In the Laboratory
Analysis of Fatty Acid Methyl Esters in Egg Yolk Using GC–MS
Lisa T. Alty
Department of Chemistry, Washington and Lee University, Lexington, VA 24450; altyl@wlu.edu
Recently fatty acid methyl esters (FAMEs) have been in the
news because they are the chemicals in biodiesel. Articles in this
Journal have addressed biodiesel as an alternative, renewable, fuel
(1–5) and have described the process of creating, identifying,
and measuring FAMEs from natural sources, including vegetable
oil (3, 6–8), olive oil (4), peanut oil (2, 6), nutmeg (6), bacteria
(9), leaves (10, 11), and eggs (12–14). Previous articles in this
Journal examined the fatty acid content of egg yolk (14), or of
phosphatidylcholine isolated from egg yolk (12, 13), but none
of these articles examined gas chromatography coupled with
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mass spectrometry (GC–MS). In those experiments, FAMEs
were separated using gas chromatography and identified using
their retention times.
The hen egg is a convenient and inexpensive source of
lipids for biochemistry laboratory experiments. Egg yolk
contains phosphatidylcholine, phosphatidylethanolamine,
triglycerides, sphingomyelin, and small quantities (1% or less) of
phosphatidylserine, phosphatidylinositol, phosphatidylglycerol,
lysophosphatidylcholine, lysophosphatidylserine, lysophosphati-
dylethanolamine, galactosylceramide, and cholesterol esters
(15); all of these contain fatty acids. The relative fatty acid ratio
J. Chem. Educ. 2009.86:962.
in eggs can be altered by manipulating the hens’ diet (16–21).
Two polyunsaturated fatty acids (PUFA) are essential in the
human diet, and these essential fatty acids and others synthe-
sized in the body from them are linked to health benefits (22).
Foods containing these particular fatty acids, called omega-3
and omega-6 fatty acids, “are believed to be beneficial for heart
health, brain, and eye function, infant development, and alleviat-
ing hypertension” (23).
18:1
10
Response / (106 Count)
16:0
8 18:2
6 m/z = 55, diunsaturated FAMEs at m/z =  67, and tri- and
4 18:0
2
16:1
14:0
0 ChemStation software. They then convert the integration values
15 20
Elution Time / min
Figure 1. Gas chromatogram of egg yolk FAMEs.
962 Journal of Chemical Education  •  Vol. 86  No. 8  August 2009  •  www.JCE.DivCHED.org  •  © Division of Chemical Education
In the experiment described here, students determine the
relative fatty acid content of egg yolk lipids, in both store-brand
large eggs and large eggs from a commercial supplier of eggs
stated to be higher in PUFA, using GC–MS. This experiment
could also be used in an instrumental analysis or food science
laboratory course.
Experimental Methods
Eggs are purchased at a local chain supermarket and the
large eggs used in the experiment are the store brand. Students
obtain the egg yolk using an egg separator. They generate FAMEs
using 100 mg of egg yolk and the acid-catalyzed transesterifi-
cation procedure of Schultz and Pugh (9). Working in pairs,
students prepare two samples for GC–MS analysis: one from
a store-brand large egg yolk and one from a higher PUFA egg
yolk.
Students inject 1 μL of each sample into the GC–MS, a
Hewlett-Packard 5890 GC interfaced with a 5971A mass se-
lective detector and equipped with the ChemStation software
package and the NBS54k library. We purchased a Supelco
SP-2380 column [poly(90% biscyanopropyl/10% cyanopropy-
lphenyl siloxane) phase, 100 m × 0.25 mm i.d. × 0.20 μm film
thickness)] to separate FAMEs, but shorter and less expensive
columns can be used (6–9, 11–13). This column allows for
resolution, detection, and identification of small quantities of
FAMEs, including methyl myristate, methyl palmitoleate, and
methyl arachidonate, not reported by Hodges (12).
Students identify individual FAMEs primarily by matching
mass spectral data to the library database but also by compar-
ing GC retention time with standards run by the instructor
previously (Figure 1). Not all molecules in the prepared egg
yolk sample are FAMEs, so if student data do not match a
library entry, students must examine the MS data to determine
whether the base peak and other fragment ions correspond
to those typically found in FAMEs. Using an electron ioniza-
tion source, saturated FAMEs produce a base peak, m/z = 74,
which results from a McLafferty rearrangement, a prominent
m/z = 87 fragment resulting from cleavage between the β and
γ carbons, as well as detectable m/z  =  57 and 43 fragments
from fragmentation at the terminus of the fatty acid (Figure 2).
Unsaturated FAMEs produce a base peak from cleavage near
the terminus: monounsaturated FAMEs have a base peak at
tetraunsaturated FAMEs at m/z = 79. In addition, all FAMEs
exhibit the characteristic clumps of peaks resulting from series
of “losses of 14”, due to fragmentations differing in mass by a
methylene group, −CH2−, m/z = 14, as illustrated in Figure 3
18:3 20:4
by methyl palmitate.
Students integrate their peaks using the Hewlett-Packard
25 30 to a mass percent value and compare their data to the literature.
Only peaks identified as FAMEs are included in the denomina-
tor for the mass percent calculations.
 
