SEPARATION OF THE HIGHER FATTY ACIDS

BY EDWARD H. AHRENS, JR.,* AND LYMAN C. CRAIG

(From the Laboratories of The Rockefeller Institute for Medical Research, New York, New York)

(Received for publication, September 4, 1951)

Previous papers from this laboratory (1, 2) reported the development of systems in which the normal saturated fatty acids up to Cl8 had par- tition ratios sufficiently different from one another to permit their separa- tion by counter-current distribution. However, the quantitative aspect of the distribution of the Cl0 to Cl8 acids was impaired by the occurrence of skewing. It appeared desirable to develop selective systems promoting more ideal behavior of the long chain saturated and unsaturated acids.

A skewed distribution is known to result (3) when the partition ratio varies with the concentration of the solute. The cause can be ascribed to the tendency of the solute to exist in solution as a mixture of associated molecules. The proportion of monomers, dimers, trimers, etc., is strongly dependent on concentration and on the environment. Since the environ- ment in each of the two phases in equilibrium is necessarily different, the relationship of the association equilibrium to concentration also is different in the two phases. Under such a complex relationship the simple dis- tribution law relating to ideal solutes does not hold.

It is not surprising that the higher fatty acids give a skewed pattern, in view of their outstanding ability to associate and form micelles over a considerable range of pH. In attempting to overcome skewing, two ap- proaches toward simplification of the distribution equilibrium were ex- plored: reduction of the pH and the provision of a component in the system which would inhibit the association of similar molecules. Such an agent might act by virtue of its having sufficient attraction for the higher fatty acid so that, for example in the case of palmitic acid, palmitic-solvent association would be promoted and palmitic-palmitic association sup- pressed. Incorporation of a high concentration of acetic acid in the system appeared to serve both purposes. The two-phase system formed by mix- ing n-heptane, glacial acetic acid, formamide, and methanol in the propor- tions 3: 1: 1: 1 gave partition ratios (K) for the higher fatty acids which were largely independent of the concentrations of these solutes. Systems containing acetic acid were excellent from every technical point of view, except that the presence of this acid interfered with the plotting of dis-

* Senior Fellow of the National Research Council and of The National Foundation for Infantile Paralysis, Inc.

299

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300 SEPARATION OF HIGHER FATTY ACIDS

tribution patterns by direct titration. However, distributions were suc- cessfully analyzed by means of a recently described microgravimetric pro- cedure (4) in which the solvents, including acetic acid, were completely volatilized and the residue weighed.

Separations of the Cl2 to Cl8 saturated acids and of three Cl8 unsatu- rated acids are presented in this report. In addition, the extension of these studies to the resolution of a complex mixture of fatty acids from a natural fat exemplifies the possible application of this approach to the study of the fatty acid composition of biologically interesting lipides.

Materials and Methods

Distributions were carried out in the 220 cell, all-glass, fully automatic machine described by Craig et al. (5). Solvents were analytical grade reagents redistilled in an all-glass apparatus before use. Formamide was not redistilled but was decolorized with charcoal.

The Cl2 to Cl* saturated fatty acids were samples of high purity kindly furnished by Dr. H. J. Harwood of Armour and Company. He reported the following freezing points: CE, 44.07”; CM, 54.33”; CM, 62.71”; Cls, 65.59”. Oleic, linoleic, and linolenic acids (Hormel Institute) were stated to have iodine numbers (Wijs) at the time of preparation of 89.56, 180.48, and 272.5 respectively. Upon receipt, and according to the modified Yasuda (6) method used in this laboratory, their iodine numbers were 88.9, 164, and 246.5. All iodine numbers in this report were determined in triplicate on 1 to 5 mg. samples. Measurement of the trans acids in these samples by the infra-red spectrophotometric method of Shreve et al. (7) showed 0, 35, and 29.3 per cent of these isomers, respectively. No attempt was made to remove the trans acids by fractional crystallization before use in the present experiments.

