

Traditionally, the methods approvedby such organizations have been "pre-scriptive;" that is, the write-up of themethod describes the analytical con-cept, specifies the reagents, and containsa set of step-by-step instructions, there-by providing all the informationrequired to perform the analysis.However, as analytical methodologiesbecome more sophisticated and increas-ingly involve proprietary elements [e.g.,a patented reagent or concept, a propri-etary calibration, etc.), many new meth-ods are nor amenable to this prescrip-tive approach, making it necessary forthe methodology approval process tofocus on performance-based validation.This approach has been in place forsome rime at the AOAC Internationalfor instrumental methods, as exempli-fied by IR milk analysis. Any instrument

Technology push, a.na.lytical pullEven though substantial research hasgone inro developing mR fats and oilsanalysis methodology, its practicalimplementation as a process controlTool is minimal at this point in time. To

some degree, this state of affairs is relat-ed to inertia and reluctance to moveaway from the standard methods andthe unfamiliarity of oil chemists withITIR spectroscopy, as well as theunproven nature of the methodology,which is related to the validation issuesnoted above.

The ability of the FDA to proposetrans labeling legislation to a largedegree hinged on the availability of thenew FrIR trans method, in that it sim-plifies trans analysis. However, likeevery new technology, implementationof this method has a cost associatedwith it, and the required initial invest-ment can be recouped more rapidly bytaking advantage of the multifunctionalcapabilities of FfIR spectroscopy. Themultifunctional potential of this tech-nique was demonstrated early on withthe development of a cis!tra"s/JV!SNpackage, which allowed for the simulta-neous determination of these four para-meters from the FrIR analysis of a neatoil sample. More recently, the single-bounce horizontal attenuated totalreflectance (SB-HATR) trans analysishas been modified to yield IV data con-currently, and it is likely that the relativeamounts of sarura red, mono- andpolyunsaturated fatty acids making upan oil also can be determined simulta-neously.

Future outlookAlthough the routine use of quantitativeITIR spectroscopy for the analysis offats and oils is not yet a reality, there isnow a substantial body of literatureindicating that a range of commonAOCS methods can be implemented asITIR procedures. The FDA tram label-ing proposal, if adopted, will prod theindusrry to become more familiar withITIR spectroscopy, even if only toimplement the SB-HATR isolated trans

method. Although this relatively unso-phisticated method does not fully exem-plify the power of ITIR spectroscopy asa qualiry-ccnrrol tool, it does serve todemonstrate the benefits of a solvent-free analysis, the superior sample han-dling characteristics, and analytical timesavings that attend this new technology.Instrument manufacturers are begin-ning to market dedicated FfIR edibleoil analysis systems and, with proactiveparticipation by AOCS in validation ofthe methodology, ITLR spectroscopyshould evolve inro the platform for thenext generation of oil analysis method-ologies available to the edible oil indus-cry.

Bibliography

From the Journal of the AmericanOil Chemists' Socidy:

-Fatty acidsChe Man, Y.B., M.H. Moh, and F.R.

van de Voort, Determination of FreeFatty Acids in Crude Palm Oil andRefined-Bleached-Deodorized PalmOlein Using Fourier TransformInfrared Spectroscopy (76:485-490,1999).

Ismail, A.A., F.R. van de Voort, and J.Sedman, Rapid QuantitativeDetermination of Free Fatty Acidsin Fats and Oils by FnRSpectroscopy (70:335-341,1993).

-Iodine valueChe Man, Y.B., G. Senowary, and F.R.

van de Voort, Determination ofIodine Value of Palm Oil by FourierTransform Infrared Spectroscopy(76,693-699, 1999).

Li, H., F.R. van de Voon, A.A. Ismail,j. Sedman, R. Cox, C. Simard, and

."H. Buijs, Discrimination of EdibleOil Products and QuantitativeDetermination of Their Iodine Valueby Fourier Transform-Near InfraredSpectroscopy (77:29-36, 2000).

van de voort, F.R., j. Sedman, G. Emo,and A.A. Ismail, Rapid and DirectIodine Value and SaponificationNumber Determination of Fats andOils by Attenuated TotalReflectance/Fourier TransformInfrared Spectroscopy(69:1 I 18-1123, 1992).

-PeroxidationChe Man, Y.B., and G. Setiowary,

Determination of Anisidine Value inThermally Oxidized Palm Olein byFourier Transform InfraredSpectroscopy (76:243-247, 1999).

Dubois, j., F.R. van de vcorr, J.Sedman, A.A. Ismail, and H.Ramaswamy, Quantitative FfIRAnalysis of Anisidine Value andAldehydes in Thermally StressedOils (73:788-794, 1996).

