COMMERCI AL FAT S AN D O IL S

Official Method Ce 1j-07

Revised 2017

cis-, trans-, Saturated, Monounsaturated,

and Polyunsaturated Fatty Acids in
Extracted Fats by Capillary GLC
DEFINITION This method provides a gas–liquid chromatography (GLC) procedure for the determination of the fatty acid
composition, including the trans fatty acid isomers of extracted fats. The fatty acid methyl esters (FAME)
are separated on a capillary gas chromatography column having a highly polar stationary phase, according to
their chain length (CL), degree of unsaturation, and geometry and position of the double bonds [DB(s)].

SCOPE
This method evaluates, by a single capillary GLC procedure, the levels of trans isomers, saturated fatty
acid (SFA), cis- and trans-monounsaturated fatty acid (MUFA), and cis- and trans-polyunsaturated fatty
acid (PUFA) in fat samples where the source of the fat is unknown or is of dairy or ruminant origins. The
method is not designed to provide detailed definitive isomer composition that may be desired for nutritional
and/or biochemical purposes. To obtain more detailed isomeric composition information prior argentation
chromatographic separations and/or additional GC analyses are required. For nutritional labeling purposes
All rights reserved. No part of this method may be reproduced or transmitted in any form or by any means without written permission of the publisher.

the total fat, saturated, cis-monounsaturated, cis-polyunsaturated and trans fatty acid contents are to be
determined. This method may also determine cis-polyunsaturated fatty acids (PUFA), including arachidonic
acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA).

   This method utilizes a triacylglycerol (13:0 TAG) internal standard (IS) for determining the concen-
tration of the individual fatty acids in the oil samples after methylation. The method is applicable to fats
derived from dairy and ruminant products. This method is not applicable to products containing mixtures
of both dairy and vegetable fats as the trans linolenic acid (18:3) isomers will coelute with the gondoic acid
(20:1) isomers. In that case both this method coupled with AOCS Ce 1h-05 would be required for analysis.
Conjugated linoleic acids (CLAs) will be present in dairy and ruminant fats and may be quantitated with
this method, however for nutritional labeling purposes CLA are not included either as cis- or trans-PUFA
(References 1 and 2). There is minor co-elution of cis- and trans-fatty acid isomers, particularly in the 16:1,
17:1, and 18:1 regions, using this technique.

   Theoretical Correction Factors (TCFs) are used to quantitate all saturated, monounsaturated, and poly-
unsaturated fatty acids (PUFA) of 18 carbons. TCFs are also used for fatty acids, which lack pure standards.
Empirical Correction Factors (ECFs) are used for very long chain PUFA of 20 carbons or more and three or
more double bonds for which standards are readily available.

APPARATUS
1. Gas chromatograph: suitable for use with a capillary column equipped with:
(a) Thermostatic chamber for the capillary column, capable of maintaining the desired temperature to ± 0.1 °C.
(b) Temperature-controlled split mode injection unit.
(c) Flame-ionization detector (FID), amplifier, and electronic recorder-integrator.
2. Capillary column: Fused silica capillary 100 m, and 0.25 mm i.d., coated with SPTM-2560 or CP-SilTM 88,
100% cyanopropylsilicone stationary phase, to a thickness of 0.20 µm. Commercially prepared columns are avail-
able from: SPTM-2560 (Part Number 24056, Supelco, Inc. Bellefonte, PA, USA) and CP-SilTM 88 (Part Number
CP7489, Varian, Walnut Creek, CA, USA; Chrompack, Middleburg, The Netherlands).
3. Microsyringe for gas chromatography—10 µL delivery, with a hardened needle.
4. Carrier gas—hydrogen or helium, 99.99% pure or better, gas chromatography quality, dried, and oxygen removed
by suitable filters.1
5. Flame gases—hydrogen and air, gas chromatography quality.
6. Make-up gas—nitrogen or helium, gas chromatography quality.
7. Injection port split liner—4 mm i.d., 6.3 mm o.d. × 78.5 mm base deactivated precision (Focus) split liner with
glass wool (e.g., Catalog #21022-211.5, Restek; P/N 092002, SGE or equivalent).2

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REAGENTS (see AOCS Laboratory Safety)

Unless otherwise stated, use only reagents as specified in ISO 6353 (Parts 2 and 3) (Reference 3) if listed there; if not,
then use reagents of recognized analytical grade and water of at least grade 3 as defined in ISO 3696 (Reference 4).
1. Reference FAMEs—Methyl esters of pure fatty acids, in particular cis- and trans isomers of octadecenoic (oleic),
trans isomers of octadecadienoic (linoleic) and octadecatrienoic (α-linolenic) acids. Wide ranges of cis- and trans
octadecenoic methyl ester isomers are available on the market (e.g., from Alltech Associates, Inc., Deerfield, IL,
Nu-Chek-Prep, Elysian, MN, Supelco Inc., Bellefonte, PA, and Sigma Chemical Co., St. Louis, MO). trans
Geometrical isomers of linoleic and a-linolenic acids can be prepared in the laboratory with the aid of p-toluene-
sulfonic acid (Reference 5) and are commercially available from Supelco, Inc.
2. Triacylglycerol (TAG) IS solution (for calculating fatty acid data as mg per 100 g oil or 100 g of food sam-
ple)—13:0 TAG—tritridecanoin (purity >99%) 5.0 mg/mL in n-hexanes or n-heptane. This TAG IS solution is
stable indefinitely if precautions are taken to eliminate the loss of n-hexanes or n-heptane and therefore a change
in the concentration of the internal standard. For example, store the solution in refrigerator in well-sealed amber
bottle when not in use. Pure tritridecanoin is available on the market (e.g., from Nu-Chek-Prep). Purity of the IS
should be confirmed by thin-layer chromatography, high-performance liquid chromatography, and gas chromatog-
raphy analysis or by any other appropriate technique.
3. GLC-714, 20 mg/mL in n-heptane or n-hexanes, 100 mg ampoule (Nu-Chek-Prep, Inc.). This mixture is used
to calculate the Empirical Correction Factors (ECFs). A Certificate of Analysis (COA) must be requested from
Nu-Chek-Prep for every lot of the standard mixture used in determining ECFs. The actual amounts weighed into
the ampoule and purities are listed on the COA and are used to calculate the actual amount of each FAME standard
present in the mixture. The actual amount is necessary for determining the response factor (area/amount) for each

All rights reserved. No part of this method may be reproduced or transmitted in any form or by any means without written permission of the publisher.
FAME in the mixture. This mixture (100 mg) is diluted in 5 mL of n-heptane or n-hexanes with a 5 mL Class
A volumetric pipette. The ampoule is rinsed with n-heptane or n-hexanes to ensure complete and homogenous
transfer of the mixture. The final concentration should be approximately 20 mg/mL. The fatty acid methyl esters
(FAMEs) standards in GLC-714 were those commercially available chosen to represent the majority of fatty acids
present in edible vegetable, dairy, and ruminant fats, marine and single cell oils. Included were the internal stan-
dards used in AOCS Ce 1h-05, AOCS Ce 1i-07, and AOCS Ce 1j-07.

PROCEDURE
1. Preparation of FAMEs:
(a) GLC-714—Analyze the 20 mg/mL preparation to determine ECFs.
(b) From oils and fats—Use a standard procedure for example, Alcoholysis (Reference 6), ISO 5509 (Reference 7),
or transmethylation (References 8 and 9). Prior to methylation, add enough IS solution to the reaction flask,
so that after the oil or fat is added, the concentration of the final solution is between 0.05 and 0.10 mg IS/mg
oil or fat (e.g., 5 mL of a 2 mg/mL TAG IS solution per 100 mg of oil).
(c) Dissolve the prepared FAME in n-hexanes or n-heptane, concentration should be approximately 15–20
mg/mL n-hexanes or n-heptane.
2. Chromatography
(a) Operating conditions are as follows:
Injection port temperature: 235 °C
Detector temperature: 325 °C
Oven temperature program: Isothermal at 180 °C (32 min) ramped at 20 °C/min to 215 °C (hold 31.25 min)
Carrier gas: hydrogen, constant flow rate 1.0 mL/min; 26 cm/s; 24.63 psi; split ratio, 100:1.
Carrier gas: helium; constant flow rate; flow rate 2.0 mL/min; 28 cm/s; 63.29 psi; split ratio, 100:1
(b) Inject 1 µL, which is equivalent to 15 to 20 µg of total FAMEs (Table 1 and Figs. 1–32).

Performance check: (System Suitability Mix)

1. Column performance is checked using fatty acid methyl esters (FAMEs) of tallow, anhydrous butterfat, and
Certified Reference Material (BCR) 163, respectively, covering the range of fatty acids under investigation (Figs.
2–16 and 18–32). Since commercial GC designs are different, and the separation obtained may not be identical to
the example chromatograms, small changes in the sample size, sample concentration, or oven temperature may be
required. If so, adjust the sample size, sample concentration, or oven temperature until the best separation results
are obtained. If the column oven temperature needs to be adjusted, it should be adjusted with small increments,
preferably by steps of 1 °C. Authentic standards of fatty acid esters, if needed for peak identification, are available.
On SPTM-2560, CP-SilTM 88, or any other cyanopropylsilicone capillary columns, the column temperature
has profound effect on the elution pattern of 13t+14t-18:1, 16t-18:1, 14c-18:1, 9c,12c,15t-18:3, 11c-20:1, and
9c,12c,15c-18:3 (Reference 10). From the chromatograms of the butterfat:
(a) Baseline separation between 9c-18:1 and 11c-18:1 should be obtained.

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(b) Resolution greater than 1.0 but less than 1.5 between 11c-20:1 and 9c,12c,15c-18:3 should be obtained
(Reference 11).
2. The Empirical Correction Factors (ECFs) are determined by analysis of GLC-714 at approximately 20 mg/mL
and plotted as the percent difference between the ECF and Theoretical Correction Factors (TCFs) divided by the
TCFs (Fig. 34). Fig. 34 shows the plots from four different laboratories. Different laboratories will most likely have
different plots and therefore should be determined for each laboratory. An example calculation follows:
ECFFAMEx − TCFFAMEx
% Difference = 100 ×
TCFFAME X
GLC-714 can also be used to help determine if a chromatographic system is operating properly. Operation under
the GC parameters listed in the method should result in a similar trend line with the long chain PUFA ECFs devi-
ating furthest from TCFs, while all saturated and monounsaturated FAME deviate from the TCFs the least. Ideally
if the % Difference is three or less for the saturated and monounsaturated FAME, then the GC system is considered
to be operating properly and TCFs can be used for those FAME. ECFs will need to be calculated and used for the
long chain PUFA (≥ 20 carbons and three double bonds). Fig. 33 shows the plot of three different methods AOCS
Ce 1h-05, Ce 1i-07, and Ce 1j-07 within one laboratory.
3. Peak identification—the individual FAMEs are identified by their retention times, which are compared with the
FAME reference standards and reference oils. The FAME reference oils are tallow and anhydrous butterfat. See
Figs. 2–11 and 18–27 for sample chromatograms of both. When peaks of unknown identity are observed, attempt
to identify such peaks using appropriate procedures that usually involve GC-MS, FTIR, silver-ion chromatography,
and classical chemical methods (References 12 and 13). Peaks of unknown identity should not be included in the
summation of peak areas when quantifying the concentrations of total fat, saturated, cis- or trans-monounsaturated,
All rights reserved. No part of this method may be reproduced or transmitted in any form or by any means without written permission of the publisher.

cis- or trans-polyunsaturated, AA, EPA, or DHA, unless they have been confirmed to be fatty acids.

