AOCS脂肪酸检测方法

SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILSAOCS Official Method Ce 1f-96Reapproved 1997 • Revised 2001 Determination of cis-and trans-Fatty Acids in Hydrogenated and Refined Oils and Fatsby Capillary GLCDEFINITIONThis method consists of the gas–liquid chromatography (GLC) conditions optimized to identify and quantify the trans fatty acid isomers in vegetable oils and fats (References, 1). The fatty acid methyl esters (FAME) of the sample are separated on a capillary gas chromatography column having a high-ly polar stationary phase, according to their chain length (CL), degree of (un)saturation, and geome-try and position of the double bonds [DB(s)].SCOPEThis method is specially designed to evaluate, by a single capillary GLC procedure, the level of trans isomers as formed during (high-temperature) refining or during hydrogenation of vegetable oils or fats (see Notes, 1 and 2). The method may also be used to report all other fatty acids, for example to obtain saturated fatty acid (SAFA), monounsaturated fatty acid (MUFA), and polyunsaturated fatty acid (PUFA) levels from the same sample and same analysis.APPARATUS1.Gas ch ro m at ograph—equipped with a cap i l l a ry injectionsystem (pre fe rably split mode, operated at a split ratio of ap p r ox i m a t e l y 1:100) and flame ionization detector (FID), cap able of meeting the fo l l owing re q u i re m e n t s: injection port temperat u re, 250°C; detector temperat u re, 250°C; oven temperat u re conditions as given in Table 1.Typical results with these described conditions are show n in example ch ro m at ograms (Fi g u res 1–5).2.Column—highly polar stationary phase, such as one ofthe following:(a)CP™-Sil 88, 100 or 50 m×0.25 mm i.d., 0.20 µmfilm (Chrompack, Middelburg, The Netherlands).(b)SP-2650, 100 m ×.025 mm i.d., 0.20 µm fi l m(Supelco Inc., Bellefonte, PA, USA).(c)SP-2340, 60 m× 0.25 mm i.d., 0.2 µm fi l m(Supelco Inc.).(d)BPX-70, 120 m or 50 m×0.22 mm i.d., 0.25 µmfilm (SGE Inc., Austin, TX, USA).3.Recording instrument.4.Electronic integrator or chromatography software. REAGENTSUnless otherwise stat e d, use only re agents as specified in ISO 6353 (parts 2 and 3) (Refe rences, 2) if listed there; if not, then use re agents of re c og n i zed analytical grade and water of at least grade 3 as defined in ISO 3696 (Refe rences, 3).1.Carrier gas—helium, nitrogen, or hydrogen, GC quali-ty, dried, and oxygen removed by suitable filters.2.Internal standard (for calculating fatty acid data as mgper g oil)—tridecanoin, 5.0 mg/mL in chloroform. This solution is stable up to 1 week if stored in refrigerator in well sealed amber bottle. (See Notes, 3). PROCEDURE1.Sample preparation—(a)P r ep a r e the methyl esters from the tri g l y c e r i d e sf r om the oils or fats to be analy z e d, using theb o r on tri fl u o ride method as descri b e d, for ex a m-ple, in AOCS Official Method Ce 2-66 or IUPAC2.301 (References, 5 and 6).(b) B e f o r e test portions are taken from samples, thesamples should be mixed thoro u g h ly. Solid samplesshould be melted to ensure proper mixing.2.C h ro m at ograp hy—(a)Set up the gas ch ro m at ograph with the temperat u reand column as described in Table 1. Measure theave r age carrier gas linear velocity as indicated inTable 1, with a split ratio of ap p rox i m at e ly 1:100.(b)Inject 0.5 to 1 µL of the methyl esters (concentra-tion approximately 7 mg/mL) from the test sampleinto the gas ch r o m a t o graph. Compare the re s u l twith the example chromatograms (Figures 1–5). Ifthe sep a r ation obtained is not identical to theTable 1Proposed optimal GLC conditions for identification and quantification of trans isomers in refined and hydrogenated veg-etable oil samples (see References, 3).Stationary phase SP-2340SP-2560CP™-Sil 88BPX-70 Temperature conditions Isotherm 192°C Isotherm 170°C Isotherm 175°C Isotherm 198°C Column head pressure (kPa)125125130155Linear velocity of carrier gas (He)15 cm/sec16 cm/sec19 cm/sec17 cm/secPage 1of 6Page 2of 6SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILSCe 1f-96 • Determination of cis-and trans-Fatty Acids in Hydrogenated and Refined Oils and Fatsexamplech r o m a t o grams, small ch a n g es in ove n t e m p e r at u r e may be re q u i r e d . If so, decrease or i n c r ease the oven temperat u r e with subsequent s t e ps of 1°C until a good sep a r ation is obtained.These small corrections might be re q u i r ed to correct for batch differences between columns and i n s t r ument temperat u r e control and ge n e r a l l y fa l l within a ra n g e of only a few degrees (plus or minus) at maximum from the indicated value. The 20:1c peak will elute earlier relative to 18:3ccc if the oven temperature is increased (see Notes, 4).3.Performance check—(a)If the GLC system is set up properly, the separa-tion obtained should allow identifi c a tion of the small amount of the nat u r a l l y present 18:1 11c i s isomer next to the 18:1 9cis peak in (high-temper-at u r e) re f ined oils such as soybean oil. The two 1 8:1c i s o m e r s should be cl e a r ly sep a r ated (see Figures 1–5).