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A new n.m.r. method for the analysis of fatty acid methyl ester mixtures with Eu(DPM)3 in carbon disulfide
134 ~2 Elsevicr
SHORT
Scientific
Publishing
Ardyrica Chimica Acta. 66 ( 1973) 134-138 Amsterdam - Printed in The Netherlands
COMMUNICATION
A new n.m.r. method for the analysis Eu(DPM), in carbon disulfide
DOUGLAS
Company.
of fatty
acid methyl
ester
mixtures
with
B. WALTERS
Animul Products Laboratory, Russell (Keceivcd 15th January 1973)
Reserrrch Cemrr.
ARS.
USDA,
Athm~,
Cu. 30604 (U.S.A.)
The use of carbon disulfide as a solvent for Eu(DPM), has, for the first time, made possible the analysis of fatty acid methyl ester mixtures by n.m.r. spectroscopy. This paper describes further uses of carbon disulfide as a solvent for Eu(DPM),, beyond those stated in a previous paper I. The increased solubility of Eu(DPM), in CS,, as compared to Ccl, or CDCI,, permits addition of enough shift reagent to a solution of a mixture of aliphatic short-chain methyl esters to distinguish the individual components. Induced changes in the chemical shifts of the methoxy resonances allow the initially coalesced methoxy signals to be separated into their respective absorbances so that components of the mixture can be identified. N.m.r. spectra of an homologous series of saturated short-chain fatty acid methyl esters were run before and after addition of Eu(DPM),. The maximum chain length for which the overlapping methylene resonances were separated completely after the addition of Eu(DPM), was eight carbons. The use of n.m.r. spectroscopy by lipid chemists has been limited because the methylene protons in most long-chain compounds are often essentially magnetically equivalent, producing a broad signal of overlapping resonances which precludes their identification, integration, and coupling constant determination. Since the initial report’ on the use of n.m.r. shift reagents several authors3-’ have used these compounds in CDCI, and Ccl, for n.m.r. studies of other methyl esters. However, for the above reasons, no work has been reported on the analysis of ester mixtures. Experimerltal Spectra were obtained with a Varian HA-100 n.m.r. spectrometer at an ambient probe temperature of 30”. Calibrations were made with a Hewlett-Packard Model 52441 electronic frequency counter, and chemical shifts were measured relative to tetramethylsilane (TMS). Line positions are estimated to be accurate to + 0.5 Hz. The instrument was tuned for each sample to compensate for changes in the magnetic field due to the paramagnetic metal present. Methyl esters (Polyscience Corporation) were certified as 99.5 + “/, pure. Carbon disulfide (Matheson, Coleman and Bell) was spectroquality and all other reagents were analytical grade. Eu(DPM), was obtained from Norell Chemical Company.
SHORT
135
COMMUNICATION
Spectra were obtained of cu. 0.4-1.1 M so&ons of the substrates disulfide, to which increments of Eu(DPM), were added.
in carbon
Results
and discussion Figure 1A shows the proton n.m.r. spectrum of a solution of methyl butyrate, hexanoate and heptanoate before the addition of Eu( DPM), ; individual components of this mixture cannot be determined from this spectrum. Figure 1B shows the same
.tu
(A)
*’
*
--+ (-OCW,)
Fig. 1. 100 MHz ‘H n.m.r. spectrum of a mixture of methyl butyratc (0.288 mmole), methyl (0.204 mmole) and methyl heptanoatc (0.716 mmole). (A) Before addition of Eu(DPM),, addition of 0.3463 g Eu( DPM),.