An unknown test mixture of fatty acids was obtained by hydrolysis of a sample of pig mesentery fat. This fat was extracted from the tissue with cyclohexane. The solvent was removed in an apparatus (8) under a vacuum at a temperature never above 50”. The fat, solid at 25”, had a saponification number of 196.7, iodine number of 50.1, acid value of 0.4, thiocyanogen value of 45.5, and peroxide value 0.8 to 0.9.’

The fat was hydrolyzed on the steam bath with a 10 per cent excess of alcoholic alkali; the solution was extracted with ether and then acidified with HCl. The mixed free fatty acids were recovered by ether extraction and evaporation of the extract.

Following the distributions described below, solute concentrations were measured by sampling individual upper phases. Aliquots were evaporated

1 We are indebted to Dr. Willy Lange of The Procter and Gamble Company for these analyses.

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E. H. AHRENS, JR., AND L. C. CRAIG 301

to dryness in tared glass shells of about 0.5 gm. of weight on an ethanol bath by the technique and apparatus described by Craig et al. (4). Theo- retical recovery of all aliphatic and ethenoid acids from Clz to C18 was assured when samples were allowed to remain on the ethanol bath not longer than 1 minute after evaporation of the solvent appeared complete. For 1 ml. aliquots the entire procedure required about 2 minutes per sample. Shells and contents were weighed on a semimicro balance with an accuracy of f0.02 mg. The absence of a weighable residue in the solute-free heptane phase obviated a blank correction.

Various fractions were recovered from the equilibration train for further study. In order to recover the entire acid content from both phases without contamination by formamide, the acids were displaced into the upper phase by adding water to the lower phase and equilibrating. The diluted lower phase was extracted twice with fresh heptane and then dis- carded. The heptane extracts were pooled and washed twice with water. Entrained water was removed from the heptane with anhydrous sodium sulfate, the extracts were filtered, and the solvent evaporated at tempera- tures below 25” under a vacuum. The last traces of heptane were evapo- rated with a few ml. of chloroform. Fractions taken to volume in chloro- form were ready for determination of dry weight and iodine number. In two experiments in which thirteen and fifteen fractions were separately handled after distributions of 2200 and 3000 transfers, recoveries totaled 92.7 and 93.1 per cent of the weight of mixed solute distributed.

Experiments with Known Acids

For the sake of simplification and in order to avoid steps which might introduce structural changes in the molecules under study, it was proposed to distribute the free acids rather than the methyl esters. Partition ratios in the heptane to methanol, formamide, glacial acetic acid system are given in Table I. p values (KJKJ proved to be of the order of 2 or more. In order to give more meaning to these values, it can be added that the present equipment of this laboratory is capable of separating compounds with p values as low as 1.1.

After most of the studies reported here had been completed, another system was found which appeared promising, especially for separation of Cls from longer chain fatty acids. In this system acetonitrile was used in place of formamide, with the advantage that both phases were volatile under the conditions used in gravimetric analysis. The K values of the various acids in this system also are given in Table I.

The effect of the various components of these systems on the K and /3 values of stearic and palmitic acids can be seen from Table II. Acetic acid, which tends to produce a linear partition isotherm, lowers the /3

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302 SEPARATION OF HIGHER FATTY ACIDS

values. This can be overcome by adding methanol, which lowers the K without affecting the p value, and formamide, which raises the K and p

TABLE I Partition Ratios of Higher Fatty Acids

Fatty acid n-Heptane 3 n-Heptane 4

Formamide, MeOH, HOAc = I:i:i Acetonitrile, MeOH, HOAc = T;- 1.1.1

Stearic . Elaidic . Oleic. . Palmitic.. Linoleio Myristic.. Linolenic. Laurie . . .

.

. .

