1. Quantitative Analysis of Short Chain Fatty Acid Methyl Ester Mixtures using Proton Nuclear Magnetic Resonance Spectroscopy (NMR)
Ronald P. D’Amelia, Ling Huang, Jayni Shumila Nanette M. Wachter
Chemistry Department, Hofstra University, Hempstead, NY
Introduction
Results
Experimental
Nuclear magnetic resonance spectroscopy is one of the most important instrumental techniques used to elucidate the molecular structure of organic compounds. However, there are a few applications of using NMR for the quantitative assay of impure or mixtures of organic substances. The unique feature of NMR that allows it’s straightforward application to quantitative problems is that the signal’s intensity is directly proportional to the specific number of protons producing that signal at the specific chemical shift. As part of our efforts to increase exposure to NMR spectroscopy in the undergraduate chemistry laboratory curriculum we have developed a quantitative proton NMR experiment for use in our analytical and instrumental courses. This experiment was developed as a teaching exercise illustrating the general principles of both qualitative and quantitative NMR methodology.
The objective of the experiment is to determine the % composition of a two component mixture of short chain fatty acid methyl esters using chemical shift and integration values obtained from running solventless samples both with and without an internal reference standard. This experiment allows for both the qualitative interpretation (peak assignments and splitting spin-spin coupling ) and quantitative calculation of the amount of analyte in the mixture. Our objective is to report on the experimental methods used and the results obtained on examining the quantitative Proton NMR properties of a series of mixtures of methyl acetate (M.A.) and methyl propionate (M.P.) ranging from 100% M.A. to 100% M.P., as well as similar mixtures of methyl acetate (M.A.) and methyl butyrate (M.A.)
Figures 1,2 &3 show the proton NMR spectrum, having TMS as the internal reference standard,
of 100% methyl acetate, methyl propionate, & methyl butyrate respectively along with the
corresponding peak assignments showing the chemical shift , multiplicity, & normalized
integration values. One can see that the methyl protons (A) on the carbon atom attached to the
oxygen all are singlets and are shifting up-field or lower frequency as the molar mass of the fatty
acid methyl ester increases. These protons are shielded & sense a smaller effective magnetic field
as the numberof methylene groups are added to the ester. Therefore these protons come into
resonance at a lower frequency. These protons are in a more electron dense environment as the
compound increase the # of methylene units. Figures 4, 5,& 6 are the proton spectrum, with
TMS as the internal standard, of 1,4 MA/MP mixture, 4:1 MA/MP mixture, and a 5:5 MA/MB
mixture respectively. The normalized integration of the appropriate peaks correspond to the %
Composition of the mixtures. Shown in Fig. 7 is the linear plot of the change in chemical shift
values of the methyl proton on M.P. (without TMS) vs the composition. Shown in Figure 8 is the
linear plot of the change in chemical shift values of the methyl proton on M.B. vs the
composition. Both of these plots show the linear relationship in the up-field changes in chemical
shift values that occur when no internal reference is considered. Summarized in Figure 9 the
linear plot of the % composition of the MA/MP mixtures found by proton NMR vs the
theoretical (prepared) % composition. Shown in Fif 10 is a similar plot for the M.A./M.B
compositions
This procedure was repeated for an additional set of six samples indicated in Table 2. The masses of each of the short chain fatty acid methyl esters used in the binary mixtures were used to determine the % compositions as shown in Tables 1 &2. A one ml aliquot of each mixture was transferred to the NMR tube using a Gilson classic P1000 and then weighed and analyzed.
Table 2: % Composition of Second Set of M.A.-M.P. – M.B. Samples
Sample #
Vol. M.A. (mL)
Vol M.P. (mL)
Vol. M.B. (mL)
% M.A.
% M.P.
