SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILS

AOCS Official Method Ce 1f-96

Reapproved 1997 • Revised 2001

Determination of cis- and trans- Fatty Acids

in Hydrogenated and Refined Oils and Fats
by Capillary GLC
DEFINITION
This 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)].
SCOPE
This 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.

APPARATUS 1. Carrier gas—helium, nitrogen, or hydrogen, GC quali-

1. Gas chromatograph—equipped with a capillary injection
system (preferably split mode, operated at a split ratio of
approximately 1:100) and flame ionization detector
(FID), capable of meeting the following requirements:
injection port temperature, 250°C; detector temperature,
250°C; oven temperature conditions as given in Table 1.
Typical results with these described conditions are shown
in example chromatograms (Figures 1–5).
2. Column—highly polar stationary phase, such as one of
the following:
(a) CP™-Sil 88, 100 or 50 m × 0.25 mm i.d., 0.20 µm
film (Chrompack, Middelburg, The Netherlands).
(b) SP-2650, 100 m × .025 mm i.d., 0.20 µm film
(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 µm
film (SGE Inc., Austin, TX, USA).
3. Recording instrument.
4. Electronic integrator or chromatography software.
REAGENTS
Unless otherwise stated, use only reagents as specified in ISO
6353 (parts 2 and 3) (References, 2) if listed there; if not, then
use reagents of recognized analytical grade and water of at
least grade 3 as defined in ISO 3696 (References, 3).
ty, dried, and oxygen removed by suitable filters.
2. Internal standard (for calculating fatty acid data as mg
per 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).
PROCEDURE
1. Sample preparation—
(a) Prepare the methyl esters from the triglycerides
f rom the oils or fats to be analy ze d, using the
boron trifluoride method as described, for exam-
ple, in AOCS Official Method Ce 2-66 or IUPAC
2.301 (References, 5 and 6).
(b) Before test portions are taken from samples, the
samples should be mixed thoroughly. Solid samples
should be melted to ensure proper mixing.
2. Chromatography—
(a) Set up the gas chromatograph with the temperature
and column as described in Table 1. Measure the
average carrier gas linear velocity as indicated in
Table 1, with a split ratio of approximately 1:100.
(b) Inject 0.5 to 1 µL of the methyl esters (concentra-
tion approximately 7 mg/mL) from the test sample
into the gas chromatograph. Compare the result
with the example chromatograms (Figures 1–5). If
the sep a ration obtained is not identical to the

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

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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

Page 2 of 6

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
refined rapeseed oil sample using 50 m × 0.25 mm × 0.20
µm CP™-Sil 88 column (Chr ompack). The trans fatty acid
isomers are indicated in the chromatogram.
example chromatograms, small changes in oven
temperature may be required. If so, decrease or
increase the oven temperature with subsequent
steps of 1°C until a good separation is obtained.
These small corrections might be re q u i red to
correct for batch differences between columns and
instrument temperature control and generally fall
within a ra n ge 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 identification of the
small amount of the naturally present 18:1 11cis
isomer next to the 18:1 9cis peak in (high-temper-
ature) refined oils such as soybean oil. The two
18:1c isomers should be clearly separated (see
Figures 1–5).
(b) The 20:1 nat u ral isomer should be positioned
exactly between the last eluting 18:3 trans isomer
(trans, cis, cis) and the 18:3ccc (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
SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILS
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 trans fatty acid isomers are
indicated in the chromatogram.
for the 18:1 t ra n s i s o m e r, two ap p rox i m at e ly
equally sized 18:2 trans isomers, and 4 (some-
times 5) 18:3 trans isomers should be obtained
(see Figures 1–5).
(d) For partially hydrogenated oils and fats, the sepa-
ration of the 18:1 13t ra n s and the 18:1 9c i s
isomers should be visible on the chromatogram.
This is required for an accurate peak split between
cis and trans.
4. Peak identification—
(a) For (high-temperature) refined oils and fats, the
trans isomers are limited in number because only
geometrical isomers, with the DB(s) on the same
natural position, are fo rm e d. For C18 fatty acids
these specific isomers are 18:1 9t; 18:2 9c1 2t
and 9t12c; and 18:3 tct, cct, ctc, tcc 9, 12, 15-
isomers (in some samples the 18:2 9t12t and 18:3ttc
isomers are found as well in very small amounts).
(b) For partially hydrogenated oils and fats the trans
DB-containing isomers are identified using the
e q u ivalent 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.
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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 4 3. Precision data for low-level trans are shown in Table 6

