58 Analysis of Fats and Oils

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Analysis of fats 5md oils by SFE and SFC

Spercritical fluids (SCF) have

attracted much attention over thepast 20 years with regard to their

potential application in chemical engi-neering, industrial processing and envi-ronmental remediation. These fluids,patiicularly supercritical carbon diox-ide (SC-CO,), permit extraction andprocessing operations to be conductedat relatively low temperatures. usingnontoxic and inert gases. The resultantproducts (both extract and substrate)from these SCF-based processes aresolvent-free and minimally altered ordegraded during the extraction process.Such factors have served as the basisfor the special applications of these flu-ids in the food industry (1).

Since the early 198Os, there hasbeen a renewed interest in the analyti-cal applications of SCF (2). This haslargely been due to the developmentof suitable analytical equipment for

conducting supercritical fluid chro-matography (SFC) and supercriticalfluid extraction (SFE) on a routinebasis (3). Analytical SFE and SFCcontinue to be developed to meet the

widespread demands of many analystsin the food, environmental and ener-gy-related industries (4). These devel-opments are accelerated by new gov-ernment regulations regarding the

generation, use and disposal o f haz-ardous solvents in the laboratory envi-ronment (5).

Regardless of the scale of the SFEor SFC operation, certain fundamental

principles apply. An SCF can beviewed as a unique state of matter,intermediate between a liquid and agas, whose physical properties aredetermined by the external pressureand temperature that are applied to thefluid. If the fluid is held a t a tempera-ture and pressure above its criticalpoint (T, and PC, respectively), then itis said to be in the SCF state, and itsdensity under such conditions can bevaried substantially by increasing theapplied pressure on the system. Suf-fice it to say that at high densities such

fluids take on the solvent-like proper-ties of many organic solvents andhave the capability to dissolve a vari-ety of substances, just as normal liq-uids do.

Why then should SCFs be of par-ticular interest to the fats and oils ana-lyst? The answer lies partly in theextraordinarily high solubility exhibit-ed by lipid materials in SCF, particu-larly CO*, which readily solubilizesnonpolar solutes (6). Such a trend isillustrated by Figures I and 2, wherethe solubility of soybean oil triglyc-

erides as a function of CO2 pressure(7) is plotted. For the illustratedisotherms, it is possible to obtain lipidsolubilities ranging from a few weightpercent to over 25 wt%. depending onthe pressure and temperature cond-tions chosen. Such solubilities arcmore then sufficient for chromatogra-phy under SCF conditions. thecnh;luslivc dclipidalion of fat- and oil-cont;Iining samples hy SFE. and lhc

INFORM. Vol. 4. no 9 (September 1993)

2(X) 3(W) J(K) 5lXI h(K) 7(K) XW

I’rc.\\LIrc I;11111

Figure 1. Solubiliiy 01 soybean oil triglycerides in SC-CO, as a function of pressure



1090

INSTRUMENTATION

1 2 3 4 5 6 7 8 9 IO 11 12 I.;

Pressure x 100 (bar)

Figure 2. Solubility of soybean oil triglycerides in SC-CO2 at high pressure

I(iiiltlI’IIaS

et3tfI:tf‘

t1iIic!

1(I‘IIIII,

Other lipids. such as fatty acids,ocoplirrols. sterols. etc.. exhibit simi-,tr solubility trends as those depictedn Figures 1 and 2 (8.9). Unfortunatelyhe. high overall solubility of manyipid compounds in SCF compromiseshe molecular specificity of SFE in thebsence of an auxiliary techntque,uch as chromatography (IO). Howev-:r. the use of lower pressures and/oremperatures permits SCF to beapplied to many analytical upplica-ions that do not require such highinite lipid solubilitics, such as capil-at-y SFC. residue analysis and on-linejFE. We shall now examine some ofhese applications to illustrate the use-‘ulness of analytical SFE and SFC inapplied lipid analysis.

