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Translation Series No. 425 a

SEPARATION OF SATURATED AND UNSATURATED C4 AND 06,FATTY ACIDS BY GAS CHROMATOGRAPHY

By 3. Janà, M. Dobiéisvovd and K. Vereàe

Original title: Trennung gesatigter und ungesatigter 04 und 06 FettsâUren durch Gaschromatographie

From: Collection Czechoslov. Chem. Commun., Vol. 25, pp. 1566-1571, 1960.

Translated by the'Bureau fdr Translations, Department of the Secretary of State of Canada, 15 pp., 5 tables, 5 figures, 1960. Ç') SECRETARIAT D'ÉTAT 4e g MPARIMENT or l ift SECRETARY Or STATE ( BUREAU FORTRANSLA1 IONS BUREAU DES TRADUCTIONS

• FOREIGN LANGUAGES DIVISION DES LANGUES DIVISION CANADA ÉTRANGÈRES

PI?13 7";

TRANsLATED FROm - TRADUCTION DE INTO - A Germf.,, n Enrflish

suBJECT - SUJET

Seraration of fatty acids

AUTHOR - AUTEUR J.JanA..k, M. Dobi as ova and K..Vere.

TITLE IN ENGLISH - TITRE ANGLAIS

Serartion of saturated and unsaturated C and CIfatty acids by- gas chromatography. 4

TITLE IN FOREIGN LANGUAGE - TITRE EN LANGUE TRANGRE Trennunp.-. gesgttigter und unRes•ttigter C4 und 06 Fettsâuren durch GDschromatographie. •

REFERENCE - ReFÉRENcE (NAmE OF BOOK OR PUBLICATION - NOM DU LIVRE OU PUBLICATION ) , Collection Czechoslov, Chem. Commun. 25.

PUBLISHER - ÉDITEUR

CITY - VILLE DATE PAGES 1960 166 6-15 71

REQUEST RECEIVED FROM OUR NUMBER REQUIS PAR NOTRE DOSSIER NO 83866

DEPARTMENT • TRANSLAToR MINISTRE des Pêcheries TRADUCTEUR

YOuR NumBER DATE COMPLETED VOTRE DOSSIER NO REMPLIE LE Nov. 23 1 0 62

DATE RECEIVED REÇU LE November 15, 1962

SOs-2so-10-8 ..epnration or snturated nnd unsaturated

and p6 - fatty acids by gas chromatography.

By J.Jnk , M.Dobioy 1c1 K.Vore;' Laboratory for g!D.s- analysis, Czechoslovakian Academy of the Sciences of Brno and Biological Institute of the Czechoslovakian Academy of the Sciences of Prague

Received on August 3, 1959.

The separation of isomeric unsaturated and saturated C4 and c6 - fatty acids occurring in cell metabolism was worked out. The specific volumes of elution (chromatographic spectra) of methyl and the free acids were measured on . polypropylene glycolic adipate (Reoplex 400), silicon elastomer E 301 and Squalan. A good separation of all isomers of the acids can be achieved on Reoplex 400 at temperatures .of 180 - 200 ° at free acids and 80 - 140 ° at methyl within 10 to 30 minutes.

In the investigation of the biosynthesis and of the metabolism of the fatty acids we were drawing attention to the rdle of the , caproic acid and their unsaturated analogues. It hes been established that butyric acid and caproic acid in metabolic processes play the role of hydrogen donorS, in which . connection the problem is being studied as to which of the possible unsaturated acids (i.e. vinyl acetic acid, caproic ncid, possibly hexene-(3)-acid and Hexene-(2)- acid)are produced and in which way.

In investignting metabolics in the organism we face

the difficult task of tracinp: analytically the 04 and C6-mono- carboxylic acids, in which case there are always a series of isomers present simultaneously; the salts of butyric acid, vinyl acetic acid nd were found. and identified by Lorber and Cook 1) by means of column-type chromatography. ie.ennedy and Barker 2) seuarnted vinyl acetic acid and butyric acid paper-chromatographically. But these methods are neither sensitive enough nor satisfactorily quick and complete for a thorough investigation of the cell'metabolism.

We, therefore, tried to use the fluid-gas 3) system of chromatography, which has just been applied for the first time for the analysis of fatty acids and their ester. In this way it was possible to easily senarate the homologous . links of the fatty acid series up to C . The separation of 34 the unsaturated 3CidS from the saturated ones with equal numbers of carbon atoms in the chain already ehcountered difficulties. This problem was recently solved by Orr and L) Callen and independently of them by Lipsky Landowne and Godet 5 ' 6) who used poly-oxyalkaline (? translator) ester of 478), 6 9) the dicarboxylic acids (adipic acid ', '- as strong polar stationary phases.

