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H- NMR Spectroscopy of Fatty Acids and Their Derivatives – Non-conjugated double bonds
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H-NMR Spectroscopy of Fatty Acids and Their Derivatives
Non-Conjugated Double Bonds
The introduction of one double bond gives rise to several peaks in the NMR spectrum compared to
the saturated chains in methyl stearate or stearic acid.
In methyl oleate (methyl 9(Z)-octadecenoate; Figure 1 (appendix)), the following changes can be
observed in comparison to methyl stearate and stearic acid: Two olefinic protons (integration value
= 2) at about 5.3 ppm, four allylic protons (at C8 and C11) at about 2.05 ppm. The theoretical
integration value of the strong CH2 peak decreases to 20.
Introduction of a second double bond to give linoleic acid or methyl linoleate (methyl 9(Z),12(Z)octadecadienoate; Figure 2 (appendix)) gives rise to a peak at 2.8 ppm caused by the bis-allylic
protons located at C11. The theoretical integration value of the olefinic protons increases to four
while that of the large CH2 peak decreases further to 14.
A third double bond in linolenic acid or methyl linoleate (methyl 9(Z),12(Z)-octadecatrienoate;
Figure 3 (appendix)) does not cause any new peaks compared to linoleic acid or methyl linoleate,
only changes in the integration values. However, the triplet of the terminal methyl group (ω1) shifts
downfield slightly, to about 0.95 ppm. This shift can be used for quantification purposes in
mixtures as the terminal methyl triplet can be integrated separately from the corresponding signals
of the other fatty acids.
“Migration” of the double bond leads to shift of signals, especially when the double bond
approaches one of end of the chain. This effect was discovered in early NMR studies of a full
series of cis-octadecenoic and some acetylenic fatty acids (Gunstone and Ismail, CPL 1967). Prior
work demonstrated this effect for double bonds near the terminal methyl group (Glass and Dutton,
1964; Storey 1960). The shifts of the olefinic protons do not differ significantly, if at all, for positions
toward the middle of the chain (C8-C12 for a C18 chain). For determining such positions in a chain,
a shift reagent [Eu(fod)3d30] was used (Frost and Sies, CPL 1974; Bus and Frost Lipids 1976).
Similar work was carried out for unsaturated triacylglycerols using [Pr(fod)3] (Frost et al. CPL
1975).
The 1H-NMR spectrum of the C18 fatty acid methyl ester methyl petroselinate (Figure 4 (Appendix)),
in which the cis double bond is located at C6, however, shows minor changes in the chemical
shifts compared to methyl oleate, such as the fine structure of the signals of the olefinic protons
and a slight downfield shift of the triplet caused by the C2 protons.
Double bond configuration affects the NMR signals of the unsaturated fatty compounds. The
compounds whose spectra are depicted in Figures 1 to 4 (Appendix) all possess cis double bonds.
Figure 5 (Appendix) shows the NMR spectrum of methyl elaidate, the ∆9 E-isomer of methyl
oleate. Figure 6 (Appendix) is an enlargement of the signals of the olefinic protons of methyl oleate
and methyl elaidate. The smaller coupling constants for cis (6-15 Hz) than for trans (11-18 Hz) are
visible in the spacing of the peaks. Table 1 gives some characteristic shifts of unsaturated fatty
compounds.
G. Knothe
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H- NMR Spectroscopy of Fatty Acids and Their Derivatives – Non-conjugated double bonds
Table 1. Chemical shifts of cis-octadecenoic acids (Gunstone and Ismail 1967;
recalculated from τ values; 60 MHz NMR); solvent CCl4.