8.9 5.5 4.9 4.4 2.9 2.0 1.6 0.9

3.1

1.9 1.9 0.9 1.2 0.6 0.8

TABLE II Effects of Various Solvents on Partition Ratios of Stearic and Palmitic Acids

Solvent system*

n-Heptane/HOAc (97.5%) n-Heptane HOAc (3)

(4) / Formamide (I) Isooctane Formamide (1)

(2) / MeOH (1) n-Heptane Formamide (1)

(3) /

HOAc (1) MeOH (1)

n-Heptane Acetonitrile (1) (4)

/ HOAc (1) MeOH (1)

n-Heptane Acetonitrile (1) (3)

/ Formamide (1) HOAc (1)

n-Heptane (11) /

Acetonitrile (5) HOAc (5)

n-Heptane Acetonitrile (3.3) 01)

/ HOAc (3.3) Methyl cellosolve (3.3)

--

-

K, stearic

4.96 24

4.04

8.9

K, palmitic

3.35 12

2.0

4.4

B

1.48 2

2.02

2

3.14 1.87 1.68

25.4 6.2 4.1

5.92 3.61 1.64

3.8 2.36 1.61

* The numbers in parentheses give the relative volumes in ml. of each component.

values. The use of acetonitrile instead of formamide in the second system produces lower K and lower p values. Methyl cellosolve appears com- parable to methanol in its effect on /3, but gives slightly higher K values.

Separation of ArtiJicial Mixture of Saturated Acids-A synthetic mixture of lauric, myristic, palmitic, and stearic acids (300 mg. of each) was taken

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E. H. AHRENS, JR., AND L. C. CRAIG 303

up in 70 ml. of each previously equilibrated phase sufficient to charge the first seven cells in the equilibration train. After 220 transfers, analysis showed complete separation of the two lower homologues, but incomplete separation of the two higher homologues. Tube 219 was connected to Tube 0, and the contents of the train were recycled until 400 transfers had been accomplished. The distribution of the four acids illustrated in Fig. 1 demonstrates almost complete separation of all components.

Recoveries of the four acids were calculated from Fig. 1 to be 93, 99, 96, and 98 per cent in order of increasing chain length. The low recovery of lauric acid was probably due to volatilization during weight analysis. A small deviation from ideality with skewing to the right is seen in all cases. Fractions were recovered from the peaks and left limbs of all

160 200 240 280 320 360 * No. of tube

FIG. 1

K)

curves in order to determine whether skewing was due to the presence of isomers. Melting points of the recovered products and infra-red spectro- grams of mulls of these eight fractions were compared with the four start- ing materials. The failure to demonstrate differences indicated the prob- able homogeneity of the initial compounds, although the presence of very closely related isomers cannot be ruled out on the basis of this evidence and may be suspected.

Separation of ArtiJicial Mixture of Cl8 Unsaturated Acids-Preliminary K and p values of ‘the Cl* unsaturated acids in the same solvent system (Table I) indicated that these acids could be separated easily by counter- current distribution. However, the possibility of oxidation of these com- pounds posed a problem. To settle this problem, 300 mg. of linoleic acid were distributed alone. Considerable skewing to the right after 100 trans- fers and significant differences in partition ratios at similar concentrations on either side of the curve were found. These findings, indicating lack of

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304 SEPARATION OF HIGHER FATTY ACIDS

homogeneity of the original sample, were confirmed by the demonstration of trans isomers (7) in the peak and right limb and their absence in the left limb of the curve. Iodine numbers of recovered fractions matched that of the original sample, however, indicating that the sample had not been oxidized during its distribution and recovery. Similar experiments were carried out on linolenic acid and with the same results.

A mixture of oleic, linoleic, and linolenic acids (500 mg. of each) was then distributed for 650 transfers with complete separation of the three acids, as shown in Fig. 2. Considerable skewing in the 2- and 3-double bond acids was due to the cis-trans mixture of the original samples. Since infra-red spectrograms showed trans acids only in fractions isolated from the peak and right limbs of these curves, it would appear that trans acids have higher partition ratios than cis acids. Indeed, in actual distributions

2

350 370 390 410 430 450 470 490 510 530 550 510

Tube No. FIG. 2

of equivalent amounts of oleic and elaidic acids, K values were 4.88 and 5.52, respectively. However, the skewing of the oleic acid curve in Fig. 2 probably is caused by a non-linear partition isotherm, since fractions from this band showed no trans contamination. Evidence that oxidative changes had not taken place during this distribution was given by com- parison of iodine numbers of fractions recovered at the peak and at the left limb of all curves with the original acids. Also, infra-red spectrograms failed to show the absorption bands which Shreve et al. (9) have stated are characteristic of oxidized fatty acids.

