FATTY ACID COMPONENTS OF GANGES RIVER
DOLPHIN OIL
HIDEO TSUYUKI* AND SHINGO ITOH*ABSTRACT

The purpose of the present work is an examination of chemical components of Ganges river dolphin oil by TLC, CC, and GLC.
The analysis by TLC and CC showed the presence of triglycerides,
phospholipids, sterol esters, diglycerides, hydrocarbons, monoglycerides,sterols and free fatty acids. A substantial part in all fractions was 80.6% of
triglycerides fraction contained a small amount of free fatty acids.
The analysis by GLC showed the presence of fatty acids with chain lengths
from 10 to 22 carbon atoms and with zero to six double bonds. The Ganges
river dolphin oil was found to be rich in unsaturated fatty acids (72. 73%),
particularly octadecamonoenoic (32.30%) and hexadecamonoenoic (24.47%).
On the other hand, saturated fatty acids were contained in small amounts(27.27%) in Ganges river dolphin oil.

INTRODUCTION

Although we have reported on the fatty acid composition of the oil of Finless porpoise (Tsuyuki and Itoh, 1969a) and Many-toothed pilot whale (Tsuyuki and Itoh,l 969b) which are cetaceans in salt-water, the literatures on the oil of fresh-watercetaceans are few.
Expedition of the Ganges river dolphin (Platanista gangetica) was done in East
Pakistan on the various field of study from Oct. 1969 through March 1970 organized
by Prof. M. Nishiwaki of the Ocean Research Institute, University of Tokyo.
The materials were brought us through Prof. M. Nishiwaki.
This study is reported as a part of the examination of Platanistidae which has
been continuing to date by organized Japanese scientists.
It was a pleasure for us to join in the study.
MATERIAL AND METHODS

Material
Ganges river dolphin used in the present investigation was caught at the Ganges
River in East Pakistan, in January 1970 by Prof. Dr. Nishiwaki. Then sample oil
was extracted by boiling the blubber of Ganges river dolphin with water. The
chemical properties of sample oil are shown in Table 1.

* Department of Food Engineering, College of Agriculture & Veterinary Medicine, Nihon University.
Sci. Rep. Whales Res. Inst.,
No. 23, 1971. 141-147

TSUYUKI AND !TOH

142

TABLE !.

PROPERTIES OF GANGES RIVER DOLPHIN OIL

nn'oAcid value
Iodine value (Wijs)
Saponification value
Unsaponifiable matter (o/o)

1.4635
1.3
104.4
195.3
I.OJ

Thin-layer chromatograplry
TLC was carried out qualitatively according to the method of Doizaki andZieve (1962). Commercially available silica gel (Wakogel B-5, Wako Pure Chemical Industries, Ltd. in Tokyo) was applied in 0.25 mm thickness on 5 x 20 cm glassplate. The plate was dried in air for 15 min after spreading, and activated for
1 hr at 110° just before use. The sample oil was applied on 1.5 cm place from thebottom of the plate with a capillary tube. The chromatograms were developed in
the glass chamber saturated with petroleum ether-ethyl ether-glacial acetic acid,90: 10: 1 (v/v/v). The developed plate was sprayed with 50% solution of sulfuric
acid in water, and the separated spots were identified by comparison with the eachstandard mixture which was developed by the same procedure.
Silicic acid column chromatography
To separate Ganges river dolphin oil quantitatively, silicic acid column chromatography was carried out according to the modified method of Barron andHanahan (1958). A glass column (height 70 cm, ID 20 mm) was packed with 60 g
silicic acid (Wakogel C-200, Wako Pure Chemical Industries, Ltd. in Tokyo) prepared by activation at 120° for 1 hr and washing with hexane. 0.526 g of sample oil
was placed on the column as 10% solution in hexane, and constituents in sample oil
were eluted. Also the flow rate was adjusted to 1.5-1.8 ml/min, and then eachfraction was respectively collected in amount of 20 ml. The solvent mixture wasreplenished from a reservoir filled consecutively with 120 ml of hexane, 180 ml of 15%
benzen in hexane, 660 ml of 5% ether in hexane, 320 ml of 15% ether in hexane,
280 ml of 30% ether in hexane, 240 ml of 90% ether in hexane and 320 ml of 20%
methanol in ether. Each fraction was identified by chromatographed known
compounds on silicic acid column by the same procedure.
Preparation ef methyl ester
Fatty acid methyl ester was prepared with boron triftuoride-methanol reagent
according to Duron and Nowotny (1963). 100 mg of Ganges river dolphin oil wasadded 10 ml of boron triftuoride-methanol reagent (140 g BF 3 per liter of methanol)
under nitrogen atmosphere, and the conical beaker was closed with the screw cap.
Then, the beaker was heated in a boiling water bath for 45 min. After the reaction,
the ester was extracted by adding 2 volumes of ethyl ether, so 1 volume of water andshaking. The ethyl ether solution was dehydrated with anhydrous sodium sulfate
and then evaporated. Finally the methyl ester was obtained in amount of 92 mg.
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GANGES RIVER DOLPHIN OIL