 In the Laboratory

Hazards γ α McLafferty
O rearrangement O
Raw eggs may contain Salmonella. Therefore after handling M ≡ R β
raw egg, students should wash their hands with soap. If sample O H O
preparation is carried out in a well-ventilated hood, there are
no significant hazards in using these chemicals. The instructor m/z ∙ 74
adds acetyl chloride. Toluene, methanol, and acetyl chloride are
flammable liquids and flammable vapors. Toluene and methanol
cause eye, skin, and respiratory tract irritation. Methanol is a O
poison and may be fatal or cause blindness if swallowed. Acetyl
chloride can cause eye, skin, digestive, and respiratory tract O
burns. It reacts violently with water, releasing toxic and corrosive m/z ∙ 43 m/z ∙ 57 m/z ∙ 87
fumes of hydrogen chloride and aqueous hydrochloric acid.
FAMEs may cause eye and skin irritation and respiratory and Figure 2. Common fragments produced from a saturated FAME.
digestive tract irritation.

Results and Discussion

Forty-eight students, working in pairs, have conducted this 74
100
experiment. Data from eight student groups, collected in fall of
2006, are shown in Table 1. The mean mass percent of the eight
groups is reported with a 95% confidence limit. The mean mass

Fraction of Base Peak (%)

percent data from the three years are shown in Table 2, which 75

illustrates the variability in results from year to year. Student 43

67

data consistently deviate from the literature, primarily in the

ratio of unsaturated to saturated fatty acids in store-brand eggs. 50
Store-brand large eggs have unsaturated:saturated fatty acid 55
270

ratios ranging from 1.5 (2004 data) to 1.1 (2006 data), while 143
227
the literature values for store-brand eggs have a ratio of 1.7. 25
Presumably this variation is due to changes in the diet local hens 185
129
are fed from year to year. 213 271
In lecture students learn the IUPAC, common, and simpli-
fied nomenclatures for fatty acids. In simplified nomenclature 0

arachidonic acid is 20:4(Δ5,8,11,14), which indicates a 20-carbon 100 200 300 400 500

fatty acid with 4 double bonds at carbons number 5, 8, 11, and m/z
14. In the lab students learn the alternative “omega minus” or Figure 3. Mass spectrum of methyl palmitate.

Table 1. Student Data Compared to Literature Data

Exp Large Egga Lit Large Eggb Lit Large Eggc Exp PUFA Egga
Fatty Acid
(mass %) (mass %) (mass %) (mass %)

Methyl myristate 14:0 0.44 ± 0.08 0.37 0.25 0.47 ± 0.07

Methyl palmitate 16:0 26.00 ± 2.18 26.50 26.43 24.75 ± 1.64

Methyl palmitoleate 16:1(Δ9) 3.30 ± 0.63 3.32 3.53 4.08 ± 0.78

Methyl stearate 18:0 13.66 ± 1.61 8.90 9.37 12.36 ± 1.10

Methyl oleate 18:1(Δ9) 34.69 ± 1.34 44.66 44.57 33.76 ± 0.63

Methyl linoleate 18:2(Δ9,12) 18.90 ± 1.34 11.45 14.44 18.12 ± 0.46

Methyl linolenate 18:3(Δ9,12,15) 0.76 ± 0.34 0.17 Not available 2.00 ± 0.46

Methyl eicosenoate 20:1(Δ11) — 0.20 Not available —

Methyl dihomo-γ-linolenate 20:3(Δ8,11,14) — 0.27 Not available —

Methyl arachidonate 20:4(Δ5,8,11,14) 2.25 ± 0.40 1.63 1.41 2.35 ± 0.31

Methyl docosahexenoate 22:6(Δ4,7,10,13,16,19) — 0.27 Not available —

a b
Data from 2006. Data from ref 24. cData from ref 25.

