nmranalysis lipids · 2013. 11. 28. · pilion al 0 10 20 30 40 50 60 70 80 0 6 12 18 lesvos 0 6 12...
TRANSCRIPT
1H ΝΜR spectroscopyOlive oil adulteration with vegetable oils
0.760.800.840.880.920.961.001.041.081.12
(ppm)
Olive oil
Soya oil
Linolenic ester
CH3
COO~
NMR spectroscopy and Chemometrics
• Quality Control of olive oil (composition, age)
• Geographical and votanical classification
• Year of production variations
• Detection of adulteration with vegetable oils and low quality olive oils
Food Chemistry and Analysis
Chromatographic techniques for lipid research
� GC-MS
� LC-MS
� SEC
Low LOD (limit of detection) but time consuming and require standards
NMR spectroscopyNMR spectroscopy
• Non-destructive
• Fast (15-20 min)
• Quantitative
• Does not require standards
144.4 144.8 145.2 145.6 146.0 146.4 146.8 147.2 147.6 148.0 148.4 148.8
(ppm)
144.4 144.8 145.2 145.6 146.0 146.4 146.8 147.2 147.6 148.0 148.4 148.8
(ppm)
1-MG
144.4 144.8 145.2 145.6 146.0 146.4 146.8 147.2 147.6 148.0 148.4 148.8
(ppm)
1-MG
2-MG
1,2-DG
1,3-DG
CH
CH2OCOR
CHOCOR
CH2OH
CH2OCOR
CHOH
CH2OCOR
SterolsAcidity
CH2OCOR
CHOH
CH2OH
CH2OH
CHOCOR
CH2OH
O
OPR-OH + HCl +
O
OPOR
I II
31Ρ NMR spectrum of olive oilGlyceride composition
A. Spyros, P. Dais, J. Agric. Food. Chem., 48, 802 (2000)
DGsin virgin olive oils
• Virgin oils contain 1-3% total DGs
• Fresh virgin oils contain mainly 1,2-DGs
D=1,2-DGs/DGs > 0.9
• D is further reduced during storage, thus it is a good index of aging
DGsin low quality olive oils
• Refined oils: 4-5 % total DGs
• Deodorized oils: 5-10% total DGs
• Low quality oils contain mainly 1,3-DGs
D=1,2-DGs/DGs ~ 0.33
• D can be used for authentication of virgin oils
Ratio D for ~140 olive oils
Determination of olive oil age
• There exists no official method
• Olive oil age is important for the consumers, olive oil bottling companies and the international olive oil trade in general, because the organolepticcharacteristics of olive oil are affected by prolonged storage.
• Can we use DGs for that ?
Kinetics of diglyceride, DG, isomerizationin olive oil
1,2-DGs 1,3-DGsk
k'
CH2OCOR
CHOCOR
CH2OCOR
CH2OCOR
CHOCOR
CH2OH
CH2OCOR
CHOH
CH2OCOR
RCOOH
TGs
H
1,2-DGs
1,3-DGs
1,2-
DG
s [µ
mol
/g]
5D
10
20
30
40
50
AD
10
20
30
40
50
Pilion
AL
10
20
30
40
50
0 6 12 18
Lesvos0 6 12 18
Sitia0 6 12 18
Iraklion0 6 12 18
time [months]
1,3-
DG
s [µ
mol
/g]
5D
-5
10
25
40
55
AD
-5
10
25
40
55
Pilion
AL
-5
10
25
40
55
0 6 12 18
Lesvos0 6 12 18
Sitia0 6 12 18
Iraklion0 6 12 18
Evolution of 1,2-DGs and1,3-DGs with aging
Evolution of total DGs and acidityΗ with aging
time [months]
Fre
e ac
idity
[µm
ol/g
]
5D
0
10
20
30
40
50
AD
0
10
20
30
40
50
Pilion
AL
0
10
20
30
40
50
0 6 12 18
Lesvos0 6 12 18
Sitia0 6 12 18
Iraklion0 6 12 18
tota
l DG
s [µ
mol
/g]
5D
01020304050607080
AD
01020304050607080
Pilion
AL
01020304050607080
0 6 12 18
Lesvos0 6 12 18
Sitia0 6 12 18
Iraklion0 6 12 18
tHH 0 hk+=
tkh+[DGs]=[DGs]0
Kinetics of diglyceride, DG, isomerizationin olive oil
1,2-DGs + H 1,3-DGs + Hk
k'
CH2OCOR
CHOCOR
CH2OCOR
CH2OCOR
CHOCOR
CH2OH
CH2OCOR
CHOH
CH2OCOR
RCOOH
TGs
H
1,2-DGs
1,3-DGsTGs
kh
tHH 0 hk+=
tk[DGs]DGs h0 +=
0 5 10 15 20
10
20
30
40
50
60
70
80
90
a
1,3-DGs
1,2-DGs
H
DGs
µmol/g
time [months]
0
10
20
30
40
50
60
70
kh
0 0.2 0.5 1
µmol/g
Evolution of DGs andΗ with time
A. Spyros, A. Filippidis, P. Dais, J.Agric. Food. Chem., 52, 157 (2004)
0 5 10 15 20
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
0 0.2 0.5 1
b
kh
D
time [months]
DGs
DGs2,1D
−=
tkke
kk
kk
kk
kD
H])[H'(2
1
0
00 0
])[DGs]'(
DGs][1,3'DGs],21[[)
'
'(
++−
⋅+
−−−+
+=
[µmol * month/g]
H*t
D
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
-100 100 300 500 700 900
PilionLesvosSitiaIraklion
[µmol * month/g]
H t
D/D
0
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
0 100 200 300 400 500
PilionLesvosSitiaIraklion
k+k’= 17.4 x 10-3 g mol-1 month-1
k+k’= 1.4 x 10-3 g mol-1 month-1
0 2 4 6 8 10 12 14 16 18 20
0
2
4
6
8
10
12
14
16
18
20
age[months]
time[months]
set A set B set C
Storage at room temperature
H*0165.0
)62.0
32.0Dln(
age
−−
=
Application to commercial extra virgin olive oils
• Eight (8) virgin olive oils were obtained from supermarkets. Four (4) of them were found to contain olive oil from the previous crop season, against labeling information.