Subsequent to these experiments linoleic acid has been distributed in the same system for 750 and oleic acid for 3000 transfers without evidence of oxidation, as judged by iodine numbers and infra-red spectrograms.

Separation of Unknown Mixture of Fatty Acids

The difficulty of separating the fatty acids from pig mesentery may be predicted from the data (Table III) of Hilditch (10) on the composition

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E. H. AHRENS, JR., AND L. C. CRAIG 305

of the closely related perinephric fat. It was anticipated that considerable overlapping of certain individual components would result.

The distribution of 7.38 gm. of fatty acids recovered from the hydrolysis of pig mesenteric fat is shown in Fig. 3. Analyses were made at 745, 1260, 2250, and 3044 transfers. At each stage fractions were removed as shown in order to give space in the distribution train for spreading of the remaining solutes during recycling. In this manner the original charge was divided into fifteen fractions totaling 6.63 gm., representing 90 per cent recovery, or 93 per cent recovery counting the weight of solute with- drawn for gravimetric analyses. In order to characterize the constituents of the different fractions, a variety of analytical methods were used, in-

TABLE III

Fatty Acid Composition (Weight Percentages) of Pig Mesenteric Fat

Myristic ............. Palmitic .............. Stearic ................ Oleic ................. Linoleic. .............. C&2 unsaturated. ....

Linolenic ............. Conjugated dienes.

Hitditch’

3.9 26.7 47.2

1 17.6 35.7 13.7

1.3

T

Spectrosc;o$ analysis,

1 46.3

.44.0 4.8 0.32 (Arachi-

donic) 0.23 0.11

Present studies

4.0 28.3 49.9

1 17.6 42.3 6.1 0.5 (C&z-diene)

Not calculated “ ‘I

* Original data given for pig perinephric fat in molar percentages (10). t Data furnished by Dr. Willy Lange, The Procter and Gamble Company, on an

aliquot of the lard used in the present experiments.

eluding melting points, infra-red spectrophotometry, iodimetry, and frac- tional crystallization. Trans acids were absent in all fractions (7) as well as in the original triglyceride.

The solute in Tubes 480 to 580 of the upper pattern of Fig. 3 appeared to represent overlapping bands of at least two components, probably myris- tic and linoleic acids. This was confirmed by fractional crystallization from acetone of cuts at -20” and identification by melting point and io- dine numbers. A degree of association is indicated by the K calculated from the run, 2.14 and 2.52, as compared to individually determined K (2.0 and 2.9). Iodine numbers ranging from 47.4 to 149.7 permit estima- tion of the degree of overlapping and the composition of the mixed band, 40 per cent myristic and GO per cent linoleic acid.

The remaining solute in Tubes 990 to 1108 was recycled until 3044 transfers had been accomplished; yet there was no clear resolution into individual components. The mixed band was divided into four fractions.

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306 SEPARATION OF HIGHER FATTY ACIDS

0

2400 24’20 2440 24EO 2480 2500 2520 2540 2560 25% 2603 2620 Tube No.

FIG. 3

The highest iodine number achieved, 90.1, essentially that of oleic acid, was found in the cut of highest K, while the lowest gave a value of 14.6. Calculations based on iodine numbers indicated that the entire band could be 60 per cent oleic acid and 40 per cent palmitic acid. Although the presence in major part of these acids was suggested by melting points and

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E. H. AHRENS, JR., AND L. C. CRAIG 307

iodine numbers after fractional crystallization from acetone at -20°, the presence of other acids in this band has not been excluded.