143

Gas-liquid chromatography
GLC was analyzed with a Shimadzu Gas Chromatograph Model GC-lC using
a flame ionization attachment. A 180 cm long, 3 mm ID, U shaped stainless steel
column was packed with Shimalite W (60-80 mesh), coated with 10% diethylene
glycol succinate (commercially available DEGS made by Shimadzu Seisakusho
Ltd, in Kyoto). Column temperature was kept constantly at 200°, and temperature
at injection port and detector block were respectively 275° and 240°. Flow rates
for air, nitrogen as carrier and hydrogen were 600, 70 and 75 ml/min respectively.
Chromatographic peaks were identified either by comparison of retention
times with those of standards or from a graph representing the relationship between
log retention time and the number of carbon atoms by applying the methods ofAckman (1963), Nelson and Freeman (1960).
Each peak was quantitated by the method of Ettre and Kabot (1963). All
fatty acids were calculated as weight percentages of the total known fatty acids
present.
RESULTS AND DISCUSSION

Fig. 1 shows the fractionation of Ganges river dolphin oil by a TLC plate qualitatively. The obtained fractions were from above, as follows: hydrocarbons, sterolesters, triglycerides, free fatty acids, sterols, diglycerides, monoglycerides and phos-crn

Cl02105003567Fig. I. Thin-layer chromatogram of Ganges river dolphin oil.
I. Hydrocarbons
5. Sterols
2. Sterol esters
6. Diglycerides
3. Triglycerides
7. Monoglycerides
4. Free fatty acids
8. Phospholipids

Sci. Rep. Whales Res. Inst.,
No. 23, 1971.

144

TSUYUKI AND ITOH

10080bi>

E60_::·c;

0.....

.cbO~40

2200320

440

660

780

100Fraction numberFig. 2.
No.
I234567

Separation of Ganges river dolphin oil by silicic acid column.
Fraction
Eluting solvent
Hydrocarbons
HexaneSterol esters
15% benzen in hexane
5% ether in hexane
Triglicerides & free fatty acids
Sterols
15% ether in hexane
Diglycerides
30% ether in hexane
90% ether in hexane
Monoglycerides
20% methanol in ether
Phospholipids

pholipids. It was seemed that triglycerides fraction was contained in sample oil atthe largest quantity as compared to other fractions, because of the spot color wasdarker than others. The seven fractions except triglycerides seem to be contained
respectively only trace amount as compared to triglycerides fraction.
Fig. 2 shows the fractionation of Ganges river dolphin oil by a silicic acidcolumn quantitatively. The seven fractions were eluted as follows: hydrocarbons,sterol esters, triglycerides and free fatty acids, sterols, diglycerides, monoglycerides,and phospholipids. The largest proportion was 80.6% as triglycerides and freefatty acids fraction, and other proportions were as follows: 4.9% phospholipids,
4.1 % sterol esters, 3.2% diglycerides, 2.8% hydrocarbons, 2.5% monoglycerides and1.9% sterols.
Sci. Rep. Whales Res. Inst.,

No. 23, 1971.

GANGES RIVER DOLPHIN OIL
TABLE 2.

