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How to analyse fatty acids
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1
Analysis of Fatty Acids
2
AOAC Official Methods
GC-IRcis- and trans-Octadecenoic isomers and general fatty acid composition
994.15
GC-FIDtrans-Fatty acid in margarines985.21etc
GC-FIDEncapsulated fish oils991.39
GC-FIDFat (total, saturated and unsaturated) in foods
996.06
Preparation methyl esters
Fatty acids in oils and fats969.33
3
Typical analysis
Fat and fatty acids are extracted from food by hydrolytic method.
Fat is extracted into ether, then methylatedto fatty acid methyl esters (FAMEs).
FAMEs are quantitatively measured by gas chromatography.
4
Typical analysis
5
Typical analysis
FatFat HydrolysisHydrolysis
Methylatedto FAMEsMethylatedto FAMEs
GC-FID(quantitation)GC-FID
(quantitation)
6
Typical analysis
Hydrolysis
Weigh sampleand pyrogallicacid into the Mojonnier flask
7
Typical analysis
Hydrolysis
Add internalstandard, ethanoland 8.3 M HCl.
8
Typical analysis
Hydrolysis
Hydrolyse thesample at 70 –80 oC
9
Typical analysis
Hydrolysis
Remove flasksfrom water bath.Cool to roomtemperature.
10
Typical analysis
Extraction
Add ethanol, Ether and shake.
11
Typical analysis
Extraction
Centrifuge sample.(allow contents tosep. at least 1 h if centrifuge is notavailable)
12
Typical analysis
Extraction
Decant the etherlayer into a tube.
13
Typical analysis
Extraction
Evaporate etheron water bathusing nitrogenstream to aidin evaporation.
14
Typical analysis
Extraction
Dissolve the residue in chloroform.
15
Typical analysis
Methylation
Transfer mixture to a glass vial. Evaporate todryness.
16
Typical analysis
Methylation
Add 7% BF3
reagent and toluene.
17
Typical analysis
Methylation
Seal vials withscrewcap andheat them at 100 oC.
18
Typical analysis
Methylation
Allow vials to coolto roomtemperature.
19
Typical analysis
Methylation
Add water (5 mL),hexane (1 mL),Na2SO4 (1 g).Shake
20
Typical analysis
Methylation
Allow layers to separate and transfer top layer to anothervial containing Na2SO4 (1 g).
21
Typical analysis
Methylation
Top layer shouldcontain FAMEsand internal standard.
22
Typical analysis
GC Determination
Transfer to autosampler vialfor GC analysis.
23
Fatty acids (saturated)
14:0 Myristic12:0 Lauric
10:0 Capric11:0 Undecanoic
8:0 Caprylic6:0 Caproic4:0 Butyric
24
Fatty acids (saturated)
15:0 Pentadecanoic
24:0 Lignoceric22:0 Behenic20:0 Arachidic18:0 Stearic17:0 Margaric16:0 Palmitic
25
Definition
Trans fatty acids means the sum of all unsaturated fatty acids which contains at least one nonconjugated and trans double bond.
