FISHERIES AND MARINE SERVICE. Translation Series No Determination of volatile fatty acids in rumen fluid by gas chromatography.

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1 ARCHIVES FISHERIES AND MARINE SERVICE Translation Series No Determination of volatile fatty acids in rumen fluid by gas chromatography By- CC Dimitrov Original title: Izsledvane no letlivite mastni kiselini v tyrbushna technost c pomoshchta no gazova khromatografiya From: Zhivotnovud. Nauki 11: , 1974 Translated by the Translation Bureau( TP) Multilingual Services Division Department of the Secretary of State of Canada Department of the Ehvironment Fisheries and Marine Service Halifax Laboratory Halifax, N.S pages typescript

2 D'EPARTMENT OF THE SECRETARY OF STATE TRANSLATION BUREAU KitM' SECRÉTARIAT D'ÉTAT BUREAU DES TRADUCTIONS MULTILINGUAL SERVICES elpê DIVISION DES SERVICES DIVISION CANADA MULTILINGUES TRANSLATED FROM TRADUCTION DE INTO EN English Bulgarian AUTHOR - AUTEUR Jimitrov,G. TITLE IN ENGLISH - TITRE ANGLAIS Determination of volatile fatty acids in rumen fluid. by gas chromatography TITLE IN FOREIGN LANGUAGE (TRANSLITERATE FOREIGN CHARACTERS) TITRE EN LANGUE ÉTRANGÉRE (TRANSCRIRE EN CARACTÉRES ROMAINS) Izsledvane na letlivite mastni kiselini v tyrbushna technost c pomoshchta na gazova khromatografiya REFERENCE IN FOREIGN LANGUACI - (NAME OF BOOK OR PUBLICATION) IN FULL. TRANSLITERATE FOREIGN CHARACTERS. RÉFÉRENCE EN LANGUE ÉTRANGÉRE (NOM DU LIVRE OU PUBLICATION), AU COMPLET, TRANSCRIRE EN CARACTÉRES ROMA:NS. Zhivotnovydni Nauki REFERENCE IN ENGLISH - RÉFÉRENCE EN ANGLAIS Animal iciences PUBLISHER - ÉDITEUR "G.Jimitrov" A2ricultural Academy PLACE OF PUBLICATION LIEU DE PUBLICATION i ofia/b ulgaria YEAR ANNÉE DATE OF PUBLICATION DATE DE PUBLICATION VOLUME 11 ISSUE NO. NUMÉRO 1 PAGE NUMBERS IN ORIGINAL NUMÉROS DES PAGES DANS L'ORI GINAL NUMBER OF TYPED PAGES NOMBRE DE PAGES DACTYLOGRAPHIÉES 8 REQUESTING DEPARTMENT TRANSLATION BUREAU NO. Environment MINISTàRE-CLIENT NOTRE DOSSIER N 0 BRANCH OR DIVISION DIRECTION OU DIVISION PERSON REQUESTING DEMANDE. PAR YOUR NUMBER VOTRE DOSSIER N 0 DATE OF REQUEST DATE DE LA DEMANDE Fisheries iervice Office of the Editor Dr.R.G.Ackman February 28, 1975 TRANSLATOR (INITIALS) TRADUCTEUR (INITIALES) UNEDITED I1AMISLATIC)t4 For ir.eiorrnari only TRADUCTION NON REVI.GEE Inforrnalion sculoment T.P. SOS (REV, 2/138) APR

3 Elf PARTMENT OF THE SECRETARY OF STATE TRANSLATION BUREAU MULTILINGUAL SERVICES DIVISION relt frïe4jv CANADA SECRÉTARIAT D'ÉTAT BUREAU DES TRADUCTIONS DIVISION DES SERVICES MULTILINGUES CLIENT'S NO. DEPARTMENT ' DIVISION/BRANCH CITY N 0 DU CLIENT MINISTÈRE DIVISION/DIRECTION VILLE Environment Fisheries Jervice Office of the Editor BUREAU NO, LANGUAGE TRANSLATOR (INITIALS) N DU BUREAU LANGUE TRADUCTEUR (INITIALES) Ottawa Bulrarian T.P. Apil Zhivotnovydni Nauki (Animal jciences),vol.xi, No.1, 1974, pp o Determination of volatile fatty acids unedited TRANSLATION in rumen fluid by gas chromatorraphy For informa1i -en only by TRADUCTION NON REV:SEE lit oma! Dimitrov, G. ion sr,wicrrient At the Kostinbrod Institute of Zootechny Volatile fatty acids are an important source of energy for the ruminant animals. It is therefore important to know not only their total amount contained in the complex stomach but the quantitative ratio of the various acids as yell, which is in direct relation to the use of energy and to the animal production. Gas chromatography is the most rapid and accurate method for the qualitative and quantitative analysis of volatile fatty acids. Different techniques were proposed for detcrmination of rumen fluid volatile fatty acids. Kromann et al. /14/, Fenner et al. /9/, Faichney /8/ and other authors applied steam distillation of volatile fatty acids in order to obtain their sodium salts which were decomposed and the free fatty acids dissolved in organic solvents were injected LI31 $0S

