.Analytical Distillation of Vitamin Ain the vVhale Liver Oil
Seiichi Ishikawa, Yoshimori Omote, Yoshitaka Soma.
Introduction.
The presence of ordinary vitamin A and a vitamin A possessing different characteristics has been confirmed through research by Willstaedt and• Jensen1l and Nakamiya, et aFJ but recently, it has been reported by Baxter,
et aP that kitol, a kind of provitamin A has been isolated from the unsaponifiable matter in whale liver oil, which has twice the molecular weightof vitamin A and which can be decomposed by heat into vitamin A. Theauthor:; carried on an analysis of vitamin A in the unsaponifiable matterof the Antartic whale liver oil (it is not certain whether the whale was afin or blue whale but it is certain that it was from one of the two) by
m1le cular distillation.
2. Analytical Distillation of Unsaponifiable Matter in Tunny Liver Oil.
First, in order to obtain an analytical distillation curve of a standard
vitamin A, unsaponifiable watter of tunny liver oil, which is rich in ordinary vitamin A was used, to \vhich was added mixed glycerides obtainedby synthesis, as co~trolled yield oiPJ (abbreviated to C. Y. 0.) and the result
indicated in Table 1. was obtained. Graphically, it is as shown in Fig. 1. .
According to this graph, elimination maximum (abbreviated to E. M.)
appears in the vicinity of 185"C and it is assumed that this is ordinary
vitamin A.
Experiment 1. Synthesis of C. Y. O.
Coconut oil fatty acid is supplemented with acetic acid, butyric acid,and capric acid and equi-molar ratio mixture of fatty acid of the various
carbon atom numbers were prepared, glycerine added and esterified in thepresence of decalin. The mixed glycerides thus obtained were distilled ina cyclic still and divided into various fractions through a range of 13028ff'C. Next, equal quantity of the various fractions were mixid and used
as C. Y. 0. and residue above 280°C used as R. 0. (residue oil). Temperature determination was made in the middle of the heating oil bath within theevaporating cylinder. During the process, the bath was well stirred so it
is assumed that it is approximately the temperature of the evaporating sur1.

36Seiichi Ishikawa, Yoshimori Omote, Yoshitaka Soma

face. Also, the temperature of the residue oil which flowed down theheating cylinder and dropped from the bottom was also determined, fromwhich it was discovered that the value was approximately 60"C lower thanthat of the oil bath. Vacuum was 10-~-10-"mm. and the distance botween
the evaporating surface and condensing surface was 0.5 cm. Temperature
was raised every lOc'C and. at each · range, distillate for each cycle wasremoved. These conditions were used for all analytical distillation explained
hereinafter.
Experiment 2. Analytical Distillation of Unsaponifiable Matter in TunnyLiver Oil.
Analytical distillation was carried on with 76 g. of oil of 102 liver oil
unit, obtained by adding 35 g. of C. Y. O. and 35 g. of R. 0. t'.) unsaponifiable matter from 25 g. of tunny liver oil for injection purpose (400 liverTable 1. Analytical Distillation of Unsaponifiable Matter in Tunny Liver Oil- - - ---· - ·Ll.Ver-Oif ---1%-of RecoveredlI Temp. ( C)Distillate (g) II Liver Oil Unit ; UnitxWeight 'Vitamin
A:---------~No.

I0--r

- 1--

123456789101112131415I

I
IIIII:

:

130
140
150
160
170
180
190
200
210
220
230
240
250
260
270

--------

--

2.4
2.1
2.5
1.8
1.8
2.8
2.0
2.5
2.6
1.9
1.2
1.2
1.2
1.1
1.7

II
II

I

---

I

248
233

103
109
310
400
585
598
462

788
729
1060
1643
903
852
533
270
156
77
52
93
91

341
202
143
130
65
4564
55

---------~--·~.

_r \I>

e~ 0t

IUO

I ·-·

II

I;

I

150''I'I ' ,~•-,;-rj'2~0·250

I:;noTemperature (°C)
Fig. 1. Analytical Distillation Curve of Unsaponifiable Matterin Tunny Liver Oil. ·----32
3.0
10.2
9.4
13.7
21.2
11.2
11.0
6.4
3.5
2.0
1.0
0.7
0.9
1.2

Analytical Distillation of Vitamin A in the Whale Liver Oil

37oil unit). Oil distilling over at each lOcC (1 cycle) was taken, its weight
and liver oil unit determined and the percentage of recovered vitamin A
was calculated by the following formula :.9£ of recovered vitamin A=[(liver oil unit x weight of each distillate)/(liver oil unit x weight of oil before distillation)] x 100,9i).
Shown graphically, the experimental result is as indicated in Fig. 1.
3.