hexanoatc (B) after
136
SHORT
COMMUNICATION
mixture afterothe addition of 0.3463 g of Eu(DPM),. The three methyl esters are now distinguishable by their respective methoxy singlets. (This technique is described in an earlier paper6.) The butyl, hexyl, and heptyl methoxy singlets absorb at 7.72, 7.66, 7.62 p.p.m. respectively whereas, initially all three signals coalesced at 3.53 p.p.m. Although the rest of the spectrum remains complex even after the. addition of Eu(DPM),, the methylene region provides information by separating a unique absorbance for each of the individual constituents of the mixture. The spinning sidebands observed in Fig. 1B result from the rert-butyl group ( -0.72 p.p.m.) of Eu(DPM),, as noted previously’. The example in Fig. 1 represents a severe test for the analysis of methyl ester mixtures, because the chain lengths are so close to one another. When the mixture is composed of esters differing by more than one carbon atom, it becomes progressively easier to distinguish the individual components of the mixture. The above results prompted a study to determine the longest saturated unbranched methyl ester for which all the nearly equivalent methylene protons could be separated by addition of Eu(DPM),. Table I lists an homologous series of methyl esters. The methylene regions of methyl acetate, propionate and butyrate were already resolved so addition of Eu(DPM), was unnecessary. The methylene regions of methyl hexanoate, heptanoate, and octanoate were resolved after the addition of amounts of EG(DPM), shown in parentheses in column 3. It was not possible to resolve the methylene region of methyl nonanoate. These results are shown in Figs. 2-4 in which the 7
0
CH3CH~CH~CHaCH2~0 654321
CH3
0
CHJCHICHaCH~CH~CH~iS’OCH~ 7L543
m
a1
m
a”2
b-
b Abm
5-
5
Abs
‘I-
Ab5
‘-Ab. 0.2
0.4
0.6
0.5
CUfDCM)3r
1.0
,.a
14 .
16 .
15 .
EdDIM)5/
5U55TRATE
Fig. 2. Variation
in the chdmical
shifts of the protons
of methyl
hexnnoate
Fig. 3. Variation
in ~hc chemical
shifts of the protons
of methyl
heptanoate
SUISTRATE
(0.234 mmole) (0.566
mmole)
in CS,. in CSI.
137
S.HORT.COMMUNlCATION TABLE
I
RESOLUTION
OF THE
METHYLENE
GROUPS
OF VARIOUS
METHYL
Methyl
esters
Methylene region beyore addition o/ Eu(DPM),
Methylene region after addition of. Eu(DPM),~
Methyl Methyl Methyl Methyl Methyl Methyl Methyl Methyl
acetate propionatc butyrate pcntanoate hexanoate heptanoate octanoatc nonanoate
Resolved Resolved Resolved Not available Not resolved Not resolved Not resolved Not resolved
Not available SResolved (0.0705) Resolved (0.1738) Resolved (0.3626) Not resolved
” Numbers
in parentheses
indicate
amount
of Eu(DPM),.
in g, added to achieve
ESTERS
resolution.
2 E”bPM)2~SUBStbl*TE Fig.
4.
Variution
in the chemical
shirts
of the protons
of methyl
octanoate
(0.502
mmole)
in cs2.
change in chemical shifts, A.6 (the difference in chemical shift before and after addition of Eu( DPM),), uersus the mole ratio of shift reagent to substrate is plotted. For methyl hexanoate, resolution was obtained after addition of cn. $0 mg of Eu(DPM), (cu. 0.4 mole ratio). For methyl octanoate, resolution occurred only after ad-
138
SHORT
COMMUNICATION
dition of cn. 300 mg Eu(DPM), ( cu. 0.9 mole ratio). Values for methyl heptanoate accordingly fell between these two compounds. Since the solubility of Eu(DPM), is CU.600 mg/OS-ml in CS, and CLI.130 mg/0.5 ml in Ccl4 or CDCI,‘, CS2 was necessarily the solvent of choice. This study illustrates,the advantages that Eu(DPM), in carbon disulfide offer the analytical lipid chemist. It should be noted that when esters larger than C, are examined, shift reagents are still of great value since the presence of other functional groups, such as double bonds6 or a unique absorbance like the methoxy group, enable the analysis to be performed and provide valuable structural information which cannot be obtained by other methods. The Laboratory, work.
author E.P.A.,
is indebted to Dr. L. H. Keith and for extending the use of the instrument
the Southeast for completion
REFERENCES 1 2 3 4 S 6
D. B. Wnltcrs. Am/. C/rim &to. 60 (1972) 421. C. C. Hinklcy. J. Amer. Chent. SW.. 91 (1969) 5160. J. K. M. Sanders and D. H. Williams, J. Amer. Clrenr. SW., 93 (1971) 641. D. Swern and J. P. Wineburg, J. Amer. Oil Chet~r. kc., 48 (1971) 371. G. J. Boudrcaux, A. V. Bailey and V. W. Tripp, J. Amer. Oil Chem. SW., 49 (1972) D. B. Walters and R. J. Horvat. hul. Chiul. Acrcr, 65 (1973) 198.
