j.

FISHERIES RESEARCH BOARD OF CANADA

Translation Series No. 1348

Researches on the isomers of monounsaturated fatty acids of vegetable oils by chromatography-mass

spectrometry.

By C.M. Zorzut and P. Capella

Original title: Ricerche sugli isomeri degli acidi grassi monoinsaturi di oli vegetali mediante gascromatografia - spettrometria di massa.

From: La rivista italiana delle sostanze Grasse, XLVI (Feb.): 66-72, 1969.

Translated by the Translation Bureau(KQA) Foreign Languages Division

Department of the Secretary of State of Canada

Fisheries Research Board of Canada Halifax Laboratory

Halifax, N.S.

1969

21 Pages typescript

.1 73 1 7' '1

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Italian Znglish

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L'ORIGINAL DATE OF PUBLICATION DATE DE PUBLICATION

PUBL ISH ER - ÉDITEUR

Stazione Sperimentalè per l'in-dustrie degli olite dei grassi

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1969 XLVI Feb. Milan 21.

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AUTHOR - AUTEUR

C.M. Zorut and P. Capella.

TITLE IN ENGLISH - TITRE ANGLAIS

Researches on the isomers of monounsaturated fatty acids of vegetable oils by chromatography7muukFearomerv

Title in foreign language- it z -oreign- charaeters)

RicerChe sugli isomeri degli acidi rr rassi monoinsaturi di oli vezetali mediante gascromatbgrafia-spettrometria di massa

REURENCE IN FOREIGN I7 ANGUAGE (NAME OF BOOK OR PUBLICATION) IN FULL. TRANSLITERATE FOREIGN CHARACTERS.

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La. Rivista Italiana delle sostanze Grasse

REFERENCE IN ENGLISH - RÉFÉRENCE EN ANGLAIS

The Italian Review of Fatty Substances

769-18-14 YOUR NUMBER VOTRE DOSSIER N°

SOS-200-10-6 (REV. 2/88)

CANADA

OEPARTMENT OF THE SECRETARY OF'STATE

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3837 Italian K.Q.A. 3, DEC, 69

• SOS-200....10-31

RESEARCHES ON THE ISOMERS OF 'MONOUTU-:-.(ATED FATTY

ACIDS OF VEGETABLE OILS BY CEROETC.GRAi(iY. -

MASS SPECTROMETRYW

by C.M. Zorzut and P. Capella Institute Of Agrarian Chemistry and Agrarian Industry of the

University of Bologna.

RESEARCHES ON THE ISOMERS OF MONOUNSATUHATED FATTY ACIDS C£, VEGETABLE OILS BY CHROMATOGRAPHY MASS SPECTROMETRY

The isomerssof monounsaturated fatty acids of some vegetable

oils were studied by the combination of gas chromatography-mass

spectrometry after conversion of the monounsturated fatty

acids into the corresponding trimethylsilyloxy (TMSO) der-

ivatives.

The results allowed ustoshow that - with the only exception of

parsley seed oil - the most widely represented isomers are the

9-cis-hexadecenoic, the 9-cis-octadecenoic, the 11-cis-eico-

senoic and the 13-cis-docosenoic. While in general the data

previously known in the literature have been confirmed the

presence of isomers not yet reported has been demonstrated,su as

the 6-cis-hexadecenoic,.the 8-cis and 9-cis-eicosenoic in par-

sley seed oil, and the 13-cis-octadecenoic in rapeseed and

mustard seed oil.

The results are then discussed in the light of the present

knowledge on unsaturated fatty acid biosynthesis.

• Paper read at the IX Italian Congress on Studies or Fatty Substances, Bologna, 10-12 June, 1968.

UNED;TED 177:1-sy'::SLATION

Introduction:

The stùdy Cf the structure of monounsaturated fatty acids

of oils and of natural fats has led to the conclusion that,

except for rare exceptions, these have the double bond in pos-

itions 9, 10 (acids of 16 and 18 carbon atoms) or in the pos-

ition 11, 12 and 13, 14 (acids of 20 and 22 carbon atoms

respectively).• The knowledge with regard to the presence of

their isomers of position is both inadequate and fragmentary.

An examination of the most recent.literature has led us

to the conclusion that numerous insoluble structural compo-

nents are widely diffused in the nature]. oils (1-3).