Double bond
position
CH2 COOH
2
2.65 (C2, C4)
3
3.01
2.08
4
2.30
2.10
CH CH
6.28, 6.10 (C3),
5.70 (C2)
5.52
CH3
0.90
0.91
5.37
0.91
5
2.10
5.36
0.90
6
2.10
5.35
0.90
7
2.10
5.34
0.90
8
2.10
5.32
0.91
9
2.10
5.32
0.91
10
2.10
5.32
0.90
11
2.10
5.32
0.91
12
2.10
5.32
0.91
13
14
2.10
2.10
5.31
5.32
0.91
0.92
15
2.10
5.31
0.96
16
2.10
5.36
1.62
17
2.15
4.95 (C18),
5.60 (C17)
Methyl oleate
Methyl elaidate
G. Knothe
CH2 CH CH
2.3
2.0
5.27
0.9
2.3-2.4
2.15
5.40
0.95
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H- NMR Spectroscopy of Fatty Acids and Their Derivatives – Non-conjugated double bonds
Table 2. Chemical shifts of cis- and trans-octadecenoic acids and esters (Frost and
Gunstone, Chem. Phys. Lipids, 1975; solvent, carbon tetrachloride (CCl4); 220MHz). This
paper also features a discussion of shielding and deshielding effects and depicts various
peak patterns.
CH2 COOR
Double
bond
CH2 CH CH
CH CH
Acid
Ester
Acid
Ester
Acid
Ester
2 cis
—
—
2.645
2.625
6.285 (C3),
5.735 (C2)
6.145 (C3),
5.680 (C2)
2 trans
—
—
2.21
2.18
7.01 (C3),
5.75 (C2)
6.85 (C3),
5.72 (C2)
3 cis
—
3.06 (C2)
—
3.06 (C2),
2.03 (C5)
—
5.51
2.995 (C2)
2.930 (C2)
2.015 (C5)
2.015 (C5)
5.50
5.47
—
2.27 (C2,
C3)
—
2.270 (C2,
C3), 2.025
(C6)
—
5.31
2.33 (C2, 3)
—
2.33 (C2,
3), 1.95 (6)
—
5.40
—
3 trans
4 cis
4 trans
5 cis
5 trans
5.32
2.28
—
6 cis
6 trans
2.295
2.215
7 cis
7 trans
2.290
—
1.945 (C7),
2.025 (C4)
—
1.995 (C8),
2.04 (C5)
1.990 (C8),
2.02 (C5)
1.950 (C9),
1.99 (C5)
1.950 (C9),
1.975 (C5)
1.985 (C9),
2.01 (C6)
1.980 (C9),
2.0005 (C6)
1.935 (C9),
1.960 (C6)
—
8 cis
5.34
—
5.29
5.28
5.28
8 trans
Frost and Gunstone CPL 1975, ∆12-∆15 determinable by effect on terminal methyl:
For cis: ∆12: 0.890 ppm; ∆13: 0.895 ppm; ∆14: 0.905 ppm; ∆15: 0.950 ppm; ∆16: 1.57 ppm
(doublet).
∆17: C16: 2.01 (broad quartet). 4.88, 4.94, 5.72 ppm for protons on C17.
For trans: ∆14: 0.885 ppm; ∆15: 0.945 ppm. Deshielding for trans extends over only two double
bonds, therefore less informative than for cis. ∆16 1.620 ppm.
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H- NMR Spectroscopy of Fatty Acids and Their Derivatives – Non-conjugated double bonds
Table 3. Chemical shifts in methyl cis,cis-octadecadienoates (Gunstone et al.
Chem. Phys. Lipids, 1969). Solvent CCl4.
CH2 CH CH
CH CH
7, 15
1.96, 2.01
5.26
8, 15
1.96, 2.01
5.26
5, 12
1.96, 2.01
5.27
9, 15
1.96, 2.01
5.26
6, 12
1.99
5.28
7, 12
1.97, 2.02
5.28
6, 11
1.99, 2.04
5.30
8, 12
2.03
5.29
6, 10
2.02
5.29
9, 12
1.99, 2.03
5.26
6, 9
2.04
5.28
10, 12
2.09, 2.16, 2.20
5.32 (C10, C13),
6.12 (C11, C12)
6, 8
2.1-2.5
5.1-5.9
Double bond
positions
CH2 COOMe
The differences in the coupling constants between cis and trans can be visualized by expanding
the peaks of the olefinic protons:
The cis and trans isomers can be distinguished by the coupling constants as well as by some
chemical shifts. For example, the signals of the allylic protons in elaidic acid are shifted slightly
upfield (by about 0.05 ppm) compared to oleic acid. On the other hand, the signals of the olefinic
protons of elaidic acid are slightly downfield (by about 0.03 ppm) compared to oleic acid. Thus, the
shift difference between the peaks of the allylic and the olefinic protons is suitable for distinguishing
cis / trans isomers, with the difference being greater for trans. This effect has been discussed in
the literature (Frost and Gunstone, 1975), where also the δ-values for each methylene group were
calculated.