Analytical DistiUation of Unsaponifiable Matter in Whale Liver Oil.
Analytical distillation was carried on with unsaponifiable matter ofAntarctic whale liver oil under the entirely similar conditions as in thecase of tunny liver oil explained above. The result is as indicated in Fig.
2. As can be clearly seen from the graph, E. M. of No. 1. is in the vicinity of 180"C and entirely coincides ·with the E. M. as in the case of tunny
liver oil. That is, there is 'no mistake that this is vitamin A. Next, in thecase of whale liver oil, there is a tendency of a second E. M. to appear,
but in the case of Fig. 2., since the same condition as in Fig. 1. wasfollowed strictly, the distillate of C. Y. 0. above 280°C was small and thedistillation was stopped here. Thus, in order to obtain the second E. M.,
C. Y. O. and R. O. above 28ff'C was newly added and distilled.
As aresult of this, the second E. M., as can be clearly seen in Fig. 3., appeared. The first E. M. was in the vicinity of 185°C, the same as in theprevious case, so that there is no question, but the shape of the second E.
M. is somewhat vague, a shape which is formed by several peaks of single

substance overlapping and its temperature ranges from 270-290°C. According to Hickman") natural vitamin A ester has E. M. at a temperatureof about 90"C higher than free vitamin A, and 11-carotene has an E. M. ata temperature about 30°C higher.
E. M. of kitol itself, comparable to that of vitamin A, could not beobserved but from the fact that its molecular weight is twice that of vitamin A, it can be assumed that it has an E. M. somewhat near that of 11carotene. However, kitol decomposes into vitaniin A with heat so that towhat extent the transformation into vitamin A took place during the analytical distillation still remains a problem.
Experiment 3. Analytical Distillation of Unsaponifiable Matter in Whale
Liver Oil.
( 1 ) The whale liver oil sample was obtained during whaling in theAntarctic Ocean and was informed as blue whale liver oil.
Its general

Seiichi Ishikawa, Yoshimori Omote; Yoshitaka Soma

38

characteristic is as follows :Specific gravity df 0.9230, refractive index nf 1.4842, acid value 0,saponification value 166, unsaponifiable matter 11.5%, liver oil unit 312.
Unsaponifiable matter was obtained from 50g. of this liver oil and 80g.
of oil of liver oil unit 66 was obtained by adding 35g. of C. Y. 0. and 35g.
of R. 0., the same as before and analytical distillation carried on under thesame condition as before. (Table 2 and Fig. 2)
Table 2. Analytical Distillation of Unsaponifiable Matter in Whale Liver Oil (1)NumberTemp. (0 C)- - - --·--- - -1234
56789101112
13
14
15
16

130
140
150
160
170
180
190
200
210
220
230
240
!::50
260
270

rno

----Distillate (g)
;

1.3
2.2

I%

Liver Oil Unit I Liv_er Oil.
o_f Ri:covered
1- _lJmtxWe1ght _ V1tamm A
38
50
1.062
1136i
2.6
111242:
4.6
212
382,
9.3:'..97651:
12.4341I
885
16.8!
333
865
16.5
205
552
10.5
179
358
6.3
106
159
3.0
59
140
2.7
69
117
2.2
72
94
1.8
78
100
1.9
75
127
2.4
95
:.::00
3.8

_II

2.2

1.8
2.2
2.6
2.6

2.7
2.6
1.5
2.4
1.7
1.3
1.3
1.7
2.1

Fig. 2. Analytical Distillation Curve of Unsaponifiable Matterin Whale Liver Oil (1)I\I \>

g,.,,/· /\~->C--)(-->f.--x-~/.t~oo~_.__._"""'-"'.'1~so'.""'""-~...........~2~00::-'-"""'-"'.'-'-'~2~5-o...._,__.__,_3~00
Temperature (°C)

( 2 ) 20 g. of new sample of the same liver oil as before was taken,unsaponifiable matter obtained from this, and to 240 g. of oil (liver oil unit188) obtained by adding 35 g. of C. Y. 0., 35 g. of R. 0. and a further 60 g.
of R. O. (C. Y. 0. above 280?C) and 70 g of molecular distillation residue ofAnalytical Distillation of Vitamin A in the Whale Liver Oil

39·oil-shark liver oil and analytical distillation carried on (Table 3 and Fig. 3).
Table 3. Analytical Distillation of Unsaponifiable Matter in Whale Liver Oil (2)Number12

3
4
56789
1011
1213
1415
161718192021
22

I Temp. ('C)
130
140
150
160
170
180
190
200
210
220
230
240
250
260
270
280
290
300
310
320
330
340!