26
trans-Fatty acids
18:2 trans 9-Linolelaidic18:2 trans 12-Linolelaidic
18:2 trans-Linolelaidic18:1 trans 11-Vaccenic 18:1 trans-Elaidic18:1 trans 6-Petroselenic 16:1 trans-Palmitelaidic14:1 trans-Myristelaidic
27
trans-Fatty acids
*Three 18:2 linoleic – conjugated fatty acids were listed in method 996.06
22:1 13-trans Docosenic20:1 Eicosenic trans 11
18:2 cis-9, trans-11-Octadecadienic18:2 Linoleic – conjugated*
28
trans-Fatty acids (18:3 trans)
18:3 cis-9, trans-12, cis-15-octadecatrienoic18:3 trans-9, cis-12, cis-15-octadecatrienoic
18:3 cis-9, trans-12, trans-15-octadecatrienoic18:3 cis-9, cis-12, trans-15-octadecatrienoic
18:3 trans-9, cis-12, trans-15-octadecatrienoic
18:3 trans-9, trans-12, trans-15-octadecatrienoic18:3 trans-9, trans-12, cis-15-octadecatrienoic
29
FAME (saturated and trans)
min10 15 20 25 30 35 40 45 50 55
pA
20
30
40
50
60
70
FID1 A, (090715\Q6F.D)
Area: 8
4.6241
Area:
94.96
42 A
rea: 12
2.222 A
rea: 148.277
Area:
524.4
83 A
rea: 146.8
38
Area: 1
62.932
Area:
58.77
83
Area:
183.4
51 A
rea: 15
4.217
Area:
48.7972
Area: 1
52.833
Area:
164.2
95
Area:
50.3563
Area:
42.95
81
Area:
49.8512
Area:
163.4
66
Area:
50.14
78
Area:
63.84
96
Area: 5
8.5977
Area:
164.4
3
Area: 5
0.2154
Area:
171.1
59
12.
610
- C
4:0
14.
673
- C
6:0
18.
189
- C
8:0
22.
973
- C
10:0
25.
601
- C
11:0
28.
203
- C
12:0
33.
268
- C
14:0
34.
633
- C
14:1
T (9
-tra
ns)
35.
670
- C
15:0
37.
968
- C
16:0
39.
063
- C
16:1
T (9
-tran
s)
40.
173
- C
17:0
42.
289
- C
18:0
43.
129
- C
18:1
T (6
-tra
ns)
43.
204
- C
18:1
T (9
-tra
ns)
43.
313
- C
18:1
T (1
1-tr
ans)
44.
673
- C
18:2
TT (
9, 1
2-tra
ns)
46.
270
- C
20:0
47.
111
- C
20:1
T (1
1-tr
ans)
48.
037
- C
18:2
T (9
-cis
, 11
-tra
ns)
48.
333
- C
18:2
T (1
0-tr
ans,
12-
cis)
49.
952
- C
22:0
50.
740
- C
22:1
T (1
3-tr
ans) 5
3.54
1 -
C24
:0
SP-2560, 100m x 0.25mm ID, 0.2 µm, flow rate: 0.7 mL/min
30
FAME (18:1 trans)
min43 43.1 43.2 43.3 43.4
pA
15
20
25
30
35
40
45
FID1 A, (090715\Q6F.D)
Area: 5
0.3563
Area: 4
2.9581
43.
129
- C
18:1
T (6
-tra
ns)
43.
204
- C
18:1
T (9
-tra
ns)
43.
313
- C
18:1
T (1
1-tr
ans)
transtrans trans
31
FAME (20:1 trans and 18:2 trans)
min46.75 47 47.25 47.5 47.75 48 48.25 48.5
pA
15
20
25
30
35
40
45
50
FID1 A, (090715\Q6F.D)
Area: 5
0.1478
Area: 6
3.8496
Area: 5
8.5977
47.
111
- C
20:1
T (1
1-tr
ans)
48.
037
- C
18:2
T (9
-cis
, 11
-tra
ns)
48.
333
- C
18:2
T (1
0-tr
ans,
12-
cis)
transcis
trans
transcis
32
FAME (18:2 trans)
min25 30 35 40 45
pA
15
20
25
30
35
FID1 A, (090813\CHK-2F.D)
Area:
497.3
21
Area:
15.15
09
Area:
6.327
9
Area:
6.490
96
25.
568
- C
11:0
44.
633
- C
18:2
TT (9
, 12-
tran
s) 4
5.07
9 -
C18
:2T
(9-c
is,
12-t
rans
) 4
5.25
0 -
C18
:2T
(9-tr
ans,
12-
cis)
33
FAME (18:2 trans)
min44.4 44.6 44.8 45 45.2
pA
14
16
18
20
22
24
26
28
FID1 A, (090813\CHK-2F.D)
Area:
15.1509
Area:
6.3279
Area:
6.43129
44.