• “Minerva”, the biggest olive oil bottler in Greeceused for two years this methodology to verify the freshness of the olive oil supplied by local agricultural cooperatives from all over the country.
1H NMR spectrum of olive oil
P. Fronimaki, A. Spyros, S. Christoforidou, P. Dais, J. Agric. Food. Chem., 50, 2207 (2002)
Fatty acid composition
Oleic acid
Linoleic acid
COOH
COOH
Linolenic acid
COOH2
3
4
5
6
7
8
9
1011
12
1314
15
16
17
18
1
COOH
COOH Palmitic acid
Stearic acid
O
OCH2
CH
CH2
O
O
O
OTripalmitin
Oleic acid
Linoleic acid
COOH
COOH
Linolenic acid
COOH2
3
4
5
6
7
8
9
1011
12
1314
15
16
17
18
1
COOH
COOH Palmitic acid
Stearic acid
O
OCH2
CH
CH2
O
O
O
OTripalmitin
Oil composition in:•saturated•monounsaturated•di-unsaturated•polyunsaturated fatty acids•sterols•Iodine Value
ChemometricsApplication of multivariate statistical methodsin the analysis of large volumes of data
What to do with all those data?
x~500 oil samples
>6000 experimental data
1,3-DGs
Total DGs
Ratio D
Acidity
Sterols
1,2-DGs
31PNMR
1,3-DGs
Total DGs
Ratio D
Acidity
Sterols
1,2-DGs
31PNMR
Iodinenumber
SFA Oleic
Linoleic
Linolenic
1Η
NMR
Iodinenumber
SFA Oleic
Linoleic
Linolenic
1Η
NMR
Geographical classification of Greek virgin olive oils
Κρήτη
Πελοπόννησος
Λέσβος
Πήλιο
Root 1
Roo
t 2
-7
-6
-5
-4
-3
-2
-1
0
1
2
3
4
5
-6 -4 -2 0 2 4 6 8 10 12 14 16
Crete
Peloponese
LesvosLesvos
Pelion
Classification of Greek virgin olive oils according to olive fruit variety
Κορωνέικη
Κολοβή
Πηλίου
Root 1
Roo
t 2
-6
-5
-4
-3
-2
-1
0
1
2
3
4
5
6
-6 -4 -2 0 2 4 6 8 10 12 14 16
Koroneiki
Kolovi
Pilioy
Classification of Greek virgin olive oils according to production year
2000-2001
2001-2002
2002-2003
Root 1
Roo
t 2
-6
-5
-4
-3
-2
-1
0
1
2
3
4
5
-8 -6 -4 -2 0 2 4 6
Classification of 13 types of vegetable oils
-40 -20 0 20 40 60 80-20
-15
-10
-5
0
5
10
15
20
25
30
virgin olive soybean hazelnut corn sesame groundnut sunflower almond safflower palm coconut walnut rapeseed
Roo
t 2
Root 1
Adulteration of olive oil with vegetable oils
Root 1
Roo
t 2
-15
-10
-5
0
5
10
15
20
25
-30 -20 -10 0 10 20 30
virgin olive
hazelnut
sunflower
soybean
corn
mhazelnut
msun
msoybean
mcorn
G. Vigli, A. Filippidis, A. Spyros, P. Dais, J.Agric. Food. Chem., 51, 5715 (2003)
Adulteration of virgin olive oil with low quality olive oils
G. Fragkaki, A. Spyros, G. Siragakis, A. Salivaras, P. Dais, J.Agric. Food. Chem., 53, 2810 (2005)