Comprobación de la Hipótesis

- adapted from Rubin and Paisley

[1960]

Olive Oil Fatty Acids

1256 g

uct

_{1 : 1 : 3}

Add

� FA:urea :MeOH

[Al]

removal

of 16:0,18:0,

20:0

Filtrate

[F1]

843 ₉

1 : 0·75 : 3

removal

of 18:2,16:1

+traces of

Precipitate

16:0,18:3

F5

p6

192 ₉

99+% 18:1

Urea Adduct

Formation

Acid Soap

Crystallisation

80% MeOH ; 0°C

39

were developed by the ascending method in saturat ed chromat ographic

tanks. Part ial glycerides were separate d on silica gel G layers impreg­ nat ed with H3Bo3 using a solvent syst em of hexane-diethyl ether ( 50 : 50 }

or chloroform-methanol ( 150 : 3 ). For TGs the thin layers were developed in either hexane-diethyl ether ( 85 : 1 5 and 50 : 50 ) or dichloromet hane.

Lipids were det ected by fluorescence ( spray reagent : 2g/l of 2 , 7- dichlorofluoroscein in methanol ) or charring (5�6 ( v/v ) sulphuric aci d ; 150°C for 1h ) . The dichlorofluoroscein spray reagent was mainly used for the det ection of fatty acid� which are not stained by the sulphuric

ac id spray.

For the determinat ion of glyceride purity , conditions were chosen so that expected impurities c ould be det ected at the 1% leve l . This detect ion level was confirmed by the examinat ion of pure standards tn whi ch increasing levels of spec ific impurit ies had been added .

2.2.2 Colwnn

With the except ion of saturated TGs of high molecular weight , all TGs were purified by column chromatography on alwnina according t o the convenient procedure of Jensen et al. ( 1 966 ) .

Column criTomatography was also used t o separat e unreacted 1 -

st earoylglycerol from 1 -butyryl-3-st earoylglycerol since the t wo compounds were not readily separat ed by crystallisat ion ( cf . Feuge and Lovegren ,

1956). To prepare the adsorbent , chromatographic grade neutral alumina

(90g ) was added t o a solut ion of H3Bo3 in methanol ( 1 0g in 200ml ) in a

21 flut ed flask and the suspension was evaporated t o dryness on a rotary vacuum evaporator at 35-40° C. After 30 min the free-flowing powder was

0

transferred t o a beaker , dried overnight at 1 1 0 C and then stored in a

dessicator. The c olumn was packed by adding the adsorbent ( 2g/g of compound ) in successive small amount s to a chromatographic tube ( 2.5cm i . d. ) filled with developing solvent ( hexane-diethyl ether 25 : 75). The crude 1,3-diacylglycerol, dissolved in 1 vol . of developing solvent , was applied t o the t op of this column and then rapidly eluted ( < 1 5 min ) with 20 vol . of developing solvent . Examination of the eluant by TLC showed that all the 1 -acylglycerol had been retained on the c olumn and t hat little further isoinerisat ion o f the 1 , 3-diacylglycerol had occurred. Based on the original mass of the reaction product applied to the column , the recovery of the part ially purified 1 -butyryl-3-stearoylglyc erol was

2.2.3

Gas-liquid chromatography ( GLC ) was used t o determine the purity of the fatty acid start ing mat erials and t he fatty acid composition of

40

the synthetic TGs and their lipolysis product s . Analyses of fatty aci d methyl esters were performed on a Varian Aerograph ( Walnut Creek , Cali­ fornia , U .S .A . ) Series 1 520 chromatograph fitted with a flame ionisat ion det e c t or and a linear t emperature prograrrmer. Long chain glyceride s and fatty aci ds were c onvert ed to met hyl esters by the method of Van

Wi jngaarden ( 1 967 ) while butyryl TGs were int eresterified by t he proce dure of Shehat a , de Han and Alexander ( 1 970) . Nethyl esters were analysed on a stainless steel column (2.4m x 3.2mrn i . d. ) packed with 1 2% diethylene glycol succinat e on Chromosorb �/ (60-80 mesh , acid washed and DHCS

treat e d ) . Gas flow rat es were 25 , 250 and 20 ml/min of nitrogen carrier gas , air and hydrogen respect ively. Injector and det ector t emperatures

0 .

were 230 and 250 C and the column oven was operated � sothermally at -190°C ( long chain esters ) or progrrunmed from -60 to 1 90°C at 1 0°C/min (mixtures of short and long chain est ers). Peak areas were measured by triangulat ion (height x width at half height ) or by an electronic

int egrator ( Varian Hodel 480 ) and the relat ive proportions of esters were obtained using experimental vteight response fac t ors ( Shehata

�

al . 1 1970).

Butyric ac id was analysed for purity by chromatography of the free ac id on

a

glass column

( 2.4m

x 3.2mm i .d.) packed with 1 0% ( w/w ) diethylene glycol adipat e and

2%

( w/w ) phosphoric acid on

60-30

mesh Chromosorb W ( ac i d-washed and L'\vJ.CS treat e d ) . The oven was maintained

0

at 1 00 C . The gas flow rates were the same as those for the methyl est er column although a formic ac id bubbler was fitted in the carrier gas line to reduce tailing of the peru(s.

2.2.4

Struct ural of

Posit ional and stereospecific analyses were performed on selected TGs by Dr I . horri son at Hassey Universit y (Ph.D. Thesis , 1 976). The proce dures were e ssent ially those described by Luddy et ( 1 964) and Chris t i e and Ho ore ( 1 969 ).

2.3 of Racemic

The general scheme adopted for the preparat ion of racemic TGs is shown in Fig. 2-2. Triacid TGs were synthesised via 1 - ( mono )acylglycerol

(1-HG)

and 1 , 3-diacylglycerol ( 1 , 3-DG ) by direct est erificat ion with the

In document EL JUEGO TEATRAL EN LA EXPRESIÓN ORAL DE LOS NIÑOS (página 75-80)