GAS-LIQUID CHROMATOGRAPHIC ANALYSIS OF THE TRIGLYCERIDE COMPOSITION OF MOLECULAR

DISTILLATES OF BUTTER OIL1

Abstract A quantitative gas-liquid chromatographic analysis of the triglycerides of

butter oil and its distillates has shown that molecular distillation can effect a significant fractionation of butterfat. The most volatile 2.5y0 distillate con- tained nine triglyceride types (CZ4-C40), six of which were present in concentra- tions representing 3- to 20-fold enrichments, when compared with the original oil. Eighty-six percent of the C30 and shorter triglycerides occurred in the most volatile fractions, which comprised 5yo of the total fat. The residue, which re- presented SOYo by weight of the oil, contained 10 triglyceride types (C~~CF,?), seven of which were present in concentrations exceeding those in the original oil. This residue contained 93y0 of the triglycerides longer than C46. The other distil- lates contained triglycerides the mean molecular weights of which increased with decreasing volatility. In all cases the triglyceride composition of the butter-oil distillates was markedly different from that of butterfat.

Introduction I t has been shown (2, 3) tha t glyceride oils can be completely distilled a t

relatively low temperatures without noticeable decomposition when these distillations are performed from thin films in special apparatuses a t pressures of a few microns. This process is known as molecular distillation and numerous attempts (4, 5, 6) have been made t o use it for the segregation of natural triglyceride mixtures. The results, however, have been disappointing as the separations obtained by molecular distillation have been poor in comparison with those achieved by countercurrent distribution (7) and low-temperature crystallization (8). The advantage found in molecular distillatioil has been the ease of separation of the unsaponifiable portion and free fatty acids from the bulk of the fatty material.

The utilization of molecular distillation for the preparatioil of butter-oil fractions of varying content of uilsaponifiable material for dietary testing had resulted in an accumulation of various distillation cuts in our laboratory. Subsequent detailed chemical investigations of these materials, stimulated by the observed differences (9) in their hypercholesterolen~ic activity, suggested tha t there had been some fractionation of the constituent triglycerides as well as the unsaponifiable material. A determination of the saponification numbers and the fatty acid composition, however, was not sufficient to determine the type or the extent of this separation. The recently developed gas-liquid chromato- graphic technique (10) for the fractionation of natural triglyceride mixtures

'Manuscript received July 11, 1962. Contribution from the Department of Biochemistry, Queen's University, Kingston,

Ontario. This work was performed with the aid of grants from the Medical Research Council of Canada and the Ontario Heart Foundation. A preliminary report has appeared (1).

Canadian Journal of Biochemistry and Physiology. Volume 40 (1962)

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1694 CANADIAN JOURNAL OF BIOCHEMISTRY AND PHYSIOLOGY. VOL. 40, 1962

by carbon number appeared ideally suited for a reexamination of the trigly- ceride composition of these butter-oil distillates and for an evaluation of mole- cular distillation as a means of triglyceride separation.

Materials and Methods

Standards and Samples Purified preparations of the simple saturated triglycerides froin trilaurin

to tristearin were purchased from the California Corporation for Biochemical Research, Los Angeles, California. Trioctanoin and trideca~loin were obtained from Eastman Kodak Company, Rochester, New York. On gas chroma- tography these showed only minor amounts of contaminants and were not further purified. The methyl esters of the saturated C6 to Cls straight chain fatty acids and methyl oleate were obtained from the Applied Science Laboratories, State College, Pennsylvania. These compounds yielded only single peaks on gas chromatography.

The molecular distillates of butter oil were obtained through the courtesy of Distillatioil Products Industries, Rochester, New York. The pressures and the feed and residue temperatures for the original distillation of 777 pounds of butter oil are given in Table I. The most volatile 10yo fractioil (D-1) was further redistilled into four approximately equal fractioils coded R-1, R-2, R-3,

TABLE I Distillation conditions employed in the original fractionation of butter oil*

First pass Second pass Residue

Code % charge Pressure (microns) Feed temperature ( O C) Residue temperature (O C)

*Total charge 7 7 7 pounds of butter oil. The distillation constants were determined by Distillation Products Industries, Rochester, New York.

and R-4 in decreasing order of volatility. The distillation data for this separa- tion are given in Table 11. These fractions provided the samples for analyses. For ease of reference they may be described as the first most volatile 2.5y0 cut (R-1), the second most volatile 2.5y0 cut (R-2), the third most volatile 2.5% cut (R-3), the fourth most volatile 2.5y0 cut (R-4), the next most volatile 40y0 cut (D-2), and the SOYo residue (D-3). In addition a reference butter-oil sample was analyzed. This was prepared in the laboratory by petroleum extraction of commercial butterfat supplied by a local dairy. With the excep- tion of the most volatile distillate (R-1) none of the materials were purified further. For gas chromatography, these lipids were dissolved in carbon disulphide to give solutions of lyo to 5yo (wlv). The most volatile fraction contained significant amounts of free fatty acids, unsaponifiable matter, and di- and mono-glycerides, all of which interfered with the gas chromatography

