Analysis of Fatty Acids
1
AOAC Official Methods
969.33
Fatty acids in oils and fats
Preparation
methyl esters
996.06
Fat (total, saturated and unsaturated) GC-FID
in foods
991.39
Encapsulated fish oils
GC-FID
994.15
cis- and trans-Octadecenoic isomers
and general fatty acid composition
GC-IR
985.21
etc
trans-Fatty acid in margarines
GC-FID
2
Typical analysis
„
Fat and fatty acids are extracted from food
by hydrolytic method.
„
Fat is extracted into ether, then methylated
to fatty acid methyl esters (FAMEs).
„
FAMEs are quantitatively measured by gas
chromatography.
3
Typical analysis
4
Typical analysis
Fat
Fat
Hydrolysis
Hydrolysis
Methylated
Methylated
to
to FAMEs
FAMEs
GC-FID
GC-FID
(quantitation)
(quantitation)
5
Typical analysis
Hydrolysis
Weigh sample
and pyrogallic
acid into the
Mojonnier flask
6
Typical analysis
Hydrolysis
Add internal
standard, ethanol
and 8.3 M HCl.
7
Typical analysis
Hydrolysis
Hydrolyse the
sample at 70 –
80 oC
8
Typical analysis
Hydrolysis
Remove flasks
from water bath.
Cool to room
temperature.
9
Typical analysis
Extraction
Add ethanol,
Ether and shake.
10
Typical analysis
Extraction
Centrifuge sample.
(allow contents to
sep. at least 1 h if
centrifuge is not
available)
11
Typical analysis
Extraction
Decant the ether
layer into a tube.
12
Typical analysis
Extraction
Evaporate ether
on water bath
using nitrogen
stream to aid
in evaporation.
13
Typical analysis
Extraction
Dissolve the
residue in
chloroform.
14
Typical analysis
Methylation
Transfer mixture
to a glass vial.
Evaporate to
dryness.
15
Typical analysis
Methylation
Add 7% BF3
reagent and
toluene.
16
Typical analysis
Methylation
Seal vials with
screwcap and
heat them at
100 oC.
17
Typical analysis
Methylation
Allow vials to cool
to room
temperature.
18
Typical analysis
Methylation
Add water (5 mL),
hexane (1 mL),
Na2SO4 (1 g).
Shake
19
Typical analysis
Methylation
Allow layers to
separate and transfer
top layer to another
vial containing
Na2SO4 (1 g).
20
Typical analysis
Methylation
Top layer should
contain FAMEs
and internal
standard.
21
Typical analysis
GC Determination
Transfer to
autosampler vial
for GC analysis.
22
Fatty acids (saturated)
4:0 Butyric
6:0 Caproic
8:0 Caprylic
10:0 Capric
11:0 Undecanoic
12:0 Lauric
14:0 Myristic
23
Fatty acids (saturated)
15:0 Pentadecanoic
16:0 Palmitic
17:0 Margaric
18:0 Stearic
20:0 Arachidic
22:0 Behenic
24:0 Lignoceric
24
Definition
„
Trans fatty acids means the sum of all
unsaturated fatty acids which contains at
least one nonconjugated and trans double
bond.
25
trans-Fatty acids
14:1 trans-Myristelaidic
16:1 trans-Palmitelaidic
18:1 trans 6-Petroselenic
18:1 trans-Elaidic
18:1 trans 11-Vaccenic
18:2 trans-Linolelaidic
18:2 trans 9-Linolelaidic
18:2 trans 12-Linolelaidic
26
trans-Fatty acids
18:2 cis-9, trans-11-Octadecadienic
18:2 Linoleic – conjugated*
20:1 Eicosenic trans 11
22:1 13-trans Docosenic
*Three 18:2 linoleic – conjugated fatty acids were listed in method
996.06
27
trans-Fatty acids (18:3 trans)
18:3 trans-9, trans-12, trans-15-octadecatrienoic
18:3 trans-9, trans-12, cis-15-octadecatrienoic
18:3 trans-9, cis-12, trans-15-octadecatrienoic
18:3 cis-9, trans-12, trans-15-octadecatrienoic
18:3 cis-9, cis-12, trans-15-octadecatrienoic
18:3 cis-9, trans-12, cis-15-octadecatrienoic
18:3 trans-9, cis-12, cis-15-octadecatrienoic
28
50
40
60
10
15
pA
70
20
30
25
30
3A3.26 8 - C14 :0
re
a:
16
3A4.63 3 - C14 :1T (9-tra ns )
2.
re
a:
32 0 - C15 :0
39A5.67
58
re
.7
a:
78
18
3
3.