Xl’-line SFEtiechanistically, analytical SFE istpplied in either an off- or on-linenode. Off-line extraction usuallymplies that the sample of interest is:xtracted in a discrete operation in,vhich the extract is first isolated andhen independently analyzed by any)ne of a variety of techniques. Withinlimits. the extraction and temperatureand pressure can be varied to control:he composition of the lipid extract;nowever. it is common when extract-ing lipid matter from different samplematrices to do an exhaustive extrac-tion. However, even when perfotmingextractions at high pressures and tem-peratures (700 bar, 8O”C), an excellentseparation can be achieved betweenphospholipids and nonpolar lipids(I 1). The former can easily be solubi-lized in SCF by the additj.on of acosolvent, such as ethanol or m ethanolto the SC-CO2.

An example of a selective extrac-tion of interest to the lipid analyst isthe isolation of cholesterol from oil orfat matrix, such as cod liver oil matrix(12). Adjustment of the CO2 densityto 0.40 g/mL (120 atm, 60°C) allowsthe cholesterol to be isolated from theoil as shown in Figure 3. A higherCO2 density, 0.93 g/mL (350 atm,40°C). permits extrac tion of thetriglycerides, as indicated by the upper

INFORM, Vol. 4, no. 9 (September 1993)



1092

INSTRUMENTATION

IO

9

8

I

6 ! Triglycerides/fats

3

2 5

4

3

2

I

02 4 6 8

Time (min)

Figure 3. HPLC of SFE fractions of cod liver oil

(conrimrcdfrompage 1090)

ultraviolet detector trace (210 nm)from high-performance liquid chro-matographic analysis of the twoestracts (Fig. 3). This simple fraction-ation was accomplished on a Hewlett-Packard Model 7680A SCF extractor,using a CO2 flow rate of 4 mL/min for10 min. after an initial static hold of 1min. The cod liver oil sample (500FL) was initially mixed with adiatomaceous earth sorbent.

ticide-containing meat productscould be affected simultaneously insix samples by using the apparatusshown in Figure 4. Quantitative lipidand pesticide extractions wereobtained at 340-680 atm at 60°Cusing 5-10 L/min CO2 flow rates(ambient conditions). Today, com-mercial instrumentation exists thatwill permit the analysis of up to 8,24 or 44 samples, either simultane-ously or in a serial mode o f extrac-tion.

On-line SFEIn contrast to off-line SFE, on-lineSFE involves conducting an extrac-tion, followed by transfer of theextract to the analysis instiument, allin a sequential fashion. The analysisinstrument of choice is frequently agas or liquid chromatograph, followedby mass or infrared spectrometry foridentification of the separated com-pounds. The technique has certainadvantages and disadvantages, whichare worth enumerating.

Off-line SFE preparation and One particular application of off- On-line SFE frequently requires theanalysis of multiple samples is cur- line SFE deserves special mention: the use of switching and sampling valvesrently available, due in part to the determination of fats and oils levels in to transport the extract from theinitial pioneering efforts of raw materials and/or processed food extraction stage to the analysis step.researchers at NCAUR (13). Rapid products. This application is becom- The extract is deposited and concen-SFE (15 min) of lipid phases in pes- ing critically important as analysts trated during a defined period of

INFORM, Vol. 4, no. 9 (September 1993)

,cck :II~ alternative to the classic:“ancicnt”) and oltcn-varlcd Soshlct

zxtrsction technique. which utilizes

Drganic and sometimes flammable and:arcinogenic solvents. There remainslittle doubt that analytical SFE canyield equivalent results to extractions

nn the same sample using nonpolarorganic solvents. This recently hasbeen demonstrated by researchers atNCAUR for the quantitative extrac-tion of oil from three different oilseed1ypes ( 14).

Perhaps of more interest are therecent results for the extraction of fatfrom different food matrices by Hop-per (as cited in 15) as noted in TableI. In this case, the SFE results weredetermined by a simultaneous m ulti-sample SFE. using a instrumentdesigned for large samples that is a

prototype of an earlier unit developedby researchers at NCAUR (13). Notethat the SFE results from this off-linetechnique using high pressure CO? arecomparable to two methods using liq-uid solvents. Method 960.39 is aSoxhlet extraction using petroleumether as specified by the AOAC Inter-national (16), while the results in thecolumn labeled PAM 1 are a sequen-tial solvent extraction using ethylether, as designated in the PesticideAnalytical Manual of the FDA (17).Despite these encouraging results, col-

laborative studies need to be undenak-en to verify the reproductivity of theSFE method, particularly in lieu of itsimportance in nutrient analysis ( 18).



