Feb., 19511 HILDITCH, PATEL AND RILEY 81 The Spectrographic Determination of Linoleic and Linolenic Acids BY T. P. HILDITCH, C. B. PATEL AND J. P. RILEY The values of the extinction coefficients (EiZ) for the ultra-violet absorption bands at 234 mp and 268 mp, which are developed when linoleic and linolenic acids are subjected to isomerisation with alkali under the conditions specified by Hilditch, Morton and Riley, have been re-investigated. Pure linoleic and linolenic acids that had been isolated from natural sources by physical methods alone were employed, in addition to specimens prepared respectively by debromination of tetrabromostearic and hexabromostearic acids, For linoleic acid prepared either by physical or chemical methods, and isomerised with alkali at 180" C for 60 minutes, the mean observed value of EiL at 234 mp was 908, in close agreement with the earlier value of 906.For linolenic acid prepared by debromination, the values were also usually close to those observed in the earlier work, b u t linolenic acid isolated by physical methods of separation gave somewhat different values, namely : after isomerisation at 170" C for 15 minutes, Ei:m at 268 mp = 555, and after isomerisation at 180" C for 60 minutes, Ei$'m at 234 mp = 575. It is considered that these should be used in place of the earlier values of 532 (268mp) and 569 (234mp). IN 1945, Hilditch, Morton and Rileyf recommended that, in the spectrophotometric determina- tion of linoleic and linolenic acids after alkali isomerisation as originally proposed by Mitchell, Kraybill and Zscheile,2 isomerisation to conjugated dienes should be carried out at 180" C for 60 minutes, and isomerisation to conjugated trienes (for linolenic acid) at 170" C for 15 minutes.Employing specimens of linoleic and linolenic acids prepared by debromination of the crystalline tetrabromo- or hexabromostearic acids obtained from the natural acids, these workers observed the following values of EtL with a Hilger E3 Quartz Spectrograph with sector photometer for the pure acids- E: & h Linolenic acid, alkali-isomerised at 170" for 15 minutes . . 532 268 mp Linolenic acid, alkali-isomerised a t 180" for 60 minutes . . 569 234 mp Linoleic acid, alkali-isomerised at 180" for 60 minutes . . 906 234 mp In recent and current investigations a Beckman or a Unicam photo-electric spectro- photometer has been employed, and it is now possible to isolate natural linoleic and linolenic acids by solely physical means (crystallisation or chromatographic adsorption) without recourse to the chemical procedure of bromine addition and subsequent debromination.In view of the extensive use now made of the spectrophotometric methods for the determination of linoleic and linolenic acids in natural fats, it was considered desirable to re-investigate the above reference values for Ei yk. Accordingly, specimens of linoleic acid from sunflower, safflower and Niger seed oils, and of linolenic acid from linseed and conophor oils, have been prepared by both the older chemical and the newer physical procedures and, after the standard isomerisations with alkali, have been examined in a Beckman photo-electric spectrophoto- meter and (in one instance) in the Hilger E3 spectrograph.ISOLATION OF LINOLEIC AND LINOLENIC ACIDS (a) B Y CHEMICAL METHODS- The procedure used was that described in the earlier paperf (p. 69). Tetrabromostearic acid was prepared from concentrates of linoleic acid from sunflower or safflower seed oils and debrominated to methyl linoleate by the method of R ~ l l e t t , ~ the crude ester being then fractionated in a vacuum. Hexabromostearic acid was prepared from unsaturated concen- trates of the acids of linseed or conophor oils and purified by crystallisation from xylene followed by washing with boiling ether. The purified acid was debrominated to linolenic acid by the method of Kaufmann and Mestern.* The crude acid was methylated and thea2 HILDITCH, PATEL AND RILEY: THE SPECTROGRAPHIC [Vol.76 methyl ester fractionally distilled. Six or seven fractions of distilled esters were obtained, those of maximum iodine value being used in the further investigation. The characteristics of the specimens of esters finally obtained and subsequently used for alkali isomerisation and spectrophotometric examination are given in Tables I and 11. TABLE I LINOLEIC ACID (METHYL LINOLEATE) PREPARED BY THE CHEMICAL METHOD Methyl linoleate Tetrabromostearj c (-Ap, Linoleic acid Distilled fraction acid froin & studied fraction. Yield, ",.p.