I

Distillate (g)

fI

II

0.4
1.2
2.3
1.6
1.9
2.3
2.6
2.8
2.0
3.2
3.4
3.6
4.8
8.2
5.0
3.6
6.4
5.2
5.4
5.9
6.3
7.2

I% of Recovered'L'
0-l U . 1 Liver Oiliver 1 mt ! UnitxWeightVitamin A-·--·---- ··-- I .. -·.
.
5367
61
103
140
138
110
57
55
33172122303425
199 .
631978
136
162
270
319
284
162
110
105
5874
105
128
141
122
159
99
46
36
172

11

28·

0.5
2.1
3.6
4.3
7.4
8.4
7.5
4.3
2.9
2.8
1.5
2.0
2.8
3.4
3.7
3.2
4.2
2.6
1.2
1.0
0.5
0.3

Fig. 3. Analytical dittillation curve of unsaponifiable matter
in whale liver oil (2)Temperature (°C)

4. Absorption Spectrum of Analytical Distillate of Unsaponifiable Mat~ter in Whale Liver Oil.
As a means of accertaining whether or. not the two E. M. 's obtained
as a result of analytical distillation of unsaponifiable matter in whale liver
oil are due to vitamin A and kitol, absorprion spectrum of its E. M. distil,.
late was taken. As a result of this, the first E. M. distillate showed a

Seiichi Ishikawa, Yoshimori Omote, Yoshitaka Soma40

typical vitamin A absorpition (Fig. 4) but in the second E. M. distillate,absorption was between the maximum absorption of vitamin A, 328 mµ, tothe maximum absorption of kitol, 290 mµ, and somewhat stronger on the290 mµ side (Fig. 5). An explanation for this may be that kitol waspresent in this whale liver oil and a part of it was decomposed by heat
during analytical distillation. (Fig. 4 and 5 ).
Experiment 4.

Absorption Spectrum of the Distillate of Analytical Dis-~ tillation of Unsaponifiable Matter in Whale

Fig.
nlOlLiver Oil
Fig_45mm~­lOC c

JOO;9'i&'

6~'50.
40;;~

ds do Cmµ.)
Molecular Distillate at 180°C ofWhale Liver Oil Unsaponifiable.

I:1~8I~:JO (n1µ.)

Molecular Distillate at 28D°C ofWhale Liver Oil Unsaponifiable.

From the various distillations of (2) of Experiment 3, distillate of two
E. M., 180°C and 280°C were taken as representative ones, a fixed quantity of the unsaponifiable matter after sapcmification was extracted withether and absorption spectrum of this ether solution photographed.
Concentrations appropriate for vitamin A in the 180°C distillate was 1/20,000mol and 175,000 mol for the 280°C distillate. In the former, absorption
appeared at 345, 328, 310, 300 and 290 mp, being the strongest. In the latter, many vague absorption appeared from 328 mµ to 290 mµ and it was noticed that it was somewhat stronger on the 290 mp than on the 328 mµ side.

...

5. Conclusion.
As a result of molecular distillation of unsaponifiable matter in whale
liver oil, it was observed that the elimination maxima ·appeared at twoplaces, 185°C and 270-290°C.
The first maximum coincided with themaximum of unsaponiable matter in tunny liver oil and is ordinary vitamin
A. The second, as observed from the absorption spectrum, absorption
occured from 290 mp, the absorption maximum of kitol, to 328 mp, absorp·tion maximum of vitamin A. Therefore, it is conculuded that kitol is present
in whale liver oil and a part of it decomposed by heat to vitamin AAnalytical Distillation of Vitamin A in the Whale Liver Oil

41

during analytical distillation.
References
1) lL Willstaedt, H. B. Jensen, Natttre, 143, 474 (1939).
'.::) Nakamiya, Koizumi, Kawakami,

Riken-iho, 20, 576 (1941).

3) F. B. Clough, H. M. Kascher, C. D. Robeson, J. G. Baxter, Science, 105. 436 (1947); N. D.
Embree, E. M. Shantz, f. Am. Chern. Soc., 65, 910 (1943).
4) J. G. Baxter, etc. Ind. Lizg. Chem., 29, 1113 (1937) ; K. C. D. Hickman, ibid, 968 (1937).
5) K. C. D. Hickman,

Chem. Rei·., 34, 95 (1941).