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Food Analysis
For the characterization of the lipid fraction, the and the geometric configuration (cis/trans) are
triglycerides are hydrolyzed (saponified) into glyc- also important parameters and their determination
erol and free fatty acids. Although the free fatty adds additional information to the characteriza-
acids can be analyzed directly on polar stationary tion of the lipid fraction in food.
phases (such as an HP-FFAP column), more robust
and reproducible chromatographic data are In this application note, three stationary phases
obtained if the fatty acids are derivatized to the are compared for the separation of FAMEs. The
methyl esters. For the derivatization, including first method uses DB-Wax, a polyethylene glycol
hydrolysis and methylation, different methods are column, where FAMEs from C4 (butyric acid) to
available [1]. These methods are easy to use and C24 (lignoceric acid) can be separated according to
do not require expensive reagents or equipment. A carbon number and degree of unsaturation. On
typical sample preparation method is described in these columns, no separation of cis- and trans-
the sample preparation section. isomers is obtained, and for complex mixtures,
such as fish oils, some FAMEs are difficult to sepa-
After preparation of the FAMEs, they are sepa- rate. However, the separation of FAMEs on poly-
rated according to the carbon number (number of ethylene glycol columns is widely used and are
carbon atoms in the fatty acid chain, excluding the applied to the characterization of “classical” sam-
methyl ester carbon) and the degree of unsatura- ples, such as vegetable oils from corn, maize, olive,
tion. Moreover, the position of the double bond(s) and soybean. Moreover, animal fats can also be
2
analyzed. One important application is the analy- Experimental
sis of butyric acid in milk fat. The concentration of
butyric acid in milk is an important indicator of Samples
milk quality, and its analysis is therefore very
important in milk, dairy, and chocolate products. Reference standards of FAMEs can be obtained
from different sources as solutions or as neat com-
For the analysis of complex samples, such as fish pounds. For analysis, the standards are typically
oils, additional resolution of the FAMEs is needed, dissolved in hexane at a 0.01%–0.1% (w/v)
and is obtained using a capillary column coated concentration.
with a cyanopropyl-stationary phase, such as a
DB-23. On this column, highly unsaturated fatty For column check-out, a 37-component mixture
acids, such as all cis 5, 8, 11, 14, 17-eicosapentenoic (Supelco #18919) was used. The mixture is avail-
acid methyl ester (EPA, C20:5 ω3) and all cis able as a 100-mg neat mixture, containing C4–C24
4,7,10,13,16,19-docosahexenoic acid methyl ester FAMEs (2%–4% relative concentration). The whole
(DHA, C22:6 ω3) are separated from other FAMEs. sample was diluted in 10-mL hexane (final concen-
This analysis is very important in the framework tration = 0.2–0.4 mg/mL per FAME) before use. Oil
of recent interest in omega-3 fatty acid determina- and fat samples can be prepared using different
tion. On the cyanopropyl column, separation of the methods [1–5].
cis- and trans-isomers is also possible. Due to
stronger interaction of the cis-isomer with the Sample Preparation Method [5]
cyano-dipole, the trans-isomers elute before the
cis-isomers. In this way, the determination of Weigh 100-mg sample in a 20-mL test tube (with
trans-fatty acids is also performed, however, the screw cap) or reaction vial. Dissolve the sample in
polarity of the stationary phase is not sufficient to 10-mL hexane. Add 100-µL 2 N potassium hydrox-
fully separate complex cis-trans mixtures. ide in methanol (11.2 g in 100 mL). Close the tube
or vial and vortex for 30 s. Centrifuge. Transfer the
For the separation of a complex FAME mixture clear supernatent into a 2-mL autosampler vial.
containing a relatively large amount of trans-fatty
acids, a highly polar HP-88 column is preferred. On Analytical Conditions
this highly polar column excellent separation
The analyses were performed on an Agilent 6890
between different cis- and trans-isomers is
GC equipped with a flame ionization detector
obtained, however, some higher molecular weight
(FID). Automated split injection was performed
fatty acids are more difficult to separate.
using an Agilent 7683 autosampler. The instrumen-
An overview of columns and their application area tal configuration and analytical conditions are
is summarized in Figure 1. summarized in Table 2 (DB-Wax column), Table 3
(DB-23 column) and Table 4 (HP-88 column).