The knowledge of the composition o in structural isomers of

monounsaturated fatty acids,nreents the following interesting

aspects:

1) Biolorically with regard to the natural mechanisms of their

synthesis and their relaboration and therefore of the veg-

etable and animal phYsiology in the catabolic and anabolic

processes of the fatty substances; in this context,

studies on.filo97enesis and taxonomy are also important,

especially in regard to the vegetable organisms.

2) Industrialleoth in relation to the modifications_undenTone,by the fatty -

/acids in the course of the processes of preparation of the

edible oil products (4) and also the exploitation of sources

of the composts with a particular structure.

In the study of the monounsaturated fraction of the fatty

methods most commonly used acids, previously isolated, the

-3-

are base:, on the *oxidated breaking-up of the ,nolecule in the

positions of the double bonds and gnstromatoraphic analysis

on chromaography of the oxidized fragments (2, 5-12). The

aforesaid methods are complicated, above sll in their quan-

titative spect, with regard to the possiblity of formation

of products with«further oxidative domolition of the orimary

fragments and from the fact that acids with different lumbers

of carbon atoms but having the same position of unsaturation,

give - ;.ace to a'product in common; the method of the oxidative

domolition, moreover, often imposes difficult processes of en-

riching and purification, -%Yllen the msnounsfiturribed fatty acids

present in small percentages in the mixture must be isolated.

Other methods (13) provide for the use of the tubular open

column (capillary) for gastromatoraphic analysis of the , sters

of the monounsaturated fatty acids the determination

is in this case possible only for the ocids hs,ving the double

bonds close to the aliphatic extremity of the molecule.

New prospects for a brilliant solution of the problem have

opened up with the application of mass spectrometry in the stu-

, dy of the fatty acids.

This method of experimentation has produced definite re-

sults in the definition of the structure of saturated esters

(14-18), while It has been halted_when faced with the esters of

the monounsaturated fatty acids in cases suchœ: the molecule

undergo/ in fact, under an electronic impact, phenomena of

breaking up of the double bond which leads to a constant sta-

tistical distribution of the position of unsaturation along the

molecule. Numerous research workers have us a consequence

turned their attention to the various derivatives of the es-

ters of monounsaturated fatty acids obtaining, in some cases

a good spectral characterization of the composts witha dif-

ferent position of the double bond andW . ,, different steric

configuration ( .19-22). The examination of the spectrums of

the mass of pure composts did not however &ive any hope for the

possibility of determining 'either .the presence or the structure

of composts represented in small percentages in a mixture of

fatty acids. .

Recent . research (23) undertaken by us on the suitable

derivatives of the esters of some oure monounsatsrated fatty

acids has enabled . us to determine how far these derivatives could

be similar 1h order to determine the position of the double

bond and has suggested to us the possibility of defining the

structure of their isomers present in complex mixtures.

The procedure adopted requires the preparation of di-

trimethyl silyloxy (TMS0) derivatives of the methyl esters

of monounsaturated fatty acids, according to the following

reaction method.:

1 0sO 4 — > C = C <

2 I Na 2S01 I OH OH

HMDS I

TMCSC C I I

TMSO OTMS

clove HMDS = esametildisilasatio; l'AACQ trinirtitrinrOSilanO.

where HMS = hexamethyllic41asane TMCS trimethylchlorosylane

- 5-

These composts were subsequently an-Jlysed by mass -.)ent-

rometry. .Plie . main fragmentation too lacebecase the

breakiniz ._kp of the CC bond in the original nosftion of the

double bond, as illustrated in the followinF formula:

CH,— [CH2 ], —CH — CH —(CH2 ), COOR 1 TMSÔ ; ÔTMS

The mass spectrums show 9 in fct, two peaks of nigh

intensity corresponding to the fragments a c b of each isomer.

In figure;1, 2, and 3 there are reproduced, for exa.nrqe, the

mass spectrums of TMSO derivatives of the methyl esters of oleic

acid, palmitoleic and petroselineic

In these mass spectrums, which facilitate conSiderably

the determination of the position that the double bond occupied .

prior to oxidization of the fatty acids, the intensity of the

main fragments (a & b) is always sufficient for the pur-

poses of their identification, even in Cases where the product

analysed contains numerous isomers of position and when these

are present in small percentages. It is interesting further to

note, in order to make a quantitative examination of the spec-

trums of thé . bixturè of the monounsaturated fatty acids, that

the relative intensity of the ions a and b varies

ly with the position of the double bond, and therefore in a

only slight-

-6-

manner which we consider to be regulated by a precise law.