CALCULATIONS
1. Calculation of grams (g) of individual fatty acids and total fat per 100 g of sample:
The food labeling regulations in Canada (Reference 1) and the USA (Reference 2) define the total fat as the
sum of fatty acids originating from all the lipid classes present in the food matrix and is expressed as TAG equiva-
lents. This requires converting the GC measured FAMEs into TAG equivalents. The fatty acids listed in Tables
1 and 2 cover most of the fatty acid chain lengths (from 4:0 to 26:0) and double bonds (from 0 to 6) that are
encountered in dairy and ruminant fats. The factors for conversion of the individual FAMEs into TAG equivalents
are given in Table 3.
The food labeling regulations in Canada (Reference 1) and the USA (Reference 2) require mandatory declara-
tion of total fat and the amounts of saturated fatty acids (SFAs) and trans fatty acids. Declaration of the amounts
of cis-monounsaturated fatty acids (MUFAs), n-6, and n-3 cis-polyunsaturated fatty acids (PUFAs) is not manda-
tory, however, they could be declared in the nutrition facts table on a voluntary basis. Furthermore, the amounts
of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) could also be declared on the label, but their values
should be displayed outside the nutrition fact table. For nutritional labeling purposes the total fat, saturated, cis-
monounsaturated, and cis-polyunsaturated fatty acid contents are to be determined with this method. For the
determination of vegetable oil fatty acid concentrations the AOCS Ce 1h-05 method is recommended. For the
determination of marine oil or single cell oils (SCOs) fatty acid concentrations the AOCS Ce 1i-07 method is
recommended. In contrast to total fat, fatty acids should be expressed as free fatty acids (FFAs). The factors for
conversion of the individual FAMEs into FFAs are given in Table 2.
(a) Calculate, the amount (in g) of individual fatty acids, expressed as FAME (WFAMEx) and TAG (WTAGx) equiva-
lents as follows.
A x × WTAG is × 1.0059 × R x
WFAMEx =
A is

WTAGx = WFAMEx × FTAGx

Where Ax = area counts for fatty acid x; WTAG is = weight of 13:0 TAG IS (in g) added to the oils; Ais = peak
area counts of the IS; 1.0059, conversion of IS in sample;3 FTAGx = conversion factor for FAMEs to TAGs
for individual fatty acids (Table 3) and Rx = theoretical flame ionization detector correction factor (TCF) for
FAMEs relative to 13:0 FAME IS, respectively (Table 4)4 or the Empirical Correction Factor (ECF) as deter-
mined via GLC-714 (Table 4). TCF should be applied to the analytical data for optimum accuracy (References
14–16) and to minimize variation between laboratories because of differences in calculating response factors.
TCFs are also used for fatty acids where standards are not available. ECFs are needed due to the large deviation

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from TCFs for long chain PUFA of 20 carbons or more and three or more double bonds. The TCFs and ECFs
of a number of FAMEs that are commonly encountered in dairy and ruminant fats are listed in Table 4. The
TCF were calculated using the following formula:

MWx
TCFx =
( N x − 1) × ( AWC ) × 1.4625
Where TCFx = theoretical flame ionization detector response factor for fatty acid x (as methyl ester) with
respect to 13:0 FAME IS; MWx = molecular weight of component x; Nx = number of carbon atoms in the
FAME of component x; AWC = atomic weight of carbon (12.011); 1.4625 = TCF for 13:0 FAME.5,6
   ECFs are required for long chain PUFA FAME of 20 carbons or more and three or more double bonds of
which standards are readily available. The ECFs are calculated using the Certificate of Analysis (COA) sup-
plied with each lot of GLC-714 from Nu-Chek-Prep, Inc. The COA will list both the purity (P) and amount
(AmtFAMEx) of each FAME used to make up the standard. The actual amount (AAmtFAMEx) is calculated by
the following equation:

AAmtFAMEx = P × AmtFAMEx

The response factor (RF) for each peak is determined by the following equation:

RFFAMEx = AreaFAMEx/AAmtFAMEx

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Each RF is then made relative to the 13:0 RF by the following equation:

RRFFAMEx = RFFAMEx/RF13:0

Where RRF = Relative Response Factor

The Empirical Correction Factor (ECF) for each FAME is then calculated by the taking the inverse of the
RRF:

ECFFAMEx = 1/RRFFAMEx

(b) Calculate the amount of total fat in sample (sum of all fatty acids; expressed as TAGs) as follows:

Total fat (g/100 g portion sample) = (∑WTAG/WTS) × 100

Where WTAG = weight of the sum of all TAG, g and WTS = weight of test sample, g

(c) Calculate weight (in g) of each individual fatty acid (Wx) as follows:

Wx (g per sample) = WFAMEx × FFAx

Where WFAMEx = individual fatty acids expressed as FAME and FFAx = conversion factors for conversion of
FAME to their corresponding fatty acids (Table 3).

(d) Calculate weight of saturated fats (sum of all saturated fatty acids) as follows:

Saturated fat (g per 100 g sample) = (∑Saturated Wx/WTS) × 100

Where ∑Saturated Wx = sum of all saturated fatty acids (4:0; 5:0; 6:0; 7:0; 8:0; 9:0; 10:0; iso 10:0; 11:0; 12:0;
iso 12:0; 13:0; anteiso and iso 13:0; 14:0; iso 14:0; 15:0; anteiso and iso 15:0; 2,6,10,14-tetramethyl 15:0; 16:0;
iso 16:0; 3,7,11,15-tetramethyl 16:0; 17:0; anteiso and iso 17:0; 18:0; iso 18:0; 19:0; 20:0; iso 20:0; 21:0; iso
21:0; 22:0; 24:0; 25:0 and 26:0) and WTS = weight of sample, g

(e) Calculate weight of cis-monounsaturated fat, fatty acids containing one double bond, as follows:

cis-monounsaturated fat (g per 100 g sample) = (∑cis-monounsaturated Wx/WTS) × 100

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Where ∑cis-monounsaturated Wx = sum of all cis-monounsaturated fatty acids (11:1; 14:1; 15:1; 16:1; 17:1;
18:1; 19:1; 20:1; 22:1; and 24:1) and WTS = weight of sample, g

(f ) Calculate weight of trans fat as follows:

trans fat (g per 100 g sample) = (Total trans Wx/WTS) × 100

Where total trans Wx = sum of all trans fatty acids excluding trans isomers with a conjugated double bond and
WTS = weight of sample, g

(g) Calculate weight of cis-polyunsaturated fat, fatty acids containing two or more double bonds, as follows:

cis-polyunsaturated fat (g per 100 g sample) = (∑cis-polyunsaturated Wx/WTS) × 100

Where ∑cis-polyunsaturated Wx = sum of all cis-polyunsaturated fatty acids (18:2; 18:3; 18:4; 20:2; 20:3;
20:4; 20:5; 22:2; 22:3; 22:4; 22:5; and 22:6).

(h) Calculate weight of EPA or DHA as follows:

EPA or DHA fat (g per 100 g sample) = (∑EPA or DHA/WTS) × 100

Where WTS = weight of sample, g

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(i) Calculate weight of n-3 PUFAs as follows:

n-3 PUFA (g per 100 g sample) = (∑n-3 PUFA Wx/WTS) × 100

Where ∑n-3 PUFAs Wx = sum of all n-3 PUFAs (9c,12c,15c-18:3; 6c,9c,12c,15c-18:4; 8c,11c,14c,17c-20:4;
5c,8c,11c,14c,17c-20:5; 7c,10c,13c,16c,19c-22:5 and 4c,7c,10c,13c,16c,19c 22:6) and WTS = weight of sam-
ple, g.

(j) Calculate weight of n-6 PUFAs as follows:

n-6 PUFAs (g per 100 g sample) = (∑n-6 PUFAs Wx/WTS) × 100

Where ∑n-6 PUFAs Wx = sum of all n-6 PUFAs (9c,12c-18:2; 6c,9c,12c-18:3; 8c,11c,14c-20:3;
5c,8c,11c,14c-20:4; 7c,10c,13c,16c-22:4; and 4c,7c,10c,13c,16c-22:5) and WTS = weight of sample, g.

PEAK IDENTIFICATION
Identification of the all the FAME components in complex matrices such as dairy and ruminant fats can be quite chal-
lenging. Identification of the peaks in this method has been done by running numerous standards such as; GLC-463
and GLC-714 (Nu-Chek Prep, Elysian, MN), Pamolyn 380 (Hercules, DE), as well as many other individual pure
standards available from a variety of different vendors. In addition, there have been a number of papers published that
use more detailed techniques like argentation chromatographic separations and/or additional GC analyses to separate
out and identify many of the different isomers (References 17–24). These papers all have excellent chromatograms that
were used to identify some of the more difficult peaks in the fats that have been analyzed in this method.

PRECISION
Precision data determined from a 2011 AOCS collaborative study by 9 laboratories analyzing 24 samples are given in
Table 5. Samples were prepared according to Ce 2b-11. Reproducibility and repeatability values are calculated according
to the AOAC/IUPAC Harmonized Protocol for Collaborative Study.

QUALITY ASSURANCE AND CONTROL

1. Blank sample—The first sample in an analysis batch is always a blank (n-hexanes and/or n-heptane). No peaks
should be detected in the blank run except for the internal standard. Analyze a blank sample with every run.
2. Control sample: TBD.

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3. Proficiency test—For external reference the method is tested each year by participation in the AOCS Laboratory
Proficiency Program or any other suitable ring test.

NOTES
1 Nitrogen gas is not acceptable as a carrier gas for this method.
2 No other liner types (e.g., straight through, etc.) should be used with this method.
3 1.0059 is the factor for conversion of the weight of the IS (which is 13:0) from the TAG form to its corresponding

weight of the FAME form. In calculating the factor, hydrolysis (and transmethylation), TAG yields three moles of
FAME. So that the general formula for the conversion factor is as follows:

MW of 13:0 FAME × 3/MW of 13:0 TAG = 228.363 × 3/681.058 = 1.0059

4 1.4625 is the theoretical flame ionization detector response correction factor (TCF) for 13:0 FAME. It is calculated as
follows.

TCF of 13:0 FAME = MW of 13:0/(Nx–1) (Atomic weight of carbon)

= 228.363/(14–1) × 12.011 = 1.4625

Here Nx is the number of carbon atoms in 13:0 FAME, which is 14. 12.011 is the atomic weight of carbon.

All rights reserved. No part of this method may be reproduced or transmitted in any form or by any means without written permission of the publisher.
5 It
is important to understand the difference (and hence the relationship) between TCF and TRF.
(a) Theoretical flame ionization response factor (TRF) is actually the proportion (with respect to the total weight,
which is the molecular weight) of the total amount of carbon atoms that are responding to the FID. For fatty acids,
the carbons that are responding to FID are CH, CH2, and CH3 units. Carbonyl carbon and ester carbonyls carbon
do not respond to FID. So that in FAME, all the carbon atoms are active, except the ester carbon. Thus, there are
Nx–1 active carbon atoms in a FAME. So that the TRF for a FAME would be:

TRF = (Nx–1) (Atomic Weight of Carbon)/MW of FAME

TRF of 13:0 FAME = 13 × 12.011/228.363 = 0.6837

(b) Since the FID response depends on the proportion of active carbon atoms, to determine the actual weight of a FAME,
the experimental measured area (or the weight) needs to be corrected. This factor is termed as TCF and it is equal to
1/TRF. In other words formula for TCF is as follows:

TCF = MW/(Nx–1) (Atomic Weight of Carbon)

The TCF of 13:0 FAME is 1.4625

6 TheTRF of 18:0 FAME is 0.7243. Therefore, its TCF is 1/0.7243 = 1.3806. Thus TCF of 18:0 FAME with respect
to 13:0 FAME = 1.3806/1.4625 = 0.9440 (Table 3).

REFERENCES
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Claims). Department of Health, Canada Gazette, Part 11. January 1, 2003.
2. Department of Health and Human Services. Food and Drug Administration. 21 CFR Part 101 [Docket No. 94P-
0036]. Food Labeling: Trans Fatty Acids in Nutrition Labelling, Nutrient Content Claims, and Health Claims.
Washington, DC. July 2003, pp. 254.
3. ISO 6353, Reagents for Chemical Analysis, Part 2 (1983) and 3 (1987); Specifications.
4. ISO 3696, Water for Analytical Laboratory Use—Specifications and Test Methods (1987).
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6. S.W. Christopherson and R.L. Glass, J. Dairy Sci. 52:1289–1290 (1969).
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270–300.
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J.L. Sebedio and E.G. Perkins, AOCS Press, Champaign, IL, 1995, pp. 277–289.
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Chromat. A, 1145, 222–228, 2007.
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382, 2007.
21. F. Dionisi, P.A. Golay, and L.B. Fay, Analytica Chimica Acta, 465, 395–407, 2002.
22. D. Precht and J. Molkentin, Eur. J. Lipid Sci. Technol. 102–113 (2000).
23. J.K.G. Kramer, M. Hernandez, C. Cruz-Hernandez, J. Kraft, M.E.R. Dugan, Lipids 43:259–273 (2008).
24. J.A.G. Roach, M.P Yurawecz, J.K.G. Kramer, M.M. Mossoba, K. Eulitz, and Y. Ku, Lipids, 35:797–802 (2000).
All rights reserved. No part of this method may be reproduced or transmitted in any form or by any means without written permission of the publisher.