FAME Analysis
3
Table 2. DB-Wax Method 1
Instrumentation
Chromatographic system: Agilent 6890 GC
Inlet Split/Splitless
Detector FID or Agilent 5973 MSD
Automatic Sampler Agilent 7683
Liner Split liner (p/n 5183-4647)
Column 30 m × 0.25 mm ID, 0.25 µm DB-Wax (J&W 122-7032)
Experimental Conditions GC-FID
Inlet temperature 250 °C
Injection volume 1 µL
Split ratio 1/50
Carrier gas Hydrogen
Head pressure 53 kPa constant pressure (36 cm/s at 50 °C)
Oven temperature 50 °C, 1 min, 25 °C/min to 200 °C, 3 °C/min to 230 °C, 18 min.
Detector temperature 280 °C
Detector gases Hydrogen: 40 mL/min; Air: 450 mL/min; Helium make-up gas: 30 mL/min.
4
Results
A typical chromatogram for the analysis of the
37-compound FAMEs reference standard, obtained
on the DB-Wax column is shown in Figure 2.
C10:0
Norm.
C8:0
C12:0
C16:0
100
C6:0
C14:0 DB-wax
C18:1 c+t
90
80
C4:0
70 C18:0
C11:0
C18:2 c+t
C13:0
C15:0
C20:0
C14:1
C16:1
C17:0
C15:1
50
C22:0
C17:1
C18:3 n6
C22:6 + C24:1
C18:3 n3
C20:1
C20:3 n3
C20:2
40
C24:0
C22:1
C20:4
C22:2
C20:5
C23:0
30
20
0 5 10 15 20 25 30
Time (min)
Figure 2. GC-FID analysis of 37-component FAMEs standard mixture on a 30 m × 0.25 mm ID, 0.25 µm DB-Wax column
using Method 1. (See Table 2).
5
C14:0
C16:0
C18:1 c+t
Norm.
100
DB-wax
90
C18:0
80
70
C12:0
60
C10:0
50
C4:0
C6:0
40
C18:2 c+t
C8:0
C16:1
C15:0
C14:1
C18:3 n3
30
C17:0
C15:1
20
4 6 8 10 12 14 16
Time (min)
Figure 3. GC-FID analysis of milk fat (CRM 164) fatty acids on a 30 m × 0.25 mm ID, 0.25 µm DB-Wax column using Method 1,
Table 2.
Norm.
C16:0
100
C12:0
C10:0
DB-23
C14:0
90
C8:0
C18:1, cis
80
C6:0
C18:0
70
C18:1, trans
C20:0
C11:0
C13:0
60
C22:0
C14:1
C20:3 n3 C20:4 n6
C15:0
C18:2, trans
C24:0
C21:0
C18:2, cis
C15:1
C16:1
C17:0
C17:1
C4:0
C20:1n9
50
C18:3 n6
C18:3 n3
C22:1
C20:3 n6
C22:2
C24:1
C20:2
C23:0
C20:5
40
C22:6
30
20
4 6 8 10 12 14 16 18 20 22
Time (min)
Figure 4. GC-FID analysis of FAMEs standard mixture on a 60 m × 0.25 mm ID, 0.15 µm DB-23 column using Method 2,
Table 3.
6
Using these conditions, all compounds in the stan-
dard mixture are well resolved. Important is the
separation of the cis/trans isomers and the separa-
tion of EPA (19.15 min) and DHA (22.38 min) com-
ponents. This method is very useful for the
analysis of fatty acid in complex mixtures, and
especially for the determination of omega-3 fatty
acids (such as EPA and DHA). An example of the
separation obtained for a mixture of polyunsatu-
rated fatty acids from a marine source is given in
Figure 5. EPA and DHA can easily be detected and
quantified.