Gunstone and Jacobsberg (1972) gave the chemical shifts of all isomers of 9,12-diunsaturated C18
acids. See Table 4 for the values of those containing only double bonds.
G. Knothe
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H- NMR Spectroscopy of Fatty Acids and Their Derivatives – Non-conjugated double bonds
Table 4. Chemical shifts in 9,12-dienoic C18 fatty acids (Gunstone and
Jacobsberg, 1972; solvent probably CCl4).a
Isomer
CH CH
HC CH2 CH
CH CH CH2
9c, 12c
5.27
b
5.25
2.71
b
2.68
1.99
b
2.10-2.20
9c, 12t
5.31
2.67
1.97
9t, 12c
5.31
2.68
1.97
9t, 12t
5.32
2.62
1.96
a) Values originally reported on the τ scale, converted to δ scale here.
b) Kannan et al. (1974) in either CCl4, CDCl3 or pyridine, depending on suitability.
Literature
13
Bus, J. and Frost, D.J. Determination of the positions of double bonds in unsaturated fatty acids by C and
proton NMR spectrometry. Lipids, [Invited Lect. Symp. Int. Congr. Fat Res. (1974)] Vol. 2, 343-350.
(pub. 1976).
Frost, D.J. and Gunstone, F.D. The PMR analysis of non-conjugated alkenoic and alkynoic acids and esters.
Chem. Phys. Lipids, 15, 53-85 (1975).
Frost, D.J. and Sies, I. PMR Analysis of alkenoic esters using shift reagents. Chem. Phys. Lipids, 13, 173177 (1974).
Glass, C.A. and Dutton, H.J. Determination of beta-olefinic methyl groups in esters of fatty acids by nuclear
magnetic resonance. Anal. Chem., 36, 2401-2404 (1964).
1
13
Gunstone, F.D. H- and C-NMR spectra of six n-3 polyene esters. Chem. Phys. Lipids, 56, 227-229 (1990).
Gunstone, F.D. and Ismail, I.A. Fatty Acids, Part 15. Nuclear magnetic resonance spectra of the cis
octadecenoic acids and of some acetylenic acids. Chem. Phys. Lipids, 1, 337-340 (1967).
Gunstone, F.D., Lie Ken Jie, M. and Wall, R.T. Fatty Acids. 23. Nuclear magnetic resonance spectra of some
octadecadiynoic acids and of some methyl cis,cis- and trans,trans-octadecadienoates. Chem. Phys.
Lipids, 3, 297-303 (1969).
Gunstone, F.D. and Jacobsberg, F.R. Fatty Acids, Part 36. The synthesis, silver ion chromatographic, and
nmr spectroscopic properties of the nine 9,12-diunsaturated n-C18 acids. Chem. Phys. Lipids, 9, 112122 (1972).
Hashimoto, T., Nukada, K., Shiina, H. and Tsuchiya, T. On the structure of highly unsaturated fatty acids of
fish oils by high resolution nuclear magnetic resonance spectral analysis. J. Am. Oil Chem. Soc., 40,
124-128 (1963).
Kannan, R., Subbaram, M.R. and Achaya, K.T. NMR studies of some oxygenated, halogenated, and
sulphur-containing fatty acids and their derivatives. Fette, Seifen, Anstrichm., 76, 344-350 (1974).
1
Lie Ken Jie, M.S.F. and Lam, C.C. H-Nuclear magnetic resonance spectroscopic studies of saturated,
acetylenic and ethylene triacylglycerols. Chem. Phys. Lipids, 77, 155-171 (1995).
Sacchi, R., Medina, I., Aubourg, S.P., Addeo, F. and Paolillo, L. Proton nuclear magnetic resonance rapid
and structure-specific determination of ω-3 polyunsaturated fatty acids in fish lipids. J. Am. Oil Chem.