(b)The 20:1 nat u r al isomer should be positionedexactly between the last eluting 18:3 trans isomer (t r ans, cis, cis ) and the 18:3c c c (linolenic acid)peak in (high-temperature) refined oils.(c)If the separation is sufficient for this type of analy-sis, in (high-temperature) refined oils a small peak for the 18:1 t r a n s i s o m e r , two ap p r ox i m a t e l y e q u a l l y sized 18:2 t r a n s i s o m e r s, and 4 (some-times 5) 18:3 t r a n s i s o m e r s should be obtained (see Figures 1–5).(d)For partially hydrogenated oils and fats, the sepa-ration of the 18:1 13t r a n s and the 18:1 9c i s i s o m e r s should be visible on the ch r o m a t o gra m .This is required for an accurate peak split between cis and trans .4.Peak identification—(a )For (high-temperat u r e) re f ined oils and fats, thet ra n s i s o m e rs are limited in number because only ge o m e t r ical isomers, with the DB(s) on the same n a t u r al position, are fo r m e d . For C 1 8fatty acids these specific isomers are 18:1 9t ; 18:2 9c 1 2t and 9t 1 2c ; and 18:3 t c t , c c t , c t c , t c c 9, 12, 15-i s o m e rs (in some samples the 18:2 9t 12t and 18:3t t c i s o m e rs are found as well in ve ry small amounts).(b)For part i a l l y hy d r oge n a ted oils and fats the t ra n sDB-containing isomers are identified using the e q u i valent chain length (ECL) concept (Refe r -ences, 7; see Table 2). For accurate peak identifi-cation with this system, the ECL values have to be determined after suitable calibration with available cis and trans fatty isomer standards.SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILSCe 1f-96 • Determination of cis-and trans- Fatty Acids in Hydrogenated and Refined Oils and FatsPage 3of 6Figure 4.Chromatogram of methyl esters from a physicall y refined rapeseed oil sample using 50 m ×0.25 mm ×0 .20µm CP™-Sil 88 column (Chr o m p a c k). The t r a n s fatty acid isomers are indicated in the chromatogram.Figure 5.Chromatogram of methyl esters from a high-tem -perature-refined rapeseed oil sample, using 50 m ×0.22 mm ×0.25 µm BPX-70 (SGE). The t ra n s fatty acid isomers are indicated in the chromatogram.SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILSCe 1f-96 • Determination of cis-and trans- Fatty Acids in Hydrogenated and Refined Oils and Fats ArrayPage 4of 6SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILS Ce 1f-96 • Determination of cis-and trans- Fatty Acids in Hydrogenated and Refined Oils and FatsSAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILSCe 1f-96 • Determination of cis-and trans- Fatty Acids in Hydrogenated and Refined Oils and Fats2.D u r ing (high-temperat u r e) re f i n i n g, only ge o m e t r i c a li s o m e r s of the mono- and poly u n s a t u r ated fatty acidsare formed; that is, the DBs remain on the same, natur-al position. During hy d r oge n a tion, on the other hand, both positional and geometrical isomers are formed.3.If quantitation of fatty acids is re q u i r ed (mg/g), thei n t e r nal standard must be added prior to methy l a t i o n.The addition of a known quantity will allow the calcu-lation of fatty acid content by simple proportions. If ac o m p l e x mat e r ial is being examined for indiv i d u a lfatty acid content for labeling purposes, the intern a l s t a n d a r d should be added to the test sample befo r e extraction commences.4.The elution profile of the BPX-70 column [Apparatus,2(c)] is somewh a t diffe r ent; the 20:1c peak alway s elutes after the 18:3ccc peak using these conditions. REFERENCES1.This method parap h r ases one submitted by Dr. GuusS.M.J.E. Duch ateau of Unilever Research Lab o rat o ri e s,V l a a rd i n gen, The Netherlands, November 1995.2.ISO 6353, Reagents for Chemical Analysis, Pa r t 2(1983) and 3 (1987); Specifications.3.ISO 3696, Water for Analytical Lab o r at o r y Use—Specifications and Test Methods (1987).4.D u c h a teau, G. S. M.J.E., H.J. van Oosten, and M.A.Vasconcellos, Analysis of c i s- and t r a n s- F atty AcidI s o m e rs with Cap i l l a ry GLC in Hydroge n ated and Refi n e dV egetable Oils, J. Am. Oil Chem. Soc. 73:275 (1996).5.AOCS Official Method Ce 2-66, Preparation of MethylEsters of Long-Chain Fatty Acids.6.I U P AC, S t a n d a r d Methods for Analysis of Oils, Fat sand Derivat i ve s,B l a c k w ell Scientific Publ i c a t i o n s;IUPAC Method 2.301.7.J. Am. Oil Chem. Soc. 58:662 (1981).8.Th i r d Unilever interl a b o r at o r y test on the determ i n a-tion of low trans levels by capillary GC. Visser, R.G., P.A. Zandbelt, Y.S.J. V eldhuizen.9.G a r f i e l d, F.M., Quality Assurance Principles fo rAnalytical Laboratories, AOAC International, 1995.Page 6of 6。