On Diethylene Glycol Succinate Monoester as a Stationary Liquid Phase in Gas-chromatography of Methyl Esters of Fatty Acids and on the Chromatographic Study of Horse-mackerel Oil.

On Diethylene Glycol Succinate Monoester as a Stationary Liquid Phase in Gas-chromatography of Methyl Esters of Fatty Acids and on the Chromatographic Study of Horse-mackerel Oil.

Shojiro MIYAHARA and Yoshiaki TABATA

The purpose of the work described below was to analyze the methyl esters of fatty acids by means of the stationary liquid phase of diethylene glycol succinate monoester in gas chromatography.

Since high vacuum grease1) had been used as the stationary liquid phase for analysis of methyl esters of fatty acids by gas-liquid-chromatography, the various.

liquids have been studied by several groups of workers in recent years. Afterwards it has been found that polyesters 2)3) and polyglycols4) give good results respectively.

Above all, polyvinyl acetate 5) and polyesters of diethylene glycol or butanediol.

succinate 6)7), glutarate 8)9) and adipate 10)11) are now in use frequently. Although, in this country of past days, these compounds were difficult to obtain, and the synthetic methods were troublesome to go through. From the result of our experiments about the stationary phase which we used in place of these com- pounds for analysis of methyl esters of higher fatty acids, it was found in our laboratory that, when we used diethylene glycol succinate monoester, methyl esters of fatty acids containing carbon atoms from 16 to 22, could be completely separated in accordance with number of carbon atoms, and unsaturated degree.

Experiments and Results 1. Preparation of diethylene glycol succinate monoester

The calculated value of well dried powder of sodium succinate was poured slowly into 2,2' -dichioro diethyl ether, and the mixture reacted to change into.

sticky liquid feverishly. Next, sodium sulfate anhydride was added rather exces- sively to the liquid. Leaving it alone overnight, its upper layer liquid was, analyzed and the result obtained was as follows.

From 0.1878g of this substance, 0.3487g of CO2 and 0.1105g of H2O was obtained.

calculated (as C81-11205) found

C 51.1% C 50.7%

H 6.4% H 6.5%

0 42.5%

100.0

2

       molecular weight 188 190・v200

    1n view of this result， it is evident that the above−mentioned sticky liquid js diethylene glycol succinate monoester．

L2． Conditions of gas chromatography

  2．1 Stationary phase

    The above．mentioned diethylene、glycol succina七e monoester Was dissolved in rnethylene chloride， and celite 545 （80t一一100 mesh） ee was added and mixed up thoroughly． Finally， methylene chloride was evaporated at 700C． ln the course of this experimeri七， the ratio of celi七e and ester was kept in 4 to 1．

  2．2 Methyl． esters of fatty acids

    Methyl esters of palmi七ic， s七earic， oleic， linolic， linolenic and erucic、 acids−

ob七ained commercially were prepared by boron七rifluoride method12）．

  2．3 Apparatus， column temperature and flow rate， etc．

    According to the appara七us and condi七ions lis七ed in Table 1，the me七hyl es七ers

．of七he six fatty acids were．submitted to chroma七〇graphy on七he column using

．．di6七hyl・n・gly・・1・ucci・at・m・n…七・・a・a・t・ti・na・y ph・・e．

      Table 1． ：Appara七τ！s and Condi七ions．

AppaTattu．s

Yanagimo七〇Gas。

 chroma七〇graph     GCG−200

Hitachi Gas一 Yanagimoto Gas−chroinatograph

ch「o ﾘ鵬aph但、gh、en、、，、認？葺10f1。m。，y，。）

       packing

Column length

       I． D．

Temperature

   （oC）

，Carrier gas

Flow rate

（cc／min．）

Sensitlvity ，

20％synthetic die七hylene．．91ycol succinate monoes七er in celi七e 545      2m

     smm      200

      H2 ・

      54

（写ath襟ete「）

        2m   2m O．6m         411ml 5mm 2 mm

       200 200 200

 （col． ．chqmber ．220）

         N2， N2． N2， H2．

         60 40 16 22

．（Kath T「） （Sens 1 ・ 補間）

3． Results

    Tenμ1 Qf each of七he me七hyl esters Was chroma七〇graphed and the separation curve asミhown in Fig．11was ob七amed． Although：Fig．1shows only the resu1七〇f separation under the condition listed in the extremely left file of Table 1． The ratio of retention time of each of the methyl esters separated under other condi−

tions coincided with the result of Fig． 1． Next， from the relationship between

ee for ga．s chroma七〇graphy， Yanagimoto Co．，：L七d．， Kyo七〇．

⑪のqo山u︒o﹄お唱08

C16

C18

C｛8一

CIs＝

CI8三

C22一

       ロ

0      8      ｛6        ．24

      Time（mi11．）

   Fj9・1．Separa七ion Qf me七hyl esters of fa七ty acids on 2m columll with       diethylene glycol succina七e monoes七er． Temp．200℃；carrier       gas H2；flow ra七e 54cc／min．