Copyright © 2017 by the AOCS Page 7 of 58

C ommercial fats and O ils

Ce 1j-07 ■ cis-, trans-, Saturated, Monounsaturated, and Polyunsaturated Fatty Acids

TABLE 1 Example elution order of fatty acid methyl esters (FAME). Separations were on a SPTM-2560 (100 m x 0.25 mm x 0.20
µm). Column temperature was 180 °C (32 min) to 215 °C @ 20 °C/min (hold 31.25 min). Carrier gas was hydrogen at 1.0 mL/min
(constant flow) with a linear velocity of 26 cm/s. Pressure was 24.63 psi @ 180 °C. Injection and FID temperatures were 235 °C and
325 °C, respectively. Split ratio was 1:100. Please note that different columns will have different retention times and sometimes the
order of elution of the fatty acids can change for later eluting compounds. Fatty acids in parentheses coelute with the peaks listed
in the same cell for this column.
Tr Tr Tr
Fatty Acids (min) Fatty Acids (min) Fatty Acids (min)

2:0 7.750 4t-18:1 19.640 9t,12c,15c-18:3 29.950

4:0 7.909 5t-18:1 19.934 11c-20:1 29.979
5:0 8.000 6t-8t-18:1 20.158 9c,12c,15c-18:3 30.377
6:0 8.108 9t-18:1 20.256 ct-6,8-18:2 30.010
7:0 8.249 10t-18:1 20.387 7c,9c-18:2 31.388
8:0 8.433 11t-18:1 20.446 9c,11t-18:2 31.466
9:0 8.674 12t-18:1 20.615 8t,10c-18:2 31.600
iso 10:0 8.800 13t-14t-18:1 (6c-8c-18:1) 20.751 10c,12t-18:2 31.700
10:0 8.984 9c-18:1 (18:1n-9) (15t-10c-18:1) 21.037 9t,11c-18:2 31.962
11:0 9.442 11c-18:1 21.268 11c,13t-18:2 32.100
iso 12:0 9.618 12c-18:1 21.508 10t,12c-18:2 32.355

All rights reserved. No part of this method may be reproduced or transmitted in any form or by any means without written permission of the publisher.
12:0 9.917 13c-18:1 21.794 21:0 32.371
iso 13:0 10.215 16t-18:1 21.952 8c,10c-18:2 32.500
anteiso 13:0 10.390 14c-18:1 22.148 9c,11c-18:2 32.687
13:0 (IS) 10.607 15c-18:1 (19:0) 22.309 10c,12c-18:2 32.750
iso 14:0 10.993 t,t-NMID_1 22.500 11c,13c-18:2 32.800
14:0 11.505 t,t-NMID_2 22.622 t,t-conj.-18:2_1 33.000
12c-13:1 11.705 t,t-NMID_3 22.800 t,t-conj.-18:2_2 33.281
iso 15:0 12.011 9t,12t-18:2 22.941 12t,14t-18:2 33.300
anteiso 15:0 12.212 9t,13c-18:2/8t,12c-18:2 23.227 11t,13t-18:2 33.489
9t-14:1 12.289 17-cyclo 23.487 t,t-8,10;9,11;10,12-18:2 33.599
9c-14:1 (14:1n-5) 12.616 9c,12t-18:2/8t,13c-18:2 23.743 7t,9t-18:2 33.738
15:0 12.666 16c-18:1 23.847 6c,9c,12c,15c-18:4 33.904
iso 16:0 13.340 9t,12c-18:2 24.302 11c,14c-20:2 34.133
10c-15:1 (15:1n-5) 13.983 11t,15c-18:2 24.403 22:0 35.425
16:0 14.237 9c,12c-18:2 24.794 8c,11c,14c-20:3 (20:3n-6) 36.134
iso 17:0 15.079 9c,15c-18:2 25.190 13c-22:1 (22:1n-9) 37.164
9t-16:1 15.206 9t,12t,15t-18:3 26.578 11c,14c,17c-20:3 (20:3n-3) 37.337
11t-12t-16:1 15.384 20:0 26.604 5c,8c,11c,14c-20:4 (20:4n-6) 37.732
7c-13t-16:1/anteiso 17:0 15.530 9t,12t,15c-18:3 27.885 23:0 38.000
9c-16:1 (16:1n-7) 15.730 9t,12c,15t-18:3 27.971 13c,16c-22:2 39.980
10c-16:1 15.868 6c,9c,12c-18:3 28.249 5c,8c,11c,14c,17c-20:5 (20:5n-3) 41.430
11c-16:1 16.026 9c,12t,15t-18:3 + 9c,12c,15t-18:3 28.578 24:0 41.655
17:0 16.177 6t-10t-20:1 28.885 15c-24:1 43.196
13c-16:1 16.308 11t-20:1 28.949 13c,16c,19c-22:3 43.667
iso 18:0 17.347 12t-13t-20:1 29.000 7c,10c,13c,16c-22:4 44.760
9t-17:1 17.602 14t-20:1 29.400 25:0 45.232
9c-17:1 (17:1n-7) 18.000 9c-20:1 29.549 7c,10c,13c,16c,19c-22:5 49.443
18:0 18.845 9c,12t,15c-18:3 29.549 4c,7c,10c,13c,16c,19c-22:6 51.719

Page 8 of 58 Copyright © 2017 by the AOCS

C ommercial fats and O ils

Ce 1j-07 ■ cis-, trans-, Saturated, Monounsaturated, and Polyunsaturated Fatty Acids

TABLE 2 Example elution order of fatty acid methyl esters (FAME). Separations were on a SP™ -2560 (100 m × 0.25 mm × 0.20
µm). Column temperature was 180 °C (32 min) to 215 °C @ 20 °C/min (hold 31.25 min). Carrier gas was helium at 2.0 mL/min
(constant flow) with a linear velocity of 28 cm/s. Pressure was 63.29 psi @ 180 °C. Injection and FID temperatures were 235 °C and
325 °C, respectively. Split ratio was 1:100. Please note that different columns will have different retention times and sometimes the
order of elution of the fatty acids can change for later eluting compounds. Fatty acids in parentheses coleute with the peaks listed
in the same cell for this column.
Tr Tr Tr
Fatty Acids (min) Fatty Acids (min) Fatty Acids (min)

2:0 6.400 4t-18:1 16.446 9t,12c,15c-18:3 25.000

4:0 6.542 5t-18:1 16.684 11c-20:1 25.230
5:0 6.615 6t-8t-18:1 16.869 9c,12c,15c-18:3 25.479
6:0 6.709 9t-18:1 16.990 ct-6,8-18:2 25.976
7:0 6.800 10t-18:1 17.043 7c,9c-18:2 26.284
8:0 6.982 11t-18:1 17.098 9c,11t-18:2 26.342
9:0 7.100 12t-18:1 17.246 8t,10c-18:2 26.400
iso 10:0 7.300 13t-14t-18:1 (6c-8c-18:1) 17.372 10c,12t-18:2 26.500
10:0 7.446 9c-18:1 (18:1n-9) (15t-10c-18:1) 17.642 9t,11c-18:2 26.714
11:0 7.788 11c-18:1 17.810 11c,13t-18:2 27.163
iso 12:0 7.830 12c-18:1 17.998 10t,12c-18:2 27.250
All rights reserved. No part of this method may be reproduced or transmitted in any form or by any means without written permission of the publisher.

12:0 8.232 13c-18:1 18.230 21:0 27.367

iso 13:0 8.479 16t-18:1 18.377 8c,10c-18:2 27.750
anteiso 13:0 8.621 14c-18:1 18.563 9c,11c-18:2 27.886
13:0 (IS) 8.813 15c-18:1 (19:0) 18.667 10c,12c-18:2 28.100
iso 14:0 9.136 t,t-NMID_1 18.800 11c,13c-18:2 28.200
14:0 9.569 t,t-NMID_2 18.947 t,t-conj.-18:2_1 28.400
12c-13:1 9.820 t,t-NMID_3 19.000 t,t-conj.-18:2_2 28.455
iso 15:0 9.995 9t,12t-18:2 19.204 12t,14t-18:2 28.700
anteiso 15:0 10.227 9t,13c-18:2/8t,12c-18:2 19.435 11t,13t-18:2 28.898
9t-14:1 10.350 17-cycclo 19.670 t,t-8,10;9,11;10,12-18:2 29.159
9c-14:1 (14:1n-5) 10.496 9c,12t-18:2/8t,13c-18:2 19.871 7t,9t-18:2 29.300
15:0 10.545 16c-18:1 19.929 6c,9c,12c,15c-18:4 29.500
iso 16:0 11.115 9t,12c-18:2 20.272 11c,14c-20:2 30.383
10c-15:1 (15:1n-5) 11.648 11t,15c-18:2 20.380 22:0 33.176
16:0 11.846 9c,12c-18:2 20.785 8c,11c,14c-20:3 (20:3n-6) 33.624
iso 17:0 12.582 9c,15c-18:2 21.082 13c-22:1 (22:1n-9) 34.681
9t-16:1 12.622 9t,12t,15t-18:3 22.200 11c,14c,17c-20:3 (20:3n-3) 34.945
11t-12t-16:1 12.655 20:0 22.309 5c,8c,11c,14c-20:4 (20:4n-6) 35.880
7c-13t-16:1/ anteiso 17:0 12.962 9t,12t,15c-18:3 23.200 23:0 36.010
9c-16:1 (16:1n-7) 13.140 9t,12c,15t-18:3 23.300 13c,16c-22:2 36.756
10c-16:1 13.250 6c,9c,12c-18:3 23.616 5c,8c,11c,14c,17c-20:5 (20:5n-3) 38.441
11c-16:1 13.368 9c,12t,15t-18:3 + 9c,12c,15t-18:3 23.800 24:0 38.954
17:0 13.532 6t-10t-20:1 24.216 15c-24:1 40.687
13c-16:1 13.606 11t-20:1 24.412 13c,16c,19c-22:3 41.375
iso 18:0 14.498 12t-13t-20:1 24.500 7c,10c,13c,16c-22:4 41.921
9t-17:1 14.697 14t-20:1 24.700 25:0 45.232
9c-17:1 (17:1n-7) 15.038 9c-20:1 24.834 7c,10c,13c,16c,19c-22:5 45.431
18:0 15.732 9c,12t,15c-18:3 24.900 4c,7c,10c,13c,16c,19c-22:6 47.345

Copyright © 2017 by the AOCS Page 9 of 58

C ommercial fats and O ils

Ce 1j-07 ■ cis-, trans-, Saturated, Monounsaturated, and Polyunsaturated Fatty Acids

TABLE 3 Factors for conversion of FAME to fatty acids and TAG equivalents.
Fatty Acida FFAxb FTAGxc Fatty Acida FFAxb FTAGxc

4:0 0.8627 0.9868 18:1 0.9527 0.9955

5:0 0.8792 0.9884 18:2 0.9524 0.9954
6:0 0.8923 0.9897 18:3 0.9520 0.9954
7:0 0.9027 0.9907 18:4 0.9517 0.9954
8:0 0.9114 0.9915 19:0 0.9551 0.9957
9:0 0.9186 0.9922 20:0 0.9570 0.9959
10:0 0.9247 0.9928 20:1 0.9568 0.9959
10:1 0.9239 0.9927 20:2 0.9565 0.9958
11:0 0.9300 0.9933 20:3 0.9562 0.9958
11:1 0.9293 0.9932 20:4 0.9560 0.9958
12:0 0.9346 0.9937 20:5 0.9557 0.9958
12:1 0.9339 0.9937 21:0 0.9588 0.9961
13:0 0.9386 0.9941 22:0 0.9604 0.9962
13:1 0.9380 0.9941 22:1 0.9602 0.9962
14:0 0.9421 0.9945 22:2 0.9600 0.9962
13:1 0.9380 0.9941 22:3 0.9598 0.9961

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14:1 0.9417 0.9944 22:4 0.9595 0.9961
15:0 0.9453 0.9948 22:5 0.9593 0.9961
15:1 0.9449 0.9947 22:6 0.9590 0.9961
16:0 0.9481 0.9950 23:0 0.9620 0.9964
16:1 0.9477 0.9950 24:0 0.9633 0.9965
17:0 0.9507 0.9953 24:1 0.9632 0.9965
17:1 0.9503 0.9952 25:0 0.9646 0.9966
18:0 0.9530 0.9955 26:0 0.9658 0.9967
aOnly one factor is given for all positional and geometric isomers and for branched-chain FAME, as the factors are dependent only on the
content of carbon to which hydrogen is bonded.
bF
FAx is the conversion factor for conversion of FAMEs to corresponding fatty acids.
cF
TAGx is the conversion factor for conversion of FAMEs to corresponding TAG equivalents.

