FISFiEY2IES AND MARINE SERVICE. Seher, and H. Vogel

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1 ARCHIVES FISFiEY2IES AND MARINE SERVICE Tranalation Series No Study of sterol mixtures IV: The retention behaviour of free sterols in gas chromatography by A. Seher, and H. Vogel Original title: Untersuchung von Steringemischen IV. Das Retentions - verhalten freier Sterine bei der Gas-Chromatographie _ T'ron : Fette, Seifen, Antrichm. 78(3): , 1976 Translated by the Translation Bureau( SM ) Multilingual Services Division Department of the Secretary of State of Canada Department of the Environment Fisheries and Marine Service Halifax Laboratory Halifax, N.S pages typescript

2 DEPARTMENT OF THE SECRETARY OF STATE TRANSLATION BUREAU MULTILINGUAL SERVICES DIVISION FaMen 2e.e rqigeet CANADA SECRÉTARIAT D'ÉTAT BUREAU DES TRADUCTIONS DIVISION DES SERVICES MULTILINGUES TRANSLATED FROM - TRADUCTION DE German AUTHOR - AUTEUR A. Seher and H.'Vogel INTO - EN English 0,1( TITLE IN ENGLISH - TITRE ANGLAIS Study of sterol mixtures IV: The retention behaviour of free sterols in gas chromatography. TITLE IN FOREIGN LANGUAGE (TRANSLITERATE FOREIGN CHARACTERS) TITRE EN LANGUE ÉTRANGÉRE (TRANSCRIRE EN CARACTÈRES ROMAINS) Untersuchung von Steringemischen IV: Das Retentionsverhalten freier Sterine bei der Gas-Chromatographie REFERENCE IN FOREIGN LANGUAGE (NAME OF BOOK OR PUBLICATION) IN FULL. TRANSLITERATE FOREIGN CHARACTERS. RÉFÉRENCE EN LANGUE ÉTRANGÈRE (NOM DU LIVRE OU PUBLICATION), AU COMPLET, TRANSCRIRE EN CARACTÈRES ROMAINS. Fette-Seifen-Anstrichmittel REFERENCE IN ENGLISH - RÉFÉRENCE EN ANGLAIS (Fats-Soaps-Paints and Varnishes) PUBLISHER - ÉDITEUR not shown PLACE OF PUBLICATION LIEU DE PUBLICATION not, si own YEAR ANN SE DATE OF PUBLICATION DATE DE PUBLICATION VOLUME ISSUE NO. NUMÉRO 3 PAGE NUMBERS IN ORIGINAL NUMÉROS DES PAGES DANS LORI GINAL NUMBER OF TYPED PAGES NOMBRE DE PAGES DACTYLOGRAPHIÉES 6 REQUESTING DEPARTMENT MINISTÈRE-CLIENT Environment TRANSLATION BUREAU NO. NOTRE DOSSIER N BRANCH OR DIVISION Fisheries and Marine Service TRANSLATOR (INITIALS) S.M. DIRECTION OU DIVISION TRADUCTEUR (INITIALES) PERSON REQUESTING DEMANDÉ PAR YOUR NUMBER VOTRE DOSSIER NO Allan T. Reid for Dr.M.Kovacs Y76 DATE OF REQUEST DATEIDE LA DEMANDE 16 August 1976 SOS-20C-I C-6 (REV. 2/68)

