AOCS Ce 1f-96 PDF
AOCS Ce 1f-96 PDF
AOCS Ce 1f-96 PDF
Table 1
Proposed optimal GLC conditions for identification and quantification of trans isomers in refined and hydrogenated veg-
etable oil samples (see References, 3).
Stationary phase SP-2340 SP-2560 CP™-Sil 88 BPX-70
Temperature conditions Isotherm 192°C Isotherm 170°C Isotherm 175°C Isotherm 198°C
Column head pressure (kPa) 125 125 130 155
Linear velocity of carrier gas (He) 15 cm/sec 16 cm/sec 19 cm/sec 17 cm/sec
Page 1 of 6
SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILS
Ce 1f-96 • Determination of cis- and trans- Fatty Acids in Hydrogenated and Refined Oils and Fats
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SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILS
Ce 1f-96 • Determination of cis- and trans- Fatty Acids in Hydrogenated and Refined Oils and Fats
Figure 4. Chromatogram of methyl esters from a physicall y Figure 5. Chromatogram of methyl esters from a high-tem -
refined rapeseed oil sample using 50 m × 0.25 mm × 0.20 perature-refined rapeseed oil sample, using 50 m × 0.22 mm
µm CP™-Sil 88 column (Chr ompack). The trans fatty acid × 0.25 µm BPX-70 (SGE). The trans fatty acid isomers are
isomers are indicated in the chromatogram. indicated in the chromatogram.
example chromatograms, small changes in oven for the 18:1 t ra n s i s o m e r, two ap p rox i m at e ly
temperature may be required. If so, decrease or equally sized 18:2 trans isomers, and 4 (some-
increase the oven temperature with subsequent times 5) 18:3 trans isomers should be obtained
steps of 1°C until a good separation is obtained. (see Figures 1–5).
These small corrections might be re q u i red to (d) For partially hydrogenated oils and fats, the sepa-
correct for batch differences between columns and ration of the 18:1 13t ra n s and the 18:1 9c i s
instrument temperature control and generally fall isomers should be visible on the chromatogram.
within a ra n ge of only a few degrees (plus or This is required for an accurate peak split between
minus) at maximum from the indicated value. The cis and trans.
20:1c peak will elute earlier relative to 18:3ccc if 4. Peak identification—
the oven temperature is increased (see Notes, 4). (a) For (high-temperature) refined oils and fats, the
3. Performance check— trans isomers are limited in number because only
(a) If the GLC system is set up properly, the separa- geometrical isomers, with the DB(s) on the same
tion obtained should allow identification of the natural position, are fo rm e d. For C18 fatty acids
small amount of the naturally present 18:1 11cis these specific isomers are 18:1 9t; 18:2 9c1 2t
isomer next to the 18:1 9cis peak in (high-temper- and 9t12c; and 18:3 tct, cct, ctc, tcc 9, 12, 15-
ature) refined oils such as soybean oil. The two isomers (in some samples the 18:2 9t12t and 18:3ttc
18:1c isomers should be clearly separated (see isomers are found as well in very small amounts).
Figures 1–5). (b) For partially hydrogenated oils and fats the trans
(b) The 20:1 nat u ral isomer should be positioned DB-containing isomers are identified using the
exactly between the last eluting 18:3 trans isomer e q u ivalent chain length (ECL) concept (Refe r-
(trans, cis, cis) and the 18:3ccc (linolenic acid) ences, 7; see Table 2). For accurate peak identifi-
peak in (high-temperature) refined oils. cation with this system, the ECL values have to be
(c) If the separation is sufficient for this type of analy- determined after suitable calibration with available
sis, in (high-temperature) refined oils a small peak cis and trans fatty isomer standards.
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SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILS
Ce 1f-96 • Determination of cis- and trans- Fatty Acids in Hydrogenated and Refined Oils and Fats
Table 2
ECL values for the most important fatty acid isomers, obtained on the three highly polar stationary phases under proposed
optimal conditions; for reference, some literature data (References, 7) are listed as well.
Isomer SP-2340, 192°C CP™-Sil 88, 175°C BPX-70, 198°C Literature data
18:1 9c 18.68 18.66 18.46 18.61
10c 18.64
11c 18.76 18.74 18.53 18.70
12c 18.80 18.75
13c 18.87 18.84
9t 18.49 18.46 18.28 18.41
10t 18.49 18.48
11t 18.52 18.49
12t 18.57 18.54
13t 18.61 18.59
18:2 9c12c 19.62 19.63 19.14 19.51
9c12t 19.40 19.40 18.94 19.32
9t12c 19.48 19.49 19.02 19.40
9t12t 19.26 19.20 18.69 19.13
12c15c 19.92 19.81
18:3 6c9c12c 20.28 20.15
9t12t15t 20.04 19.94
9t12c15t 20.23 20.26 19.42 20.15
9c12c15t 20.35 20.36 19.61 20.38
9c12t15c 20.53 19.51 20.31
9t12c15c 20.57 20.56 19.82 20.42
9c12c15c 20.68 20.67 19.93 20.52
5. Calculation and expression of results— At = the sum of the corrected areas under all the
(a) Correct the area of each peak to compensate for the peaks, excluding the solvent peak
flame ionization detector (FID) response for each (c) Determine the total trans level as defined in Notes,
component. The FID correction factors are calcu- 1. The amount should be reported to the nearest
lated from the molecular weight of the FAME as tenth of a percent.
follows:
MWx
FIDx = Table 3
(nx − 1)(AWC)(FID16:0) List of FID response factors.