Page 10 of 58 Copyright © 2017 by the AOCS

C ommercial fats and O ils

Ce 1j-07 ■ cis-, trans-, Saturated, Monounsaturated, and Polyunsaturated Fatty Acids

TABLE 4 Theoretical flame ionization detector theoretical and empirical correction factors (TCFs/ECFs) for fatty acid
methyl esters (FAME)a
FAME TCF FAME TCF FAME TCF/ECF FAME TCF/ECF

4:0 1.4534 14:0 0.9856 18:3 0.9249 22:1 0.9124

5:0 1.3224 14:1 0.9774 18:4 0.9185 22:2 0.9071
6:0 1.2351 15:0 0.9731 18:5 0.9122 22:3 0.9019
7:0 1.1727 15:1 0.9655 19:0 0.9364 22:4 0.8967
8:0 1.1259 16:0 0.9622 19:1 0.9303 22:5 ECFb
9:0 1.0896 16:1 0.9550 20:0 0.9295 22:6 ECFb
10:0 1.0604 16:2 0.9479 20:1 0.9237 23:0 0.9124
10:1 1.0490 16:3 0.9407 20:2 0.9180 24:0 0.9076
11:0 1.0366 16:4 0.9335 20:3 ECFb 24:1 0.9029
11:1 1.0262 17:0 0.9526 20:4 ECFb 25:0 0.9033
12:0 1.0168 17:1 0.9458 20:5 ECFb 26:0 0.8993
12:1 1.0072 18:0 0.9440 21:0 0.9232
13:0 (IS) 1.0000 18:1 0.9377 21:5 0.8959
13:1 0.9912 18:2 0.9313 22:0 0.9176
All rights reserved. No part of this method may be reproduced or transmitted in any form or by any means without written permission of the publisher.

aAtomic weights used: carbon 12.011; hydrogen, 1.0079; oxygen, 15.994. Factors are relative to 13:0, which has a factor of 1.0000 by
definition. Only one factor is given for all positional and geometric isomers and for branched-chain FAME, as the factors are dependent only on
the content of carbon to which hydrogen is bonded. Calculations were adopted from Reference 8.
bECFs are to be calculated from GLC-714. Any positional or geometrical isomers will have the same TCFs and ECFs.

TABLE 5 Results of cis-, trans-, Saturated, Monounsaturated, and Polyunsaturated fatty acid determination by
9 laboratories in a collaborative study organized by AOCS in 2011. FAMEs were analyzed using AOCS Ce 1j-07.
Total Fat Total Fat Total Total Total Total cis Total Total cis Total n-3 Total n-6
(TAG%)* (FA%)* Saturated trans MUFA MUFA PUFA** PUFA** PUFA** PUFA**

Anyhydrous milk fat

n 9 9 9 9 9 9 9 9 9 9
Mean 93.59 88.92 58.27 5.11 26.39 21.59 3.78 3.25 0.53 2.64
Repeatability (Sr) 2.48 2.39 1.32 0.41 1.21 0.27 0.39 0.20 0.06 0.04
%RSDr 2.65 2.69 2.26 8.06 4.60 1.27 10.23 6.08 11.21 1.60
r (2.8×Sr) 6.94 6.69 3.69 1.15 3.40 0.77 1.08 0.55 0.17 0.12
Reproducibility (SR) 2.48 2.39 2.39 0.67 1.53 0.73 0.54 0.20 0.14 0.24
%RSDR 2.65 2.69 4.10 13.14 5.81 3.38 14.28 6.08 26.83 8.94
R (2.8×SR) 6.94 6.69 6.69 1.88 4.29 2.05 1.51 0.55 0.40 0.66

Tallow
n 9 9 9 9 9 9 9 9 9 9
Mean 99.68 95.21 45.44 7.14 46.53 39.31 2.65 1.99 0.33 1.66
Repeatability (Sr) 1.01 0.96 1.03 0.19 0.78 0.33 0.12 0.05 0.01 0.04
%RSDr 1.01 1.01 2.26 2.73 1.67 0.84 4.43 2.41 3.80 2.33
r (2.8×Sr) 2.82 2.69 2.88 0.55 2.18 0.92 0.33 0.13 0.04 0.11
Reproducibility (SR) 1.66 1.58 1.36 0.30 1.71 0.80 0.34 0.24 0.07 0.18
%RSDR 1.66 1.66 2.99 4.20 3.68 2.03 12.74 12.24 22.64 11.13
R (2.8×SR) 4.64 4.42 3.81 0.84 4.80 2.24 0.94 0.68 0.21 0.52

(continued)

Copyright © 2017 by the AOCS Page 11 of 58

C ommercial fats and O ils

Ce 1j-07 ■ cis-, trans-, Saturated, Monounsaturated, and Polyunsaturated Fatty Acids

TABLE 5 Continued

Total Fat Total Fat Total Total Total Total cis Total Total cis Total n-3 Total n-6
(TAG%)* (FA%)* Saturated trans MUFA MUFA PUFA** PUFA** PUFA** PUFA**

Chocolate cake mix

n 9 9 9 9 9 9 9 9 9 9
Mean 10.81 10.34 4.44 0.90 3.21 2.47 2.65 2.49 0.23 2.27
Repeatability (Sr) 0.23 0.22 0.11 0.03 0.06 0.04 0.07 0.06 0.01 0.05
%RSDr 2.09 2.09 2.37 3.29 1.79 1.72 2.77 2.60 2.46 2.20
r (2.8×Sr) 0.63 0.61 0.30 0.08 0.16 0.12 0.21 0.18 0.02 0.14
Reproducibility (SR) 0.42 0.40 0.25 0.07 0.10 0.09 0.12 0.10 0.01 0.08
%RSDR 3.92 3.91 5.68 7.43 3.25 3.45 4.67 4.20 4.63 3.62
R (2.8×SR) 1.19 1.13 0.71 0.19 0.29 0.24 0.35 0.29 0.03 0.23

Cheese Powder
n 9 9 9 9 9 9 9 9 9 9
Mean 29.78 28.38 13.49 7.27 14.08 6.95 1.24 0.97 0.14 0.83
Repeatability (Sr) 0.43 0.41 0.22 0.12 0.22 0.12 0.04 0.02 0.00 0.01
%RSDr 1.43 1.44 1.62 1.68 1.55 1.66 3.10 2.22 3.22 1.60
r (2.8×Sr) 1.19 1.14 0.61 0.34 0.61 0.32 0.11 0.06 0.01 0.04

All rights reserved. No part of this method may be reproduced or transmitted in any form or by any means without written permission of the publisher.
Reproducibility (SR) 2.39 2.28 0.71 0.37 0.75 0.57 0.12 0.02 0.03 0.05
%RSDR 8.02 8.02 5.24 5.04 5.31 8.18 9.54 2.46 19.48 6.58
R (2.8×SR) 6.69 6.38 1.98 1.03 2.09 1.59 0.33 0.07 0.08 0.15

DHA/EPA fortified infant formula

n 9 9 9 9 9 9 9 9 9 9
Mean 28.94 27.58 12.07 0.15 10.15 10.07 5.21 5.14 0.53 4.62
Repeatability (Sr) 0.66 0.63 0.07 0.02 0.20 0.20 0.13 0.12 0.01 0.09
%RSDr 2.29 2.29 0.59 15.01 1.96 2.02 2.43 2.41 2.16 2.01
r (2.8×Sr) 1.86 1.77 0.20 0.06 0.56 0.57 0.35 0.35 0.03 0.26
Reproducibility (SR) 1.48 1.42 0.45 0.12 0.65 0.65 0.39 0.38 0.02 0.33
%RSDR 5.11 5.14 3.77 78.47 6.39 6.46 7.52 7.48 3.49 7.08
R (2.8×SR) 4.14 3.97 1.27 0.33 1.82 1.82 1.10 1.08 0.05 0.92

Extruded dog food (high protein and high fat)

n 9 9 9 9 9 9 9 9 9 9
Mean 22.04 21.06 6.72 0.31 9.75 9.31 4.76 4.72 0.33 4.36
Repeatability (Sr) 1.49 1.42 0.43 0.11 0.64 0.61 0.31 0.30 0.03 0.28
%RSDr 6.75 6.75 6.38 34.60 6.53 6.60 6.44 6.41 9.24 6.41
r (2.8×Sr) 4.17 3.98 1.20 0.30 1.78 1.72 0.86 0.85 0.09 0.78
Reproducibility (SR) 1.49 1.42 0.43 0.11 0.64 0.61 0.31 0.30 0.04 0.28
%RSDR 6.75 6.75 6.38 34.97 6.53 6.60 6.44 6.41 11.77 6.41
R (2.8×SR) 4.17 3.98 1.20 0.31 1.78 1.72 0.86 0.85 0.11 0.78
Oatmeal Cookie
n 9 9 9 9 9 9 9 9 9 9
Mean 19.19 18.33 8.95 0.05 7.12 7.08 2.25 2.20 0.05 2.16
Repeatability (Sr) 0.27 0.26 0.20 0.02 0.08 0.08 0.02 0.02 0.00 0.02
%RSDr 1.42 1.42 2.23 28.55 1.13 1.11 1.03 1.02 2.41 0.99
r (2.8×Sr) 0.76 0.73 0.56 0.04 0.22 0.22 0.06 0.06 0.00 0.06
Reproducibility (SR) 0.72 0.68 0.39 0.02 0.31 0.31 0.09 0.10 0.00 0.09
%RSDR 3.74 3.72 4.30 44.84 4.38 4.40 4.00 4.33 4.89 4.36
R (2.8×SR) 2.01 1.91 1.08 0.07 0.87 0.87 0.25 0.27 0.01 0.26

(continued)

Page 12 of 58 Copyright © 2017 by the AOCS

C ommercial fats and O ils

Ce 1j-07 ■ cis-, trans-, Saturated, Monounsaturated, and Polyunsaturated Fatty Acids

TABLE 5 Continued

Total Fat Total Fat Total Total Total Total cis Total Total cis Total n-3 Total n-6
(TAG%)* (FA%)* Saturated trans MUFA MUFA PUFA** PUFA** PUFA** PUFA**
Evaporated milk
n 9 9 9 9 9 9 9 9 9 9
Mean 6.28 5.97 3.93 0.33 1.78 1.47 0.23 0.19 0.03 0.16
Repeatability (Sr) 0.29 0.27 0.17 0.03 0.09 0.08 0.02 0.01 0.00 0.01
%RSDr 4.61 4.57 4.44 8.45 5.11 5.17 7.06 4.73 8.53 4.38
r (2.8×Sr) 0.81 0.76 0.49 0.08 0.25 0.21 0.05 0.03 0.01 0.02
Reproducibility (SR) 0.29 0.27 0.22 0.05 0.09 0.08 0.03 0.02 0.01 0.02
%RSDR 4.61 4.57 5.54 15.89 5.11 5.17 14.13 10.75 32.47 10.66
R (2.8×SR) 0.81 0.76 0.61 0.15 0.25 0.21 0.09 0.06 0.03 0.05

Peanut butter
n 9 9 9 9 9 9 9 9 9 9
Mean 54.01 51.69 10.11 0.06 30.28 30.13 11.23 11.17 0.08 10.79
Repeatability (Sr) 1.17 1.12 0.33 0.01 0.70 0.74 0.29 0.40 0.02 0.26
%RSDr 2.16 2.16 3.29 21.71 2.31 2.47 2.61 3.62 21.36 2.37
r (2.8×Sr) 3.26 3.12 0.93 0.03 1.96 2.08 0.82 1.13 0.05 0.72
All rights reserved. No part of this method may be reproduced or transmitted in any form or by any means without written permission of the publisher.