The perception that the selectivity in the case of .this ester deuends on the donor-accepter reciprocal action of the molecules of the stationary phase with the Ti'- electrons of the dcuble bond (possibly of the aromatic and heterocyclic rinz systems 9) Of the chromatographed substances, made us assume that in the case of the C4 and C6 -acids it would not only be possible to secarate the saturated from the unsaturated acids, but also to differentiate between the individusl unssturated acids on the basis of the position of the double bond.

• • • /3 75-

Experimental part.

Acrartus_ani._mP.terial.

The measurments were c9r ,"ied out nt the instrument from Griffin and George, type II. .The properties of this instrument have alrerldy been described elsewhere 10) The - parameters of these measurements rire found workinp in the corresponding Tables in the texte. As filling material we used the polar polypropyleneglycolic adipate (Reoplex 400, Geigy, Chem. Co, Manchester) ., the unpolar hydrocarbon Squalan (May and Baker Ltd., London) and the silicon elastomer E 301 (Griffin & George Ltd., London). In all cases 25 per weight of stationary phase of Celit 545 (Johns Manville, London) were used. The model-substances were Prepared syntheticallà , . * via chloro-iSocratonic acid Isocrotonic acid was prepared according to Fittig, Kochs, Michael and Schu1thess 11 12) But; we did not succeed in producing the pure substance accordinF to this method. The methyl esters of the butyric acid, isobutyric acid and crotonic acid were prepared by esterification of the acids by means of methanol in acid solutionlq l-) .. The methyl esters of the vinylacetic acid, isocrotonic acid, , caproic acid, hexene-(2)- acid and hexene-(3)-acid were prepared by methylation of the acid by means of diazomethane in ether solution according to 14) Arndt . Their physical characteristics could rot be deter- mined, because the quantities at our disposal were too small/ Judging by the chromatographic results, the prepared methyl esters were pure. •0nly in. the ease of the methylester of the isocrotonic acid we received as product a.mixture of at least

7 subst:m.nues. Apart from the vinylacetic-acid ester with the relative elution volume 1,60, and crotonic acid 2.09, the

Plense see p. II -4-

mixture contained a fr?ction 'Aith the relative elution volume that should corres-ond f.o the isocrotonic acid ester, and fractions with the relative elution volume 2.83, 3.45, 4.00 and 4.87 (temperature 110 ° C.) which did nob correspon to any of the tested model-substances. The boilinF point values of the used substances are summarized in Table I, to-' gether with the boiling points deduced from the chromatographic elution data.

Specific elution volumes (chrematograPhic spectra)

The positions of the individual acids or:their methyl esters,respectively, in the chromatographic spectrum were measured at temperatures 190 to 200 0 C.. and 80 to 190 0 C., respectively. The specific eiution volumes (chromatographic spectra) of the > methyl esters on three stationary phases are listed in Table II and those of the free acids in Table III.

Findinp:s and Discussion.

From the elution data for the fatty acids on poly- prophyleneglycolic adipate, which by its carbonyl and ether functional groups acts as a strong electron-accepter, the 'selective effect of the double bond is conclusively perceptible, as described by Orr and Callen 4) for fatty acids with 16 to 8) 20 carbon atoms in the chain, and by Stoffel and Ahrens for

the n -acids with a larger number of double bonds. The , ' 16 presentation of the ratios of theEution volumes and the differences of boiling points show that clearly (Table IV). All pairs with approximtely ecual . boiling points obviously . possess different elution periods in the sense that the un- s , turated acid is always more readily absbrbed. Simultaneously

ir WI Of ) we cnn observe tblt a compritivelY considerable difference in the boiling points of the substances does by far not . cause n similnrly lore chane in the rntios of the elution volumes. ,Je, therefore, dedicated ourselves .more recently to the 7tudy of the influence of the arrangement of the molecules in the neiqhbourhood of the double bond, as compared with the isolated carbonyl groups of the acid (vinylacetic acid, hexene-(3)-acid) and the conlIgated ones (methacrylic acid, crotonic acid and hexene-(2)-acid), on the chromatograPhic behaviour: The Tr-electron . pair of the double bond, in the first case, is wholly available for the reciprocal action between chromograPhic substance and stationary phase. In the case of the 0(43-relation of the double bond, as donor of the 1 1' -electrons to the carbonylic group of the acid as electron . accepter, there exists, therefore, a certain impairment of the external activity of the T1' -electrons, depending on the degree of the internal polarization. In both cases then, alkyl groups create a. steric hindering and suppress the already • mentioned selective reciprocal effect. From all acid s. with a double bond in ai(3,Ï -position or in a position that is furthe r. the carbonylic group, respectively,we can, therefore, away from expect, as a general principle, an increase in absorption and a corresponding separability which is equal for all corresponding isomers (i.e. such which do not possess an alkyl group in the carbon atom with the double bond). The straight separation of the system butyric acid-vinylacetic acid or crotonic acid- .hexene-(3)-acid confirm this.