633
- C
18:2
TT (
9, 1
2-tr
ans)
45.
079
- C
18:2
T (9
-cis
, 12-
trans
)
45.
250
- C
18:2
T (9
-tra
ns, 1
2-ci
s)
34
Hard margarine
min10 15 20 25 30 35 40 45 50 55
pA
20
30
40
50
60
FID2 B, (GC-FTIR\080919\366.D)
Area: 0
.568315
Area: 1
.76648 A
rea: 195.537
Area: 3
.70447
Area: 4
8.169 A
rea: 8.47994
Area: 3
85.024
Area: 2
72.04
Area: 4
.21389
Area: 3
5.3004
Area: 0
.237679
Area: 2
.80935 A
rea: 7.33718
Area: 0
.91372 A
rea: 0.372423
17.
424
- C
8:0
22.
163
- C
10:0
24.
749
- C
11: 0
27.
367
- C
12:0
32.
423
- C
14:0
34.
822
- C
15:0
37.
162
- C
16: 0
38.
242
- C
16:1
T (9
-tra
ns)
39.
331
- C
17:0
41.
485
- C
18: 0
42.
300
- C
18:1
T (6
-tra
ns)
42.
425
- C
18:1
T (9
-tra
ns)
42.
472
- C
18:1
T (1
1-tr
ans)
43.
792
- C
18:2
TT (
9, 1
2-tr
ans)
45.
418
- C
20:0
47.
115
- C
18:2
T (9
-cis
, 11
-tra
ns)
49.
093
- C
22:0
52.
597
- C
24:0
35
Hard margarine
min25 30 35 40 45
pA
22
24
26
28
30
32
34
36
38
FID2 B, (GC-FTIR\080919\366.D)
Area: 1
95.537
Area: 0
.237679
Area: 3
.63294
Area: 3
.72232
24.
749
- C
11:0
43.
792
- C
18:2
TT (
9, 1
2-tr
ans)
44.
191
- C
18:2
T (9
-cis
, 12
-tra
ns)
44.
439
- C
18:2
T (9
-tra
ns,
12-c
is)
36
Hard margarine
min42 42.5 43 43.5 44 44.5 45 45.5
pA
22
24
26
28
30
32
34
36
38
FID2 B, (GC-FTIR\080919\366.D)
Area: 4
.21389 A
rea: 35.3004
Area: 0
.237679
Area: 2
.80935
42.
300
- C
18:1
T (6
-tran
s)
42.
425
- C
18:1
T (9
-tran
s) 4
2.47
2 -
C18
:1T
(11-
tran
s)
43.
792
- C
18:2
TT (
9, 1
2-tr
ans)
45.
418
- C
20:0
trans
trans
trans
37
Hard margarine
min42 42.5 43 43.5 44 44.5 45
pA
22
24
26
28
30
32
FID2 B, (GC-FTIR\080919\366.D)
Area: 0
.237679
Area: 3
.62988 A
rea: 3.51442
43.
792
- C
18:2
TT (
9, 1
2-tra
ns)
44.
187
- C
18:2
T (9
-cis
, 12-
trans
)
44.
434
- C
18:2
T (9
-tra
ns,
12-c
is)
38
Corn oil and 18:3 trans
39
Olive oil and 18:3 trans
40
Points to note (1)
Definition of “0”Saturated fatty acids ≤0.5/100 gTrans 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
41
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.
42
Points to note (3)
Availability of FAME standards for calibration is crucial to this test method.
Insufficient FAME standards wouldunderestimate the level of saturated fat and trans fat.
43
Availability of FAME standards
Single and/or mixed FAMEs are available from different suppliers, including:
1. Supelco2. Fluka3. Nu-check Prep4. Chem Service5. Sigma
44
Proficiency test
FAPAS
AOAC
LGC
45
Thank You
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