The mass ppectrums of the derivatives of monounsaturated

fatty acids'stereoisomers do not show. such :_jnificant differen-

ces as to define their geometrical structure.

This however does not constitute a handicap in the method,

si:ace the isomers cis-trans can be easily separated for pur-

poss of chromatography on a thin strata •4), before the

preparation of the derivatives.

The favourable aspects of fragmentation under el-

ectronic impact of the , composts examined in the aforementioned

research has induced us to study the isomers of position and

the geometrical ones,of the monounsaturated acids present in

the following oils: rapeseed, parsley, soya, olive, flax,

mustard, sunflower, maize, peanuts and hemp.

Lx_p_primental Part.

The samples of the oils analysed were extracted with hex-

ane from seed- or from the oily fruit. The fatty acids, obtain-

ed for purposes of saponification, were then esterified with

methanol- BF3 at 12%.

By means of chromatography on a thin strata of silica gel

G/AgNO3 (2L) the corresponding fractions were then separated

from the methyl esters of the cis-monounsaturated and trans-

monounsaturated acids.

spectrometry.

PM 445

s 10

combination

100

50

-7-.,

Thse fractions were then transformed into the corres-

ponding di-trimethylsilyloxy derivatives according to the method

described previously (23) and analysed directly by means of the

of gaschromatography-mass

• n

Ue$10ertstr41100:111»»

11411

cid

(C) 332

T r --t

415 I

r• 400 rn>s

it 1 it?

Figure 1 • Spettro dl massa del TMSO derlvato dell'oleato dl metlle.

Figure 1 - Mass spectrum of TM() derived from methyl oleate.

Figure 2 • Spettro di mess. del TMS0 derivate del petrosellnato dl metile.

- Mass spectrum of TMSO derived from methyl-petro selinate.

Figure

13

—r- t

-8-

It •

CH,f01 1,—r--1-.1H-KeytœrtCH,

TM: LI

100

na 4/4

1111

200 300

10

/143 451

,

1 4o0 1 ' 'fr• T'ut

11 14,1,41, 1)

• CO CO 14? 158

332

Id 11.3

(bi 2511

'figure 3 • Spettro cil mess* del TMSO derivato del palmItoleato dl motile,

Figure -3 - Mass spectrum of TM'S° derived from methyl palmytholeate.

For this purpose a_ gaschromatoi!.:raph -mass spectrometer

LKB model 9,000 was utilized (°). The Fastromatographic ap-

paratus for the introduction of the samples was furnished with

a spiral column in glass measuring 2 m X 3 mm, filled with SE

30 at 2.5% on das Chrom P, 100-120 mesh previously washed with

acids and silanated. Temperature of the column: 220 degrees

C; temperature of the evaporator: 270 degrees C; carrier gas:

He, 30 ml/min. The molecular separater was maintained at 240

degrees C and the ionizing source at 310 degrees C. Power

of ionization 70 eV; current of . ionization 20 A. The spec-

trums were registered in 5 sec (m/e 1 - 400) on an apex of

• gasoromatographic peaks. •

(*) Apparatus in use at the Centre of mass spectrometry of the University of Milan. Ne wish to offer our thanksto the directors of the Centre for their kind permission to use the above and Dr. Tito Salvatori for his courteous col-laboration.

-9-

Results an-.\ review

Figure • is a reproduction of a gascromatogram of TMS0

derived from the monounsaturated acids of rapeseed oil. The

separation c.f the derivatives with differing numbers of carbon

atoms is total and it is therefore possible to rcord the

spectrums 'f the mass of single peaks without Interference.

cI

elt

z_o

Figure 4 - Gascromatogramma del TMSO derivati degli este. ri nieIlikI degli acldi 91-Hsi monoinsaturi dell'olio dl colza. :.;

Figure 4 - Gascromatogram of TMSO derivatives of the methyl esters of monounsaturated fatty acids of rapeseed oil.