Repeatability results, obtained with HP 5890 II a. (References, 9).
Results Repeatability GC BCR reference
QUALITY ASSURANCE AND CONTROL
BCR 162 FA equipment (mean, SD) value (± 95% CI)
1. Blank sample—The first sample in an analysis batch is
16:0 10.60, 0.030 10.65 ± 0.17 always a blank (n-heptane). No peaks should be detected
18:0 2.91, 0.042 2.87 ± 0.07 in the blank run. Repeat this test after every ten samples.
18:1 24.17, 0.031 24.14 ± 0.28 2. C o n t rol sample BCR 162—At least one BCR 162
18:2 55.97, 0.070 56.65 ± 0.54 sample is analyzed per analysis batch (= methylation
18:3 4.77, 0.020 4.68 ± 0.21 p e r fo rmed in one bat ch). The results for fatty acid
aBCR = Joint Research Center, Institute for Reference Materials (FA) 16:0, 18:0, 18:1, 18:2, and 18:3 are logged in a
and Measurement, Retieseweg, B-2440 Geel, Belgium. Shewhart-type control chart (References, 8). The FAME
of the BCR are reinjected after each set of ten samples.
3. Proficiency test—For external reference the method is
tested each year by part i c i p ation in the AO C S
METHOD PRECISION Laboratory Proficiency Program or any other suitable
1. R ep e at ab i l i t y — The rep e at ability of the GC part as ring test.
determined with manually prepared FAME is available
for the Certified Reference Material (CRM) BCR 162 NUMBERED NOTES
( Joint Research Center, Institute for Refe re n c e 1. The trans content in this procedure is defined as:
Materials and Measurement, Retieseweg, B-2440 Geel, (a) For (high-temperature) refined oils and fats, the
Belgium; see Table 4). sum of the 18:1t, 18:2t, and 18:3t components.
2. Reproducibility—The reproducibility of the complete (b) For partially hydrogenated oils and fats, the sum
method, including the methylation step, manual and of all trans DB-containing components.
automatic, is derived from the long-term results for The t ra n s content as obtained with this
BCR 162 (see Table 5). The results are well within the method may not agree with the trans content as
limits as specified for CRM 162 by IRRM. obtained using other methods.

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.

Sunflower oil Rapeseed oil Olive oil

trans trans trans Total trans trans trans Total trans trans trans Total
C18:1 C18:2 C18:3 trans C18:1 C18:2 C18:3 trans C18:1 C18:2 C18:3 trans
n 31 51 30 50 33 48 47 47 31 36 22 40
Mean 0.029 0.154 0.033 0.205 0.032 0.074 0.406 0.521 0.023 0.021 0.016 0.055

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
isomers of the mono- and polyunsaturated fatty acids
are formed; that is, the DBs remain on the same, natur-
al position. During hydrogenation, on the other hand,
both positional and geometrical isomers are formed.
3. If quantitation of fatty acids is required (mg/g), the
internal standard must be added prior to methylation.
The addition of a known quantity will allow the calcu-
lation of fatty acid content by simple proportions. If a
c o m p l ex mat e rial is being examined for indiv i d u a l
fatty acid content for labeling purposes, the internal
standard should be added to the test sample before
extraction commences.
4. The elution profile of the BPX-70 column [Apparatus,
2(c)] is somewhat different; the 20:1c peak always
elutes after the 18:3ccc peak using these conditions.
REFERENCES
1. This method paraphrases one submitted by Dr. Guus
S.M.J.E. Duchateau of Unilever Research Laboratories,
Page 6 of 6
Vlaardingen, The Netherlands, November 1995.
2. ISO 6353, Reagents for Chemical Analysis, Pa rt 2
(1983) and 3 (1987); Specifications.
3. ISO 3696, Water for Analytical Lab o rat o ry Use—
Specifications and Test Methods (1987).
4. D u ch ateau, G. S. M . J.E., H.J. van Oosten, and M.A.
Vasconcellos, Analysis of cis- and t ra n s-Fatty Acid
Isomers with Capillary GLC in Hydrogenated and Refined
Vegetable Oils, J. Am. Oil Chem. Soc. 73:275 (1996).
5. AOCS Official Method Ce 2-66, Preparation of Methyl
Esters of Long-Chain Fatty Acids.
6. IUPAC, Standard Methods for Analysis of Oils, Fats
and Derivatives, B l a ck well Scientific Publ i c at i o n s ;
IUPAC Method 2.301.
7. J. Am. Oil Chem. Soc. 58:662 (1981).
8. Third Unilever interlaboratory test on the determina-
tion of low trans levels by capillary GC. Visser, R.G.,
P.A. Zandbelt, Y.S.J. Veldhuizen.
9. G a r fi e l d, F.M., Quality Assurance Principles fo r
Analytical Laboratories, AOAC International, 1995.