Special Properties of Fats, AACC Method 58-18

Oils, and Shortenings Page 1 of 4

Fatty Acid Composition by

Gas Chromatography
Final approval October 24, 1974; Reapproval November 3, 1999

Objective
This method permits quantitative separation of mixtures containing saturated
and unsaturated methyl esters. It is applicable to methyl esters of fatty acids
having 8–24 carbon atoms and to animal fats, vegetable oils, and fatty acids after
their conversion to methyl esters. Conditions specified are not suitable for
determining epoxy or oxidized fatty acids nor for fatty acids that have been
polymerized. This method is unsuitable for quantitation of fatty acids for labeling
purposes. It is equivalent to Official Method Ce 1-62 of the American Oil
Chemists’ Society (AOCS).

Apparatus
1. Gas chromatographic instrument consisting of:
a. Column oven, capable of constant temperature operation to ±1.0°, at
temperature between 170 and 210°.
b. Sample inlet port, independently heated to about 50° higher temperature
than column oven.
c. Columns, 1.5–3 m (5–10 ft) long, 3–6 mm (1/8–1/4 in.) outside diameter,
made of stainless steel, glass, copper, or aluminum tubing. See Note 1.
Columns of 3 mm (1/8 in.) outside diameter are packed with 8–12%
polyester liquid phase coated on 80–100-mesh acid-washed Chromosorb
W or equivalent. Columns of 6 mm (1/4 in.) outside diameter are packed
with 15–20% polyester liquid phase coated on 60–80-mesh acid-washed
Chromosorb W or equivalent. Diethylene glycol succinate is recom-
mended liquid phase, although other liquid phases are used for specific
separations. The 3 mm columns are usually employed with flame
ionization detector (FID) and the 6 mm columns with the thermal-
conductivity detector (TCD). It is recommended that analysis be made on
two columns, each with different separating properties (polyesters of
differing polarity) to check for coincident peaks.
d. TCD or FID detectors, if separately thermostatted, should be maintained
at column temperature or up to 50° hotter than column oven.
e. Recorder, with range of 0 to 1.0, 2.5, or 5.0 mV, 1 sec full-scale deflec-
tion with chart speed of 1.25–2.5 cm (1/2–1 in.)/min. Attenuator switch to
change recorder range as required. The 0–1.0 mV recorder is used when
TCD is employed; or any of the three may be used with FID.
f. Gases
(1) Carrier
(a) TCD: Helium, minimum purity 99.95 mol %
(b) FID: Helium, nitrogen, or argon, minimum purity 99.95 mol %
Special Properties of Fats, AACC Method 58-18
Oils, and Shortenings Page 2 of 4

Fatty Acid Composition by Gas Chromatography (continued)

(2) FID: Hydrogen, minimum purity 99.95 mol %

Air, dry, dew point –60° (–75° F) maximum, hydrocarbon-free.
2. Syringe for injecting sample. A 1- or 5-µl capacity with known and repro-
ducible volume. Hamilton 7000 Series syringe or equivalent is recommended.

Procedure
Preparation of methyl esters
Method 58-17 is recommended.

Chromatographic procedure
1. With carrier gas flowing through apparatus, adjust to operating temperature
and record baseline to check for stability of instrument. With FID, it is recom-
mended that apparatus be operated at medium sensitivity rather than near its
maximum sensitivity. Condition new column by holding it about 10° above
operating temperature with helium flowing at least 24 hr. Disconnect column
from detector during conditioning period.
2. Proper gas-flow rate and temperature will permit elution of methyl linoleate,
other C18S, and shorter chain-length esters in less than 30 min. When esters of
fatty acids of greater chain length are present, increase gas flow and/or tempera-
ture so that retention time of last component is reduced. Use largest sample size
consistent with attenuation requirements (see step 3, below) in order to better
detect and quantitate these slow-moving esters. Maintain constant gas flow
throughout analysis. When using TCD, measure gas flow at exit with soap-
bubble flow meter or other suitable device. It is more difficult to measure flow in
instrument equipped with FID; it is best to follow manufacturer’s instructions.
3. Measure methyl ester sample in syringe. When using TCD, usual sample
size is between 0.5 and 4 µl. For FID, about 0.1–0.01 µl of actual esters are
measured, but this is usually in suitable solvent so that 0.5–4 µl of solution is
injected. Pierce septum of sample inlet port and quickly discharge sample.
Withdraw needle and note on recorder chart the small peak due to air (when
using TCD) or solvent (when using FID) that marks sample-introduction refer-
ence point. Adjust sample size so that major peak is not attenuated more than
eight times, preferably less.
4. Watch recorder pen to see that peaks do not go off scale. Change setting of
attenuator as necessary to keep peaks on chart paper. Mark attenuator setting on
chart.
5. After all peaks have been traced and pen has returned to baseline, remove
chart for calculation.