Where— FAME MW n−1 FID factor Correction factor
FIDx = the FID factor for component x
4:0 102.13 4 2.126 1.51
MWx = molecular weight of component x
6:0 130.19 6 1.807 1.28
nx = the number of carbon atoms in the FAME of
8:0 158.24 8 1.647 1.17
component x
9:0 172.27 9 1.594 1.13
AWC = the atomic weight of carbon (12.01)
10:0 186.30 10 1.551 1.10
FID16:0 = the FID correction factor for 16:0 (1.407)
11:0 200.32 11 1.516 1.08
All other FID correction factors used in the calcu- 12:0 214.35 12 1.487 1.06
lation are re l at ive to FID 16:0. For example, the 13:0 228.37 13 1.463 1.04
c o rrection factor for 10:0 becomes 1.10. FID 14:0 242.40 14 1.442 1.02
correction factors are listed in Table 3. 15:0 156.42 15 1.423 1.01
(b) C a l c u l ate the (re l at ive) perc e n t age x of each 16:0 270.46 16 1.407 1.00a
component by determining the corrected area of 17:0 284.49 17 1.393 0.99
the corresponding peak relative to the sum of the 18:0 298.52 19 1.370 0.97
corrected areas of all the peaks, as follows: 19:0 312.52 19 1.370 0.97
20:0 326.57 20 1.360 0.97
Ax 21:0 340.57 21 1.350 0.96
x=
At 22:0 354.62 22 1.342 0.95
23:0 368.62 23 1.334 0.95
Where—
24:0 382.68 24 1.328 0.94
Ax = the corrected area of the peak corresponding
to component x aReference value.
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SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILS
Ce 1f-96 • Determination of cis- and trans- Fatty Acids in Hydrogenated and Refined Oils and Fats
Table 5
Reproducibility results, obtained using HP 5890 II a.
Results Manual BF3/CH3OH Automatic HP-PrepStation BCR reference value
BCR 162 FA (mean, SD) (mean, SD) (± 95% CI)
16:0 10.60, 0.069 10.58, 0.087 10.65 ± 0.17
18:0 2.91, 0.014 2.90, 0.010 2.87 ± 0.07
18:1 24.17, 0.060 24.11, 0.027 24.14 ± 0.28
18:2 55.97, 0.146 56.04, 0.103 56.65 ± 0.54
18:3 4.77, 0.080 4.81, 0.022 4.68 ± 0.21
aBCR = Joint Research Center, Institute for Reference Materials and Measurement, Retieseweg, B-2440 Geel, Belgium.
Table 6
Precision data taken from Reference 8.
Repeatability
Sr 0.005 0.009 0.007 0.015 0.005 0.012 0.015 0.027 0.005 0.007 0.005 0.012
RSDr 18.47 5.80 21.65 7.49 15.63 15.93 3.69 5.21 23.29 34.01 29.02 22.08
r 0.015 0.025 0.02 0.043 0.014 0.033 0.042 0.076 0.015 0.02 0.013 0.034
Reproducibility
SR 0.014 0.030 0.013 0.048 0.009 0.020 0.058 0.081 0.009 0.011 0.009 0.030
RSDR 46.80 19.25 38.96 23.17 29.02 27.03 14.16 15.63 40.37 51.02 53.57 54.55
R 0.038 0.083 0.036 0.133 0.026 0.056 0.161 0.228 0.026 0.03 0.024 0.084
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SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILS
Ce 1f-96 • Determination of cis- and trans- Fatty Acids in Hydrogenated and Refined Oils and Fats
2. During (high-temperature) refining, only geometrical Vlaardingen, The Netherlands, November 1995.
isomers of the mono- and polyunsaturated fatty acids 2. ISO 6353, Reagents for Chemical Analysis, Pa rt 2
are formed; that is, the DBs remain on the same, natur- (1983) and 3 (1987); Specifications.
al position. During hydrogenation, on the other hand, 3. ISO 3696, Water for Analytical Lab o rat o ry Use—
both positional and geometrical isomers are formed. Specifications and Test Methods (1987).
3. If quantitation of fatty acids is required (mg/g), the 4. D u ch ateau, G. S. M . J.E., H.J. van Oosten, and M.A.
internal standard must be added prior to methylation. Vasconcellos, Analysis of cis- and t ra n s-Fatty Acid
The addition of a known quantity will allow the calcu- Isomers with Capillary GLC in Hydrogenated and Refined
lation of fatty acid content by simple proportions. If a Vegetable Oils, J. Am. Oil Chem. Soc. 73:275 (1996).
c o m p l ex mat e rial is being examined for indiv i d u a l 5. AOCS Official Method Ce 2-66, Preparation of Methyl
fatty acid content for labeling purposes, the internal Esters of Long-Chain Fatty Acids.
standard should be added to the test sample before 6. IUPAC, Standard Methods for Analysis of Oils, Fats
extraction commences. and Derivatives, B l a ck well Scientific Publ i c at i o n s ;
4. The elution profile of the BPX-70 column [Apparatus, IUPAC Method 2.301.
2(c)] is somewhat different; the 20:1c peak always 7. J. Am. Oil Chem. Soc. 58:662 (1981).
elutes after the 18:3ccc peak using these conditions. 8. Third Unilever interlaboratory test on the determina-
tion of low trans levels by capillary GC. Visser, R.G.,
REFERENCES P.A. Zandbelt, Y.S.J. Veldhuizen.
1. This method paraphrases one submitted by Dr. Guus 9. G a r fi e l d, F.M., Quality Assurance Principles fo r
S.M.J.E. Duchateau of Unilever Research Laboratories, Analytical Laboratories, AOAC International, 1995.
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