Reproducibility (SR) 3.10 2.96 0.79 0.04 1.57 1.60 1.46 1.48 0.08 1.14
%RSDR 5.73 5.73 7.83 75.73 5.17 5.32 13.02 13.21 98.99 10.58
R (2.8×SR) 8.67 8.30 2.22 0.12 4.38 4.49 4.10 4.13 0.22 3.19

Yogurt (plain)
n 9 9 9 9 9 9 9 9 9 9
Mean 5.80 5.51 3.60 0.32 1.65 1.37 0.23 0.19 0.03 0.15
Repeatability (Sr) 0.55 0.53 0.39 0.03 0.13 0.11 0.03 0.02 0.01 0.01
%RSDr 9.54 9.55 10.90 7.94 8.09 8.26 12.67 13.02 42.60 9.08
r (2.8×Sr) 1.55 1.47 1.10 0.07 0.37 0.32 0.08 0.07 0.04 0.04
Reproducibility (SR) 0.55 0.53 0.39 0.03 0.15 0.14 0.03 0.02 0.02 0.01
%RSDR 9.54 9.55 10.90 7.94 9.23 9.92 12.83 13.02 59.74 9.08
R (2.8×SR) 1.55 1.47 1.10 0.07 0.43 0.38 0.08 0.07 0.05 0.04

Canned cat food

n 9 9 9 9 9 9 9 9 9 9
Mean 5.70 5.44 1.71 0.05 2.40 2.01 1.25 1.24 0.42 0.76
Repeatability (Sr) 0.66 0.64 0.15 0.01 0.25 0.23 0.12 0.11 0.05 0.07
%RSDr 11.59 11.67 8.83 13.55 10.58 11.68 9.22 9.19 11.58 9.53
r (2.8×Sr) 1.85 1.78 0.42 0.02 0.71 0.66 0.32 0.32 0.14 0.20
Reproducibility (SR) 0.80 0.77 0.26 0.03 0.46 0.93 0.23 0.22 0.24 0.14
%RSDR 14.09 14.11 15.08 49.55 19.14 46.30 18.07 17.88 56.88 18.16
R (2.8×SR) 2.25 2.15 0.72 0.08 1.29 2.60 0.63 0.62 0.68 0.39

DHA/EPA fortified orange juice

n 9 9 9 9 9 9 9 9 9 9
Mean 0.12 0.11 0.03 0.00 0.03 0.03 0.03 0.03 0.02 0.01
Repeatability (Sr) 0.02 0.02 0.00 0.00 0.00 0.00 0.01 0.01 0.00 0.01
%RSDr 15.70 17.14 13.26 0.00 8.92 16.09 29.64 29.64 25.75 46.97
r (2.8×Sr) 0.05 0.05 0.01 0.00 0.01 0.01 0.03 0.03 0.01 0.02
Reproducibility (SR) 0.02 0.02 0.02 0.00 0.00 0.01 0.02 0.02 0.01 0.01
%RSDR 19.39 20.56 51.60 0.00 12.41 19.13 58.12 58.12 67.03 58.62
R (2.8×SR) 0.06 0.06 0.05 0.00 0.01 0.01 0.05 0.05 0.03 0.02

(continued)

Copyright © 2017 by the AOCS Page 13 of 58

C ommercial fats and O ils

Ce 1j-07 ■ cis-, trans-, Saturated, Monounsaturated, and Polyunsaturated Fatty Acids

TABLE 5 Continued

Total Fat Total Fat Total Total Total Total cis Total Total cis Total n-3 Total n-6
(TAG%)* (FA%)* Saturated trans MUFA MUFA PUFA** PUFA** PUFA** PUFA**

Butter
n 9 9 9 9 9 9 9 9 9 9
Mean 71.12 67.76 36.52 2.49 22.31 20.23 8.77 8.36 1.73 6.91
Repeatability (Sr) 1.62 1.54 1.00 0.13 0.63 0.54 0.44 0.39 0.14 0.14
%RSDr 2.28 2.28 2.73 5.34 2.81 2.68 4.99 4.72 8.25 2.05
r (2.8×Sr) 4.54 4.32 2.80 0.37 1.75 1.52 1.23 1.10 0.40 0.40
Reproducibility (SR) 4.74 4.52 2.32 0.43 1.94 1.74 0.89 0.88 0.15 0.50
%RSDR 6.66 6.68 6.35 17.29 8.69 8.62 10.15 10.50 8.89 7.17
R (2.8×SR) 13.26 12.66 6.49 1.21 5.43 4.88 2.49 2.46 0.43 1.39

Whole egg powder

n 9 9 9 9 9 9 9 9 9 9
Mean 40.24 38.47 14.34 0.43 16.73 16.35 7.36 7.31 0.37 6.84
Repeatability (Sr) 1.20 1.15 0.41 0.02 0.56 0.56 0.22 0.21 0.02 0.20
%RSDr 2.98 2.98 2.85 4.79 3.33 3.41 2.95 2.92 5.99 2.88
r (2.8×Sr) 3.36 3.21 1.14 0.06 1.56 1.56 0.61 0.60 0.06 0.55

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Reproducibility (SR) 1.20 1.15 0.50 0.06 0.56 0.56 0.35 0.33 0.03 0.38
%RSDR 2.98 2.98 3.52 12.99 3.33 3.41 4.79 4.50 9.04 5.56
R (2.8×SR) 3.36 3.21 1.41 0.16 1.56 1.56 0.99 0.92 0.09 1.06

Full-fat soy flour flakes

n 9 9 9 9 9 9 9 9 9 9
Mean 23.05 22.05 3.70 0.02 4.73 4.74 13.49 13.48 1.61 11.69
Repeatability (Sr) 0.23 0.22 0.11 0.00 0.12 0.05 0.39 0.39 0.03 0.12
%RSDr 0.98 0.98 3.02 22.12 2.55 1.10 2.91 2.91 1.61 1.07
r (2.8×Sr) 0.63 0.61 0.31 0.01 0.34 0.15 1.10 1.10 0.07 0.35
Reproducibility (SR) 0.66 0.62 0.39 0.01 0.14 0.12 0.39 0.39 0.15 0.25
%RSDR 2.85 2.83 10.42 73.10 2.93 2.57 2.91 2.91 9.34 2.13
R (2.8×SR) 1.84 1.75 1.08 0.04 0.39 0.34 1.10 1.10 0.42 0.70

Encapsulated DHA/EPA
n 9 9 9 9 9 9 9 9 9 9
Mean 56.05 53.66 18.18 0.68 14.09 13.33 21.02 20.63 18.25 2.19
Repeatability (Sr) 1.31 1.26 0.43 0.08 0.37 0.35 0.47 0.46 0.41 0.09
%RSDr 2.34 2.34 2.36 12.13 2.65 2.64 2.23 2.21 2.25 4.24
r (2.8×Sr) 3.68 3.52 1.20 0.23 1.05 0.98 1.31 1.28 1.15 0.26
Reproducibility (SR) 3.32 3.15 0.95 0.23 0.81 0.67 1.83 1.75 1.46 0.93
%RSDR 5.92 5.87 5.22 33.82 5.72 5.01 8.71 8.50 8.01 42.40
R (2.8×SR) 9.29 8.83 2.66 0.64 2.26 1.87 5.13 4.91 4.09 2.60

Creamy ranch dressing

n 9 9 9 9 9 9 9 9 9 9
Mean 46.18 44.16 7.12 0.24 9.66 9.63 25.68 25.51 3.40 23.18
Repeatability (Sr) 1.91 1.82 0.32 0.01 0.52 0.52 0.98 0.98 0.11 0.92
%RSDr 4.13 4.13 4.51 3.71 5.39 5.40 3.83 3.84 3.18 3.98
r (2.8×Sr) 5.34 5.11 0.90 0.02 1.46 1.46 2.75 2.74 0.30 2.58
Reproducibility (SR) 1.91 1.82 0.32 0.16 0.52 0.52 2.36 2.37 0.11 1.16
%RSDR 4.13 4.13 4.51 65.50 5.39 5.40 9.17 9.30 3.18 5.02
R (2.8×SR) 5.34 5.11 0.90 0.44 1.46 1.46 6.60 6.64 0.30 3.26

(continued)

Page 14 of 58 Copyright © 2017 by the AOCS

C ommercial fats and O ils

Ce 1j-07 ■ cis-, trans-, Saturated, Monounsaturated, and Polyunsaturated Fatty Acids

TABLE 5 Continued

Total Fat Total Fat Total Total Total Total cis Total Total cis Total n-3 Total n-6
(TAG%)* (FA%)* Saturated trans MUFA MUFA PUFA** PUFA** PUFA** PUFA**

Potato chips
n 9 9 9 9 9 9 9 9 9 9
Mean 36.01 34.44 6.35 0.22 14.16 14.02 13.70 13.59 0.12 13.45
Repeatability (Sr) 1.75 1.67 0.31 0.06 0.74 0.72 0.63 0.61 0.02 0.60
%RSDr 4.85 4.85 4.95 26.22 5.21 5.14 4.57 4.50 13.30 4.50
r (2.8×Sr) 4.89 4.68 0.88 0.16 2.07 2.02 1.75 1.71 0.04 1.69
Reproducibility (SR) 1.75 1.67 0.31 0.14 0.74 0.72 0.63 0.61 0.04 0.60
%RSDR 4.85 4.85 4.95 62.69 5.21 5.14 4.57 4.50 37.66 4.50
R (2.8×SR) 4.89 4.68 0.88 0.39 2.07 2.02 1.75 1.71 0.12 1.69

Cheese powder duplicate

n 9 9 9 9 9 9 9 9 9 9
Mean 30.10 28.69 13.35 7.20 13.85 6.96 1.31 0.98 0.13 0.85
Repeatability (Sr) 0.45 0.43 0.27 0.10 0.22 0.11 0.05 0.02 0.01 0.02
%RSDr 1.49 1.48 2.05 1.36 1.59 1.56 4.14 2.12 5.11 2.18
r (2.8×Sr) 1.25 1.19 0.77 0.27 0.62 0.30 0.15 0.06 0.02 0.05
Reproducibility (SR) 1.18 1.11 0.30 0.31 0.37 0.21 0.05 0.10 0.04 0.08
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%RSDR 3.91 3.88 2.22 4.27 2.70 3.04 4.14 10.08 29.05 9.25
R (2.8×SR) 3.30 3.12 0.83 0.86 1.05 0.59 0.15 0.28 0.11 0.22

Frozen cheese pizza

n 9 9 9 9 9 9 9 9 9 9
Mean 8.06 7.66 3.56 0.37 2.25 1.68 1.33 1.29 0.05 1.23
Repeatability (Sr) 0.67 0.64 0.30 0.04 0.20 0.16 0.13 0.12 0.01 0.12
%RSDr 8.32 8.37 8.54 10.04 9.10 9.76 9.45 9.46 20.01 9.39
r (2.8×Sr) 1.88 1.80 0.85 0.10 0.57 0.46 0.35 0.34 0.03 0.32
Reproducibility (SR) 1.59 1.51 1.63 0.07 0.45 0.77 0.24 0.24 0.03 0.23
%RSDR 19.72 19.76 45.76 18.70 19.92 45.85 18.19 18.38 56.05 18.41
R (2.8×SR) 4.45 4.24 4.56 0.19 1.25 2.15 0.68 0.66 0.07 0.64