> Yield, r-A-, Iodine Source % (w/w) C yo (w/w) No. Iodine value value* Safflower seed oil . ... . . 55 115.5 33 3 171.4 180.1 Sunflower seed oil concentrates (I.V. 169) . . . . . . . . a4 116 41 4 171.0 179.2 TAEZE I1 LINOLENIC ACID PREPARED BY THE CHEMICAL METHOD Methyl Hexabromos tearic acid linolenate. Linolenic Crude Purified crude fraction studied from I o d i n e Iodine Source yo (w/w) C % (w/w) C yo (w/w) value* No. value* value* A f 1 Linolenic acid Distilled acid - -7 - Yield, y.p., Yield, F.p., Yield, Iodine fraction. Linseed acid concentrates (I.V. 231) . . 65 178 35 181 33 265.3 3 259.4 270.6 270.7 Conophor mixed acids (I.V. 210). . 50 178 29 181.4 33 264.0 4 { iii:: 269.8 Conophor mixed acids (I.V. 210). . 44 178 33 181.2 32 264.2 4 259.9 271.0 * It was noticed (cf. McCutcheone) that, at high concentrations of linolenic acid or ester, iodine values determined by the Wijs method required 3 hours contact with the Wijs reagent before a constant maximum absorption was reached.For this reason 3 hours (instead. of the usual half-hour) contact with the reagent was employed for both the linoleic and the linolenic compounds dealt with in this paper when approximate purity was being approached. These 3-hour contacts arc: recorded as "iodine value*." Theoretical iodine values : linoleic acid 181.4, linolenic acid 274.1. (b) BY PHYSICAL METHODS- Attempts were made to find conditions whereby linoleic or linolenic acids could be effectively separated from small proportions of accompanying unsaturated acids by crystallisa- tion of the lithium salts from acetone, but without success. In other experiments the use of lithium or barium hydroxides in amounts sufficient only to neutralise a portion of the fatty acids present was attempted, but in none was the salt of linoleic (or linolenic) acid obtained in a condition approaching purity, whether from.solutions in alcohol or acetone or from mixtures of the two solvents. The procedure, recommended by Nicholson and Formo,s of employing a mixture of two bases (e.g., sodium and barium hydroxide) also failed to lead to the isolation of the pure acids. Recourse was therefore had to crystallisation of the acids themselves from appropriate solvents a t low temperatures. Following the general procedure recommended by Brown and Frankel6 we were able by repeated crystallisation of acids rich in linoleic acid, first from acetone at -60" to -70" C, and finally from light petroleum at -65" to -70" C, to isolate specimens of linoleic acid of high purity; but we also confirmed the experiences of Shinowara and Brown' that linolenic acid cannot be prepared free from contamination by about 10 per cent.of linoleic acid by low-temperature crystallisation methods alone. Riemenschneider, Herb and Nichols,s however, have recently described an adsorption method for the isolation of pure linolenic acid, and we found that application of their procedure to concentrates rich in linolenic acid produced by preliminary use of low-temperature crystallisation enabled us to prepare almost pure specimens of natural linolenic acid.Feb., 19511 DETERMINATIOX OF LINOLEIC AND LINOLENIC ACIDS 83 Linoleic acid-Specimens of the pure acid were obtained from the mixed acids of sunflower, safflower and Niger seed oils by crystallisation from acetone and light petroleum (b.p.40" to 60" C) at low temperatures. As an example, the isolation of linoleic acid from the mixed acids of Niger seed oil may be briefly described. The mixed acids (100 g, iodine value 139.6) were first crystallised from 10 per cent. solution in acetone at -60" C, the deposited acids being further crystallised from acetone a t -55" C, and this process was repeated twice at -55" C. The acids left in solution a t each stage were: at -60" C, 17.9 g, iodine value 157.1; at -55" C (l), 17.8 g, iodine value 169.7; at -55" C (2), 17*0g, iodine value 173.4; at -55°C (3), lO.Og, iodine value 173.6. The first batch of acids contained most of the unsaponifiable matter from the original oil, but the three succeeding batches (44 g) were united and further crystallised as below; from this stage onwards linoleic acid was concentrated in the deposited solids and not in the acids left in solution.The acids deposited at each stage, and the conditions of crystallisation, were as follows- Cry stallisation - Deposited acids r I o d i n e v a i u e * Solvent of solute, n / Temperature, O P 6 Concentration ;'(I Acetone . . . . . . . . 