The material in Tubes 1108 to 1170 at the 1260 transfer stage, Fig. 3, was almost entirely stearic acid. Iodine numbers of 3.95 and 7.10 sug- gested a degree of unsaturation but, because of the inaccuracy of the method in this range and the unknown nature of the unsaturated con- taminant, percentages cannot be given. The fraction was redistributed in the acetonitrile system detailed in Table I. After 215 transfers only a single band was demonstrated (Fig. 4). Since the band was slightly skewed, partition ratios were determined in Tubes 155 and 171. The

6 ”

Steapic acid fraction (1.15 gm.)

5 5

0 0 20 20 40 40 60 60 80 80 100 100 120 120 140 140 160 160 180 180 200 200 Tube No. Tube No.

FIG. FIG. 4 4

finding of the same K at the same concentration level indicates rather poor resolution. The completely volatile system permitted easy recovery by evaporation (1.025 gm.). A melting point 3” below that of pure stearic acid and an iodine number of 4.8 indicated the persistence of contamina- tion by unsaturated acids. Fractional cryst.allization at -20” suggested contamination by a small amount of unsaturated acid, probably at least a diene. The percentage composition of the band calculated on the basis of a mixture of C&z-diene and stearic acid was 3.2 and 96.8 per cent re- spectively.

In summary, at least six fatty acids were indicated in this mixture. Other experiments with larger as well as smaller amounts of starting ma- terial demonstrated that there were definite advantages in working with smaller quantities. This can be illustrated in Fig. 5, in which 3 gm. of the

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308 SEPARATION OF HIGHER FATTY ACIDS

fatty acid mixture were distributed. Better separation between linoleic and pahnitic acids and between oleic and stearic acids was achieved in this distribution in 750 transfers than at a comparable stage in the distri- bution of 7.38 gm. (Fig. 3), which in turn was superior to still another distribution of 9.0 gm. On the basis of the present experience it can be reasonably predicted that a distribution of 2 gm. of starting material would produce more clean cut results than those presented in Figs. 3 and 4, and still allow adequate mater&l in the various fractions for identifica- tion of the components by other techniques.

c ., Fatty acids of pig mesenteric fat Oleic

750 ti?ansfePa

490 510 530 550 570 590 610 630 650 670 690 710 TubeNo.

FIG. 5

The studies at the present stage permit the calculation of the percentage composition by weight of the various acids in pig mesenteric triglycerides. These data are compared in Table III with the figures for pig perinephric fat achieved by Hilditch (10) mainly by fractional distillation, and with the figures derived from iodine numbers, thiocyanogen values, and ultra- violet absorption after alkali isomerization. The data of the present re- port are based on the assumption that only six components are present, but with full recognition of the uncertainties of this assumption.

DISCUSSION

The technical improvements reported here indicate that considerable advantage will be gained in the investigation of natural fats by the addi- tion of counter-current distribution to current fractionation techniques.

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E. H. AHRENS, JR., AND L. C. CRAIG 309

It has been demonstrated that a mixture of saturated acids differing only in chain length is readily and almost ideally separable, as are mixtures of acids differing only in degree of unsaturation. The partition ratios Of cis-trans isomers differ significantly. Since partition ratios of fatty acids (11) are markedly affected by hydroxyl groups, it is to be expected that hydroxylated fatty acids can be separated from the normal acids in very few transfers. That peroxides, epoxy compounds, and hydroperoxides also can be isolated by this technique is further indicated by the recent report of Fugger et al. (12).

The present studies indicate that considerable simplification of complex mixtures of fatty acids, differing both in chain length and in saturation, can be effected by counter-current distribution. Whether or not it would be more profitable to apply other fractionat.ion methods before counter- current distribution, or vice versa, cannot be stated at present. Separation of the mixed acids of pig mesenteric triglycerides into saturated and un- saturated groups by crystallization in the cold could have been followed by an evaluation of the efficiency of this group separation by counter- current distribution of the two fractions. However, because of the likeli- hood of mutual contamination of the two fractions, the alternative course was followed.