Soc., 70, 225-228 (1993).
Storey, W.H. Fatty acids analysis by high resolution nuclear spin resonance. A preliminary evaluation. J. Am.
Oil Chem. Soc., 37, 676-678 (1960).
G. Knothe
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H- NMR Spectroscopy of Fatty Acids and Their Derivatives – Non-conjugated double bonds
Gerhard Knothe
National Center for Agricultural Utilization Research, Agricultural Research Service,
U.S. Department of Agriculture, Peoria, IL, USA
th
Last updated: January 16 , 2006
Appendix
CH3 O CO CH2 CH2 (CH2)4 CH2 CH CH CH2 (CH2)6
a
b
c
d
e
f
f
e
CH3
d
g
d
a
g
b
e
f
c
1.96
5.5
3.00
5.0
4.5
4.0
2.00 3.99 2.00 20.20
3.5
3.0
2.5
Chemical Shift (ppm)
2.0
1.5
3.00
1.0
0.5
0
Figure 1. 1H-NMR spectrum of methyl oleate.
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H- NMR Spectroscopy of Fatty Acids and Their Derivatives – Non-conjugated double bonds
CH3O CO CH2
a
b
CH2
(CH2)4
c
d
CH2
e
CH CH CH2 CH CH CH2 (CH2)3
f
g
f
f
f
CH3
d
e
h
a
d
h
b
e
g
f
c
3.96
5.5
2.99
5.0
4.5
4.0
3.5
2.00 4.04 1.98 14.34 3.02
2.00
3.0
2.5
2.0
1.5
1.0
0.5
0
Chemical Shift (ppm)
Figure 2. 1H-NMR spectrum of methyl linoleate.
G. Knothe
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H- NMR Spectroscopy of Fatty Acids and Their Derivatives – Non-conjugated double bonds
CH3 -O-CO-CH2-CH2-(CH2 )4 -CH2-CH=CH-CH 2 -CH=CH-CH2-CH=CH-CH 2-CH3
f g
f g
h
a
b
c
e
f
d
f
f
f e
a
h
d
f
b
g
e
c
5.82
5.5
3.00
5.0
4.5
4.0
4.01
2.03 3.99 2.16 8.32 2.89
3.5 3.0 2.5 2.0
Chemical Shift (ppm)
1.5
1.0
0.5
0
Figure 3. 1H-NMR spectrum methyl α-linolenate.
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H- NMR Spectroscopy of Fatty Acids and Their Derivatives – Non-conjugated double bonds
CH3 O CO CH2 CH2 CH2 CH2 CH CH CH2 (CH2)9
a
b
c
d
f
e
f
e
d
d
CH3
g
a
g
b
e
f
2.99
2.00
5.5
5.0
4.5
4.0
2.00 4.01
3.5
3.0
2.5
Chemical Shift (ppm)
2.0
c
2.00
20.20
1.5
3.00
1.0
0.5
0
Figure 4. 1H-NMR spectrum of methyl petroselinate.
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H- NMR Spectroscopy of Fatty Acids and Their Derivatives – Non-conjugated double bonds
CH3 O CO CH2 CH2 (CH2)4 CH2 CH CH CH2 (CH2)6
a
b
c
d
e
f
e
f
CH3
g
d
a
d
g
b
f
e
c
1.97
5.5
2.94
5.0
4.5
4.0
2.00 4.00 2.00 20.24 3.00
3.5 3.0 2.5 2.0
Chemical Shift (ppm)
1.5
1.0
0.5
0
Figure 5. 1H-NMR spectrum of methyl elaidate.
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H- NMR Spectroscopy of Fatty Acids and Their Derivatives – Non-conjugated double bonds
b)
a)
5.48 5.46 5.44 5.42 5.40 5.38 5.36 5.34 5.32
Chemical Shift (ppm)
5.48 5.46 5.44 5.42 5.40 5.38 5.36 5.34 5.32
Chemical Shift (ppm)
Figure 6. Expansion of the signals of the olefinic protons in the 1H-NMR spectra of a) methyl
oleate and b) methyl elaidate.
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