Calculations
1. Identify peaks by relative position on chart. Esters appear on chromatogram
in order of increasing number of carbon atoms and of increasing unsaturation for
Special Properties of Fats, AACC Method 58-18
Oils, and Shortenings Page 3 of 4

Fatty Acid Composition by Gas Chromatography (continued)

same number of carbon atoms. That is, C16 is ahead of C18, and C18 esters appear
in the order stearate, oleate, linoleate, and linolenate. C20-saturated (arachidic)
ester usually appears before C18:3 on some columns. (Coincident peaks are usu-
ally revealed by repeating analysis at different column temperature or employing
second column with different polyester.) With constant operating conditions,
retention times (or chart distances) from air peak to various sample component
peaks can be used for identification of peaks. However, relative retentions are
more reproducible. Relative retentions are determined by dividing observed
retention time for each peak by retention time observed for peak of methyl
palmitate (or other peak if some other basis is desired). Compare observed
retention times or relative retention times with those calculated from known
mixtures run periodically on same column under same conditions.
2. Determine area of each peak. If instrument is equipped with electrome-
chanical or electronic integrator, area is best measured by following manufac-
turer’s instructions. When integrator is employed, baseline must remain constant
or integrator must be equipped with baseline corrector. Otherwise, area is
obtained by drawing lines tangent to sides of peak and intersecting baseline.
Determine area of resulting triangle by multiplying height × 1/2 the base. For
attenuated peak, outer sides of peak must be full chart span and tangents drawn
intersecting baseline should use upper 2/3 to obtain peak width. Determine area
by multiplying height × 1/2 the base. Height must be correct for attenuation
including correction required if baseline is not recorder zero. Divide area of each
component by its calibration factor. Calculate percent of each component from
ratio of each area to sum of areas under all component peaks and report as
percent by weight.
3. Calibration factors are determined relative to methyl palmitate to correct for
nonlinearity of instrument response and for molecular weight differences. Such
factors are determined by analyzing known mixtures preferably having composi-
tion similar to that of unknown sample. Divide area of each peak by true weight
percent of that component; then obtain calibration factors by dividing each value
by value for methyl palmitate.
4. Monitor instruments and column performance by noting separation of oleate
and stearate ester peak, which is expressed as peak resolution.
2Y
Peak resolution =
S+O
where Y = distance between peak maxima for stearate and oleate esters, S = base
width of stearate peak, O = base width of oleate peak.
Determine these values on sample size containing approximately equal quanti-
ties of oleate and stearate esters using sample size such that peak heights are 25–
50% of chart width. If peak resolution is ≥1.0, column and instrument are in sat-
Special Properties of Fats, AACC Method 58-18
Oils, and Shortenings Page 4 of 4

Fatty Acid Composition by Gas Chromatography (continued)

isfactory condition. All columns, when used, will show gradual loss in peak
resolution; when value becomes less than 1.0, install new column.

Precision
1. Two single determinations of major components (5%) performed in one
laboratory shall not differ by more than 1.0% unit.
2. Two single determinations performed in different laboratories shall not dif-
fer by more than 3.0% units.

Notes
1. If polyunsaturated components with more than three double bonds are pres-
ent, they may decompose in a stainless-steel column.
2. It is recommended that chromatographers read Standard Recommended
Practice for General Gas Chromatography Procedures, ASTM Designation
E260-73; Standard Recommended Practice for Gas Chromatography Terms and
Relationships, ASTM Designation E355-77; and Standard Recommended Prac-
tice for Testing Flame Ionization Detectors Used in Gas Chromatography,
ASTM Designation E594-77.
3. In 1990, several laboratories reported the following:
a. Using a medium polarity column packing in a 2.44 m long, 6.3 mm
diameter column produces better separation, especially for the C18:3 and
C20:1 fatty acids.
b. The initial column temperature appears to affect the final result more
than any other temperature variable.
c. The GC operating conditions should be adjusted to give satisfactory sepa-
ration and recovery of a suitable fatty acid reference material, e.g., a
Smalley GC reference sample.

References
1. American Oil Chemists’ Society. 1998. Official Methods and Recommended Practices, 5th ed.
Methods Ce 1-62 and Ce 2-66.
2. AOAC International. 1998. Official Methods of Analysis of AOAC International, 16th ed., 4th
rev. Methods 996.01 and 996.06. The Association, Gaithersburg, MD.