1093

extraction, either on some form of a

retention gap or at the head of thechromatographic column. This pre-vents contamination of and loss of theextract prior to the analysis step.

However, the analyst employingon-line SFE also loses the freedom IOchoose the analytical method once theextraction module ih fixed in the sys-tem. Considerable “replumbing” maybe necessary IO mate the SFE step withan alternative analytical technique.The sample sizes that can be analyzed

Figure 4.

multi-sari

extractor

AUR

SCF

by on-line SFE are often small (mg) in

the case of lipid-containing substrates,since the high solubility of lipid com-pounds in the SCF tends to lead to col-umn overloading in the case of micro-bore and capillary columns. The care-less handling of extraction cells canalso lead to analysis artifacts, such aslipids from fingerprints, which showup on the resultant chromatograms.

However. the ability of on-lineSPE to extract small samples for sub-sequent analysis is also an attractive

Table 1

Percent fat extracted (%RSD, n = 6)

Pork snusafe 30.s5 (4.7.5) 29.83 ( I .55) 29.83 (I .32)

Pc;inul hurter so.32 (0.39) 49.29 (0.60) 492 I (0.44)

Cheddsr cheese 33.8s (3.13) 33.94 t.: 16) 33.X11.14)

Corn chip il.32 (0.62) 3 I .x0 (0 76) 3 I.5 I (0.46)



1094

INSTRUMENTATION

I I 1 pressure I - ICC-MS 1

Figure 5. Schematic of SFElGClhlS system for volatile analysis

feature of the technique. King (19) hasshown that the lipid content of singleseeds and insects can be characterizedby coupling on-line SFE with SFC.Volatiles and semi-volatile compo-nents from the degradation of fats andoils also can be identified and quanti-

tated by cdupling on-line SFE withSFC or gas chromatography. Recentstudies by Snyder (20) have shown

that low temperature/pressure extrac-tion of volaliles/semi-volatiles from aslittle as IO J.ILof oxidized oil offers animproved method of characterizingthe oil decomposition products. Theexperimental apparatus is very simple.as depicted in Figure 5, consisting of a

high pressure syringe pump in-linewith a thermostatted macro-extractioncell connected to the injection part of

$*‘I\ ~lil-olll~iltrgra1,1 1. Gcnllc c\Ir.icIIon2.

at I02 ;itm ;III~I 50°C limit5 rhc cxlr;ic-110111‘high nioIccuI;ir wci$il compo-ncnts and minimizes the dccornposl-tion of hydraperoxides to lowermolecular weight artifacts. thcreblproviding ;I more accurate analysis of

volatile components in the osidizcdoil.

Recently on-line SFE has beencombined with :I precolumn chemicalreaction followed by gas and/or SFC

for the analysis of reaction products.Such ;I technique allows the study 01reactions in SCF media. bur pcrhapbmore importantly. the anslyssl~ot‘ ana-lytically-useful derivatives. Berg andco-workers (3 I) have synthesized bothmethyl and butyl esters from inter-esterification of triglycerides In cdiblcfats using an immobilized lipase in the

extraction cell at 1.50 atm and 50°C.Similar analytically-useful resultshave been obtained by King and co-

INFORM,Vol. 4. no. 9 (September 1993)



1095

workers (22), using an aluminumoxide sorbent in the extraction cell forthe production of methyl esters. Par-tial conversion to methyl esters wasobtained at 200 atm and 40°C on sin-gle plant seeds. thereby allowing seed

viability to be maintained after on-lineanalysis of the methyl esters by gaschromatography.

SFCSFC offers the lipid analyst some veryinteresting options that are not easilyachieved by using other types of chro-matography. Unfortunately SFC isoften perceived as a technique that isapplicable to only a few niche applica-tions that cannot be solved by gaschromatography (GC) or high-perfor-mance liquid chromatography

(HPLC). This is not true when oneexamines the versatility of SFC inapplied lipid analysis, as well asadvantages of the technique itself.