10 Light petroleum . . . . . . 5 Light petroleum . . . . . . 5 Light petroleum . . . . . . 6 Light petroleum . . .. . . 6 Light petroleum . . .. . . 3 Light petroleum . . . . . . 6 L - 70 38.0 173.5 - 60 33.1 176.2 - 60 28.2 177.1 - 60 23.2 177.7 - 60 17.1 180.0 - 60 11.7 179.9 - 60 5.5 179.5 The specimens with iodine values* 180 and 179.9 (theory 181.4) were used for spectro- The specimens of purified linoleic acid thus prepared and employed for spectrophotometric photometric examination after alkali isomerisation.analysis are summarised in Table 111. TABLE I11 SPECIMENS OF LINOLEIC ACID PREPARED BY LOW-TEMPERATURE CRYSTALLISATION Source of acid Iodine value* of acid studied Sunflower seed oil . . .. .. . . . . 176.9 SafRower seed oil . . . . . . .. . . 179.3 Niger seed oil . . . . .. . . . . . . 180.0 Niger seed oil . . . . . . .. . . .. 179.9 Linolenic acid-Application of the cryst allisation procedure used to isolate linoleic acid to the mixed acids of oils (linseed or conophor) rich in linolenic acid failed to give a pure product, a mixture of more or less constant composition (about 85 per cent. of linolenic and 15 per cent.of linoleic acid) being reached, which could not be further separated by crystallisa- tion alone. Thus, the mixed acids (iodine value 215) of conophor oil (iodine value 205) left in solution in acetone at -70" C about 80 per cent. of fatty acids of iodine value 242. These were crystallised nine times from 5 per cent. solutions in light petroleum at -60" to -70" C, but the acids that separated from the last three recrystallisations remained at a practically constant iodine value* of 256. Specimens of approximately pure linolenic acid were, however, obtained by submitting the methyl esters of polyethenoid concentrates of the acids of conophor or linseed oils obtained by low-temperature crystallisation to separation by chromatographic adsorption, employing the technique of Riemenschneider, Herb and Nichols.* The process may be illustrated with reference to fatty acids from linseed oil.The mixed acids (188 g, iodine value 192) from linseed oil (iodine value 182.7) were first crystallised from 10 per cent. solutions in acetone at -60" C, when 132 g of acids (iodine value 235) were left in solution. These were converted to methyl esters (iodine value 226), which were fractionated in a vacuum through an electrically-heated and packed column : from 37.5g of esters a distilled fraction (20*3g, iodine value 231-0) was obtained for use in the separation over the silica gel adsorbent.84 HJLDITCH, PATEL AND RILEY: THE SPECTROGRAPHIC [Vol.76 The adsorbent was prepared (cf. Riemenschneider, Herb and Nichols*) by thoroughly mixing 4 parts of silica gel (previously ground to pass a 30-mesh sieve and washed with aqueous hydrochloric acid and then with water until free from acid and fine particles of gel) with 1 part of Hyflo "Supercel" in a mortar and activating the mixture by heating for 2 hours at 220" C in a gentle current of carbon dioxide. 'The prepared adsorbent was cooled and at once transferred to the adsorption apparatus (a tube 4.3 cm in diameter filled to a height of 40 to 45 cm with a column of the adsorbent). The column was electrically heated and, after filling, was kept at 72" C for 3 hours in a current of carbon dioxide. It was then cooled in the stream of carbon dioxide and the packed column was wetted with light petroleum (b.p.40" to 60" C). The apparatus was so designed that throughout the separation (which occupied several days) a slight positive pressure of carbon dioxide was maintained in the system. A solution of the unsaturated esters (1945 g, iodine value 231.0) in light petroleum (97.5 