4 2 into the apparatus. Other authors (Cottyn et al. /5/, Iahadevan et al. /16/, Erwin et al. /7/, Emery et al. /6/, Kellog /13/, jtorry et al. /20/ and others) did not apply steam distillation and injected into the gas chromatography apparatus an aqueous solution of the previously deproteinized rumen fluid containing volatile fatty acids. In all methods using aqueous solutions, a flame ionization detector was used, whereas when working with organic solvents, a flow detector was sometimes used. Jeparation was performed with different stationary phases. Most frequently used were Tween 80, PEGA and LM-550 silicone oil. Ackman et al. /1/ used PGA. Kuzdzal-Savoie et al. /15/, Hamada et al. /11/ Uikelley /17/ and other authors used OG3E, whereas Kello gg et al. /16/ and others uses Porapak Q. /13/, Mahadevan Almost all authors added a small percentage of phosphoric or stearic acid to the stationary phase in order to enhance the separation and to obtain clear peaks. In some of the above mentioned works, the quantitative results were not sure and in others separation of straight- and branchedchain acids was not satisfactory. In this paper, a aps chromatography method for volatile fatty acids determination is described. This method follows that of Eoos et al. /19/ which we however modified in soie essential points. Versamid 900 was used as stationary nhase which provided a high quality chromatographic separation.

5 .t^:-per.,imentation procedure 3 5 cm3 of rumen fluid ^trained through gauze was ^3? rn7.xed t ;ith 5 cm3 of a.3at;.arated 3olution of I^_9.304 in 2,'j 1I2j041 in or-der to deproteinize the fluid. Then the fluid underwent steam distillation in a P ar. kham aptaaratuâ. 250 cm3 of distillate containin,^-, total volatile fatty acids were collected and irirlediately titrated v.rith 1 N NaOH, usin.` phenolphtalein as an indicator. The di:3tillate was evaporated in a vacuum evaporator or in a drier at until dry sodium sa.1t3 of the contained volatile fatty acids were obtained. At this moment, the samples could be stored in a desiccator for some time. In. this research, 220 mr-, of 1^fH41".3OO 1HO mg of volatile fatty acid salts and 20 i,ag of anhydrous 1 ;a230`+ were introduced into a test tube of 7 cm in length and a diameter of 0,8 cm. The test tube was c_lozied with. a silicone stopper through which a;iasj tube was inserted. One drop of distilled water was added into the test tube which was then agitated. Afterwards, 0,2 cm3 of he-cane t'er. e add-^d and the test tube was agitated again. a'fter alloz*.^in^ a:^hort ti7i^e f or àettlin1g, 1^.a of the cleared layer of hexane was extracted with a microsyringe through the stopper arid injected into the apparatus. A mo.ïel GD û,,rlo Er. ba as chromatog,raphy : pparatti.), Z.i.th flame ionization detector was u.3ed. Its 3piral column of 3tainle3s steel had a length of 2 m and a diameter of 4x2 nnn. The column was packed with 10/,? Versamid ,j H3i'04 on mesh Chrolllojorb W. The temperature of the

6 thermostat was of 170 and that of the evaporator of 270. Carrier gas was nitrogen and its flow was of 20 cm 3/min ; the oxygen flow waa of 20 cm 3 /min and the air flow of 200 cm3/min. A Dpeedom x G recorder was used. Analysis time was of 12 min until the appearance of valeric acid. The column packing was prepared as follows. The given amount of Versamid 900 and H 3 P0 was dissolved in a 2:1 per volume mixture of chloroform and methanol. Then the inert carrier was added. Afterwards, under continuous mixing, the solvents were evaporated on a steam bath. Versamid 900 i3 a polymeric product of ethylenediamine and dimeric acid (epoxy rubber). The dimeric acid is a dimerization product of linoleic acid and dicarbonic acid (036). Versamid 900 can be used at temperatures ranging from 50 to 275. It is of medium polarity. The described procedure provided chromatograms with very clear symmetric peaks which were identified by using staneards of pure acids. For the quantitative interpretation of the results, the peaks areas were measured and then expressed in relative weighted per cent (wei-;hted = Ai \. The obtained values for each peak were then E A 1 I corrected with a corresonding. correction factor. In order to compute the correction factors, a mixture of pure acids, the same as those under study, in accurate weight relations as near as possible to those of the rumen fluid, was transformed into sodium salts, processed as described and run 4