Peanut butter duplicate

n 9 9 9 9 9 9 9 9 9 9
Mean 51.50 49.29 9.66 0.05 30.84 30.55 8.78 8.77 0.08 8.55
Repeatability (Sr) 3.97 3.79 0.88 0.03 2.40 2.26 0.59 0.59 0.01 0.59
%RSDr 7.70 7.70 9.13 53.93 7.79 7.41 6.76 6.77 6.55 6.91
r (2.8×Sr) 11.10 10.62 2.47 0.08 6.73 6.34 1.66 1.66 0.02 1.65
Reproducibility (SR) 4.43 4.24 0.92 0.05 2.60 2.89 1.51 1.50 0.09 1.22
%RSDR 8.61 8.61 9.51 85.63 8.42 9.46 17.22 17.15 105.13 14.32
R (2.8×SR) 12.41 11.88 2.57 0.13 7.27 8.09 4.23 4.21 0.24 3.43

Dry cereal fortified with flax

n 9 9 9 9 9 9 9 9 9 9
Mean 1.91 1.82 0.28 0.00 0.36 0.36 1.20 1.20 0.52 0.64
Repeatability (Sr) 0.03 0.02 0.02 0.00 0.01 0.01 0.03 0.03 0.01 0.02
%RSDr 1.34 1.35 6.75 0.00 2.04 2.04 2.53 2.53 2.48 3.42
r (2.8×Sr) 0.07 0.07 0.05 0.00 0.02 0.02 0.08 0.08 0.04 0.06
Reproducibility (SR) 0.27 0.26 0.04 0.00 0.06 0.06 0.09 0.09 0.03 0.09
%RSDR 14.17 14.19 14.49 0.00 16.81 16.81 7.26 7.26 6.12 13.26
R (2.8×SR) 0.76 0.72 0.12 0.00 0.17 0.17 0.24 0.24 0.09 0.24

(continued)

Copyright © 2017 by the AOCS Page 15 of 58

C ommercial fats and O ils

Ce 1j-07 ■ cis-, trans-, Saturated, Monounsaturated, and Polyunsaturated Fatty Acids

TABLE 5 Continued

Total Fat Total Fat Total Total Total Total cis Total Total cis Total n-3 Total n-6
(TAG%)* (FA%)* Saturated trans MUFA MUFA PUFA** PUFA** PUFA** PUFA**

Horse feed
n 9 9 9 9 9 9 9 9 9 9
Mean 3.22 3.07 0.72 0.00 0.76 0.75 1.58 1.58 0.21 1.38
Repeatability (Sr) 0.17 0.16 0.04 0.00 0.05 0.04 0.07 0.07 0.01 0.06
%RSDr 5.31 5.26 6.22 0.00 6.27 5.89 4.52 4.52 3.57 4.68
r (2.8×Sr) 0.48 0.45 0.13 0.00 0.13 0.12 0.20 0.20 0.02 0.18
Reproducibility (SR) 0.19 0.18 0.07 0.00 0.06 0.04 0.09 0.09 0.02 0.07
%RSDR 5.86 5.82 9.52 0.00 7.85 5.89 5.54 5.54 8.39 5.36
R (2.8×SR) 0.53 0.50 0.19 0.00 0.17 0.12 0.25 0.25 0.05 0.21

Gamebird feed
n 9 9 9 9 9 9 9 9 9 9
Mean 3.00 2.87 0.57 0.00 0.64 0.63 1.65 1.65 0.13 1.52
Repeatability (Sr) 0.07 0.07 0.02 0.00 0.02 0.02 0.02 0.02 0.00 0.02
%RSDr 2.25 2.29 4.27 0.00 3.40 3.47 1.49 1.44 1.68 1.50
r (2.8×Sr) 0.19 0.18 0.07 0.00 0.06 0.06 0.07 0.07 0.01 0.06

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Reproducibility (SR) 0.40 0.38 0.07 0.00 0.08 0.07 0.26 0.26 0.03 0.23
%RSDR 13.39 13.41 12.91 0.00 12.14 10.97 15.48 15.46 19.07 15.19
R (2.8×SR) 1.12 1.08 0.21 0.00 0.22 0.19 0.72 0.71 0.07 0.64

*NLEA (Nutritional Labeling and Education Act) according to the Office of Nutritional Products, Labeling, and Dietary Supplements in the Center
for Food Safety and Applied Nutrition at the U.S. Food and Drug Administration
**Does not include CLA

Page 16 of 58 Copyright © 2017 by the AOCS

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FIGURE 1a GC/FID profile of GLC-714. Separations were on a SPTM-2560 (100 m × 0.25 mm × 0.20 µm). Column temperature was 180°C (32 min) to 215 °C @ 20 °C/min (hold 31.25
min). Carrier gas was hydrogen at 1.0 mL/min (constant flow) with a linear velocity of 26 cm/s. Pressure was 24.63 psi @ 180 °C. Injection and FID temperatures were 235 °C and
325 °C, respectively. Split ratio was 1:100.

Page 17 of 58
Ce 1j-07 ■ cis-, trans-, Saturated, Monounsaturated, and Polyunsaturated Fatty Acids
C ommercial fats and O ils
Page 18 of 58
C ommercial fats and O ils

Ce 1j-07 ■ cis-, trans-, Saturated, Monounsaturated, and Polyunsaturated Fatty Acids

FIGURE 1b GC/FID profile of GLC-714. See Figure 1a for chromatographic parameters.

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FIGURE 2 GC/FID profile of major fatty acid methyl esters (FAME) in Tallow (References 17–24). See Figure 1a for chromatographic parameters.

Page 19 of 58
Ce 1j-07 ■ cis-, trans-, Saturated, Monounsaturated, and Polyunsaturated Fatty Acids
C ommercial fats and O ils
Page 20 of 58
C ommercial fats and O ils

Ce 1j-07 ■ cis-, trans-, Saturated, Monounsaturated, and Polyunsaturated Fatty Acids

FIGURE 3 GC/FID profile of fatty acid methyl esters (FAME) in Tallow from start to 16:0 (References 17–24). See Figure 1a for chromatographic parameters.

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FIGURE 4a GC/FID profile of fatty acid methyl esters (FAME) in Tallow from 16:0 to 20:0 (References 17–24). See Figure 1a for chromatographic parameters.

Page 21 of 58
Ce 1j-07 ■ cis-, trans-, Saturated, Monounsaturated, and Polyunsaturated Fatty Acids
C ommercial fats and O ils
Page 22 of 58
C ommercial fats and O ils

Ce 1j-07 ■ cis-, trans-, Saturated, Monounsaturated, and Polyunsaturated Fatty Acids

FIGURE 4b GC/FID profile of fatty acid methyl esters (FAME) in Tallow zoomed in to Oleic and Linoleic regions (References 17–24). See Figure 1a for c­ hromatographic
parameters.

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All rights reserved. No part of this method may be reproduced or transmitted in any form or by any means without written permission of the publisher.

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FIGURE 5 GC/FID profile of fatty acid methyl esters (FAME) in Tallow from 20:0 to the CLA region (References 17–24). See Figure 1a for chromatographic parameters.
9c,12t,15c-18:3 will coelute with 9c-20:1 (21).

Page 23 of 58
Ce 1j-07 ■ cis-, trans-, Saturated, Monounsaturated, and Polyunsaturated Fatty Acids
C ommercial fats and O ils
Page 24 of 58
C ommercial fats and O ils

Ce 1j-07 ■ cis-, trans-, Saturated, Monounsaturated, and Polyunsaturated Fatty Acids

FIGURE 6 GC/FID profile of fatty acid methyl esters (FAME) in Tallow from the CLA region to end of the chromatogram (References 17–24).
See Figure 1a for chromatographic parameters.

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FIGURE 7 GC/FID profile of major fatty acid methyl esters (FAME) in Anhydrous Butterfat (References 17–24) . See Figure 1a for chromatographic parameters.

Page 25 of 58
Ce 1j-07 ■ cis-, trans-, Saturated, Monounsaturated, and Polyunsaturated Fatty Acids
C ommercial fats and O ils
Page 26 of 58
C ommercial fats and O ils

Ce 1j-07 ■ cis-, trans-, Saturated, Monounsaturated, and Polyunsaturated Fatty Acids

FIGURE 8 GC/FID profile of fatty acid methyl esters (FAME) in Anhydrous Butterfat from start to 16:0 (References 17–24) .
See Figure 1a for chromatographic parameters.

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FIGURE 9a GC/FID profile of fatty acid methyl esters (FAME) in Anhydrous Butterfat from 16:0 to 20:0 (References 17–24). See Figure 1a for chromatographic parameters.

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Ce 1j-07 ■ cis-, trans-, Saturated, Monounsaturated, and Polyunsaturated Fatty Acids
C ommercial fats and O ils
Page 28 of 58
C ommercial fats and O ils

Ce 1j-07 ■ cis-, trans-, Saturated, Monounsaturated, and Polyunsaturated Fatty Acids

FIGURE 9b GC/FID profile of fatty acid methyl esters (FAME) in Anhydrous Butterfat zoomed in to Oleic and Linoleic regions (References 17–24). See Figure 1a for
chromatographic parameters.

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FIGURE 10 GC/FID profile of fatty acid methyl esters (FAME) in Anhydrous Butterfat from 20:0 to the CLA region (References 17–24). See Figure 1a for chromatographic
parameters. 9c,12t,15c-18:3 will coelute with 9c-20:1 (21).

Page 29 of 58
Ce 1j-07 ■ cis-, trans-, Saturated, Monounsaturated, and Polyunsaturated Fatty Acids
C ommercial fats and O ils
Page 30 of 58
C ommercial fats and O ils

Ce 1j-07 ■ cis-, trans-, Saturated, Monounsaturated, and Polyunsaturated Fatty Acids

FIGURE 11 GC/FID profile of fatty acid methyl esters (FAME) in Anhydrous Butterfat from the CLA region to end of the chromatogram (References 17–24).
See Figure 1a for chromatographic parameters.

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FIGURE 12 GC/FID profile of the major fatty acids in BCR-163 fatty acid methyl esters (FAME) (References 17–24). See Figure 1a for chromatographic parameters.

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Ce 1j-07 ■ cis-, trans-, Saturated, Monounsaturated, and Polyunsaturated Fatty Acids
C ommercial fats and O ils
Page 32 of 58
C ommercial fats and O ils

Ce 1j-07 ■ cis-, trans-, Saturated, Monounsaturated, and Polyunsaturated Fatty Acids

FIGURE 13 GC/FID profile of fatty acid methyl esters (FAME) in BCR-163 from start to 16:0 (References 17–24). See Figure 1a for chromatographic parameters.

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FIGURE 14a GC/FID profile of major fatty acid methyl esters (FAME) in BCR-163 from 16:0 to 20:0 (References 17–24). See Figure 1a for chromatographic parameters.

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C ommercial fats and O ils
Page 34 of 58
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Ce 1j-07 ■ cis-, trans-, Saturated, Monounsaturated, and Polyunsaturated Fatty Acids

FIGURE 14b GC/FID profile of major fatty acid methyl esters (FAME) in BCR-163 zoomed in to Oleic and Linoleic regions (References 17–24). See Figure 1a for
chromatographic parameters.

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All rights reserved. No part of this method may be reproduced or transmitted in any form or by any means without written permission of the publisher.

Copyright © 2017 by the AOCS

FIGURE 15 GC/FID profile of fatty acid methyl esters (FAME) in BCR-163 from 20:0 to the CLA region (References 17–24). See Figure 1a for c­ hromatographic parameters.
9c,12t15c-18:3 will coelute with 9c-20:1 (21).

Page 35 of 58
Ce 1j-07 ■ cis-, trans-, Saturated, Monounsaturated, and Polyunsaturated Fatty Acids
C ommercial fats and O ils
Page 36 of 58
C ommercial fats and O ils

Ce 1j-07 ■ cis-, trans-, Saturated, Monounsaturated, and Polyunsaturated Fatty Acids

FIGURE 16 GC/FID profile of fatty acid methyl esters (FAME) in BCR-163 from the CLA region to end of the chromatogram (References 17–24).
See Figure 1a for chromatographic parameters.