The uniqueness of SFC-based sep-arations derives in part from the abili-ty of the analyst to vary the mobilephase solvating power as a function ofpressure. Hence, many separations inSFC are affected in a similar mannerto gradient elution techniques inHPLC, where retention and separationare altered by varying the compositionof the mobile phase. SFC utilizes both

capillary and packed columns for lipidanalysis (23). the latter option beingcapable of producing very high col-umn efficiencies (24) by using ultra-small diameter columns. Flamc-ion-ization detection has been the mostsuccessful method to date in the anal-ysis of lipids by SFC; howcvcr. rcccntadvances in coupling the evaporativelight-scattering dctcctor with SFChave been successful (7-S). Both dctcc-tars also offer ;I universal mode ofdetection that is not readily availablewith HPLC.

Several advantages attend the useof SFC that arc missing in GC andHPLC. The use of mobile phase pres-sure/density-programming techniquescan eliminate the need for samplepreparation prior to analysis (26).since unwanted or interfering compo-nent> can be injected along with thet;lrfct analyses and simply eluted outof ~olunin by increasing the density 01II~C niohii~ phase. The rclarivcl,

benign conditions employed in SFCmake it a technique that is compatiblefor the chromatography of non-volatile, but thermally labile com-pounds, or moieties prone to oxida-tion. The ability of SFC to analyze

lipid-type compounds approaching1000 Daltons in molecular weight alsoeliminates the need for derivatization,as required in many GC-based meth-ods. SFC also eliminates or reducesthe use of solvents relative to HPLCmethods.

What are the unique applications ofSFC that are of interest to the appliedlipid analyst? As noted above, SFCoften can be directly applied to ananalysis situation without resorting tosample preparation or derivatization.-Generic applications include the direct

characterization of raw materials orreaction mixtures, the deformulationof commercial products containing awide range of lipid types and thedirect detection of product adulter-ation or deterioration. Examples ofthese applications have been providedby King (19).

SFC is also unique in its ability toseparate oligomeric mixtures of poly-mers, surfactants and other homolo-gous series of compounds. The highresolving power of capillary SFC forthese applications is due in part to the

analyst’s ability to specify complex

pressure, density or temperature pro-grams which facilitate the separationof oligomeric mixtures. Figure 6demonstrates this type of separationby SFC for an oleic acid-esterifiedpropoxylated glycerol having 5 moles

of propylene oxide/mole of glycerol(low caloric fat substitute), which wasobtained by using a asymptotic pro-grammed density ramp from 0.12 to0.6 1 g/mL (27).

Other analysts have also employedSFC to great advantage for the charac-terization of surfactant mixtures (28)and synthetic oligomers (29). Chester(30) recently advocated even higherpressures in SFC to allow the separa-tion of the higher molecular weightspecies in a oligomeric mixture.Applying this concept along with the

choice of the right stationary phaseresults in an optima1 separation of thehigher oligomers in a synthetic m ix-ture of ethoxylated steryl alcohololigomers (Brij 78). as shown in Fig-ure 7.

As in SFE, SFC can provide ageneral assessment of the lipid com-ponents in a natural product matrix,either by coupling SFE with SFC orby simply performing a solventextraction on the sample matrix, fol-lowed by injection into the SFC. An

(continued on page 1097)

2.00 4.00 6.00 X.00

Tlmc x IO’ min

Figure6. SFCof oleic acid-esterified propoxylated lycerol (EPG) with 5 moles of propy-

lene oxide/mole of glycerol

INFORM,Vol 4. no 9 (September 1993)



1OG7

Il. :Methyl Column.

IhOT

1, E30

-

Biphenyl Column.

200 “C : ,lIl””

E 6,Cyanopropyl Column.

I I I I I I I I I

I20 I80 240 300 360 420 4X0 540 600 660 am

PVXWW

I I I I I I I I I I J

0 I2 24 3h 4x h0 min

Tim

Figure 7. Advantage of high pressure for the SFC analysis of Brij 78.

Time axis is for the biphenyl chromatogram.

300

0

Twnc (mln) 0 20 30

Pressure calm)75 175 175

240

own lcnlp. (“C) 140

Figure 8. SFC separation of the lipid extract from freeze-dried hamster feces (with

permission of Journal of High Resolution Chromatography).