The failure to resolve the main band composed of Clg-monoethenoid and palmitic acids, even after 3000 transfers (Fig. 3), might be explained by the presence of oleic acid isomers, each with a slightly different parti- tion ratio. The present studies indicated that trans acids were absent. However, evidence for the existence of position isomers of oleic acid in mammalian fat has been presented by Millican and Brown (13). Such isomers would be expected to have definite differences in K, and their presence in this fatty acid mixture might explain the broadness of the curve in Fig. 3.

In the solvent system containing formamide, it appeared that partition ratios were almost as much affected by loss of two methylene groups as by iritroduction of a double bond. Thus, in counter-current distribution of a complex mixture in this system, there will be considerable overlapping of C&-saturated with C&monoethenoid and perhaps by C&-diethenoid acids. Likewise Cl4 saturated acid overlaps with Cls-diethenoid and prob- ably with C16-monoethenoid acids. While it may be unwise to carry this argument to extremes, such a state of affairs seems fortunate in that the mixtures most difficult to separate by counter-current distribution are precisely those which can be resolved readily by fractional distillation of the methyl esters and perhaps by fractional crystallization. By their nature the latter methods are most effective when applied to simplified mixtures.

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310 SEPARATION OF HIGHER FATTY ACIDS

SUMMARY

1. A mixture of the four fatty acids, lauric, myristic, palmitic, and stearic, has been clearly separated by counter-current distribution in sys- tems which give nearly linear partition isotherms. Similarly, complete separation of oleic, linoleic, and linolenic acids has been effected without evidence of oxidation taking place.

2. A complex mixture of saturated and unsaturated fatty acids obtained by hydrolysis of the triglycerides of pig mesenteric fat has been subjected to counter-current distribution. The five components known to be present in such material were separated into three groups in a distribution of 3 gm. of the mixture, and percentage composition of the fatty acid mixture could be calculated.

3. The advantages of utilizing counter-current distribution to comple- ment classical fractionation techniques for the study of the composition of fatty acid mixtures are discussed.

BIBLIOGRAPHY

1. Sato, Y., Barry, G. T., and Craig, L. C., J. Biol. Chem., 170, 501 (1947). 2. Barry, G. T., Sato, Y., and Craig, L. C., J. Biol. Chem., 188, 299 (1951). 3. Craig, L. C., Anal. Chem., 22, 1346 (1950). 4. Craig, L. C., Hausmann, W., Ahrens, E. H., Jr., and Harfenist, E., Anal. Chem.,

23, 1326 (1951). 5. Craig, L. C., Hausmann, W., Ahrens, E. H., Jr., and Harfenist, E., Anal. Chem.,

23, 1237 (1951). 6. Yasuda, M., J. BioZ. Chem., 94,401 (1941-42). 7. Shreve, 0. D., Heether, M. R., Knight, H. B., and Swern, D., Anal. Chem., 22,

1261 (1950). 8. Craig, L. C., Gregory, J. D., and Hausmann, W., Anal. Chem., 22, 1462 (1950). 9. Shreve, 0. D., Heether, M. R., Knight, H. B., and Swern, D., AnaZ. Chem., 23,

277, 282 (1951). 10. Hilditch, T. P., The chemical constitution of natural fats, 2ndeditioq London, 314

(1949). 11. Zilch, K. T., and Dutton, H. J., Anal. Chem., 23, 775 (1951). 12. Fugger, J., Zilch, K. T., Cannon, J. A., and Dutton, H. J., J. Am. Chem. Sot., 73,

2861 (1951). 13. Millican, R. C., and Brown, J. B., J. BioZ. Chem., 164, 437 (1944).

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Edward H. Ahrens, Jr. and Lyman C. CraigACIDS

SEPARATION OF THE HIGHER FATTY

1952, 195:299-310.J. Biol. Chem. 

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