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FIGURE 17a GC/FID profile of GLC-714. Separations were on a CP-SIL-88 (100 m × 0.25 mm × 0.20 µm). Column temperature was 180 °C (32 min) to 215 °C @ 20 °C/min
(hold 31.25 min). Carrier gas was helium at 2.0 mL/min (constant flow) with a linear velocity of 28 cm/s. Pressure was 63.29 psi @ 180 °C. Injection and FID temperatures were
235 °C and 325 °C, respectively. Split ratio was 1:100.

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Ce 1j-07 ■ cis-, trans-, Saturated, Monounsaturated, and Polyunsaturated Fatty Acids
C ommercial fats and O ils
Page 38 of 58
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Ce 1j-07 ■ cis-, trans-, Saturated, Monounsaturated, and Polyunsaturated Fatty Acids

FIGURE 17b GC/FID profile of GLC-714. See Figure 17a for chromatographic parameters.

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FIGURE 18 GC/FID profile of major fatty acid methyl esters (FAME) in Tallow (References 17–24). See Figure 17a for chromatographic parameters.

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C ommercial fats and O ils
Page 40 of 58
C ommercial fats and O ils

Ce 1j-07 ■ cis-, trans-, Saturated, Monounsaturated, and Polyunsaturated Fatty Acids

FIGURE 19 GC/FID profile of fatty acid methyl esters (FAME) in Tallow from start to 16:0 (References 17–24) . See Figure 17a for chromatographic parameters.

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FIGURE 20a GC/FID profile of fatty acid methyl esters (FAME) in Tallow from 16:0 to 20:0 (References 17–24). See Figure 17a for chromatographic parameters.

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Ce 1j-07 ■ cis-, trans-, Saturated, Monounsaturated, and Polyunsaturated Fatty Acids
C ommercial fats and O ils
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FIGURE 20b Blow up GC/FID profile of fatty acid methyl esters (FAME) in Tallow for the Oleic and Linoleic regions. See Figure 17a for chromatographic parameters.

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All rights reserved. No part of this method may be reproduced or transmitted in any form or by any means without written permission of the publisher.

Copyright © 2017 by the AOCS

FIGURE 21 GC/FID profile of fatty acid methyl esters (FAME) in Tallow from 20:0 to the CLA region (References 17–24). See Figure 17a for chromatographic parameters.
9c,12t,15c-18:3 will coelute with 9c-20:1 (21).

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FIGURE 22 GC/FID profile of fatty acid methyl esters (FAME) in Tallow from the CLA region to end of the chromatogram (References 17–24). See Figure 17a for
chromatographic parameters.

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All rights reserved. No part of this method may be reproduced or transmitted in any form or by any means without written permission of the publisher.

Copyright © 2017 by the AOCS

FIGURE 23 GC/FID profile of major fatty acid methyl esters (FAME) in Anhydrous Butterfat (References 17–24). See Figure 17a for chromatographic parameters.

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Ce 1j-07 ■ cis-, trans-, Saturated, Monounsaturated, and Polyunsaturated Fatty Acids

FIGURE 24 GC/FID profile of fatty acid methyl esters (FAME) in Anhydrous Butterfat from start to 16:0 (References 17–24) . See Figure 17a for
chromatographic parameters.

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All rights reserved. No part of this method may be reproduced or transmitted in any form or by any means without written permission of the publisher.

Copyright © 2017 by the AOCS

FIGURE 25a GC/FID profile of fatty acid methyl esters (FAME) in Anhydrous Butterfat from 16:0 to 20:0 (References 17–24). See Figure 17a for chromatographic
parameters.

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C ommercial fats and O ils
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FIGURE 25b GC/FID profile of fatty acid methyl esters (FAME) in Anhydrous Butterfat zoomed in to Oleic and Linoleic regions (References 17–24). See Figure 17a for
chromatographic parameters.

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All rights reserved. No part of this method may be reproduced or transmitted in any form or by any means without written permission of the publisher.
All rights reserved. No part of this method may be reproduced or transmitted in any form or by any means without written permission of the publisher.

Copyright © 2017 by the AOCS

FIGURE 26 C/FID profile of fatty acid methyl esters (FAME) in Anhydrous Butterfat from 20:0 to the CLA region (References 17–24). 9c,12t,15c-18:3 will coelute with
9c-20:1 (21). See Figure 17a for chromatographic parameters.

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Ce 1j-07 ■ cis-, trans-, Saturated, Monounsaturated, and Polyunsaturated Fatty Acids

FIGURE 27 GC/FID profile of fatty acid methyl esters (FAME) in Anhydrous Butterfat from the CLA region to end of the chromatogram (References 17–24).
See Figure 17a for chromatographic parameters.

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All rights reserved. No part of this method may be reproduced or transmitted in any form or by any means without written permission of the publisher.

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FIGURE 28 GC/FID profile of the major fatty acids in BCR-163 fatty acid methyl esters (FAME) (References 17–24). See Figure 17a for chromatographic parameters.

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FIGURE 29 GC/FID profile of fatty acid methyl esters (FAME) in BCR-163 from start to 16:0 (References 17–24). See Figure 17a for chromatographic parameters.

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All rights reserved. No part of this method may be reproduced or transmitted in any form or by any means without written permission of the publisher.

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FIGURE 30a GC/FID profile of major fatty acid methyl esters (FAME) in BCR-163 from 16:0 to 20:0 (References 17–24). See Figure 17a for chromatographic parameters.

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C ommercial fats and O ils
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Ce 1j-07 ■ cis-, trans-, Saturated, Monounsaturated, and Polyunsaturated Fatty Acids

FIGURE 30b C/FID profile of major fatty acid methyl esters (FAME) in BCR-163 zoomed in to Oleic and Linoleic regions (References 17–24). See Figure 17a for
chromatographic parameters.

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All rights reserved. No part of this method may be reproduced or transmitted in any form or by any means without written permission of the publisher.
All rights reserved. No part of this method may be reproduced or transmitted in any form or by any means without written permission of the publisher.

Copyright © 2017 by the AOCS

FIGURE 31 GC/FID profile of fatty acid methyl esters (FAME) in BCR-163 from 20:0 to the CLA region (References 17–24). 9c,12t,15c-18:3 will coelute with 9c-20:1 (21).
See Figure 17a for chromatographic parameters.

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C ommercial fats and O ils
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FIGURE 32 GC/FID profile of fatty acid methyl esters (FAME) in BCR-163 from the CLA region to end of the chromatogram (References 17–24).
See Figure 17a for chromatographic parameters.

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All rights reserved. No part of this method may be reproduced or transmitted in any form or by any means without written permission of the publisher.
All rights reserved. No part of this method may be reproduced or transmitted in any form or by any means without written permission of the publisher.

Copyright © 2017 by the AOCS

FIGURE 33 Plot of % ECF – TCF Differences from one lab for the different AOCS methods. Values should fall between ± 3% for saturated, monounsaturated, and long chain
fatty acids with 18 or less carbons and three or less double bonds.

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FIGURE 34 Plot of % ECF—TCF Differences from four different labs. Values should fall between ± 3% for saturated, monounsaturated, and long chain fatty acids
with 18 or less carbons and three or less double bonds.