‘conrinued from pag e 1095)

example of this type of analysis asapplied to a study on the absorption ofdietary fats is illustrated in Figure 8,where the lipid components in ham-ster feces have been separated by SFC(31). The separation depicted in Fig-ure 8 was accomplished bysuperimposing both a temperature andpressure gradient during the SFC run

to effect a better separation betweenthe sterol esters and triglycerides.Similar SFC profiles could beobtained from either making a liquidinjection of a Soxhlet extract or byinserting a fete pellet into an on-lineWE module attached to the SFC.

Maturity of an analytical techniquecan often be assessed by its applica-tion to quantitative or collaborativetypes of analysis. Routine and stan-dard methods based on SFC haveemerged recently and offer not onlyimprovements in analytical methodol-ogy, but a reduction in solvent dispos-al or regeneration costs. Recently thestatus of the official AOCS methodfor a-monoglycerides has been notedin INFORM (32) and alternativemethodology suggested. SFC also canbe applied for monoglyceride determi-nation, and recent quantitative studieson commercial emulsifiers indicate

that excellent results can be obtained.Table 2 compares the results for totalmonoglycerides in a commercialemulsifier determined by HPLC usingevaporative light-scattering detection.GC of the propionyi ester and SFCwith flame-ionization detection on theunderivatized sample (33). The agree-ment between all three methods is

excellent.

Future horizons

The future application and potential ofSFE and SFC appears promising,

since regulatory concerns involvingthe use and disposal of hazardous sol-vents opens up a new vista for theabove techniques. The above uses alsowill be accelerated by the federalNutritional Labeling and EducationAct of 1990, where concern over the

lipid content of food will assure new

uses for SFE and SFC.On the horizon arc some new

applications 01’ SCFs. which contarn

INFORM. Vol 4. no 9 (September 1993)



1098

INSTRUMENTATION

Table 2Comparison of monoglyceride results for a commercial emulsifier asdetermined by the HPLC-ELSD, GC and SFC method+

Total monoglycerides, /l00 g

HPLC-ELSD (Xlderivatized SFC/underivatized

Lor IX40Mean 92.5 93.3 93.4%RSD (n) I.1 (4) 1.3 (4) 3.5 (II )

Lot 6022

Mean 94.1 94.0 95.9%RSD (n) I.5 (4) I .8 ( 4) 3.4 (12)

uAbbrcvmlwn\. HPLC-ELSD. bxgh-perlormancs tquid chrom~lo~rilphy-cvaporativc light-mnering

dcwcmr: CC. gas chronmo~raph~. SFC. wpercnlmt fluid chromnrographyr RSD. relaive standard devi-

~,Wll

TCchllical Int‘orm;~~~or~ ScrvIcc.

Springfield. Virginia. 19x9. Mc[h-ads 211.13 (C). (1-l nd (h).

18. Anonymous. INFORM 3.562( 1993).

t 9. King. J .w.. ./. c/lW~WJfO,~,-. SC;

28.9 ( I YYO).

20. Snvdcr. J.M.. INFORAI 4.533( 19-93).

21. Berg. B.E.. E.M. Hansen and T.

Greibrokk, Abstracts of the 2ndEII~O~CJUS~mposirtn~ on Supe--critical Fluid Chronlaro,~rul)lI!and Extracrion. Huethig-Verlag.Heidelberg. 1993. pp. I%-202.

22. King. J.W.. J.E. France and J.h4.Snyder, Frcsenius J. Anal. CIwn.334: 474 ( 1992).

23. Markides. K.L., and M.L Lee.SFC Applications, Brigham

elements of both analytical and pro- 8. King, J.W., J. Am. Oil Chem. Sot. Young University Press. Provo,