ml, b.p. 40" to 60" C) was introduced into the column and, immediately after they had been adsorbed, elution with the same quality o:F light petroleum was begun, the rate of efflux of the solvent being about 250 ml per hour. This was an extremely lengthy process and, as shown in Table IV, resulted in a long series of fractions of esters being recovered, the first being of very low iodine value, and the final fractions approaching closely in iodine value to that of methyl linolenate (261.0).When this point was reached, the eluant was changed to a mixture of 98 per cent. of the light petroleum with 2 per cent. of ether, which rapidly removed the remaining methyl linolenate. This fraction (usually about 4 to 5g) was then finally redistilled through a small vacuum-jacketed fractionation column, and the middle fractions of the distillate were converted to acid and used for the spectrophotometric analysis. TABLE I'V ELUTION OF ADSORBED METHYL LINOLENA'TE FROM THE ADSORPTION COLUMN (19.5 g of esters (iodine value 231.0) of unsaturated acid concentrates from linseed oil) Elution with digkt ;betroleurn Fraction 1 2 3 4 5 6 7 8 9 10 11 Eluate, ml 2500 2900 2500 1500 2300 2300 2300 2300 1500 2000 1500 Methyl esters & 6 I.V.0.10 66.3 0.05 83.6 0.05 - 0.06 31-2 0.05 - 0.1 1 92.0 0-17 103.6 0.33 105.0 0-36 112.4 0.62 125.0 2.40 127.7 Fraction Eluate, 12 1100 13 1000 14 1500 15 1000 16 850 17 1250 18 1000 19 1000 20 1000 21 1500 22 1000 ml Methyl esters & g I.V. 1.95 209.3 1-35 230.3 1.37 242.4 1.12 244.1 0-46 246.6 0.5 1 246.G 0-38 - 0-28 250-0 0.23 - 0.29 253.5 0.17 252.3 Elution with 98% of light petroleum f 2% of ether Fraction Eluate, /-Ap, Methyl esters ml g I.V. (3.09 0-12} 255.0 23 450 24 700 25 1000 4.98 256.7 26 1000 0.36 255.5 (Total weight recovered 18.0 g = 92.1%) Fraction No. 25 had an iodine value* (Wijs, 3 h.ours contact) of 260.1 and was fractionally distilled. The third and fourth fractions of the distilled esters (1.68 and 1.73 g, iodine values* 25943 and 261.0) were combined and hydrolysed, arid the resulting acids (iodine value* 271.9) were used for spectrophotometric analysis.From a similar sequence of operations conducted on the methyl esters (iodine value 200) of the mixed fatty acids of a specimen of conophoi: oil, there was finally obtained a distilled fraction of methyl linolenate, iodine value* 259.7, .which yielded a specimen of linolenic acid with iodine value* 271.8.Feb., 19511 DETERMINATION OF LINOLEIC AND LINOLENIC ACIDS SPECTROPHOTOMETRIC EXAhlINATION OF ALKALI-ISOMERISED LINOLEIC AND LINOLENIC ACIDS 85 The specimens of highly-purified linoleic and linolenic acids isolated as described in the preceding pages by both chemical and physical methods were submitted to isomerisation with caustic potash in ethylene glycol solution, a t 170" C for 15 minutes to develop triene TABLE V ISOMERISATION AT 180°C FOR 60 MINUTES EXTINCTION COEFFICIENTS (AT 234 mp) FOR LINOLEIC ACID AFTER ALKALI Extinction coefficient determinations Isolation f A Deviation Iodine & Origin value* 30.EiFm Mean Standard Chemical (debromination) . . Sunflower seed oil . . 179.2 6 906.3 10.1 13-1 Safflower seed oil . . 180.1 6 915-5 9.7 12.5 Physical (crystallisation) . . Sunflower seed oil . . 176.9 7 912-1 4.7 6.7 Safflower seed oil . . 179.3 2 903-1 4.3 - Jl80.0 2 906.3 1.3 - Niger seed oil . . * . 1179.9 2 910.9 4.5 - TABLE VI EXTINCTION COEFFICIENTS FOR LINOLENIC ACID Extinction coefficient determinations r-- & Iodine Isolation Origin value* Chemical (debromination) .. Linseed oil . . 270-6 Conophor oil . . 271.0 A t 268 mp, after alkali isomerisation at xo. Ey& 170" C f o r 15 nzinutes 12 539.0 6 538-3 I6 559.3 Physical (adsorption) . . Linseed oil . . 271.9 Conophor oil . . 271.8 9 358.9 6 552.0 At 234 mp, after alkali isomerisation at 180" C f o r 60 nzinutes Chemical (debromination) . . Linseed oil . . 270-6 6 560-7 Conophor oil . . 271.0 6 570-3 Conophor oil . . 270.7 8 589.7 Physical (adsorption) . . Linseed oil . . 271.9 Conophor oil . . 