200.
Water of this
SHORT
Scientific
Publishing
Ardyrica Chimica Acta. 66 ( 1973) 134-138 Amsterdam - Printed in The Netherlands
COMMUNICATION
A new n.m.r. method for the analysis Eu(DPM), in carbon disulfide
DOUGLAS
Company.
of fatty
acid methyl
ester
mixtures
with
B. WALTERS
Animul Products Laboratory, Russell (Keceivcd 15th January 1973)
Reserrrch Cemrr.
ARS.
USDA,
Athm~,
Cu. 30604 (U.S.A.)
The use of carbon disulfide as a solvent for Eu(DPM), has, for the first time, made possible the analysis of fatty acid methyl ester mixtures by n.m.r. spectroscopy. This paper describes further uses of carbon disulfide as a solvent for Eu(DPM),, beyond those stated in a previous paper I. The increased solubility of Eu(DPM), in CS,, as compared to Ccl, or CDCI,, permits addition of enough shift reagent to a solution of a mixture of aliphatic short-chain methyl esters to distinguish the individual components. Induced changes in the chemical shifts of the methoxy resonances allow the initially coalesced methoxy signals to be separated into their respective absorbances so that components of the mixture can be identified. N.m.r. spectra of an homologous series of saturated short-chain fatty acid methyl esters were run before and after addition of Eu(DPM),. The maximum chain length for which the overlapping methylene resonances were separated completely after the addition of Eu(DPM), was eight carbons. The use of n.m.r. spectroscopy by lipid chemists has been limited because the methylene protons in most long-chain compounds are often essentially magnetically equivalent, producing a broad signal of overlapping resonances which precludes their identification, integration, and coupling constant determination. Since the initial report’ on the use of n.m.r. shift reagents several authors3-’ have used these compounds in CDCI, and Ccl, for n.m.r. studies of other methyl esters. However, for the above reasons, no work has been reported on the analysis of ester mixtures. Experimerltal Spectra were obtained with a Varian HA-100 n.m.r. spectrometer at an ambient probe temperature of 30”. Calibrations were made with a Hewlett-Packard Model 52441 electronic frequency counter, and chemical shifts were measured relative to tetramethylsilane (TMS). Line positions are estimated to be accurate to + 0.5 Hz. The instrument was tuned for each sample to compensate for changes in the magnetic field due to the paramagnetic metal present. Methyl esters (Polyscience Corporation) were certified as 99.5 + “/, pure. Carbon disulfide (Matheson, Coleman and Bell) was spectroquality and all other reagents were analytical grade. Eu(DPM), was obtained from Norell Chemical Company.
SHORT
135
COMMUNICATION
Spectra were obtained of cu. 0.4-1.1 M so&ons of the substrates disulfide, to which increments of Eu(DPM), were added.
in carbon
Results
and discussion Figure 1A shows the proton n.m.r. spectrum of a solution of methyl butyrate, hexanoate and heptanoate before the addition of Eu( DPM), ; individual components of this mixture cannot be determined from this spectrum. Figure 1B shows the same
.tu
(A)
*’
*
--+ (-OCW,)
Fig. 1. 100 MHz ‘H n.m.r. spectrum of a mixture of methyl butyratc (0.288 mmole), methyl (0.204 mmole) and methyl heptanoatc (0.716 mmole). (A) Before addition of Eu(DPM),, addition of 0.3463 g Eu( DPM),.