C20:5 (EPA)
DB-23
C16:1
C14:0
Norm.
C20:1n9
C18:1, cis
C22:6 (DHA)
100
90
80
70
C18:2, cis
60
C16:0
C18:3 n3
50
C18:1, trans
C17:0
C24:0
C22:1
C18:2, trans
C24:1
40
C22:0
C20:4 n6
C22:2
C17:1
C23:0
C20:3 n3
C20:2
C18:3 n6
C14:1
C20:3 n6
C15:0
30
C21:0
C20:0
C18:0
C15:1
C12:0
20
8 10 12 14 16 18 20 22 24
Figure 5. GC-FID analysis of unsaturated fatty acid mixture from marine origin on a 60 m × 0.25 mm ID, 0.15 µm
DB-23 column using Method 2, Table 3.
7
Norm.
50
C16:0
C12:0
C14:0
45
HP-88
C10:0
C220 + C 20:3 n6
40
35
C24:0
C8:0
30
C18:1 cis
C18:0
C20:0
C13:0
C11:0
C20:3 n3 C22:1
C15:0
25
C14:1
C24:1
C18:1 trans
C22:2
C18:2 trans
C23:0
C15:1
C6:0
C20:4
C18:3 n3
C18:2 cis
C16:1
C21:0
C18:3 n6
C20:1
C17:1
20
C22:6
C20:2
C17:0
C20:5
C4:0
15
10
5
5 10 15 20 25 30
Time (min)
Figure 6. GC-FID analysis of 37-component FAMEs standard mixture on a 100 m × 0.25 mm ID, 0.2 µm HP-88 column
using Method 3A, Table 4A.
Norm. c11
c6
90
80
HP-88
70
c9
60
18:1 t6 t11
50
40
30
20
10
15 16 17 18 19 20
Time (min)
Figure 7. GC-FID analysis of C18:1 isomers on a 100 m × 0.25 mm ID, 0.2 µm HP-88 column using Method 3A, Table 4A.
8
Equally good separation is obtained for four C18:2
isomers (trans-trans, cis-trans, trans-cis and
cis-cis), as shown in Figure 8.
C18:2 tt
Norm.
90
HP-88
80
70
60
tc
50 ct
40
30 cc
20
10
15 16 17 18 19 20 21 22
Time (min)
Figure 8. GC-FID analysis of C18:2 isomers on a 100 m × 0.25 mm ID, 0.2 µm HP-88 column using Method 3A, Table 4.
9
pA
DB-23
100
18:1
80 trans
18:2
60
18:0
40
20
pA
HP-88
35
18:1 cis
30
25
18:0 18:1 trans
20
15
10
5
15.5 16.0 16.5 17.0 17.5 18.0 18.5
Time (min)
Figure 9. Comparison of the separation of C18:1 isomers from a hydrogenated oil obtained on a 60 m x 0.25 mm ID,
0.15 µm DB-23 column (upper window) and on a 100 m × 0.25 mm ID, 0.2 µm HP-88 column. Both analyses were
performed at 180 °C isothermally.
10
Norm.
28
26
HP-88
24
1. trans-C18:1
2. trans-C18:2
3. trans-C18:3
22
20
18
1
16
14
2
12
3
10
Figure 10. GC-FID analysis of FAMEs from a partially hydrogenated rapeseed oil on a 100 m × 0.25 mm ID, 0.2 µm HP-88 column
using Method 3A. (See Table 4).
11
www.agilent.com/chem
Norm.
14
C18:0 C18:1 C18:2
13 HP-88
12 C16:0
11
C16:1
10
C18:3
9
8 C20:0
C20:1
7
6
C18:1 trans
5
0 2 4 6 8 10 12 14 16 18 20
Figure 11. GC-FID analysis of olive oil FAMEs on a 60 m × 0.25 mm ID, 0.2 µm HP-88 column using Method 3B, Table 4.