271.8 5 572-3 6 578-5 Mean 5.8 3.7 2.2 6.2 6.8 2.1 3.2 3.1 3.6 8.0 4.0 2.5 Standard 8.0 5.5 2.9 7.4 8.9 2.8 5.5 4.4 4-6 10.0 5.4 3.4 conjugation from linolenic acid, or at 180" C for 60 minutes to develop diene conjugation from either acid. The analytical details given in the earlier paper1 (pp.69, 70) for the preparation of the alkaline glycol reagent and for the conduct of the isomerisation were carefully followed, As therein recommended, all determinations were made on the free acids and not on their esters. In some instances we employed glycerol in place of ethylene glycol 3s the isomerisation solvent (cf. Brice and Swainlo), but in our experience this led to less concordant values and we reached the conclusion that glycol is definitely preferable to glycerol for this purpose. The spectrophotometric measurements were made throughout in a Beckman spectro- photometer, but in one series, for comparison, determinations were also made on a Hilger E3 Quartz Spectrograph (as used in the earlier work). Blank determinations with the alkaline glycol solution were carried out under exactly the same conditions as the actual determinations, the spectrographic observations of the latter being made in duplicate on each isomerised specimen.After the isomerisations at 170" or 180" C, the solutions were diluted with86 HILDITCH, PATEL AND RILEY: THE SPECTROGRAPHIC [Vol. 76 purified absolute alcohol so that the values of log I,/I recorded on the Beckman apparatus fell within the range 0.3 to 0.6. Tables V and VI give a summary of the extinction coefficients recorded, respectively, for the specimens of linoleic and linolenic acid. In these tables are given the method of isolation of the acids (chemical or physical), the oils from which they were obtained, the observed iodine value of the specimen, the number of separate alkali isomerisations made in each instance, and the mean value of the extinction coejkcients observed, together with the mean and standard deviations therefrom.DISCUSSION AND CC~NCLUSIONS LINOLEIC ACID- The mean of all the observed values (Table V) for the extinction coefficient at 234mp of alkali-isomerised linoleic acid isolated by physical means from natural sources is Et& = 908. This value is within 0.3 per cent. of that formerly de terminedl (906), and we therefore consider that the latter may continue to be employed. The final mean value for linoleic acid produced chemically by debromination of tetra- bromostearic acid was very close to this figure, although the mean values for each series and the mean and standard deviations varied to a greater extent than the corresponding figures for the linoleic acids obtained by crystallisa tion.LINOLENIC ACID- Extinction coeficients at 268 mp (triene con$%gfi:tion)-Here the values of Ei& for alkali- isomerised linolenic acid isolated by purely physical (adsorption) methods differed by about 4 per cent. from that (532) observed earlier,l the final mean value being 555 (cf. Table VI). The latter value should in our opinion now be adopted as the reference value for Ei:& at 268 mp for alkali-isomerised linolenic acid. Of three specimens of linolenic acid prepared by the debromination procedure, one gave values of the same order as the above (EiL = 555 to 559), but the other two gave values similar to those observed in the earlier work for debrominated linolenic acid, namely, EiL = 538 to 539.Matthews, Brode and Brown,ll who have contributed considerably to our knowledge of the chemical properties and beha.viour of linoleic and linolenic acids alter- natively prepared by debromination or isolated by low-temperature crystallisation, have concluded that the debrominated acids may contain 12 per cent. or more of unsaturated acids that are not the same as the natural linoleic or linolenic acids. Although some of the isomers produced will presumably be geometrical (cis-trans) isomers of the natural acids, and these may isomerise on treatment with alkali at 170" to 180" C more or less similarly to the natural (wholly cis-) acids, it seems probable that, at least when linolenic acid is con- cerned, some of the chemically regenerated acid does not react to alkali to the same extent as the natural acid.It is, nevertheless, curious that in one instance the acid obtained by debromination gave the same extinction Coefficient at 268 mp after isomerisation as that given by the linolenic acids prepared by physical means. It is also evident