A Rapid Method for the Separate Determination ofVitamin A and Kitol in the Whale-liver Oil

BYYOSHIMORI 0MOTE(Received June 10, 1952)

Spectrophotometric determination of vitamin A in the whale-liver
oil is usually interfered by the existence of kitol. 1) The method todetermine vitamin A by the color reaction with glyceroldichlorohydrine
has been proposed to reject the interference of kitol, 2) but there re..:mained unknown how to determine kitol in the co-existence of vitamin A.
The author has found that the simple method of semimicro moleculardistillation is useful to separate vitamin A from kitol. The method
was used to separate quantitatively the free cholesterol from the ester
one in the blood plasm, 3) but no example has been applied to liver oilconstituents. It can be naturally assumed that both vitamin A andkitol exist in the liver oil in state of ester form, then the distilling
temperature differs each other so much that each compound can beseparated by molecular distillation. The paper describes the fundamental
experiment in detail to perform the newly found method.
Experimental Part1.

Distillation at 200° and Recovery of Vitamin A.-Semimicro

molecular distillation apparatus 4) consisting of semimicro molecular stilland Hickman's oil diffusion pump was used. The evaporation surface
was 2.5 cm. diameter, and the distance between evaporator and condenser was 0.8 cm., the condensing surface being cooled by water
current. Samples were liver oils of pollack, salmon shark, hammerhead
shark, mixed shark (mixture of several kinds of shark-liver), and whales
A and B, all being commercially prepared.
Just 0.120 cc. of the sample was micropipetted, from which thefilm thickness, one of the important factors in molecular distillation,
was calculated as 0.25 mm. At first, the temperature of oil bath waskept constant at 200° and the distillation time was changed from 2 min.
to 40 min. After each distillation, the condensing surface was takenout and washed with chloroform to recover the condensed vitamin A
into an aliquot, which was treated by antimony trichloride reagent
to measure the recovered vitamin A, the cod-liver oil unit being
conveniently calculated for the weight of original sample 111. 7 mg.

Y. 0MOTE48(calculated from mean specific gravity multiplied by 0.120) (Table 1).
Table 1.
Distillation
timemin.

Recovery of Vitamin A in Distillation at 200° (C.L.O.U.)
Liver oil of:
Pollack-----1

Salmon<sharkShark,
mixed

9.0
11.0
11.0

6.30
8.10

33.7
42.7

I

8.13

44.2

II

I

---- - - - - ----1

4.04
6.10
7.10
7.10

2
512

22
40

1

Hammerhead / Whale A
Whale Bshark-------23.o

45.2

I

99.0
140
191
215
240

80.0
105
130

Original

7.25

11.2

9.0-1

45.7

206

108

Ratio*

100

100

100

104

113124=* Ratio
R 200° (22/12): that is, % of recovered vitamin A after 22 min.
for that after 12 min.

Color reaction of the original liver oil of mixed shark, hammerhead
shark and whale considerably differed from the standard color, so thatthe ratio of recovered vitamin A after 22 min. for that after 12 min.
expressed in percentage was tentatively introduced, which divided liver
oils into two groups. To one group with the ratio of 100% belonged
the liver oil of pollack, salmon shark, and mixed shark, and to theother group with the ratio more than 100% belonged the liver oil ofhammerhead shark, mixed shark, and whale, the former containingvitamin A ester, while the latter containing kitol ester in addition tovitamin A ester. Also Table 1 shows that about 15 minutes' heatingis necessary to recover vitamin A completely by molecular distillation
at 200°.
2.

Distillation for 15 minutes and Recovery of Vitamin A.-

Secondly, the distilling time was kept constant for 15 min. and thetemperature was changed from 150° to 250° (Table 2).
In order to discriminate the liver oil containing kitol from that
free from kitol the ratio of the recovered vitamin A at 230° for thatat 200° during distillation for 15 min., expressed in percentage, wasintroduced. The ratio in case of the whale-liver oil is about 130%,
while those of pollack and salmon shark-liver oils are about 100%. The
difference 30% is attributed to that the former contains much amountof kitol. Moreover, the color test of the residual oil was found to be
useful to check the presence of kitol, blue color representing vitamin
A, and red color representing kitol. The recovery of vitamin A at49

Separate Determination of Vitamin A and KitolTable 2.