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Laboratory Safety
INTRODUCTION Nitric acid is a highly corrosive and toxic oxidizing agent. Use effective acid-resistant
fume-removal device whenever heating acids or performing reactions that liberate
The following sections do not contain complete listings of all the elements involved acid fumes. When diluting acids, always add acid to water unless otherwise directed
in laboratory safety. These precautionary notes serve as a reminder of possible hazards in a method. Keep acids off skin and protect eyes when working with acids. If acids
involved in the use of particular operations or substances, especially those items and come in contact with skin or eyes, wash immediately with large amounts of water.
materials frequently used in AOCS methods. The user of these methods should refer Do not store oxidizing acids (perchloric, nitric, sulfuric) near organic materials.
to standard texts on laboratory safety for a more complete treatment of the subject. Mixing organic materials with these acids, particularly perchloric, could result in an
Follow safety requirements and rules issued by voluntary organizations and govern- explosion.
ment agencies [Occupational Safety and Health Administration (OSHA), in particu-
lar] expert in the field of laboratory safety. Periodic acid is an oxidizing agent and is dangerous in contact with organic materials.
It is a strong irritant. It decomposes at 130°C. Do not use cork or rubber stoppers on
storage bottles.
EQUIPMENT
Sulfuric acidis a strong acid and will cause severe burns. Protective clothing should be
Blenders, grinders, electrical equipment—Motors on high-speed blenders used to mix worn when working with this acid. It is a dehydrating agent and should not be stored
flammable solvents with other material should be rated for use with the materials in the vicinity of organic materials. Use great caution in mixing with water due to
in question and in the class and division rating of the lab where the work is being heat evolution that can cause explosive spattering. Always add the acid to water, never
performed. Blend toxic or flammable liquids in an effective fume-removal area. the reverse.
Accidents involving electrical equipment may result in mechanical injury, e.g., fingers
are being caught in chopping mill knives or grinders; electrical shock, which may
ALKALIES
be due to lack of or improper grounding, defective equipment, exposed wiring, or
inadequate maintenance; and fire through ignition of flammable vapors by electrical Alkalies can burn skin, eyes, and respiratory tract severely. Wear heavy rubber gloves
sparks. Ground all electrical equipment. Installation, maintenance, and repair opera- and face shield to protect against concentrated alkali liquids. Use effective fume-re-
tions should be performed by qualified electricians. moval device or gas mask to protect respiratory tract against alkali dusts or vapors.
Atomic absorption spectrophotometer—Use effective fume-removal device to remove When working with extremely caustic materials, like sodium hydroxide and potas-
gaseous effluents from burner. Use specially designed exhausts when nitrous oxide sium hydroxide, always add pellets to water and not vice versa. These alkalies are
(N2O) is used as a fuel oxidant. If instrument has drain trap, check to ensure it is extremely exothermic when mixed with water. Take precautions to contain the caustic
filled with water before igniting burner. Explosions of fuel gas accumulated through solution in the event the mixing container breaks from the extreme heat generated.
drain traps have been reported. Potassium hydroxide can severely burn skin, eyes, and respiratory tract. Wear heavy
Compressed gas cylinders—Identify contents (by means of attached decal, stencil or tag) rubber gloves and face shield to protect against concentrated alkali liquid splash. Use
of compressed gas cylinders by name of gas contained in the cylinder rather than by effective fume-removal device or gas mask to protect respiratory tract against alkali
color codes. Secure cylinders in upright position by means of strap, chain, or nontip dusts or vapors. When working with extremely caustic materials, such as potassium
base. Use only correct pressure gages, pressure regulator, and flow regulator for each hydroxide, always add the pellets to the water and not the reverse.
size of gas cylinder and type of gas, as specified by supplier. Use toxic gases only in Sodium hydroxide can severely burn skin, eyes and respiratory tract. Protective cloth-
effective fume-removal areas. When burning gas or performing a reaction, use back ing should be worn when working with this alkali. Wear heavy rubber gloves and face
flow prevention device in gas line to prevent flame or reaction from being sucked shield to protect against concentrated alkali liquid splash. Use effective fume-removal
back into cylinder. device or gas mask to protect respiratory tract against alkali dusts or vapors. When
Distillations, extractions, evaporations—Forflammable liquids, perform operations working with extremely caustic materials, such as sodium hydroxide, always add pel-
behind safety barrier with hot water, steam, or electric mantel heating. Do not use lets to water and not the reverse.
open flames to heat flammable liquids. Use effective fume-removal device to remove
flammable vapors as they are produced. Set up apparatus on firm supports and secure SOLVENTS
all connections. Leave ample headroom in flask and add boiling chips before heating
begins. All controls, unless vapor sealed, should be located outside vapor area. For Vapors from some volatile solvents are highly toxic. Several of these solvents are read-
toxic liquids, use effective fume-removal device to remove toxic vapors as they are ily absorbed through the skin. Do not let vapors concentrate to a flammable level in
produced. Avoid contact with skin and inhalation of vapors. Store and dispose of the work area, because it is nearly impossible to eliminate all chances of sparks from
toxic solvents in the manner prescribed by the Environmental Protection Agency static electricity, even though electrical equipment is grounded. Use effective fume-re-
(EPA) and OSHA. moval device to remove solvent vapors as they are liberated.
Vacuum—Any apparatus to be used under vacuum shall be coated, taped, or other- Acetone is a highly flammable solvent. Forms explosive peroxides with oxidizing
wise treated to minimize effects of possible implosion, and a safety shield in place agents. Use effective fume-removal device. Do not mix with chloroform.
during operation. Vacuum pump drive belts must have effective guards.
Acetonitrile is a flammable solvent. There is toxic action by skin absorption and inha-
lation. A fume hood should be used at all times when using acetonitrile.
ACIDS Anilineis an organic chemical compound consisting of a benzene ring attached to
Use effective acid-resistant fume-removal device whenever heating acids or perform- an amino group. Acute exposure can cause upper respiratory tract irritation and
ing reactions which liberate acid fumes. When diluting acids, always add acid to congestion.
water, unless otherwise directed in a method. Keep acids off skin, and protect eyes Benzene is a highly toxic and highly flammable solvent. Avoid contact with the skin.
when working with acids. If acids come in contact with skin or eyes, wash immedi- Do not breathe vapors. Use effective fume-removal device. Decomposes violently in
ately with large amounts of water. Do not store oxidizing acids (perchloric, nitric, the presence of strong oxidizing agents. Reacts violently with chlorine. Benzene is a
sulfuric) near organic materials. Mixing organic materials with these acids, particu- cancer-causing agent.
larly perchloric, could result in an explosion.
Carbon disulfide is a colorless, highly-flammable poisonous liquid. It is harmful either
Acetic acid in the pure state is moderately toxic by ingestion and inhalation. It is a by inhalation, prolonged or repeated skin contact, or by ingestion. Chronic poison-
strong irritant to skin and tissue. The TLV in air is 10 ppm. ing may ensue from repeated exposure to vapor. It is a dangerous fire and explosion
Hydrochloric acid is
a strong acid and will cause severe burns. Protective clothing risk, and can be ignited by friction. Extreme precautions should be taken when using
should be worn when working with this acid. It is toxic by ingestion and inhalation this solvent. A fume hood should be used at all times when handling this solvent.
and a strong irritant to eyes and skin. The use of a properly operating fume hood is Carbon tetrachloride is a known carcinogen. It is toxic by ingestion, inhalation, and
recommended. When diluting the acid, always add the acid to the water, never the skin absorption. It is a narcotic. It should not be used to extinguish fires. It decom-
reverse. poses to phosgene gas at high temperature. It reacts violently with alkali metals. A
Hydrogen bromide gas and hydrobromic acid are toxic by inhalation and strong irritants fume hood should be used at all times when handling this solvent.
to eyes and skin. Use a properly ­operating fume hood when working with these Chlorobenzene is a colorless flammable liquid. It has a moderate fire risk. Explosive
compounds. limits are 1.8–9.6% in air. Avoid inhalation and skin contact. The TLV is 75 ppm in
air. Chlorobenzene and trichlorobenzene are toxic by ingestion and inhalation. Use a CHEMICALS
properly operating fume hood when working with this solvent.
Chlorine is a poisonous gas. The TLV is 1 ppm in air. It is a strong oxidizing agent and
Chloroform is a known carcinogen. It is toxic by inhalation and has anesthetic proper- should not be allowed to come in contact with organic materials, hydrogen, pow-
ties. Avoid contact with the skin. Prolonged inhalation or ingestion can lead to liver dered metals, and reducing agents. A fume hood should be used at all times when
and kidney damage and may be fatal. It is nonflammable, but will burn on prolonged using chlorine.
exposure to flame or high temperature, forming phosgene gas when heated to decom-
Gossypol is toxic by ingestion. Avoid contact with particulate matter when working
position temperatures. Can react explosively with aluminum, lithium, magnesium,
with standards. It is inactivated by heat.
sodium, potassium, disilane, N2O4, and sodium hydroxide + methanol. The TLV is
10 ppm in air. A fume hood should be used at all times when using chloroform. Hydrazine sulfate can cause eye, skin, and mucous membrane irritation and liver and
kidney damage. This compound is a known carcinogen in laboratory animals, caus-
Cylcohexane is a highly flammable liquid. It may be fatal if swallowed or inhaled, and
ing lung and liver tumors in rats. It is a suspected human carcinogen. Precautions
can cause skin irritation. Use effective fume-removal device. Can react vigorously
should be taken in handling this compound—use gloves, eye protection, and respira-
with strong oxidizing agents.
tory protection. Avoid the inhalation of dust and powder. Dispose of waste material
Dichloromethane (methylene chloride)is toxic and a carcinogen that will emit highly and waste solutions in a proper and safe manner.
toxic fumes and phosgene gas when heated. The TLV is 100 ppm in air. A fume hood
Lead acetate is toxic by ingestion, inhalation, and skin absorption.
should be used at all times when using methylene chloride.
Mercury vapors and compounds are extremely toxic and cumulative. Hazardous
Diethyl etheris an extremely flammable liquid, and a severe fire and explosion hazard
in contact with ammonia, halogens, and alkali. Regard spills on hot surfaces as
when exposed to heat or flame. It is a central nervous system depressant by inhalation
extremely hazardous and clean up promptly. Powdered sulfur sprinkled over spilled
and skin absorption. Store protected from the light. It will form explosive peroxides
mercury can assist in cleaning up spills. High degree of personal cleanliness is neces-
upon exposure to light. Handle empty containers, particularly those from which ether
sary for persons who use mercury. Handle only in locations that can be readily and
has evaporated, with extreme caution. Explosive limits in air are 1.85–48%. The TLV completely cleaned up. When mercury evaporation is required, use effective fume-re-
is 400 ppm in air. Can react explosively when in contact with chlorine, ozone, lithium moval device. To avoid environmental contamination, dilute liquid remaining in
aluminum hydride, or strong oxidizing agents. A fume hood should be used at all Kjeldahl digestion flasks to about 300 mL with water, cool to room temperature,
times when using ethyl ether. Avoid static electricity. and add 50 mL 30% hydrogen peroxide. (If Raney powder method is used, 6 mL
Dimethylformamide is a clear flammable liquid and a strong irritant to skin and tissue. of hydrogen peroxide is sufficient.) Warm gently to initiate reaction, let reaction go
It is toxic by skin absorption. The TLV is 10 ppm in air. to completion in warm flask, and separate precipitated mercuric sulfide. Reserve
precipitate in closed, labeled container for recovery of mercury or disposal by EPA
Ethanol (ethyl alcohol) is a clear, colorless, highly flammable liquid. Use effective
requirements.
fume-removal device when heating or evaporating.
Potassium dichromate is toxic by ingestion and inhalation. There is sufficient evidence
Ethyl etheris a highly flammable liquid and a severe fire and explosion hazard when
in humans for the carcinogenicity of chromium [+6], in particular lung cancer. It is a
exposed to heat or flame. It is a central nervous system depressant by inhalation and
strong oxidizing agent and a dangerous fire risk in contact with organic chemicals.
skin absorption. It will form explosive peroxides upon exposure to light. Handle
empty containers, particularly those from which ether has evaporated, with extreme Sylon BFT isa powerful silylating reagent, composed of mixing 1 part trimethyl chlo-
caution. Explosive limits in air are 1.85–48%. The TLV is 400 ppm in air. A fume rosilane with 99 parts of bistrimethylsilyl-trifluoroacetamide, and should be used
hood should be used at all times when using ethyl ether. only in a properly operating fume hood. This reagent is highly flammable.
Hexane is a highly flammable solvent and a dangerous fire risk. All work should be tert-Butyl methyl ether is
extremely flammable and toxic. Avoid inhalation, ingestion,
performed in a fume hood, with no open flames. The TLV for hexane is 50 ppm in and eye or skin contact. The TLV is 50 ppm in air. OSHA recommends that expo-
air. OSHA recommends that exposure not exceed 350 ng/M3 for a time-weighted sure not exceed 100 mg/M3 for a time-weighted average. Respiratory irritation, dizzi-
average. Hexane vapor causes lung irritation and produces neurotoxic effects. ness, and disorientation have been reported. A fume hood should be used at all times
when using tert-Butyl methyl ether.
Heptane is a highly flammable liquid and a dangerous fire risk. Vapors may cause lung
irritation and may produce neurotoxic effects. A fume hood should be used at all Wijs solution,iodine monochloride, causes severe burns, and the vapors can cause lung
times when using this solvent. and eye damage. Use of a fume hood is recommended. Wijs solution without carbon
tetrachloride is available commercially.
Methanol (methyl alcohol) is flammable liquid, and toxic. Avoid contact with eyes.
Avoid breathing vapors. Use effective fume-removal device. Can react vigorously with
sodium hydroxide + chloroform, potassium hydroxide + chloroform, and perchloric ADDITIONAL MATERIALS
acid.
Castor seeds are poisonous due to the presence of ricin, a highly toxic albumin, and
Methyl isobutyl ketone (MIBK) is a clear, colorless, and highly flammable liquid and a ricinine, a highly toxic alkaloid. Neither pressing nor extraction removes them; hence
dangerous fire risk. Explosive limits in air are 1.4–7.5%. Avoid inhalation and inges- both hazards remain in the pomace. They also contain an allergenic protein poly-
tion. It is absorbed by the skin. The TLV is 50 ppm in air. saccharide (CB-1A) that is among the most powerful known allergens. It is strongly
Petroleum ether is the petroleum fraction consisting of aliphatic hydrocarbons in the recommended that workers wear rubber gloves when preparing analytical samples,
boiling range 35–60º C. The term ether is only figurative, signifying extreme light- and that they avoid inhaling any of the dust arising from the castor beans by working
ness and volatility. It is extremely flammable. The explosive limits in air are 1–6%. near an air exhaust or in a well ventilated laboratory hood.
Use effective fume-removal device. Avoid static electricity. Fumonisins are hepatotoxic and carcinogenic to rats; effects on humans are not fully
Pyridine is a clear liquid with a distinct odor, is highly flammable and a dangerous fire known. Wear protective gloves to reduce skin contact with corn extracts. Any labo-
risk. The explosive limits in air are 1.8–12.4%. It is toxic by ingestion and inhalation. ratory spillages should be washed with a 5% aqueous solution of commercial sodium
The TLV is 5 ppm in air. The danger from crude pyridine is greater than from pure hypocholorite followed by H2O. (Dispose of waste solvents according to applicable
pyridine, the associated homologs and impurities being even more toxic than pyri- environmental rules and regulations.)
dine itself. Mycotoxins should be handled with extreme care because they are highly toxic sub-

Tetrachloroethylene (perchloroethylene) is a colorless, volatile, nonflammable liquid stances. Perform manipulations under a properly operating fume hood. Take partic-
chlorinated hydrocarbon that will emit toxic fumes of phosgene when exposed to ular precautions, such as the use of a glove box, when toxins are in dry form, because
sunlight or flames. It is an irritant to eyes and skin. The TLV is 50 ppm in air. of their electrostatic nature and resulting tendency to disperse in working areas. Swab
accidental spills of toxin with 5% NaOCl bleach. Rinse all glassware exposed to tox-
Tetrahydrofuran is a highly flammable liquid and a dangerous fire risk. The flammable ins with 1% NaOCl bleach solution and then wash thoroughly with warm water.
limits in air are 2–11%. It is toxic by ingestion and inhalation. The TLV in air is 200
ppm. It tends to form peroxides upon storage in air.
REFERENCES
Toluene is a highly flammable liquid and a dangerous fire risk. Explosive limits in air
are 1.27–7%. It is toxic by ingestion, inhalation, and skin absorption. The TLV is Official Methods of Analysis, Association of Official Analytical Chemists, 14th edn.,
100 ppm in air. A fume hood should be used at all times when using toluene. 1984, pp. 1010–1015.
Trichloroethane is a synthetic, light-sensitive, volatile, colorless, liquid miscible with Standard Methods for the Analysis of Oils, Fats and Derivatives, 7th edn., Blackwell
many nonpolar organic solvents. It is an irritant to eyes and skin. The TLV is 350 Scientific Publications, 1987.
ppm in air.
Hawley’s Condensed Chemical Dictionary, 11th edn., revised by N. I. Sax and R. J.
Xylene is flammable and a dangerous fire risk. The TLV is 100 ppm in air. Lewis, Jr., 1987.