Analysis of Fatty Acids · 2017. 6. 16. · 2 AOAC Official Methods cis-and trans-Octadecenoic...
Transcript of Analysis of Fatty Acids · 2017. 6. 16. · 2 AOAC Official Methods cis-and trans-Octadecenoic...
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Analysis of Fatty Acids
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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
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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.
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Typical analysis
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Typical analysis
FatFat HydrolysisHydrolysis
Methylatedto FAMEs
Methylatedto FAMEs
GC-FID(quantitation)GC-FID
(quantitation)
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Typical analysis
Hydrolysis
Weigh sampleand pyrogallicacid into the Mojonnier flask
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Typical analysis
Hydrolysis
Add internalstandard, ethanoland 8.3 M HCl.
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Typical analysis
Hydrolysis
Hydrolyse thesample at 70 –80 oC
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Typical analysis
Hydrolysis
Remove flasksfrom water bath.Cool to roomtemperature.
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Typical analysis
Extraction
Add ethanol, Ether and shake.
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Typical analysis
Extraction
Centrifuge sample.(allow contents tosep. at least 1 h if centrifuge is notavailable)
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Typical analysis
Extraction
Decant the etherlayer into a tube.
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Typical analysis
Extraction
Evaporate etheron water bathusing nitrogenstream to aidin evaporation.
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Typical analysis
Extraction
Dissolve the residue in chloroform.
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Typical analysis
Methylation
Transfer mixture to a glass vial. Evaporate todryness.
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Typical analysis
Methylation
Add 7% BF3
reagent and toluene.
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Typical analysis
Methylation
Seal vials withscrewcap andheat them at 100 oC.
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Typical analysis
Methylation
Allow vials to coolto roomtemperature.
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Typical analysis
Methylation
Add water (5 mL),hexane (1 mL),Na2SO4 (1 g).Shake
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Typical analysis
Methylation
Allow layers to separate and transfer top layer to anothervial containing Na2SO4 (1 g).
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Typical analysis
Methylation
Top layer shouldcontain FAMEsand internal standard.
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Typical analysis
GC Determination
Transfer to autosampler vialfor GC analysis.
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Fatty acids (saturated)
14:0 Myristic
12:0 Lauric
10:0 Capric11:0 Undecanoic
8:0 Caprylic
6:0 Caproic4:0 Butyric
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Fatty acids (saturated)
15:0 Pentadecanoic
24:0 Lignoceric
22:0 Behenic
20:0 Arachidic
18:0 Stearic
17:0 Margaric
16:0 Palmitic
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Definition
Trans fatty acids mean the sum of all unsaturated fatty acids which contains at least one nonconjugated and trans double bond.
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trans-Fatty acids
18:2 trans 9-Linolelaidic18:2 trans 12-Linolelaidic
18:2 trans-Linolelaidic
18:1 trans 11-Vaccenic
18:1 trans-Elaidic
18:1 trans 6-Petroselenic
16:1 trans-Palmitelaidic
14:1 trans-Myristelaidic
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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*
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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
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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:
84.6241
Area: 9
4.9642
Area: 1
22.222 A
rea: 148.2
77 A
rea: 52
4.483 A
rea: 146.8
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Area:
162.932
Area:
58.7783
Area:
183.451
Area: 1
54.217
Area:
48.79
72
Area:
152.833
Area:
164.295
Area:
50.35
63
Area: 4
2.9581
Area:
49.85
12 A
rea: 163.4
66
Area:
50.1478
Area:
63.84
96
Area: 5
8.5977
Area:
164.43
Area: 5
0.2154
Area: 1
71.159
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
-tra
ns)
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-tr
ans)
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
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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
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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-tra
ns)
48.
037
- C
18:2
T (9
-cis
, 11-
trans
)
48.
333
- C
18:2
T (1
0-tr
ans,
12-
cis)
transcis
trans
transcis
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FAME (18:2 trans)
min25 30 35 40 45
pA
15
20
25
30
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FID1 A, (090813\CHK-2F.D)
Area:
497.3
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Area:
15.15
09
Area:
6.327
9
Area:
6.490
96
25.
568
- C
11:0
44.
633
- C
18:2
TT (
9, 1
2-tr
ans)
45.
079
- C
18:2
T (9
-cis
, 12
-tra
ns)
45.
250
- C
18:2
T (9
-tra
ns,
12-c
is)
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FAME (18:2 trans)
min44.4 44.6 44.8 45 45.2
pA
14
16
18
20
22
24
26
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FID1 A, (090813\CHK-2F.D)
Area
: 15.1
509
Area
: 6.32
79
Area
: 6.431
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44.
633
- C
18:2
TT (9
, 12
-tran
s)
45.
079
- C
18:2
T (9
-cis
, 12
-tran
s)
45.
250
- C
18:2
T (9
-tran
s, 1
2-ci
s)
trans
cistrans
transcis
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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
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Hard margarine
min25 30 35 40 45
pA
22
24
26
28
30
32
34
36
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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-
trans
) 4
4.43
9 -
C18
:2T
(9-t
rans
, 12
-cis
)
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Hard margarine
min42 42.5 43 43.5 44 44.5 45 45.5
pA
22
24
26
28
30
32
34
36
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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
-tra
ns)
42.
425
- C
18:1
T (9
-tra
ns)
42.
472
- C
18:1
T (1
1-tra
ns)
43.
792
- C
18:2
TT (
9, 1
2-tr
ans)
45.
418
- C
20:0
tran
s
trans
trans
trans
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Hard margarine
min42 42.5 43 43.5 44 44.5 45
pA
22
24
26
28
30
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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-tr
ans)
44.
187
- C
18:2
T (9
-cis
, 12
-tra
ns)
44.
434
- C
18:2
T (9
-tra
ns,
12-c
is)C18
:2(9
,12-
tran
s)
C18
:2(9
-tran
s,12
-cis)
C18
:2(9
-cis,
12-tr
ans)
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Corn oil and 18:3 trans
tran
s
tran
sci
str
ans
cis
tran
s
cis
tran
str
ans
cis
cis
tran
s
cis
tran
sci
str
ans
cis
cis
cis
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Olive oil and 18:3 trans
C18
:3(9
,12,
15-tr
ans)
C18
:3(9
,12-
tran
s,15
-cis)
,C
18:3
(9-tr
ans,
12-c
is,15
-tran
s)
C18
:3(9
-cis,
12-tr
ans,
15-tr
ans)
,C
18:3
(9-c
is,12
-cis,
15-tr
ans)
C18
:3(9
-cis,
12-tr
ans,
15-c
is),
C18
:3(9
-tran
s,12
-cis,
15-c
is)
C20
:0
C18
:3(9
-cis,
12-c
is,15
-cis)
Olive oilStandard
C20
:1(c
is)
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Points to note (1)
Definition of “0”Saturated fatty acids ≤0.5 g/100 gTrans fatty acids ≤0.3 g/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
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Points to note (2)
For prepackaged product with “Free of sat fat”claim:
Sum of sat and trans fat ≤0.1 g/100 g
Limit of detection of saturated fatty acids and trans fatty acids should be better than 0.05 g/100 g respectively.
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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.
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Availability of FAME standards
Single and/or mixed FAMEs are available from different suppliers, including:
1. Supelco2. Fluka3. Nu-check Prep4. Chem Service5. Sigma
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Proficiency test
FAPAS
AOAC
LGC
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Thank You