Li, H., F.R. van de voore, A.A. Ismail,and R. Cox, Determination ofPeroxide Value by FourierTransform Near-Infrared (IT-NIR)Spectroscopy (77:137-142, 2000).

Ma, K., F.R. van de vcon, A.A. Ismail,and j. Sedman, QuantitativeDetermination of Hydroperoxidesby Fourier Transform InfraredSpectroscopy Using a DisposableInfrared Card (75:1095-1101,1998).

Ma, K., F.R. van de voort, J. Sedman,and A.A. Ismail, StoichiometricDetermination of Hydroperoxides inFats and Oils by FfIR Spectroscopy(74,897-906,1997).

Volume II • June2000 • Inform

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Moh, M.H, Y.B. Che Man, F.R. van deVoort, and W.j.W. Abdullah,Determination of Peroxide Value inThermally Oxidized Crude Palm Oilby Near Infrared Spectroscopy(76,19-23,19991.

van de vocrr, F.R., A.A. Ismail, J.Sedman, J. Dubois, and T.Nicodemo, The Determination ofPeroxide Value by FourierTransform Infrared (FfIR)Spectroscopy (71:921-926,19941.

van de Voort, ER., A.A Ismail, J.Sedman, and G. Erne, Monitoringthe Oxidation of Edible Oils byFTIR Spectroscopy (71:243-253,1994).

-Solid fatvan de Voort, F.R., P. Memon, J.

Sedman, and A.A. Ismail,Derermiunticn of Solid Fat Index byFTIR Spectroscopy (73:411-416,19961.

-ctrans Fatty acidsLi, H" E R. van de Voort, J. Sedman,

and A.A. Ismail, RapidDetermination of cis and transConrent. Iodine Value, andSaponification Number of EdibleOils by Fourier Transform Near-Infrared Spectroscopy (76:491-497,1999).

Ma, K., ER. van de voorr, J. Sed man,and A.A. Ismail, TransDetermination in Fees and Oils and

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Near Infrared Spectroscopy(76:249-254, 1999).

Margarine by FTIR SpectroscopyUsing a Disposable lR Card(76:1399-1404,1999).

Sedman, J., F.R. van de Voort, andA.A. Ismail, Upgrading the AOCSInfrared trans Method for theAnalysis of Neat Fats and Oils byFourier Transform InfraredSpectroscopy (74:907-913, 1997).

Sedman, J., F.R. van de Voorr, A.AIsmail, and P. Maes, IndustrialValidation of Fourier TransformInfrared trans and Iodine ValueAnalyses of Fats and Oils(75:33-39, 1998).

Sedman, J., F.R. van de voon, andA.A. Ismail, SimultaneousDetermination of Iodine Value andtrans Content of Fats and Oils byFourier Transform InfraredSpectroscopy Employing an Single-Bounce Horizontal Attenuated TotalReflectance Accessory (77:399-403,20001.

van de voorr, F.R., A.A. Ismail, and J.Sedman, A Rapid, AUTomated Methodfor the Determination of cis and tTallSContent of Fats and Oils by FTIRSpectroscopy (72:873-880, 1995).

OtherMoh, M.H., Y.B. Che Man, 8.5.

8adlishah, S. Jinap, M.S. Saad, andW.J.W. Abdullah, QuantitativeAnalysis of Palm Carotene UsingFourier Transform Infrared and

From other publications:Lei, Y.W., E.K. Kemsley, and R.H.

Wilson, Potential of FourierTransform Infrared Spectroscopy forthe Authentication of VegetableOils,). Agric. Food Chern.42:1154-1159 (1994).

Lai, Y.W., W. Lai, E.K. Kemsley, andR.H. Wilson, Quantitative Analysisof Potential Adulterants of ExtraVirgin Oil Using InfraredSpectroscopy, Food Chem.5H5-98 (19951.

Teo, W.B., and E.M. Goh,Determination of the TotalUnsaruraricn, Free Fatty AddContent, and Saponification Valuein Palm Oil by Fourier TransformInfrared Specrroscopy, MalaysianOil Sci. Techncl. 4(2): 178-181(19951.

van de VOOrt, F.R., J. Sedman, A.A.Ismail, and S. Dwight, MovingFTiR Spectroscopy into the QualityControl Laboratory. I. Principlesand Development, Lipid Technoi.8(4),117-119 (1996).

van de voon, F.R., J. Sedman, A.A.Ismail, and S. Dwight, MovingFTIR Spectroscopy into the QualityControl Laboratory. 2.Applications, Lipid Technel.8(5h89-91 (19961·0