Three main peaks are present due to the homologous acids

with 18, 20 and 22 carbon atoms.

The central zones of their mass spectrums are reproduced

in Figure 5.

These confirm that the peak C18 contains, side by side

with the predominant cis-9-octadecenoic acid, small quantities

of the isomerscis-11 - and cis-6-ottadecenoic. This last

23

-10-

iti;omer was not referred to in the work of Kuennel (2) whose

isearches were . undertaken using the method of oxidative dem-

olition.

The isomer cls-11-eicosenoic 9 and small quantities of the

isomer cis-13 are present, instead, in the :)eak C 20 while in the peak

C 92 there is only the isomer cis-13-docosenoic0 In this last case

we were unab-le to confirm the presence of the isomer cis-lci-

do.?,osenoic, which was found instead by Kuemmel.

1 8

“

259

C2

cl Au ) 388

là( c(à ") 315 360

1

te(Au) 315

215

'3(L1 1 ,) 181

°go) 215

78

50

25

WO

75

60

25

100

75

eo

23

100

7

50

25

• ict)

/GI

GO

• 20

u(t)...)

21:5

otA 0

gA5) C 20

p .211 !!!JA-o

287

Figura S - Spettri di masse del TAISO deriveti degli esteri metiliel degli ncidi gressi monoinsaturi dell'ollo di colza.

150 200 250 m/e 300 350 ' 400.

Figure 5 - Mass spectrums of TMS0 derivatives of the methyl esters of monounsatured fatty acids of rapeseed oil.

(M11

259 ed) b(A0

Lau

e(à,) 332

MO

75

50

• Flgerw 6 e Spettrl di massa del TMS0 derived degll esterl me.

Mick. digit «WI grass, monoinsaturl dell'ollo di prenemolo.

b(An) 0( ) ci(Ani 217 245 257

12(A1) 259

i u(6 .) b2(5A711) i 285i

+2

c(t) i c3( ‘;‘; ) 3c (6% 1 )

318

2à0 300 350 46-0 eth

u(14) „ 215 d‘tito

! 243

C o 2

150 200

Figure 6 - Mass spectrums of TMSO derivatives of methyl esters of monounsaturated fatty acids of parsley oil.

-12-

We must mention however l evenif our reservations are valid with

regard to the possibility of errorsresulting_from the method of

analysis by means of oxidative demolition in the case of small

quantities of isomers, that our research was done . on one

sample only and has, therefore, no statistical validity.

The composition of the monounsaturated fatty acids of par-

sley oil is of considerable interest. The mass spectrums of

TMS0 derivatives of the acids C1 6, Cg, C20 are reproduced

in Figure 6.

It.may be pointed out that the hexadecenoic acids are com-

posed of quantities which have more or less equal isomers cis-

6-hexadecenoic and cis-9-hexadecenoic. The presence of the cis-

6-hexadecenoic acid, though this is not surprising, was J,s fa) as

we know never traced before in oils and vegetable fats, but

only in the fat of hair (25) and in the fat of human excretion

(26). The spectrum of the acids with 18 carbon atoms, confirms,

instead, what has been stated previously (27, 28): às in all

the oils of umbelliferous seeds, thé petroselinic (cis-6-octa-

decenoic) forms the major part, together with a lesser quantity

of oleic acid (cis-9-octadecenoic). The composition of the peak

C20 = 9

present: the cis-6, the cis-8, the cis-9, and the cis-11 eico-

senoic, as can be Pointed out from the couples of peaks at m/e

285-217, 257-245, 243-259 and 215-287.

instead, appears more complex where four isomers are

:**

- 1 3-

In Figure 7 is reproduced the isomeric composition of the

acids C 16 - 3.nd C18 - of the oil of virgin olives the isomers

which are most represented, confirming the previous researches

(2), are respectively the cis-9-hexadecenoic and the cis-9-

octadecenoic together with a small quantity of the isomers cis-

11-hexadecenoic and cis-11-octadecenoic0

Figure 7 • Spettrl dl masse del TMSO derived degil este n!

metillel degil acid, grisai monoinsaturl dell'ollo dl olive.

Fig. 7 - Mass spectrums of TMSO derived from the methyl esters of the monounsaturated fatty acids of olive oil.