32

LosVR

3

2

   騨． ︐ ！！ 1／  1  ！／  ！ノ   1ノ   ︐！   ！    ノ！         1︐     ！     ︐     ノ    3ノ︐          〇     ニノ！          二     亡﹁           F     ♂げ        ワも一        3＝︐       F︑F／          ︒          ！ ₃₀₀₀ 2000

IOOO 800 600 400

200 1OO 80 60 40

︵餌﹀︶のおお︒嘱oo霞三〇＞qo唄唱80

2

i 14 16 8 20 22

       Number of carbon atoms

Fig．2． Rela七ion between retention volume and number of

       carbon．a七〇ms of esters．

retention volume and．number of carbon atoms of fatty acids， Fig． 2 was obtained．

But， for the．purpose of comparison， the retention，volume of methyl esters of

lauric and myristic acids under． the same conditiOn is also shown in Fig．  2；

4

Φの3仙の曾お唱︒8創

〆

◎6一

C。s●

C軽 C陪

1

C隠

c解5

C18雀G。●

@x2

^CがC22・ x2 ^幽1

0 ⁸

︸16 24

32       Time （min．）

   ：Fig．3． Separa七ion of me七hyl es七ers converted from fa廿y acids of horse・mackere1        （azi） oil． Condi七ions as shown ．in Fig．1． Peak heights of C18三〜C22＿一is        mul七iplied by 2 in七〇F三9・1・

一

D ln case of temperature over 200 O C

    When the sepa．ration was executed under the same conditions as listed i n Table 1 except the column temperature of 2300C． ratio of retention volume was

・・cca・ ・nally n・t・・n・㌻・nゆ七t・ 1 ・g w・・、・b・e・Y・d・a七h・・d…1y・

．5． Gas chromatography of fish oil （horse−mackerel oil）

    The oil of horse−mackerel （Trachurus juPonicus TEMwhNcK ＆ ScHLEGEL． body 胃1eng七h 18．5cm）， was obtained by the extraction of its fresh flesh with ether．

The oil was transesterified by the usual methgd and sodium methoxide method 13）．

・Gas chromatography of both of七hese esters gave the same retention time under

uthe same一／ condition as stated in 2． ，3．

    The ・result is shown in Fl g． 3． ldentification of each pelak in Fig． 3 was

：practise according七〇Fig．2． The me七hyl esters of which七he retention time

−is la「ge「． than that gf e「ucic a・id（C22）a「e no七rhown in Fig・3・

      Discussion

    Th・n・mb・・gf七b…e七i・・l pl・七…f七his c・lumn・a1・u1・t・d f・・m Fig・1i・

，about 1100 as far as methyl ester of linolic acid is eoncerned． Under other

・condition＄ in Table 1， maximum， minimum and average numbers of theoretical pla七es to each me七hyl esters are llOO，480 alld 600 respectively． Ju二dging from

      Lli

these tesults and from the 1inear function between retention volume a nd number 盾?@carbon atoms as showp in Fig． 2， it is considered that the column using

・diethylene glycol succina七e monoester has sufficient separability fbr allalysis of．

methyl esters of fat七y acids． Moreover， in case of ternpera七ure over 230。C，七he

・separa七ions were unsuccessf亡1 wi七h七hose．七ailings of peaks of七en apPearing．1七is probably because七he transi七ion of sta七ionary phase．migh七happen occasionally，

gand七he vapour pressure of stationary phase is comparatively higher． The■efo：re，

Tthe column of diethylene glygol succinate monoester is not alwaYs best for the

＄eparation of methyl esters of all fatty acids but， in general， when the separation

is practised at the temperature below 200°C, this column is sufficient to be used for analysis of methyl esters of fatty acids which contain carbon atoms up to

22. For instance, as shown in Fig. 3, in the experiment in which horse-mackerel oil is used as a sample, the separation can be practised completely.

Summary

1 . After preparation of the column of diethylene glycol succinate monoester, using this substance as stationary phase, gas chromatography of methyl esters of fatty acids which contain carbon atoms from 16 to 22, was studied.

2. This column has the sufficient separability for analysis of methyl esters of fatty acids which contain carbon atoms below 22, when the separation temperature is below 2 0 0°C.

3. Using this column, horse-mackerel oils were analyzed and the separation curve as shown in Fig. 3 was obtained.

A part of this study was reported at the Meeting of the Japanese Society of Scientific Fisheries held in autumn, 1962.

Thanks are given to Mr. Takeyasu Murata and Mr. Kinsei Ishihara for their assistance in the experimental work.

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