7 5 through the gas chromatoctaphy apparatus. Then the precise per cent relations of the mixture's colvonents were compared with the per cent relations of the same components contained in the chromatogram (table 1). The obtained factors were applied to correct the results from the chromatoe;rams for the biological samples. analyzinq blood, because of its low volatile fatty acid content, a sample of 20 cm 3 should be used for deproteinization with metaphosphoric acid. By steam distillation, 500 cm 3 of distillate should he collected. Further, the procedure is the same as the one described above. A standard mixture of known acids, having a ratio near to that of the volatile fatty acid content of blood is used. Jiscussion The described technique does not exclude steam distillation, even if it requires much time, because it provides accurate. results. Exclusion of steam distillation may cause quantitative losses, especially of the more volatile components (mainly acetic acid) during the processing of samples until they are injected. Allavena and Zacchetti /2/ assume that in this case volatile impurities damaging the chromatograms may be inserted with the samples into the apparatus. Following Foster and hurfin /10/, the presence of an excessive amount of water (in more dilute solutions) inevitably has a depressing ef:ect upon the flame ionization detector. _3y using aqueous solutions, Emery et al. /6/ observed a "ghosting effect", i.e. a

8 double peak formation of two subsequent chromato ;rams. jottyn et a]-./5/ f oun-d that formic a Cid added by :arlstr8m et al. /3/ and Van Eenaeme et al. /21/ to the aqueous solutions of volatile fatty acids prior to their injection, dimini.3hed but did not exclude co,^_ipl.etely this effect. 7-loo6 et al. /1g j and icuzdz,,,l-3avoi e et al. /15/ used Nahj O4 to decoiupoae the sodium salts and free the volatile fatty acids. In our research, instead of this salt, we used the "more acid" NIH 4 h,j 04 in order to assure the separation of all present ac-ids.' :`e did not apply H2304 used by nromann et al. /14/, Kaufrnann et al. /12/ and other authors, because of the corroding effect upon the apparatus t f the inevitable excess of H2304 contained in the -sample5. We added a minimum amount of water to the sample in order to enhance the contact of NH4H3 O4 with the sodium salts of volatile fatty Lcids. A bigger amount of water interferes with the complete transition of the freed acids ( especially of acetic acid) into the hexane layer, which causes quantitative errors. e did not use ethyl esters of volatile fatty acids despite their advantages, because ethylation with potassium ethyl sulfate applied by vescon et al. /1^/ had not been complete (kla1l3 /18/). For the quantitative interpretation of the i.-eaults, 13'- ve applied, li'ce Hamada et al. /17./, Fenner et al. /9/, Cottyn et al. /5/, Kellogg /13/ and other authors, correction factors computed on a r.lixture of pure!:icid7 with ratios near to those of the fluid under 3tudy ( f ig.l ). For a higher

9 7 accuracy, several standard mixtures were prepared with different ratios of the contained volatile fatty acids, being thus possible to chose correction factors resulting from the mixture with the nearest composition to that of the sample under study. It i3 also possible to apply an internal standard, but this i3 difficult because isobutyric acid which could be used for this purpose is contained in the sample and caproic acid and the longer-chain acids provide, in the given circumstances, diffuse peaks and higher quantitative errors. Table 1 Composition of Areas of the peaks mixture prepared on the chromato- Correction by weighting,in A gram of the stan 7 factors dard mixture,in A l C A A 2 -I - A 0 3 B1 Lid , C 5i E l Fl 0 3 B 2 02 C 4 D 2 C 5i E 2 C5 F 2 B2 3 Cl -1-TF D2 D El r. E2 F l F F2

10 ,itandard mixture -Lumen Fluid' C2 çf3 C4 C5 C51 C41 C5 C 51 C41 u 13ibliot', raph_y - 1. Aktnan R. G., R. D. Burger, Anal. Chem., 35, 647, Allavena S., G. Zaechetti, Industria Dolciaria Internazionale, 5, Carlstrôm G., W. Hallgren, B. Pehrson, O. Wallin, Acta Vet. Scand., 6, 52, Cescon J., E. BramUilla, E. Melgrati, Clin. Vet., 91, 42, Cottyn B., Ch. Boucque, J. Agr. Food Chem., 16, 1, 105, Eniery E. M., W. E. Koerner, Anal. Chem., 33, 146, Erwin E. S., G. J. Marco, E. M. Enery. J. Dairy Sci., 44, 1768, Faichncry G. H., J. Chromatog., 27, 482, Fenner H., J. M. Ellioti, J. Anim. Sci., 22, 624, Foster J. S., J. W. Murfin, Analyst, 90, 118, Hamada T., S. Omori, K. Kameoka, J. Dairy Sci., 1, 222, Kaufmann W., K. Rohr, Z. Tiert>hysioi. Tierernâhr. Futtermittelk., 22, 1, Kellog D. W., J. Dairy Sci., 10, 1690, Kromann R. P., J. H. Meyer, W. J. Sticlau, J. Dairy Sci., 1, 73, Kuzdzal-Savoic, W. Kuzdzal, Le lait, , 255, Mahadvan V., L. Sternroos, Anal. Chem., 39, 1652, Nikelly J. G., Anal. Chem., 1A. 2244, Ralls J. W., Anal. Chem 32, 332, , 515, Roos J. 13. A. \' ei'snzl. G. A. Werdmuller, Kieler Milchwirts Forschung, Storry J. E., D. ^iillard, L. Sci. ï-d. Agric., 16, 417, ,, 569, Van Eenaeme C. C., J. M. Bienfait, O. Lambo, Ann. Med. N'et.,

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