In Table 1, finally, there is a, representati7e recapitulation

of the isomers of position present in the homologous mono-

unsaturated fatty acids of all the oils examined.

A prusal of the aforesaid data enables us to show that

for acidy with 16 and 18 carbon atoms there predominate largely

the isomers with the double bond in the position 9, 10. The

octadeceno\c acids contain moreover smaller quantities of the

isomer Ai â which does not seem to be a normal nonstituent of

the hexadecenoic acids: this is present in olive oil, but has

not been fo und, for example, in the oil of parsley seeds; in

this last, Inere is Present instead, as we have examined,the

isomer A 6.

The aoids with 20 carbon atoms are comnosed e in a large part,

of the isomer 11 with small qualties of the isomer A 13.

The only tsomer A 13 was found in the acids with 22 carbon atoms.

The position of the double bonds in the monoenic acids ex-

amined so far seems to be in a large measure in accord with the

most recent yiews on the biosynthesis of unsaturated acids. The

formation of every isomer could in fact, be reasonably explain-

ed by the suggested meollanism of enzymatic de-hydroP:enation of

the corresponding saturated acids and the elongation of the

chain by addition of two carbon atoms.

The data reproduced in Table 1 seems to agree with what

has been suggested by Kishimoto and Radin (29), who have pos-

tulated the existence of two distinct de-hydrogenated enzymes,

capable of moving respectively on the positions 6 and 9 from

the carboxyl group of palmitic and stearic acids. These enzymes

i f ie dj CigA,

\ . 8 C2044 C 247 11 CzA9. • tiyAi i

kt‘e I c t

-15-

would ke into account . the formation of the acids cis-6 and

cis-9-exadecenoic, and ois-6 and cis-9-octadecenoic.

:t ma y however be noted that the knowledge on the de-

hydroinated enzymes is still somewhat limited and only from

yeast there has been isolated an enzyme which is capable of

de-hydrogenating the palmitic and stearic acids in position 9

with the formation of the palmitoleic and oleic acids (30).

The mechanism of elongation of the chain - demonstrated with

the help of the composts marked in the case of the monoenic

acids (31) and dienic acids (32) of the sphino-lipids and '

in vitro, with olein CoA (33) - would explain the formation of

the monoenic acids with 20 and 22 carbon atoms and the

isomers cis-11-octadecenoic, according to the following pattern:

„

c mà

c„C

The isomers enclosed in the squares are those formed by

de-hydrogenation of the corresponding saturated acids. It may

be pointed out that this arrangement would take into account

the formation of all the isomers, with the exception of the acids

C16 A 11 9

C20 A 6 and C20 49 for which direct de-hydrogen- ation of the corresponding saturated acid would seem to be the

a b a b a b a b d b• a bla

Cf."

3,3 9,4

C*

c,-

Cie 2,8 9,1

93 83

Ce 3,5 10,2

C 16" 88 100 77 100

ce m

Cie C„« CO"

14,2 à,) 100 83

45 42

13,2 8,5

18,2 6,0 2,5 1,7

9,5 5,8

5,1 3,7 100 89

4,1 3,3

4,2 3,4 •

5,0

12,8 7,8

100 98

90 61 14 18 38 46

100 9S

100 79 99100

100 98

94 100

97 100

100 96

100 93

100 . . '85

1,9 1,1 100 72

4,7 2,6 100.77

M 8,2

answere The acids cis-6 and cis-9-eicocenoic have been found

however, only in the oil of parsley seed.

We would like to point out, finally, that we could find no

trace in this oil àf the acid cis-8-octadecenoic, which should

have resulted .from the addition of a unit of C2 to the acid

cis-6-hexacenoic, while the acid cis-8-eicocenoié is present

and is most likely derived from the acid cis-6-octadecenoic with

a similar mechanism.