© Division of Chemical Education  •  www.JCE.DivCHED.org  •  Vol. 86  No. 8  August 2009  •  Journal of Chemical Education 963
 In the Laboratory

Table 2. Data for Store-brand Large Eggs

Exp 2006 Exp 2005 Exp 2004 Lit
Fatty Acid
(mass %) (mass %) (mass %) (mass %)
14:0 0.44 0.55 0.53 0.37

16:0 26.00 34.49 29.98 26.50

16:1(Δ9) 3.30 3.44 2.24 3.32

18:0 13.66 9.81 11.43 8.90

18:1(Δ9) 34.69 39.57 26.34 44.66

18:2(Δ9,12) 18.90 12.17 15.00 11.45

18:3(Δ9,12,15) 0.76 0 0.15 0.17

20:1(Δ11) — — — 0.20

20:3(Δ8,11,14) — — — 0.27

20:4(Δ5,8,11,14) 2.25 1.37 1.81 1.63

22:6(Δ4,7,10,13,16,19) — — — 0.27
Note: The literature data are from ref 24.

“n  minus” nomenclature used to describe the placement of Conclusion

double bonds in fatty acids. For example, an ω-3 fatty acid, or
omega-3, has a site of unsaturation on the third carbon atom
away from the final carbon (ω or n) of the fatty acid. α-Linolenic
acid, 18:3(Δ9,12,15), is an ω-3 fatty acid, because of the double
bond at carbon 15, which is 3 carbons away from the ω carbon
(carbon 18 in this fatty acid). Linoleic acid, 18:2(Δ9, 12), and
arachidonic acid, 20:4(Δ5,8,11,14), are ω-6 fatty acids.
Once familiar with this nomenclature, students are able to
address some of the commercial supplier’s claims:
• “eggs contain 25 percent less saturated fat than ordinary
eggs”
• “eggs have 100 mg of Omega 3, compared to 37 mg in or-
dinary eggs” [This is 2.7 times the quantity in an ordinary
egg (23).]
Student data do not support the first claim. Store-brand large
eggs had 40.1% saturated fat while commercial supplier’s eggs
had 37.6% saturated fat; therefore commercial supplier’s eggs
had only 6% less saturated fat than store-brand large eggs. Stu-
dent data do support the second claim. Store-brand large eggs
had 0.76% of linolenic acid, the only ω-3 fatty acid found in our
samples, while commercial supplier’s eggs had 2.00% linolenic
acid, almost exactly 2.7 times the quantity in store-brand eggs.
After calculating percent error, one student recognized
“The largest difference between experimental values and litera-
ture occurs with the 14:0 FAME. This methyl ester represents
less than 1% of the total integration; therefore a small numerical
difference is translated into a relatively large percent difference.”
In the lab report students critique the experimental design.
Students should recognize that they obtained only one sample
from a single egg and made a single GC–MS injection of that
prepared sample. Students should propose an experimental
design that would include sampling many more eggs, both store-
brand large eggs and higher PUFA eggs. Students also suggest
buying eggs from different stores.
This experiment exposes students to GC–MS analysis:
they identify peaks in the GC chromatogram using the mass
spectral library and retention time data and review fragmenta-
tion patterns. Students learn to calculate the mass percent of
the compounds in each sample and compare their data to the
literature. In addition, students become familiar with the four
nomenclature systems for fatty acids: IUPAC, common, simpli-
fied, and the “ω minus” system.
Acknowledgments
I thank Phillip Trimmer for determining the GC condi-
tions for our column and generating the retention time data on
FAMEs. I also thank the Biochemistry I laboratory students,
2004–2006, who generated the results.
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