hexanoatc (B) after
136
SHORT
COMMUNICATION
mixture afterothe addition of 0.3463 g of Eu(DPM),. The three methyl esters are now distinguishable by their respective methoxy singlets. (This technique is described in an earlier paper6.) The butyl, hexyl, and heptyl methoxy singlets absorb at 7.72, 7.66, 7.62 p.p.m. respectively whereas, initially all three signals coalesced at 3.53 p.p.m. Although the rest of the spectrum remains complex even after the. addition of Eu(DPM),, the methylene region provides information by separating a unique absorbance for each of the individual constituents of the mixture. The spinning sidebands observed in Fig. 1B result from the rert-butyl group ( -0.72 p.p.m.) of Eu(DPM),, as noted previously’. The example in Fig. 1 represents a severe test for the analysis of methyl ester mixtures, because the chain lengths are so close to one another. When the mixture is composed of esters differing by more than one carbon atom, it becomes progressively easier to distinguish the individual components of the mixture. The above results prompted a study to determine the longest saturated unbranched methyl ester for which all the nearly equivalent methylene protons could be separated by addition of Eu(DPM),. Table I lists an homologous series of methyl esters. The methylene regions of methyl acetate, propionate and butyrate were already resolved so addition of Eu(DPM), was unnecessary. The methylene regions of methyl hexanoate, heptanoate, and octanoate were resolved after the addition of amounts of EG(DPM), shown in parentheses in column 3. It was not possible to resolve the methylene region of methyl nonanoate. These results are shown in Figs. 2-4 in which the 7
0
CH3CH~CH~CHaCH2~0 654321
CH3
0
CHJCHICHaCH~CH~CH~iS’OCH~ 7L543
m
a1
m
a”2
b-
b Abm
5-
5
Abs
‘I-
Ab5
‘-Ab. 0.2
0.4
0.6
0.5
CUfDCM)3r
1.0
,.a
14 .
16 .
15 .
EdDIM)5/
5U55TRATE
Fig. 2. Variation
in the chdmical
shifts of the protons
of methyl
hexnnoate
Fig. 3. Variation
in ~hc chemical
shifts of the protons
of methyl
heptanoate
SUISTRATE
(0.234 mmole) (0.566
mmole)
in CS,. in CSI.
137
S.HORT.COMMUNlCATION TABLE
I
RESOLUTION
OF THE
METHYLENE
GROUPS
OF VARIOUS
METHYL
Methyl
esters
Methylene region beyore addition o/ Eu(DPM),
Methylene region after addition of. Eu(DPM),~
Methyl Methyl Methyl Methyl Methyl Methyl Methyl Methyl
acetate propionatc butyrate pcntanoate hexanoate heptanoate octanoatc nonanoate
Resolved Resolved Resolved Not available Not resolved Not resolved Not resolved Not resolved
Not available SResolved (0.0705) Resolved (0.1738) Resolved (0.3626) Not resolved
” Numbers
in parentheses
indicate
amount
of Eu(DPM),.
in g, added to achieve
ESTERS
resolution.
2 E”bPM)2~SUBStbl*TE Fig.
4.
Variution
in the chemical
shirts
of the protons
of methyl
octanoate
(0.502
mmole)
in cs2.
change in chemical shifts, A.6 (the difference in chemical shift before and after addition of Eu( DPM),), uersus the mole ratio of shift reagent to substrate is plotted. For methyl hexanoate, resolution was obtained after addition of cn. $0 mg of Eu(DPM), (cu. 0.4 mole ratio). For methyl octanoate, resolution occurred only after ad-
138
SHORT
COMMUNICATION
dition of cn. 300 mg Eu(DPM), ( cu. 0.9 mole ratio). Values for methyl heptanoate accordingly fell between these two compounds. Since the solubility of Eu(DPM), is CU.600 mg/OS-ml in CS, and CLI.130 mg/0.5 ml in Ccl4 or CDCI,‘, CS2 was necessarily the solvent of choice. This study illustrates,the advantages that Eu(DPM), in carbon disulfide offer the analytical lipid chemist. It should be noted that when esters larger than C, are examined, shift reagents are still of great value since the presence of other functional groups, such as double bonds6 or a unique absorbance like the methoxy group, enable the analysis to be performed and provide valuable structural information which cannot be obtained by other methods. The Laboratory, work.
author E.P.A.,
is indebted to Dr. L. H. Keith and for extending the use of the instrument
the Southeast for completion
REFERENCES 1 2 3 4 S 6
D. B. Wnltcrs. Am/. C/rim &to. 60 (1972) 421. C. C. Hinklcy. J. Amer. Chent. SW.. 91 (1969) 5160. J. K. M. Sanders and D. H. Williams, J. Amer. Clrenr. SW., 93 (1971) 641. D. Swern and J. P. Wineburg, J. Amer. Oil Chet~r. kc., 48 (1971) 371. G. J. Boudrcaux, A. V. Bailey and V. W. Tripp, J. Amer. Oil Chem. SW., 49 (1972) D. B. Walters and R. J. Horvat. hul. Chiul. Acrcr, 65 (1973) 198.
200.
Water of this