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McCARTHY E T AL.: BUTTER OIL

TABLE I1 Distillation conditions employed in the redistillation of fraction D-1 from

the original fractionation of the butter oil*

Temp., Pressure, Net Code " C P kg cut % total Color State

Charge 1 R- 1

R-2

R-3

R-4

Charge 2 R- 1

R-2 R-3 R-4

Amber Yellow Yellow Yellow

49.6 Yellow Yellow

74.0 Yellow Yellow

99.3 Yellow

,4111 ber Yellow Yellow

50.2 Yellow 75.3 Yellow

100.2 Yellow

Liquid Solid Solid Liquid Liquid Liquid Liquid Liquid Liquid

Liquid Solid Liquid Liquid Liquid Liquid

*The distillation constants were determined by Distillation Products Industries, Rochester, New York.

of the triglycerides. The acid material in the distillate was removed by extrac- tion with 5yo potassiuin carbonate solution, and the triglycerides isolated by silicic acid chroinatography of the neutral lipid (1 1).

Methods of Analyses Saponification values were determined by A.O.C.S. Official Method Cd 3-25

(12). The inethyl esters of the fatty acids were prepared by trailsesterificatioi~ (13) and chroinatographed using a Beckrnan G C 2A gas chroi~~atograph equipped with a filai~lent cell, a Minneapolis-Honeywell Brown 1-nlv recorder, and a Model K1-1 Disc Integrator. A staiilless steel colunln (6 ft X ill. O.D.) packed with 20y0 (wlw) DEGS on acid-washed firebrick (60-80 nlesh) was used. The long-chain fatty acids were deterrllined using a column temper- ature of 222" C and a helium flow rate of 100 1171 per minute. The short-chain fatty acids were estimated a t lower temperatures and flow rates (180" C and 80 ml per minute). Fa t ty acid compositions were calculated fro111 the peak areas given by the integrator record. Runs with known mixtures indicated that these estimates approxiinated the weight distributioll of the fatty acid test mixture.

Gas-liquid chromatography of the triglycerides was performed as previously described (10). For the chromatography of the most volatile distillate these conditions were altered slightly to increase the retelltion time. 111 this case the starting temperature was 190" C and the temperature increment 2.1" C per minute.

Results and Discussion The conditions of rnolecular distillatioil described above had been designed

to separate the unsaponifiable matter of butterfat fro117 the bulk of the fatty

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1696 CANADIAN JOURNAL OF BIOCHEMISTRY AND PHYSIOLOGY. VOL. 40, 1962

material and not specifically for the study of triglyceride fractionation. The choice of the cuts, however, had beell such that significant amounts of trigly- cerides had been collected in all fractions. An inspection of the weight distri- bution of the triglyceride material obtained during the original distillation and redistillation revealed a proportioning of distillate tha t was well suited for the detection of any segregation of triglycerides. If triglyceride separations had occurred these should be reflected in the coinposition of the distillates. The wide spectrum of fatty acids found in this fat made the occurrence of trigly- cerides of greatly varying molecular weight particularly likely.

A comparison of these distillates on the basis of visual appearance, iodine values, saponification numbers, and average molecular weights, however, indicated only a moderate fractionation. The saponification values and the estimates of the average nlolecular weight recorded in Table 111 for these

TABLE I11 Saponification values of butter oil and its

molecular distillates*

Saponification Average molecular Distillate number weight t R- 1 R-2 R 3 R-4 D-2 D-3 Butter oil Trilaurin

*As determined by A.O.C.S. Official Method Cd 3-25 (12). ?The following expression was used for the calculation of the average molecular

weights from the saponification numbers: average molecular weight = (3 X 56 X lOOO)/(saponification No.).

distillates indicate some concentration of the lower nlolecular weight material into the more volatile fractions. The fatty acid composition data given in Table IV for the various distillates and the original oil supports this observa- tion. Each distillate contains all the fatty acids present in the original butter- fat , any variations being found only in the relative proportions of the fatty acids. Tha t the triglyceride separations anticipated on a theoretical basis and suggested by the above analyses had actually been realized could be demon- strated by gas-liquid chromatography of the distillates. This technique (10) permits the separation of natural triglyceride mixtures on the basis of their molecular weights or carbon numbers and the recorded peak areas indicate their approximate weight ratios.