Recovery of Vitamin A in Distillation for 15 min. (C.L.O.U.)

Distillation I
temperature

Liver oil of:ocPollack

I

7.30
7.21

II-

I

Salmon
shark

I-

150
180
200
230
250

I
III
I
Ii
I-

IHammerhead

Shark,

I mixed
III

8.0
11.2
11.4

I-

I

I

1.27
4.71
8.14
9.67
9.19

I
I
I
II

shark

Whale A

Whale B-

55.1
141
206
269
240

84.6
114
147
157

28.3
44.8
53.3
53.9

-

Color reaction of the residue with antimony trichloride reagent

150
180-

I200

pale blue

230
250

I colorless

Ratio*

99-

blue
blue
blue
pale bluish pale reddish
violetvioletcolorlesspale red
pale red-I=102

blue!bluish violet
bluish violet bluish violet

red

red

red

pale red
pale red

red
blue

red
blue

131

129

119

119

I

* Ratio
R 15 min. (230/200): that is, % of recovered vitamin A at 230° forthat at 200°.

250° of whale-liver oil was less than that at 230° because a portion of
the condensed vitamin A oil dropped to the residue, which was clearlyproved by that the color test of the residual oil was blue.
3. Change of Absorption Spectrum during Distillation.- In theseparation method before mentioned the distilled vitamin A was stable
at condensing surface cooled with water, but the stability of remaining
kitol was unknown. It was proved by absorption spectrum that kitol
Table 3.

Wave
length,mµExtinction Coefficient of the Whale-liver Oil before
and after Distillation, E (1%, lcm.). .
['0 rrgma1

Distillation, 250°, 15 min.

Distillation, 200°, 15 min.

liver oil ! Distillate J Residue

T_o_t_a1__ ,_D_i_st_i_II_a_te_ _ _R_e_s_id_u_e_,l __T_o_ta_l_

1_ _

1

310
320
325
328
330
334
340
350

53.2
52.4
50.8
49 .0
47.5
43.5
35.85
22.9

I'I!

30.4
34.5
35.85
35.85
35.25
32.9
28.65
19.52

I

25.40
19.02
15.55
13.85
12.75
10.65
7.38
3.96

55.8
53.5
51.4
49. 7
48.0
43.6
36.0
23.5

I

46.6
53.1
55.0
54.9
54.1
50.5
43.6
29.45

12.80
9.14
7.05
6.10
5.52
4.44
2.74
1.14

59.4
62.2
62.1
61.0
59.6
54.9
46.3
30.6

50

Y. 0MOTE

did not decompose during distillation at 200° for 15 min. Precisely
weighed sample of the whale-liver oil was placed in a small dish on
the evaporating surface and was distilled for 15 min. at 200° and 250°.
After distillation, the absorption spectra of distillate and residue weremeasured in isopropanol by Beckman spectrophotometer, while theabsorption spectrum of the original sample was independently measured.
Each extinction coefficient was calculated from the total weight of thesample.
As shown in Table 3, in case of the distillation at 200° and for 15min., the total extinction coefficient of the distillate and residue ateach wave length after distillation was almost equal to that of theoriginal sample, on the other hand the difference clearly appeared incase of the distillation at 250° and for 15 min. Consequently, it wasproved that both vitamin A and kitol did not change during the separation process by molecular distillation at 200° and for 15 min. Separated
vitamin A and kitol can be determined by colorimetric or spectrophotometric method as usual. Simple colorimetric method to determine kitol
will be reported by the author in future.
Summary

It was found that when the whale-liver oil or other fish-liver oils
containing kitol in addition to vitamin A was distilled in a semimicro
molecular still at 200°, for 15 min., and with the film-thickness of
0.25 mm., all vitamin A ester distilled on the condenser and kitol ester
remained in the residue without decomposition.
References
1)
2)
3)
4)

Y. Omote: J. Chem. Soc. Ind. Sect. (J ap'.tn), 54, 589 (1951).
0. R. Braekkan: Anal. Chem., 21, 1530 (1949).
A. E. Koehler, E. Hill: J. Biol. Chem., 179, 1 (1949).
S. Ishikawa, Y. Omote, M. Kijim'.t, H. Okuda: Sci. Rep. Whales Research Inst.,
No. 5, 61 (1951).
(Department of Chemistry, University of Education, Tokyo)