3A7.96 8 - C16 :04 51
re
a:
3A9.06 3 - C16 :1T (9-tra ns )
15
re
4
a:
4A0.17 3 .-2 1 C17 :0
48
7
r
ea
.7
97
:1
2
52
.8 9 - C18 :0
4A2.28
re 3 3
a:
44A3.12
C18
:1T
(6-tra
3.20
C18
:1T
(9-tra
ns
4A3.31
3 9-4 --C18
:1T
(11
-tranns
s )))
16
re re a
4.
a: : 5
29
42 0 3. - C18 :2TT (9 , 12 -tran s )
4A4.67
5
re .9 53 5
a: 8 63
49 1
4A6.27 0 - C20 :0
.8
re
51
a:
4A7.11 1 -2 C20 :1T (11 -tran s )
re
1
a:4A8.03 7 - C18 :2T (9-c is , 11 -tran s ) 6 3.
4A8.33
46
5 3 - C18 :2T (10 -tran s , 1 2-c is )
re 0re.1a:
6
a: 4 76
58 83 .8
4A9.95 2 - C22 :0
.5 4 9
re
97 6
5A0.74 0 - C22
:1T (13 -tran s )
a:
7
re
16
a:
4.
50
43
.2
15
4
5A3.54 1 - C24 :0
re
a:
17
1.
15
9
2A2.97 3 - C10 :0
re
a:
14
8.
27
7
2 5.60
A r 1 - C11 :0
ea
:5
24
.4
83
2A8.20 3 - C12 :0
re
a:
14
6.
83
8
1A8.18 9 - C8:0
re
a:
12
2.
22
2
1A2.61 0 - C4:0
re
a:
84
.
1A4.67632 4 - C6:0
re
1
a:
94
.9
64
2
FAME (saturated and trans)
SP-2560, 100m x 0.25mm ID, 0.2 µm,
flow rate: 0.7 mL/min
FID1 A, (090715\Q6F.D)
20
35
40
45
50
55
min
29
40
35
25
43
43.1
30
ns
a
r
t
43.2
A4r 3. 31 3 - C18 : 1T (11 -t ran s )
ea
:4
2.
95
81
4 3. 20 4 - C18 : 1T (9-t ra ns )
A4r 3. 12 9 - C18 : 1T (6-t ra ns )
ea
:5
0.
35
63
FAME (18:1 trans)
FID1 A, (090715\Q6F.D)
pA
45
t
s
r an
ns
a
r
t
20
15
43.3
43.4
min
30
45
40
35
pA
50
30
46.75
47
47.25
ns
a
r
t
ns
tra
47.5
47.75
cis
48
A4r 8 . 3 3 3 - C 1 8 : 2 T (1 0 -t ra n s , 1 2 -c is )
ea
:5
8.
59
77
A4r 8 . 0 3 7 - C 1 8 : 2 T (9 -c is , 1 1 -t ra n s )
ea
:6
3.
84
96
A4r 7 . 1 1 1 - C 2 0 : 1 T (1 1 -t ra n s )
ea
:5
0.
14
78
FAME (20:1 trans and 18:2 trans)
FID1 A, (090715\Q6F.D)
cis
ns
a
r
t
25
20
15
48.25
48.5
min
31
30
25
20
- C18:2TT (9, 12-trans )
A44.633
re
a
- C18:2T
(9-c is , 12-trans )
A45.079
- :C18:2T
(9-trans , 12-c is )
15
re
A45.250
r ea :
.
15
a: 6
09
6..3 2
4 97
099
6
pA
35
25.568
Ar - C11:0
ea
:4
97
.3
21
FAME (18:2 trans)
FID1 A, (090813\CHK-2F.D)
15
25
30
35
40
45
min
32
26
24
22
20
18
44.4
44.6
44.8
45
- C18:2T (9-trans , 12-c is )
A45.250
re
a:
6.
43
12
9
- C18:2T (9-c is , 12 -tran s )
A45.079
re
a:
6.
32
79
- C18:2TT (9, 12-trans )
A44.633
re
a:
15
.1
50
9
FAME (18:2 trans)
FID1 A, (090813\CHK-2F.D)
pA
28
16
14
45.2
min
33
30
10
15
20
25
40
30
35
40
(9 1-t-traran ns s) )
A4r 2 . 3 0 0 - CA4128.:4172T25 (6- -tCra1n8s: 1) T (1
re
ea
a
:
:
A4r 3 . 7 94 .22 1 - C 1 8 : 2 T35T. (9 , 1 2 -t ra n s )
30
ea
38
04
:0
9
.