Furthermore, in the tested acids with . the double bond In the a,(?i-position the sequence of the elution should correspond to the steric accessibility of the double bond, on the one side, and to the degree of intensity of the interry.q.

•••/6 pô12rization of the molecule, on the other. In this case the increse of the absorption should be less than in the case

of the presence of 9 double bond in („;q -position. This lias been only partly fulfilled in rr.:'ctice: It is possible to se!-Jar?te a mixture of butyric acid, metha.crylic acid and crotonic acid in the same way as the mixture of capronic acid

anJ hexene-(2)-acid; but the crotonic acid 9s well as the hexene-(2)-acid lie behind the correspondinz :5,i- -isomers in - I 0 the chromatographic spectrum, what contradicts the presented theoretical conception of the 1Y-electron reciprocal action.

In order to eliminate also any possible selective influence of the hydroxyl groups on the absorption .of the free acids,- we carried out measurements with the methyl esters of the acids. The sequence that.is extended by the methyl ester of the isocrotonic acid corresponds to the relations of the free acids, in which case the ester of the «13-acids in the chromatograrhic spectrum again lies behind the esters of the ,N(-unsaturated acids.

It will, therefore, be necessary to assumethe presenc of a further kind of forces which are probably also the cause of the evidently higher boiling points of the steriàally . Unimpaired cy, -isomers. A chromatogrorhic experiment in the area of an unpolar stationary phase in a state approaching infinite dilution maygive us some information on the presence of tbese forces. In this case the polar forces of the chroma- tographic substance (in our case, of the «IS -isomer) are reduced to a minimum until they disapnear entirely. Tiese forces are the cause for the reciprocal association of the molecules of the substance and hence for the raising of the boiling point to a higher value than would correspond to the

• • • / 7 effect of the forces or dispersion. Since the chromaboi:raphic

absor-)tion of the free.acids in the unpolar area creates sonie difriculties (tail forMation due to the alcsorbing effect of the very slightly polar supporting medium) we carried out the experiment with the methyl ester. The djmlacements in the chromatogyranhic eectrum are clearly shown in Table II. The esters of the -acids attain lower or as a maximum, the same values as the corresponding esters of the saturated n-acids, which agrees with the elimination of the influence of the reciprocal association of the molecules of the chromatograrhed substance by the infinite dilution in the unpolar area in this experiment. The esters of the trans - S -acids evidently retain a higher elution period also under these conditions. The arrangement of the experiment excludes an explanation of the elution secuence on the basis of the vapour pressure of the substance in its pure state (i.e. due to tÉle vapour pressures corresponding to the bOiling point of the substance). The sequence of the elution then is a qualitative characteristic of the presence of forces which pertain merely to the structural arrangement of the acid molecules with a steric unimpaired double bond in -position as to carbonyl.

Illustration 1 is a graphical representation of the mutual relations of the boiling points and the elution data in the unpolar ares (compare 14),; in this way, for a series-of methyl esters the boiling points that correspond to the dispersion forces can be deduced approximately. The anomalous position of the crotonate can be seen on the dotted line. The * figure values are Fiven in the form of n Table.

Illustrutions 2-4 present the practical solution of possible cases of acid mixtures, as they will occur in the

• • • /8 investigation of the cell met!abolism.

T.ble I.

Data on the boiling points of the model substances.

Acid roint 0 C. Dolling point ofthe methyl ester ° C. literature found literature -T found 'Isobutyric acid Butyric acid Vinylacetic acid

Methacryl acid. For figures. please see Isocrotonic acid German text.' Crotonic acid

. Capronic acid Hexene-(3)-acid Hexene-(2)-acid

Table Ii.

Specific elution volumes (chromatographic spectra) of the methyl ester of the fatty acids.

Methyl ester Reoplex 400 Silicon E 301 Squalan of the acid

Isobutyric acid T'utyric acid

. Methacrylic acid For f.igures plesse see Vinylacetic acid German text. ,Isocrotonic acid Crotonic !acid •.Cnrronic acid Fexene-M 3-3cid / Hexene-W-acid

V 0 (?illegible, translator) (Butyrate) • • • /n -o--

Table III.