3 DEPARTMENT OF THE SECRETARY OF STATE TRANSLATION BUREAU SECRÉTARIAT D'ÉTAT BUREAU DES TRADUCTIONS MULTILINGUAL SERVICES DIVISION CANADA DIVISION DES SERVICES MULTILINGUES CLIENT'S NO. DEPARTMENT Div's! ON/BRANCH CITY N DU CLIENT MINISTRE DIVISION/DIRECTION VILLE Environment Fisheries and Marine Halifax BUREAU NO. LANGUAGE TRANSLATOR (INITIALS).'' _., -çi. N DU BUREAU LANGUE TRADUCTEUR (INITIALES) German S.M....xi,.. cç5') 2.1- F, Fette.Seifen.Anstrichmittel 78, 3, 106 (1976) Study of sterol mixtures IV:* 1 06 The retention behaviour of free sterols in gas chromatography** by A. Seher and H. Vogel*** from the Federal Institute for Fat Research, MUster (Westfalia) Retention behaviour of a number of free sterols vas studied by gas clromaiography using SE-30, UCCW-082, 0V-17 and 0V-25 as stationary phases. In a similar manner as tested for steryl acetate.s, the leparation factors were determined for sterol pairs which differ by one fuete.kc=r1 cast. The efficiency of sep,ration of the statibnary phases OY47 and 0V-25 were found to lie distinctly superior to those of less polar phases.. Sterols having K -h.s's'j-clouble bords are especially well separated from their saturated counterparts. Steryl acetates exhibit on all four staticmary phases longer retention limes compared to free sterols whereas the trimethylsily1 ailier derivative.s show on SE-30 and lic.c1v-982 columns longer and in 0V-17 and 0V-25 columns greatly reduced retention limes. 1. Introduction The rapidly advancing development of gas chromatographic analysis of mixtures of sterols has led to the discovery of numerous new sterols, particularly in plants * Part III: Fette.Seife.Anstrichmittel 74, 261 (1972). ** From the thesis of Dr.H.Vogel l University of Minster, *** Addresses of the authors: Prof.Dr.A.Seher Dr.H.Vogel l Bundesanstalt fr Fettforschung, Georgstrasse 40, Piusallee 68/76, D-5300 Bonn D-4400 MUster West Germany SOS West Germany

4 2 and plant oils. In order to improve these analytical procedures, numerous studies have been reported which had the aim to elucidate the relationship between chemical structure and the retention behaviour of sterols. Earlier research in this areal,2 was carried out with stationary phases, some of which are rarely used today, or by the study of sterol acetates3? 4 Both the recent development of silicon rubbers which are stable at high temperatures, as well as improvements in the apparatus, have made it possible to also work with free sterols5. However, very little information has been published in the literature concerning the retention behaviour of free sterols. For this reason we decided to determine the retention data and separation factors for as great a number of sterols as possible. `Thése values are reported here for 31 sterols and several triterpenes. II. Description of the experiments In addition to the separation phase SE-30 (dimethyl silicon rubber), which was at one time the separa-jion phase used most frequently, we utilized the vinylmethyl silicon rubber, UCC[h'-982, as well as the two phenylmethyl silicon rubbers, OV-17 and OV-25, which differ in their phenyl group content (,;0 and 75/^' respectively). The polycarboranesiloxane " Dexil 300" which is extremely temperature stable produced excellent separations only with trimethylsilyl ethers (TDIS-ethers);, the polyphenyl ether PPE 20 proved to be unsuitable for the separation of free sterols. The separations were carried out using the liquid phases UCCW-982 (3.8i' on Diatoport, 80 to 100 mesh), SE-30, OV-17 and OV-25 (each 3% on Supelcoport, 80 to 100 mesh as support material) in 3 m long glass columns with 3 mm internal diameter. These columns contained about 7 g of the coated support material. 1 R. Ÿ. Claylnrt, I3iochr,ni!;l,) 1, 3,57 [ = 1\'. Ikrkrrrrr, R. 11'rrl.nrf^'. K. Tsurln u. K. Srrkai, Analytic. Clain [195S]. ' G. W. 1'ullrrson. Aral.tic (:!:c,n (i5 [1971]. ll. E. u. Sr..^',.). J. Chion:atugr. [Arnstcrclam] ï^. ltii [19r?]. C. Grrnrtr,uld. J. Chr,na; r. [:1n, lr,d;:m] 9^. 173 [1969]. 106

5 Apparatus: Fraktometer F20 and Fil from the Perkin-Elmer firm at Lake of Constance, Germany Oven temperature: 265 C isothermal Injection block temparature: 280 C Carrier gas: N2) repurified;. the flow-through rate was adjusted for each case so that cholesterol had a retention time of 30 minutes The sterols studied came from three sources (commercially available sterols, substances isolated from plants, synthesized compounds * "). 3 III. Results 1. Relative retention times Table 1 gives an overview of the relative retention times of the sterols and triterpenes studied on the four different phases. Rating the stationary phases on a general basis according to their separation capability for a mixture of many sterols is not possible since the effect of the different functional groups produces variations in the retention behaviour on the four phases. However, usually the silicon 1C7 rubbers 0V-17 and 0V-25 allow better separation of the mixture than the two less polar phases. 2. Separation factors In order to express the different retention behaviour of two methylsterol ethers which differ only by one functional group, R. B. Claytonl calculated a separation factor. This is the quotient of the relative retention times of the two sterol ethers to be compared; k x = r (s x) r s **** We would like to thank Dr.Homberg and Dr.Schiller of Mûnster for giving us a number of pure sterols prepared synthetically.