An inspectioil of Figs. 1-7 reveals tha t the distillates differ greatly from each other and froin the original oil in their glyceride composition. While the original butter oil contains high concentrations of the C3& C38, C40, and C4& C60, and C62 triglyceride types, the distillates represent only segments of the total pattern. The degree of separation obtained is best seen from the numer-

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McCARTHY E T AL.: BUTTER OIL

TABLE IV Fatty acid composition of butter oil and its molecular distillates*

Distillate or oil

Fatty acid R-1 R-2 R-3 R-4 D-2 D-3 Orig. oilt Ref. oil1

Butyric 7 .3 8 . 3 3 . 7 2 .9 2.7 0 . 7 2.0 Caproic 6 . 5 5.2 3 . 5 3 .8 3 . 3 0 .9 2.2 Caprylic 6 .1 3 .4 2 .3 1 . 8 1 . 7 0 . 8 1 .4 Capric 9 .5 7 .2 4 .1 3 . 3 3 . 8 2 .3 3 .3 Decenoic 1 . 0 0 .4 0 .3 0 . 2 0 . 4 0 . 2 0 . 3 Lauric 9 . 1 7.3 5 . 5 3.7 3 . 9 2.7 3 . 5 Myristic 18.5 22.3 18.4 7.6 14.2 11.0 12.9 Myristoleic 2 .6 1 .5 1 .8 1 .6 2.1 1 . 9 2.0 Pentadecanoic 1.5 Trace 1 .6 1.5 1 .3 1 . 0 1 .1 Palmitic 24.9 31.3 36.2 42.2 35.4 30.0 32.5 Palmitoleic 1 .3 1.4 1 .6 1 . 5 1 . 6 1 .8 1 . 7 Stearic 4 .0 3 .8 6 . 8 10.1 10.2 13.8 11.6 Oleic 7 .7 8 .1 13.5 19.8 19.4 32.0 25.0 Linoleic Trace Trace 0 . 9 Trace Trace 0 . 9 0.5

Total 100.1 100.2 100.2 100.0 100.0 100.0 100.0

*Conditions of gas-liquid chromatography as described in the text. The percentage area distributions deter- mined and recorded here approximate those for the weight distribution of the fatty acid methyl esters in the filament cell detector.

tcalculated on the basis of the percentage area response and the percentage weight distribution of the distillates. A comparison of this calculated fatty acid composition of the origlnal oil w ~ t h the fatty acid composition of the reference butter oil indicates a loss in the unsaturated and an increase in the saturated fatty acids of the distilled oil, suggesting possible selective destruction of the triglycerides during the process of molecular distillation.

$Percentage area distributions for the fatty acid methyl esters of the reference butter oil.

FIG. 1. A gas-liquid chromatographic elution pattern recorded for the reference butter- oil sample prepared in the laboratory. Chromatography conditions as described in the text. The fatty acid carbon numbers were assigned to these peaks following co-chroma- tography with standard triglycerides.

ical values calculated from these elutioil patterns and presented in Table V. I t may be noted tha t the C2g type of triglyceride has been concentrated into the most volatile cut and represents a 26-fold enrichment. This increase is

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1700 CANADIAN JOURNAL OF BIOCHEMISTRY AND PHYSIOLOGY. VOL. 40, 1962

FIG. 6. A gas-liquid chromatographic elution pattern recorded for the 40% clistillate from the original butter oil distillation (D-2). Legends as for Fig. 1.

FIG. 7. A gas-liquid chromatographic elution pattern recorded for the 50% residue from the original butter oil distillation (D-3). Legends as for Fig. 1.

similar to that observed for vitamin A from fish liver oils (S), a c o i ~ ~ i ~ ~ o n l y quoted indicator of the efficiency of molecular distillation. The enrichments of the other lower molecular weight triglycerides in this and subsequeilt distil- lation cuts were comparable but of progressively decreasing magnitude. The separations observed in these distillations are iilfluenced by the fact that the

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McCARTHY E T AL.: BUTTER OIL

TABLE V Triglyceride composition of butter oil and its molecular distillates*

yo distribution of triglyceride types in distillate or oil - - -

Triglyceride R-1 R-2 R-3 R-4 D-2 D-3 Orig. oilt Ref. oil1

Total 100.0

*Conditions of gas-liquid chromatography as described in the text. The percentage area distributions deter- mined and recorded here have been shown to approximate those for the weight distribution of standard trigly- cerides in the hydrogen flame ionization detector (10).

?Calculated on the basis of the percentage area response and the percentage weight distribution of the distillates. $Percentage area distributions for the triglycerides of the reference butter oil.

PERCENT BUTTER OIL D I S T I L L E D

FIG. 8. Elimination curves of butter-oil triglycerides during molecular distillation. The curves are identified by the number of carbons in the fatty acid parts of the trigly- cerides.

first 10% cut from the original distillation was redistilled to obtain the four 2.5% cuts, and as such may not be comparable with the results recorded for single distillations of oils of comparable complexity (6). I t may be speculated tha t with a sufficient number of redistillations of suitable cuts and with pos- sible improvements in equipment design, the separations obtained by mole-

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1702 CANADIAN JOURNAL OF BIOCHEMISTRY AND PHYSIOLOGY. VOL. 40, 1962

cular distillation might approach those noted for gas chromatography. In both cases the separations appear to depend solely on the molecular weight of the triglyceride and appear to require only a limited number of plates in a temperature gradient.