Specific elution volumes (chromatogra .chic'spectra) of the fatty acids on Reoplex 460.

••• Ac la 192 0 200 °

Prolpionic acid 0,72 0,72 Acryl acid 0,99 0,98 IsobutyriC acid 0,77 0,78 Butyric acid • 1,00 1,00 Methacrylic acid 1,22 1,21 Crotonidacid 1,82 1,76 • Capronic acid 2,21 2,10 liexene-(3)-acid - 3,13 2,90 Hexene-(2)-acid 3,61 3,33 42 Ve° (Butyric)acid) 68

Table IV.

Reciprocal relations between the ratio of the elutionvolumes V and the differences in the boiling points of the fatty acids.

acids 2atio of the Difference elutionvolumes in boiling unsaturated saturated (V - v')/V' points ° C.

Acrylic acid 0,39 0 Methacrylic acid Butyric acid 0,22 0 Vinylacetic acid Butyric acid 0,63

Crotonic acid Butyric. acid 0,82 26 Pexene-(5)-acid Capronic acid 0,42 3 Pexene-(2)-2cid Capronic acid 0,63 12

.../l o -1 0-

111 1. Corre1.7tion of boiling points aml elution dates of the methyl ester of the fatty acids (silicon elrlstomer E 3011 t = ExPerimentally foun..1 vlues: 1 Isobutyrate, 2 Tutyrate,

3 Methcrylate, 4 Crotonate, 5 Gapronate; deduced data:

6 vinylacetate,. 7 Hexene-(3)-acid ester, 8 Hexene-(2)-acid ester, 4' uncorrected value for Crotonate, 8 1 uncorrected - value for Hexene-(2)-acid ester.

Illustration 2. Chromatogram of the mixture of the methyl ester of the aci_ds 1 Isobutyric acid, 2 Butyric acid, 3 Vinyl acetic acid and 4 Crotonic acid.• Reoplex 400, 6,51 g. 25g per weight of Celit; . Temperature of the column 80 ° C., Room temperature 24 ° C.; Supporting • gas 0,935 ml H2/s; Entering pressure 746 torr (Torricelli); . Leaving pressure 130 torr.

Illustration 3. Chromatogram of the mixture of the methyl ester of 1 Capronic acid, -2 liexene-(3)-acid and 3 Hexene- (2)-acid. • Reoplex 400, 6;51 g. 25g per weight of Celit; Temperature . • of the column 110 ° C., Hom temperature 24 ° C.; Support in gas • pressure 0,848 ml H2' /s• Entering pressure 746 torr.; Leaving 130 torr.

Illustration 4. Chromatogram of the mixture of free. acids: 1 Isobutyric acid, 2 Butyric acid, 3 Methacrylic acid, L.

Vinylacetic acid, 5 Crotonic acid, 6 Capronic acid, 7 Ilexene- (3)-acid and 8 Hexene-(2)-acid.

Reoplex 400, 6,51 g. 25g per ileight of Celit; Temperature of the column 190 ° C.; FOom temperature 24° C 4; Supporting gas 0,617 ml H 2 /s; Enterine pressure 749 torr; Leaving pressure 110 torr.

... /11 - 1 1-

*) Gur best thanks to Mrs. J.Kolin (rest of the rime translator) Riologrical institute, Czechoslovakian Academy of the Sciences of Prague, for supplying us with the vinyl2cetic acid and the two hexene

Lite rature.

1) Lorber V., Cook M. : J,?iol. Chem. 215,823 (1955).

2) Kennedy E/P., Parker H.A.: Anal. Chem. 23, 1033 (1951). 3) James A.T., Martin A.J.P.: «Piochem. J. 50, 679 (1952). 4) Orr C,H., Callen J.E.: J.Am. Chem. Soc. 80, 21., 9 (1958); Ann. N.Y. Aced. Sci. 72, 649 (1959).

5) Lipsky S.R., Landowne R.A.: Biochem. et Biophys. Acta 27, 666 (1958).

6) Lipsky S.R., Landowne R.A., Godet Diochim. et Biophys. Acta 31, 336 (1959). 7) Horning E.C., Moscatelli E.A., Swealey C.C.: Chem & Ind. (London) 1959, 751 , 8) Stoffel '4., Ahrens E.H. Jr.: J.Am. Chem. Soc. 80, 6604 (1958). 9) Janek J., likvneC M.: J.ChromatogrePhy, in the press 10) Janek J., Krejéi K., DubskY P.E.: this magazine 24, 1080 (1959); Chem. listy 52, 1099 (1958). 11) Fittig R., Kochs E.: Ann. 268, 14 (1892);