Studies on Kitol IV
Purification of Kitol by Chroinatographie
By
TADASHI TAWARA, RYUSUKE FUKAZAWA

Previously the authors separated whale liver oil into Vitamin A fraction andKitol fraction through their different solubility to the solvent. (t) This Kitol fraction showed Extinction value of 500 at 290 mµ, which is about of 70% pure, be1
ing E % (290 mµ)=707' 2) in pure Kitol. The authors purified it through1 cm
1chromatographie and obtained light yellow glassy lump of E % C290mµ)==680.
1 cm
Its melting point was 72°C1) and it showed pure scarlet colour in the Carr-Pricereaction. This colour showed its maximum some seconds later, being rather stable.
The i:;ame colour is also obtained by cone. sulphulic acid, acid clay and glyceroldichlorhydorine. It had a slight odour like terpene and was easily soluble in petro.leum ether acetone and chloroform; soluble in ethanol and methanol; but quite
insoluble in water. It was unstable in the air and light. When it was left exposed
1E $ (290 mµ) decreased to 500.
1 cm
When kept in a dark room filled with C02 gas, it showed little change even twodays later. When it was preserved in petroleum ether solution, it was so unstablethat even when kept in the dark room, it oxidized rapidly and precipitated white
insoluble matter. This precipitate was insoluble in olive oil too, 2) while easily soluble in chloroform and acetone, and soluble in ethanol and methanol. Alkaline
treatment produced a re:I substance, which could be removed from Kitol as saponifiable matter. Absorption curve of Kitol obtained by the authors is as shown in
the following figure.
The authors wish to express their gratitude to the Taiyo Fishing Company Ltd.

in powdered form in the rooni. for two days,

for providing the sample liver oil used in the present experiment.
Experimental Part
300 g. of a mixture of liver oil of blue and fin whales caught in the Antarctic (containing 100,000 I.U. of Vitamin A) was saponified with 300 cc. of 20% alcoholic potash; and 32.5 g. of unsaponifiable matter was obtained therefrom. Thisunsaponifiable matter was dissolved in I L of petroleum ether; and from this solution 90% methanol was extracted 35 times, 500 cc. at each extraction.

Most of1) Melting point of crystalized Kitol is 88-90°0. (3)2) Even when Kitol was preserved in olive solution it gave this white precipitate.

•50T. TAWARA

sterin and Vitamin A collected in the methanol layer.

After the petroleum ether

layer was washed several times with water and then drie:I, petroleum ether wasdistilled. When the residue was saponified again with a small amount of 20% alcoholic potash, unsaponifiable matter was obtained which was reddish yellow andviscous. Very thin chloroform solution of this unsaponifiable matter showe:I bluecolour with the first drop of chloroform solution of antimony trichloride; but further addition of the same solution caused the blue colour to change instantly to1cherry red. This is E % (290 mµ) = 500. 500 cc. of petroleum ether solution of
1 cm
this substance was passed into an adsorption column of aluminium oxide. Aluminium oxide used in this experiment as adsorbent was a mixture of equal amountsof "BL6" maimfactured by the Japan Aluminium Co. Ltd. and inactivated" BL6 "3).
When this adsorption column was developed by the use of petroleum ether, a yellow
layer of Vitamin A was formed at the bottom a colourless layer of Kitol in themiddle, and a light yellow layer of unknown matter at the top. When this chromatogramm column was further developed with petroleum ether, the yellow partof the Vitamin A layer finally separated from the alminium layer, moved into thevessel bellow as petroleum ether solution. The aluminium adsorbed was eluted
with petoleum ether containing a few drops of methanol and washed with waterto remove methanol. Then chromatogramm was again made and the slight amountof Vitamin A at the bottom was washed off. The top of this chromatogramm,
light yellow in colour, adsorbed a matter which showed blue in the Carr-Pricereaction. This matter was adsorbed a little easier by aluminium oxide than Kitol
and was slower than Vitamin A in the appearance of blue in the Carr-Price reaction. Namely, when antimony trichloride was added to a mixture of Vitamin Aand Kifol, blue colour appeared first; then the blue gradually changed to purple,
then finally to red.

But when antimony trichloride was adde:I to a .mixture ofthis unknown matter and Kitol, the first colour to appear was red, which gradually turned to purple. Moreover, when this mixture was dissolved in petroleum
ether and extracted again and again with pure methanol, Kitol moved gradually
to methanol, leaving this unknown matter in the petroleum ether layer. From theabove facts, this unknown matter was thought to be of larger molecule than Kitol.
After removing the upper part of chromatogramm in which this unknown matter was adsorbed, the Kitol laye'r in the middle was eluted in the sam3 way as in
the above experiment. When this was passed into a very short adsorption column
consisting of inactivated alminium oxide " BL5 " to which 10% activated alminium
had been added, most of the Kitol flowed down without. being adsorbed and the3) It was too adsorptive for "BL6" to separate Vitamin A from Kitol.

Stuufo:: on ~:itol IV

51

i_1rlknown matter which showe:l blue colour with antimony trichloride was adsorbed
in the column. After shaking 300 cc of methanol with the above petroleum ethersolution of Kitol (about 1.5 L) 4) and removing the methanol layer, the petroleum
El%Icm

700.

'~_,.

600

290 mµ328 mµ

IIi'\I

III

I

500

\'I \400I\I
300

I200

I
II

I
I
I
I

I
I
I

I

100

220

230

240

250

260

270

280

290

300

310

320

330 340

3,0ID/'

Fig. Ilayer was washed. After drying it, petroleum ether was distilled off to obtainabout 5 g. of light yellow glassy lump which had a melting point of 72"C. Thisis El% (290 mµ) = 680.5) It showed pure scarlet in the Carr-Price reaction.

References:(1) The Scientific Reports of the Whale:: Re::earch Institute No. 3 (1950), 85.
(2) F. B. Clough, H. M. Kascher, C. D. Robe::on, J. G. Baxter:

Science, 105 (1947) 436.

(3) Ditto.

4) to remove a slight amount of oxide produced in adsorption treatment.
5)

97'}~

in Kit:il purity.