TABELLA 1 Table 1 r 6

ogous, Altezze relative del frammenti a e b per grupP1 dl midi Isomeri

hel,ghts of TragmentR R and 11 for groups of _ • ... à0Ljei t

a 1; la b a b Imrermes...1.«.*

Hew

omo. isomerà of acids. loghl àe aè à* en Au

Vegetable oil 011 vegetall

Calm (Drnsmica enmpestrls)

Rapeseed' Prezzeinolo (Petroselinum sntivum)

Parsley Soia (Saki hispida) Soya Oliva (Olea europaea) Olive Lino •

9risnim uitatissimum

&nape (Sinnpis alba)

Mustard:-• Girasole (Helinnthus annuub) ecilils.n flower

'- (7..ea mays)

A rachide (& achis hypogea)

Cattapa (Cannabis sativa) nemp

-17-

Concluslohs:

The method of determining the position of the double bond

in the monoenic acids by means of gascromatography-mass spec-

trometry has been found to be simple and quick to operate and

enables one to wOrk with very small quantities, of one-two mg.

And small quantities of isomers can be readily shown up

when these are present in quantities exceeding or equal to 1%

of the most abundant isomer.

The results obtained have confirmed basically the data

already noted in the literature on the subject, but in some

cases it has been possible to show up the presence of isomers

which were not noted previously, such as the acids cis-6-

hexadecenoici cis-8 and cis-9-eicosenoic in the Pil of parsley

seed, and cis-13-octadecenoic in rapeseed and mustard oils.

The slight variations that are found in the relative in-

tensity of the fragments a and b in the different isomers of

position (23) make it necessary to determine the mass spectrum

of the pure single isomers, other than the standardization of

experimental conditions, when exact quantitative results

are to be obtained..

From an examination of the results obtained by us, it

is possible, nevertheless . , to show with a percentage error of

around 15%, the quantitative rationof the isomers present in

the different aCids,based on the ratio of the height of the main

fragments correaponding to the different isomers on the mass

spectrum.

-18- BILIOGRAPHY:•

B tELTOGRAFIA

1) MAGNEI F. C.: J. Am, 011 Chem. Soc., 42, 332 (1965). 2) KUEIMMEL D. F.: J. Am. Oil Chem. Soc., 41, 667 (1964). 3) Lou G. e PALtorrs U.: Rit'. Ital. Sost. Grasse, 41, 425 (1966).

4) Zoszur C. M. e Csrritri P.: in .corso di stampa. 3) Wt.: RUDLOFF L.:. Catt, J. Citent., 33, 1701 (1955). 6) Vox Rtrot.one L.! J. Am. Oil Chem. Soc.) 33, 126 (1956):

7) Tuaocii A. P. e Casio B. M.: J. Am, Oil Client. Soc., 41, 322 (1964),

8) JONES E. P. e DAVISON V. L.: J. Am. Oil Citent. Soc., 42, 121 (1965).

9) MA1.I.Ako T. M. e CRAIG B. M.: J. Am. Oil' Chem. Soc., 43, 1 (1966).

10) Pluviirr O. S. e NICKELL E. C.: J. Am. Oil Citent. Soc., 43, 393 (1966).

II) amt.: E., CAl'ELLA P. e VALENTINt A.: Rite, liai. Sost. Grasse, 40, 313 (1963).

12) PRIVETT O. S. C NICKELL E. C.: Lipids, I, 166 (1966).

13) ACKMAN R. G.: J. Am. Oil Chem. Soc., 43, 483 - (1 966).

14) RYIIAGE R. e SIENUAGEN E.: Arkiv. Kenti, 13, 523 (1959).

15) RYIIAGII R. e e STENIIAGEN E.: Arkiv. Kenti, 14, 483 (1959).

16) RYIIAGII R. c STENIIAGEN E.: Arkiv. Kent, 15, 291 (1960).

17) Rynnun R. e STENIIAGF.N E.: Arkiv, Kenti, 15, 333 (1960),

18) RYIIAGI: R. e STENIIAGE.N E.: Aikiv. Kemi, 15, 545 (1960).

19) DINII-NGUYEN N., RYIIAGEI R. e STALDERG-STENIIAGEN S.: Arkiv. Kemi, 15, 433 (1960).

20) KENNER G. W. C STENUAGEN E,: Acta Chem. Scand., 18, 151 (1964).

21) AUDIER H., Bon' S., FErnoti M. LONGEVIALLE P. e TOUDIANA

R.: Bull. Soc. Chin:. France, 3034 (1964).

22) MCCLOSKEY J. A. e MCCLELLAND M. J.: J. Am. Chetn. Soc., 87, 5090 (1965).

