PMR analysis of unsaturated triglycerides using shift reagents

Gtemistry and Physics of Lipids 14 (1975) 189-192 © North-Holland Publishing Company

PMR A N A L Y S I S OF U N S A T U R A T E D TRIGLYCERIDES USING SHIFT REAGENTS D.J. FROST, J. BUS, R. KEUNING and I. SIES Unilever Research, Vlaardingen, The Netherlands

Received

accepted

The addition of Pr(fod) 3 i.e. tris(1,1,1,2,2,3,3-heptafluoro-7,7-dimethyl-4,6-octanedionato) praseodymium, to trilinolein has been found to induce a difference in the chemical shifts of the absorptions from the acids on the a- and ~-positions. At 220 MHz this was observed up to 18 carbon atoms along the chain. Decoupling of the alkenyl protons at 100 MHz enabled the absorptions from the skipped methylene groups to be used to determine the position of linoleate and linolenate chains in triglycerides.

1. Introduction The currently accepted [1] method for the analysis of triglycerides involves a time consuming enzymatic hydrolysis followed by a GLC analysis o f the fatty acids liberated. The whole procedure commonly takes several hours or even days. Two recent publications [2,3] have indicated that the PMR spectra of triglycerides, upon addition of europium shift reagents, may provide the basis for a simpler analysis. The present work considerably extends the applicability of this method by combining the use o f a different shift reagent, Pr(fod)3 i.e. tris(1,1,1,2,2,3,3-heptafluoro7,7-dimethyl-4,6-octanedionato)praseodymium, together with decoupling experiments.

I1. Experimental The triglycerides were synthesized (by C.W. van Oosten) in our laboratory (purity ~95%). Pr(fod)3 d30 was obtained from the Norell Chemical Company (Landing, New Jersey). PMR measurements were obtained from solutions in CC14 at 100 MHz and 27°C on a Varian HA100, and at 220 MHz and 30°C on a Varian HR220.

III. Results and discussion The PMR spectra of unsaturated fatty acids are most clearly characterized by the

I

25

big. 1. 220 MHz I’MR spectrum

I

20

of trilinolein

15

plus I’r(fc)d)3 (mole ratio

10

1

: 3.60)

absorptions of those protons on carbon atoms adjacent to the double bonds 141, viz. CH,G and =CCH,C= groups. However. the addition of a europium shift reagent often results in these absorptions becoming overlapped by other bands. and preventing their being used effectively for locating the position of the fatty acid chains in a triglyceride. A praseodymium reagent, such as Pr(fod), , does not present this disadvantage since it causes the offending absorptions to be displaced to higher field [5]. and furthermore induces larger shifts than the europium analogue. These points are demonstrated in fig. 1 by the 6 0.7-2.7 region of the 220 MHz spectrum of trilinolein (LLL) with added Pr(fod), (mole ratio I : 3.60). A similar experiment with Eu(fod), not only causes the absorptions from the protons on C, to come between those of C,, and C,,, but this region of the spectrum is further complicated by absorptions from the C, . c6 and C, methylene groups. The assignments of the absorptions in fig. I as coming from the CY-and fl-positions on the glyceryl moiety were tnade from the 2 : 1 ratio of the integrals. It should be noted, however, that contrary to measurements witb europium shift reagents, the protons from the &acid are further shifted than those from the a-acids, with the “crossover” in this tendency found by Almqvist et al. [3] not being observed in the range studied. The difference between the lanthanide induced shifts of the protons from the (Y-and P-acids extends over a very long range, under the conditions for the spectrum in fig. 1 being 0.220 ppm at the protons on C8, 0.1 10 ppm at Cl,, 0.035 ppm at C,,, and, surprisingly, even 0.030 ppm between the terminal methyl absorptions, i.e. at C, 8. The observation of this phenomenon on the CH2C= and

D.J. Frost et al., PMR o f unsaturated triglycerides 220 MHz

LLL

100 MHz

~

LeLeLe~

191

100 MHz with decoupling

21

~tC [~Cll

231

UCl**[~C1413Cll

LeLL

~zC14 I~C11

Fig. 2. Region at about 6 2.7 of PMR spectra of trilinolin (LLL), trilinolin (LeLeLe) and ~yceryl 1-1inolenate 2,3-dilinoleate (LeLL). (Slightly different chemical shifts for ~ Cll methylene absorptions from linoleate and linolenate chains.) =CCH2C= absorptions consequently provides a powerful method for the determination of tile position of unsaturated fatty acids in triglycerides. For example, the position of all oleic acid chain may be determined from the relative position of the C 8 methylene absorption. Tile effect of Pr(fod)3 on glyceryl 1-1inolenate 2,3-dilinoleate (LeLL) is shown in fig. 2 by the spectra of the =CCH2C= region, together with the spectra from trilinolein (LLL) and trilinolin (LeLeLe). The 220 MHz spectra contain too many overlapping absorptions to allow clear assignments to be made, but irradiation at tile alkenyl region (about 6 5.3) decouples these protons and reduces the absorptions to singlets which are well resolved at 100 MHz (fig. 2). From the decoupled spectra it may be seen that the methylene absorptions from c~ C 11 and/3 C14 have virtually the same chemical shift, causing the spectrum of LeLeLe to consist of three singtets in the ratio 2 : 3 : 1. Similarly, the ratio of these peaks for LeLL was found to be 1 : 2 : 1, whilst for LLeL it is to be expected that the peak at lowest field would be

192

D.J. Frost et al., PMR oj uttsaturated trig@ceridcs

absent and the others to be in the ratio 3 : 1. The small chemical stfift (< 0.030 ppm) between the c~ CI1 methylene absorptions from linoleate and linolenate chains is expected [4] from the different deshielding effects involved. The technique is currently being developed to encompass a wider range of fatty acid moieties and to provide quantitative data on mixtures of triglycerides. The method is not suitable, however, for discriminating between long fatty acyl chains of different length, e.g. C 18 and C20, containing the same or similar unsaturated systems.

IV. Conclusion Pr(fod)3 has been shown to induce significantly different shifts in the PMR absorptions from the c~- and 13-acids in a triglyceride, extending over a distance of up to 18 carbon atoms. With the aid of 220 MHz spectra and/or decoupling experiments this phenomenon promises to provide a useful method for both qualitative and quantitative analysis of many unsaturated triglycerides, which may be carried out in less than 30 rain.

References [1] H. Brockerhoff, J. Lipid Res. 8 (1967) 167 [2] P.E. Pfeffer and H.L. Rothbart, Tetrahedron Letters (1972) 2533 [3] S.O. Almqvist, R. Andersson, Y. Shahab and K. Olsson, Acta Chem. Scand. 26 (1972) 3378 [4] D.J. Frost and J. Barzilay, Anal. Chem. 43 (1971) 1316. [5] J. Briggs, G.H. Frost, F.A. Hart, G.O. Moss and M.L. Staniforth, Chem. Commun. (1970) 749