6 4 Table 1 10^j Relative retention timos for sterols (based on cholesterol for an absolute retention time of 30 minutes) = Sterol Stationary phase l)v 17 clv-':", Cholestanol A4 -Cholestanol Cholesterol A6'-Cholestenol 47-Cholestenol 414-Cholestenol Desmosteral Campestanol Campesterol. Ergosterol Brassicasterol 24-I.ethylene Cholesterol 47 -Campestenol -'l- oo-. UerOl 47,9{n}) 22o st a tr i enol A7,22 --^:.g ostacl.lenol o8-±-irgostenol :^rg ostp,no1 e22-ergostenol Stigmastanôl Sitosterol bs.7+2-stigmastatri enol Stigmasterol D5 -Avenasterol Fucosterol 47 -Stigmastenol a7,9t"a- Stigmastadienol 47's 'Ft--Stigmastadienol A7s22-Sti-maste.dienci a7^^}^25)--stigmastadienol 47 -Avenasterol Dihydrolanosterol Lanosterol (3-Amyr in I-' _ S l.so Os ,-1, ? 1.5' ^ I f 1.`? ^ _

7 In this expression: k x. the separation factor "(s + x) = the relative retention time of a sterol with a double bond at position x r s the relative retention time of the correspending saturated sterol The magnitude of the separation factor is,dependent upon the separation column used. We were able to confirm that Clayton's expression which was developed for sterol derivatives, also applies for free sterols and for the newer stationary phases which we used. The initial separation factor is not affected by the introduction of additional isolated double bonds;, therefore the following expression applies fora non-conjugated doubly-unsaturated sterol relative to the corresponding saturated sterol: 5 r, Ls + x + y) r k k y Separation factors for other functional groups may be calculated as well as those for double bonds, for example for methyl or ethyl groups at Cali --thus hemologous sterols or for pairs such as sterol acetate/free sterol or trimethylsily1 ether/free sterol. The separation factors which we determined for free sterols are summarized in Table 2. By definition,, values for the separation factors - in the neighbourhood of 1.0 indicate that no separation was possible. The more a value differs from 1.0, the better the sterol pair is separated in columns packed with this stationary phase. The extensive additivity of the functional group increments permits one to calculate relative retention times even for complex sterols. Comparison of the retention behaviour of an unknown sterol on several of the given separation phases indicates which functional groups might be present and thus simplifies elucidation of the structljre. Received January 15, 1976.

8 4 Table Separation factors for sterol pairs on different stationary phases Sterol Pair UCCW-932 SE-30 0V-17 0V- 9 5 à4 /4-saturated à 5 /5-saturated e/6-saturated 47 /7-saturated 4e0/8-saturated e/l4-saturated e/22-saturated 45 ;7 /A 5 5 :7 /e:7,417, 9 (11)/4 d';14/a te S /24-saturated LIS to de1 24 (28)/24-saturated(C 28 ) om o.is In WW24-saturated(C 29 ) LH LH A 21f 2 er24-saturated(c 2 0, /0 27 (homologs) , /C28 (homologs) tie l.21 Acetate/free sterol TMS ether/free sterol b ) to )0.68

FISHERIES AND MARINE SERVICE. Translation Series No Investigation of the lipid composition of several species of fish

FISHERIES AND MARINE SERVICE. Translation Series No Investigation of the lipid composition of several species of fish FISHERIES AND MARINE SERVICE ARCHIVES Translation Series No. 3637 Investigation of the lipid composition of several species of fish by A.G. Rudenko, and V.I. Lisovskaya Original title: Issledovanie sostava