% M.B. 1C
3.75
1.25 -
72.79
23.9 2C
2.5
49.53
49.21 3C
22.77
75.32 4C
74.76
22.81 5C
49.99
47.94 6C
25.08
72.74
C) Analysis: The proton NMR profiles of the short chain fatty acid methyl ester mixtures were obtained on a JEOL 400 MHz model ECS-400 NMR. The JEOL Delta NMR processing and control software version (Windows) was used to analyze the individual spectra. Each sample was run as a single pulse, 1D proton NMR, no solvent, acquisition time of 4-5 sec and a resolution of 0.25 Hz. No carbon spectra were obtained. The use of an internal standard like TMS renders the integration an absolute measurement
Figure 7: Plot of change in Chemical Shift of Methyl Proton on M.P. vs % Composition of M.P. with M.A. No TMS
Figure 8: Plot of change in Chemical Shift of Methyl Proton
on M.B. vs % Composition of M.B. with M.A No TMS
Experimental
Results
A) Materials and Chemicals: The NMR sample tubes were Wilmad 5mm thin wall 7” Pyrex tubes having a round bottom and a sandblasted marking spot for labeling samples. They have a concentricity of 51 um and a camber of 25 um. Methyl Acetate (CH3COOCH3) , Methyl Propionate (CH3CH2COOCH3) , and Methyl Butyrate (CH3CH2 CH2 COOCH3 ) were purchased from Sigma-Aldrich. They are all Reagent Plus anhydrous liquids having greater than 99% purity. Tetramethylsilane (TMS) (Si(CH3)4) was also purchased from Sigma-Aldrich. TMS was ACS reagent grade having a purity of greater than 99.9%
Figure 1: Proton NMR of 100% Methyl Acetate
Figure 2: Proton NMR of 100% Methyl Propionate
Figure 3: Proton NMR of 100% Methyl Butyrate A A A B
Figure 10: % Composition of M.A./M.B.
Figure 9: % Composition of M.A/M.P. D C
B) Methods: Mixtures of methyl acetate, methyl propionate, and methyl butyrate were prepared according to the volume/weight ratios in tables 1 & 2. All mixtures had a final total volume of 5 ml. B B C
Table 1: % Composition of M.A.-M.P.-M.B. Samples
Sample #
Vol. M.A. (mL)
Vol. M.P. (mL)
Vol. M.B. (mL)
% M.A.
% M.P.
% M.B. 1B 5 -
100 2B 4 1
81.46
18.54 3B 3 2
63.83
36.17 4B
43.79
56.21 5B
26.97
73.03 6B 7B
80.91
19.09 8B
61.67
38.33 9B
41.53
58.47
10B
22.35
77.65
11B
Methyl Acetate
Methyl Propionate
Methyl Butyrate
Proton
Shift (δ)
Multiplicity
Integration A
3.596
Singlet
1.00
3.607
singlet
3.592 B
1.979
2.273
quadruplet
0.65
2.255
triplet
0.64 C
1.076
1.612
multiplet
0.67 D
0.918
Conclusions
Thus far, the data has reaffirmed the quantitative uses of the NMR. There is a strong linear correlation between the actual and theoretical compositions of each mixture as shown in the above graphs. Also, there is a direct linear correlation between the chemical shift values and how they are changed by the difference compositions of each mixture. In the future we hope to make solutions of unknown compositions and experiment with our ability to work back quantitatively from the NMR spectra in order to test the plausibility of using this experiment as a design for an undergraduate laboratory experiment.
Figure 4: Proton NMR of 1:4 M.A. – M.P.
Figure 5: Proton NMR of 4:1 M.A.-M.P.
Figure 6: Proton NMR of 5:5 M.A. – M.B.
A Mettler analytical balance with a precision of g was used to determine all masses during this experiment. Masses were obtained after every chemical addition throughout the experiment. A 7.5ml empty glass vial was labeled and weighed with Teflon lined screw caps on. To each vial, 200µl of tetramethylsilane was added using a Gilson classic pipetman model P200, followed by the appropriate volume of methyl acetate indicated in Table 1. The methyl acetate volume was added using a Gilson classic pipetman model Then, to the sample vials labeled 1B-6B, appropriate volumes of methyl propionate were added, and to the sample vials labeled 7B-11B, appropriate volumes of methyl butyrate were added. Again the volumes of M.P. & M.B. were added using a Gilson classic pipetmen model P5000.
References
Klemann,L.P., Aji, K., Chrysam, M.M., D’Amelia, R.P., Et Al. Random Nature of Triacylglycerols Produced by the Catalyzed Interesterification of Short- and Long- Chain Fatty Acid TriglyceridesJ. Agric. Food Chem. 1994, 42, pp
Support
We acknowledge the support from a Hofstra HCLAS Faculty Research & Development Grant