that, although Brown et d.ll have observed similar chemical differences between regenerated and recrystallised linoleic acid. as with the linolenic acids, linoleic acids produced by either method give similar yields of conjugated diene acids when isomerised with alkali. In one instance in Table VI it will be seen that a specimen of linolenic acid was examined, after alkali isomerisation, in both the Beckman and the Hilger instruments, with identical results, The differences in extinction coefficients that have been observed are accordingly due solely to the means employed to isolate linolenic acid, and not to any difference in the spectrographic technique.Extinction coejicients at 234 mp (diene conjugation)-The earlier figure found for the extinction coefficient at 234 mp for linolenic acid after isomerisation with alkali a t 180" C for 60 minutes was EiZ = 569. The mean value determined in the present study from eleven determinations on linolenic acid isolated from linseed or conophor oils by the adsorption procedure was 575. Although this is only about I. per cent. higher than the former figure, it is suggested that the value EiZ = 575 at 234 nip should be used for the contribution of linolenic acid to diene conjugation during alkali isomerisation.The corresponding figures for linolenic acid specimens produced by debromination are less consistent but also lead to a final mean value of EiL = 575.Feb., 19511 DETERMINATION OF LINOLEIC AND LINOLENIC ACIDS 87 We recommend that the extinction coefficients for linoleic and linolenic acids to be used in spectrophotometric determinations of these acids should be as follows- E: & h Linolenic acid, alkali-isomerised at 1TO" C for 15 minutes . . G55 (formerly 532) 268mp Linolenic acid, alkali-isonierised a t 180" C for 60 minutes . . 575 (formerly 568) 234mp L noleic acid, alkali-isomerised a t 180" C €or 60 minutes . . 906 (as formerly) 234mp On this basis, the figures based on spectrophotometric analyses recorded in our publica- tions up to the present for linolenic acid in drying oils may be about 4 per cent.too high, i e . , 2 to 3 units per cent. too high in linolenic-rich oils such as conophor or linseed, and about 1 unit per cent. high in candlenut or rubberseed oils, whilst in oils such as soya bean or hemp seed there is only a fractional difference. Similarly, the figures recorded hitherto for linoleic acid are about 1 per cent. low for oils rich in linolenic acid, but not sensibly different in other drying oils. The effect on the proportions of oleic and saturated acids is somewhat greater; in oils rich in linolenic acid (linseed, etc.) the oleic acid figures may be 4 to 5 units per cent. low, and the saturated acid figures 2 to 3 units per cent. high, as recorded on the basis of the Ei& values hitherto used. It is, however, only in the mixed acids of oils such as conophor or linseed that the differences involved exceed, at the most, about 1 unit per cent. in the component acid data. We wish to express our cordial thanks to Professor R. L4. Morton, F.R.S., for the use of the spectrographic apparatus and for his assistance and advice during the course of this work, and to Mr. R. H. Creed for assistance with many of the spectrographic measurements. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. Hilditch, T. P., Morton, R. A., and Riley, J. P., Analyst, 1945, 70, 68. Mitchell, J. H., Kraybill, H. R., and Zscheile, F. P., Ind. Eng. Chem, Anal. Ed., 1913, 15, 1. Rollett, A, 2. physiol. Chem., 1909, 62, 410. Kaufmann, H. P., and Mestern, H. E., Ber., 1936, 69, ( B ) , 2684. Nicholsen, R., and Formo, M. W., J . Amer. Oil Chem. Soc., 1949, 26, 329. Brown, J. B., and Frankel, J. S., J . Amev. Chem. Soc., 1941, 63, 1483; Frankel, J. S., Stoneburner, Shinowara, G. Y . , and Brown, J. B., Ibid., 1938, 60, 2734. liiemenschneider, R. W., Herb, S. F., and Nichols, P. L., J . Awer. Oil Chem. Soc., 1949, 26, 371. McCutcheon, J. W., Ind. Eng. Chem., Anal. Ed., 1940, 12, 465. Brite, B. A., and Swain, M. L., .J. Opt. SOC. 14nzer., 1945, 35, 532. Matthews, N. L., Rrode, W. K., and Brown, J. B., J . ,4???el/. Chem. Soc., 1941, 63, 1064. W., and Brown, J. B., Ibid., 1943, 65, 259. THE UNIVERSITY LIVERPOOL June, 1950