Figure 8 illustrates graphically the efficiency of the separations obtained in the present double distillation and indicates the potential remaining for further separations to be realized by additional redistillations. I t may be noted from these elimination curves that the distillatioil of the short-chain triglycerides (C24 to C28) is essentially complete when only 50/0 of the total oil has been distilled. Distillation of 50% of the total oil results in a recovery of only about 7y0 of the triglycerides higher than C46.

On the basis of the present data and the gas-liquid chromatographic analyses recorded elsewhere (10) it would appear tha t nlolecular distillation should produce little or no fractionation of the triglyceride mixtures found in inost seed oils. These oils contain mainly C16 and C18 fatty acids and their constit- uent triglycerides do not differ by more than four in the carboil numbers. Molecular weight differences resulting from uilsaturation would not be suffi- ciently great for differential distillation. With plant fats such as coconut and palm-kernel oils, however, molecular distillation should yield separations con~parable with those observed for butterfat, the completeness of which would depend upoil the efficiency of the apparatus and the number of redistil- lations.

The completeness, estimated by gas-chron~atography, of the eliminations of the various triglyceride types illto the different distillates depends on the quantitative interpretation of the response in the hydrogen flame ionization detector. Previous studies with saturated medium- and long-chain trigly- cerides have shown tha t the recovered area percentages recorded with this type of detector represent approximately the correct weight composition of the test mixture (10). With increasing proportions of oxygen in the shorter- chain triglycerides this relationship between the weights and recorded areas might not hold since the lower molecular weight triglycerides would be ex- pected to show lower responses per unit of weight in this detector. Gas- chromatographic fractionations performed with weighed inixtures of short- and long-chain triglycerides, however, failed to show significailt differences in the detector respoilse when allowance was made for a variation of about 5yo commonly observed in these runs. The reason for this may be an incon~plete volatilizatioil and recovery of the higher molecular weight triglycerides. This rnatter is being further investigated. For the present purposes i t is of minor importance, although the content of the short-chain rnaterial inay have been underestimated. The qualitative distribution is apparently correct and should be sufficient for the appraisal of the effectiveness of n~olecular distillation in the fractionation of butterfat triglycerides. A comparisoil of the calculated conlposition of the original oil, based on the percentage weight distributioil of the distillates and the percentage area response in the hydrogen f l a ~ ~ l e ioniza-

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McCARTHY ET AL.: BUTTER OIL 1703

tion detector, with similar area percentage response recorded for a reference butterfat, indicates that the estimated weight percentage con~positions are substantially correct. There may have been small errors in the estimates of the C24, C26, and C54 triglycerides.

References 1. M. J. MCCARTHY and A. KUKSIS. Proc. Can. Federation Biol. Soc. 5, 53 (1962) . 2 . N. D. EMBREE. Chem. Rev. 29, 317 (1941) . 3 . K . C. D. HICKMAN. Chem. Rev. 34, 51 (1944) . 4 . H. W . RAWLINGS. Oil Soap, 16, 231 (1939) . 5 . S. G. BHAT, J. G. KANE, and A. SREENIVASAN. J. Am. Oil Chemists' Soc. 33, 197 (1956) . 6 . M. NAUDET, E. SAMBURG, J. PASERO, and P. DESNUELLE. Bull. Soc. Chim. France, 718

(1959). 7. C. R. SCHOLFIELD. J. Am. Oil Chemists' Soc. 38, 562 (1961) . 8 . J. B. BROWN and D. K. KOLB. In Progress in the chemistry of fats and other lipids.

R. T. Holman, W. 0 . Lundberg, and T. Malkin (Editors). Vol. 3 . Pergamon Press, New York. 1955. p. 57.

9 . J. M. R. BEVERIDGE, W. F. CONNELL, H. L. HAUST, and G. A. MAYER. Can. J. Biochem. Physiol. 37, 575 (1959).

10. A. KUKSIS and M. J. MCCARTHY. Can. J. Biochem. Physiol. 40, 679 (1962) . 11. A. KUKSIS and J. M. R, BEVERIDGE. J. Lipid Res. 1, 31 1 (1960). 12. AMERICAN OIL CHEMISTS' SOCIETY. Official and tentative methods of the American Oil

Chemists' Society. 2nd ed. Official Method Cd 3-25. Published by the American Oil Chemists' Society, Chicago. 1951.

13. A. T . JAMES. I n Methods of biochemical analyses. D. Glick (Editor) . Interscience Publish- ers, Inc., New York. 1960. p. 18.

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