cess technologies. SFE techniques 601711 (1983). Utah, 1988, 1989.will undoubtedly be coupled with 9. Chrastil, J., J. Phys. Chem. 24. Chester, T.L., in Analyticalnumerous forms of chromatography 86:3016 (1982). Instrumentation Handbook, edit-for further fractionation of the 10. Favati, F., and J.W. King, ed by G.W. Ewing. Marcelderived extracts and with techniques Abstracts of the 4th International Dekker inc., New York. 1990, pp.such as immunoassay (34) for the Symposium on Supercritical 843-88 I.rapid screening of many samples. Fluid Chromatography and 25. Demirbuker, M.. Analysis ofThe renaissance of SFC indicates the Extraction, Cincinnati, Ohio, Lipids by Supcrcritical Fluidincreased use of packed-column May 2&22, 1992, pp. 7 l-72. Chromatography, Ph.D. thesis,methodology, for both capillary and 11. Friedrich, J.P., G.R. L ist and A.J. Stockholm University. 1992.microbore columns, as well as for Heakin, J. Am. Oil Chem. Sot. 26. King, J.W., J. Chromato‘qr. Sci.enhanced selectivity using modest 59:288 (1982). 27:355 (1989).cosolvent addition. 12. Gere, D.R., L.G. Randall, CR. 27. Lu. X.J., M.R. Myers and W.E.,

Knipe, W. Pipkin and L.C. Artz, J. Am. Oil Chem. Sot.

Doherty, Proceedings of the 9th 70.355 (1993).References Annual Waste Testing and Quali- 28. Silver. A.H., and H.T. Kalinoski.1. Rizva, S.S.H., J.A. Daniels, A.L. ry Assurance Symposium, Alexan- Ibid. 69:599 ( 1992).

Benado and J.A. Zoliweg Food dria, Virginia. July 12-16, 1993. 29. Geissler, P.R., and A.E. JohnsonTech. 40 (7): 57 (1986). 13. King, J.W.. J.H. Johnson, S.L. Jr., Ibid. 67:54 I (1990).

2. Lee, M.L., and K.E. Markides, Taylor, W.L. Orton and M.L. 30. Chester, T.L., and D.P. Innis,Analytical Supercritical Fluid Hopper, Abstracts of Pittsburgh Abstracts of rite 2nd EuropeanChromarography and Extraction, Conference on Analytical Chem- Syntpckium on Analytical Super-Chromatography Conferences istry and Applied Spectroscopy. critical Fluid Chromatography.Inc., Provo, Utah. 1990. Chicago, Illinois, March 4-8, Huethig-Verlag, Hei’delberg,

3. Wenclawiak. B., Analysis with I99 I. Abstract No. 180. 1993, pp. 16-26.Super-critical Fluids: Extraction 14. Taylor, S.L.. J.W. King and G.R. 31. Pinkston, J.D., T.E.Delaney. D.J.and Cht-onlatography, Springer- List, J. Am. Oil Chem. Sot. Bowling and T.L. Chester, J.Verlag, Heidelberg, 1988. 70:437 ( 1993). High Resolut. Chromtogr. 14:401

4. Hawthorne, S.B., Anal. Chem. 62 15. Sawyer, L.D., J. Assoc. Off. Anal. (1991).(11):633A (1990). Chem. 76(1):144 (1993). 32. Steiner, J., INFORM 4:706

5. Katauskas, T., and H. Goldner, 16. McNeal, J.E. in Oficial Methods ( 1993).Res. Del,. 33(4):40 ( I99 1). of Analysis of the AOAC-15th 33. Liu, J., T. Lee, E. Babik, Jr., M.

6. Stahl, E., K.W. Quirin and D. Ed., Vol. 2. edited by K. Helrich, Guzman-Harty and C. Hastilow,Gerard, Dense Gases for Extrac- AOAC International, Arlington, J. Am. Oil Chem. Sot. 70:343tion and Refining, Springer-Ver- Virginia, 1990, Method 960.39, (1993).lag, Heidelberg, 1988, pp. 85-94. pp. 93 l-932. 34. France, J.E., and J.W. King, J.

7. Friedrich. J.P., U.S. Patent 4.466. 17. Pesticide Analytical Manual Assoc. Off. Anal. Chem. 74923 ( 1984). (PAM)--FDA, Vol. 1. National (6j.1013 (1991). n

INFORM, Vol. 4, no. 9 (September 1993) Suppliedby U.S.Dept,of AgricuftureNatianalCenter or Agricuttural

58 Analysis of Fats and Oils

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