23) CAPELLA P. e Zoitzur C. M.: Anal. Client., 40, 1458 (1968).

24) . PALLorr* U, e MATARESE Riv. Ital. Sost. Grasse, 41, 210 (1964

• ./

25) WEITKAMP A. W., SMILMNIC, A. M. e Rcrrtimsm S.: J. Am. Chem. Soc., 69, 1936 (1947).

26) JACOO J. e GRINNER G.: J. Lipid Res., 8, 308 (1963).

27) Lam G. e BAZAN E.: La Ricerca Scientilica, 798 (1967).

28) Lorri G. c BAZAN E.: La Ricerca Scientifica, 1005 (1967).

29) Kr.sitisurro Y. e RADIN N. S.: J. Lipid Res., 5, 98 (1964).

30) Elt.00mmun 1). K. e Ewen K.: J. Biol. Citent., 225, 479 (1960).

31) KISIIIMOTO Y. e RADIN N. S.: J. Lipid Res., 4, 414 (1963).

32) MEAD J. F. e Howie: D. R.: Radioisotope Studies of Fatly Acid Metabolism, Pali: 51.56, Pergamon Press, New York, 1960.

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ITALIAN BIBLIOGRAPHY

3) G. Loi and U. Pallotta: Italian Review of Fatty Substances:

41, 425 (1966).

4) C.M. :,orzut and P. Capella: In Course of Printing.

11) E. Fedeli, P. Cepella and A. Valentini: Italian Review of

Fatty Substances: 40, 313 (1963).

23) P. Capella and C.M. Zorzut: Anal. Chem: 40, 1458 (1968).

24) U. Pallotta and L. Matarese: Italian Review of Fatty

Substances: 41, 210 (1964).

27) G. Lotti and E. Bazan: Scientific Research: 798 (1967).

28) G. Lotti and E. Bazan: Scientific Research: 1005 (1967).

<

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DISCUSSION RECORDED

PRESTDENT: PROF. CANTARELLI (Institute of Agrarian Industries of the University of Perugia).

PRESIDENT

We congratulate most heartily Dr. Zorzut for the excellent

demonstration of an extremely refined technique i which I con-

sider most u-seful since it has a very wide application. The

next time we shall have a direct quantitative tabulation. This

has been a most oriented and extremely interesting paper; and

then there are applications of a biochemical nature which are yet

another horizon that is opening up in relation to this research.

My warm thanks again.

A discussion then took place in which amoniz those takin

2ar_t_mre Prof. Privett Hormel Institute of the University of

Minnesota) and a resumLof which was provided by Prof. Capella:

PROF. CAPELLA (Institute of Agrarian Industries of the Univer-sity of Bologna).

As everybody knows, Prof. Privett has put forward a most

refined method for the determination of the position of the

double bonds by 'means of ozonlysis of the unsaturated acids and

determination by means ofgaschromatography of the fragments that

are obtained by this method. Now according to Prof. Privatt, -

his method has above all the advantage of being a very rapid one

and also à better deal than the method used by us; naturally by

deal I refer to the fact that we must have the mass spectrometer.

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am certinly in agreement with the second of these object-

ions but cannot say the same for the first; our method is

more or less equally rapid and above all the point where we

believe 3ur methoi is different from the method of ozonlysis

is this: we are not forced to proceed with the prior sep-

aration of the homologues present in a complex mixture of fatty

acids in order to study the different isomers of position of

- each homologue.

For example if we have a series from 014 to 0 20 of mono-

unsaturated acids, we can effect a gascromatography of the

derivatives -, take the mass spectrum of each r.,'eak and thus see

in each peak how many isomers and what sort of isomers . are

present.

Naturally, in the method outlined by Prof. .Privett also

which does not provide for the separation by means of chroma-

tography on a thin strata4 but provides instead a separation by

means of liquid - liquid chromeography of the different isomers

which according to him, brings the things to a parity, i.e,

we are forced to effect a chromatoreraphy on a thin strata in

order to separate the monounsaturated acids from the polyunsat-

urated ones and Prof. Privett makes a liquid-liquid chromato-

graph which separates the homologues. Thus, he states we bring

thethings to parity". I belieire that on this position of parity

we can all be satisfied.