A4r 5 243 1 8 - C 2 0 : 0
ea 76
: 2 79
A4r 7 . .18 0195 - C 1 8 : 2 T (9 -c is , 1 1 -t ra n s )
ea
: 7 35
.3
A4r 9 . 0 9 337-1 C 2 2 : 0
ea
8
:0
.9
13
72
A5r 2 . 5 9 7 - C 2 4 : 0
ea
:0
.3
72
42
3
39.331 - C17:0
A3r 2 . 4 2 3 - C 1 4 : 0
ea
:4
8.
16
9
A3r 4 . 8 2 2 - C 1 5 : 0
ea
:8
.4
79
94
3 8 . 2 4 2 - C 1 6 : 1 T (9 -t ra n s )
A2r 7 . 3 6 7 - C 1 2 : 0
ea
:3
.7
04
47
60
4 1 . 4A8 5 - C 1 8 : 0
re
a:
27
2 .0
4
3 7 . 1A 6 2 - C 1 6 : 0
re
a:
38
5 .0
24
2 4 . 7A 4 9 - C 1 1 : 0
re
a:
19
5 .5
37
pA
A2r 2 . 1 6 3 - C 1 0 : 0
ea
:1
.7
66
48
A1r 7 . 4 2 4 - C 8 : 0
ea
:0
.5
68
31
5
Hard margarine
FID2 B, (GC-FTIR\080919\366.D)
50
20
45
50
55
min
34
38
30
28
26
24
22
A4r 3 . 7 9 2 - C 1 8 : 2 TT (9 , 1 2 -t ra n s )
e aA4 4 . 1 9 1 - C 1 8 : 2 T (9 -c is , 1 2 -t ra n s )
: r0eA4 4 . 4 3 9 - C 1 8 : 2 T (9 -t ra n s , 1 2 -c is )
.a2r:e
33a7
.:637
.927
9242
32
pA
2 4 . 7A 4 9 - C1 1 : 0
re
a:
19
5.
53
7
Hard margarine
FID2 B, (GC-FTIR\080919\366.D)
36
34
32
25
30
35
40
45
min
35
36
34
32
30
28
26
24
FID2 B, (GC-FTIR\080919\366.D)
ns
a
r
t
38
22
42
42.5
43
43.5
44
A4r 5. 41 8 - C20 : 0
ea
:2
.8
09
35
pA
A4r 3. 79 2 - C18 : 2TT (9 , 12 -t ran s )
ea
:0
.2
37
67
9
A4r 2. 30 0 - C18 : 1T (6-t ra ns )
ea
:4
.2
A4r 2. 42 5 - C18 : 1T (9-t ra ns )
13
4 e2.a 47 2 - C18 : 1T (11 -t ran s )
89
:3
5.
30
04
Hard margarine
ns
tra
ns
a
r
t
44.5
45
45.5
min
36
26
24
22
28
42
42.5
43
43.5
44
A4r 4. 18 7 - C18 : 2T (9-c is , 12 -t ran s )
ea
:3
.6
29
88
A4r 4. 43 4 - C18 : 2T (9-t ra ns , 12 -c is )
ea
:3
.5
14
42
A4r 3. 79 2 - C18 : 2TT (9 , 12 -t ran s )
ea
:0
.2
37
67
9
Hard margarine
FID2 B, (GC-FTIR\080919\366.D)
pA
32
30
44.5
45
min
37
Corn oil and 18:3 trans
38
Olive oil and 18:3 trans
39
Points to note (1)
„
Definition of “0”
Saturated fatty acids ≤0.5/100 g
Trans fatty acids ≤0.3/100 g
„
Limit of detection of saturated fatty acids
and trans fatty acids should be better than
0.5 g/100 g and 0.3 g/100 g respectively
40
Points to note (2)
„
For prepackaged product with “Free of sat
fat”claim:
Sum of sat and trans fat ≤0.1/100 g
„
Limit of detection of saturated fatty acids
and trans fatty acids should be better than
0.05 g/100 g respectively.
41
Points to note (3)
„
Availability of FAME standards for calibration
is crucial to this test method.
„
Insufficient FAME standards would
underestimate the level of saturated fat and
trans fat.
42
Availability of FAME standards
Single and/or mixed FAMEs are available from
different suppliers, including:
1. Supelco
2. Fluka
3. Nu-check Prep
4. Chem Service
5. Sigma
43
Proficiency test
FAPAS
AOAC
LGC
44
Thank You
45