145

FATTY ACID COMPONENTS OF GANGES RIVER DOLPHIN OIL

No. ofcarbon atoms

No. ofdouble bonds

Weightper cent

No. ofcarbon atoms

No. ofdouble bondWeightper cent

1212
14
14
14
16
16
16
16
18
18
18

0

0.39
0.09
4.89
2.35
1.40
18.11
24.47
1.32
l. 71
1.27
32.30
4.65

181820
20
20
2020
20
22
22
22
22

3
40

0.43
0.24
2.51
0.54
0.110.290.98
0.42
0.10
0.53
0.41
0.49

l020230l2l

23450456

TABLE 3. A COMPARISON OF SATURATED AND UNSATURATEDFATTY ACIDS OF GANGES RIVER DOLPHIN OIL
Weight per cent of total fatty acids
No. ofcarbon atoms

Saturated

Unsaturated

Total

1214161820
22Total

0.39
4.89
18.11
1.27
2.51
0.10
27.27

0.09
3.75
27.50
37.62
2.34
1.43
72.73

0.48
8.64
45.61
38.89
4.85
1.53
100.00From the results of thin layer chromatogram and column chromatogram, it
was found that Ganges river dolphin oil was approximately consisted of triglycerides.
Table 2 shows the fatty acid components of Ganges river dolphin oil by quantitative GLC analysis. There were 24 kinds of fatty acids, having from 12 to 22
carbon atoms. The majority were hexadecanoic, hexadecamonoenoic and octadecamonoenoic fatty acids, because of the total of the above three fatty acids held really
74.88% of the total fatty acids. The next prominents were tetradecanoic, tetradecamonoenoic and octadecadienoic fatty acids. On the other hand, the total proportion of other fatty acids was slightly 9.72%. In comparison with saturated andunsaturated fatty acids, the proportion of total saturated fatty acids was 27.27%, andthose of total unsaturated fatty acids was 72. 73%, as shown in Table 3. Moreover,to compare with the proportion of each carbon numbers, the most prominent was
45.61 % of 16 carbon atoms fatty acids, and the next was 38.89% of 18 carbon atoms
fatty acids. The rest were as follows: 8.64% 14 carbon atoms, 4.85% 20 carbon
atoms, 1.53% 22 carbon atoms and 0.48% 12 carbon atoms.
Finally, the principal ingredient of Ganges river dolphin oil was triglycerides,
and main fatty acid components of sample oil were hexadecanoic, hexadecamonoenoic and octadecamonoenoic.
Sci. Rep. Whales Res. Inst.,
No. 23, 1971.

146

TSUYUKI AND ITOH

SUMMARY

1. The fractionation of Ganges river dolphin oil was done by thin layer
chromatography and silicic acid chromatography. The largest fraction was triglycerides and free fatty acids, and its proportion was 80.6%. The other proportions
were as follows: phospholipids 4.9%, sterol esters 4.1 %, diglycerides 3.2%, hydrocarbons 2.8%, monoglycerides 2.5%, and sterols 1.9%.
2. Fatty acid components of Ganges river dolphin oil were analyzed by gasliquid chromatography.
The results obtained were as follows:
Total saturated fatty acids
27.27%;
dodecanoic
0.39%
tetradecanoic
4.89%
hexadecanoic
18.11 %
octadecanoic
1.27%
eicosanoic
2.51 %
docosanoic
0.10%
Total unsaturated fatty acids
72.73%;
dodecamonoenoic
0.09%
tetradecamonoenoic
2.35%
te'.radecadienoic
1.40%
hexadecamonoenoic
24.47%
hexadecadienoic
1.32%
hexadecatrienoic
1. 71 %
octadecamonoenoic
32.30%
octadecadienoic
4.65%
octadecatrienoic
0.43%
octadecatetraenoic
0.24%
eicosamonoenoic
0.54%
eicosadienoic
0.11 %
eicosatrienoic
0.29%
eicosatetraenoic
0.98%
eicosapentaenoic
0.42%
docosatetraenoic
0.53%
docosapentaenoic
0.41 %
docosahexaenoic
0.49%
REFERENCES
R. G., 1963. Structural correlation of unsaturated fatty acid esters by graphycal comparison ofgas-liquid chromatographic retention times on a polyester substrate. J. Am. Oil Chemists' Soc., 40:558-564.
BARRON E.J. and D.J. HANAHAN, 1958.
Silicic acid chromatography of the neutral lipides of rat liver, beefliver, and yeast. J. Biol. Chem., 231: 493-503.
DorzAKI, W. M. and L. ZmvE, 1962. Rapid thin-layer chromatographic separation of phospholipids andAaKMAN,Sci. Rep. Whales Res. Inst.,
No. 23, 1971.

GANGES RIVER DOLPHIN OIL

147

neutral lipids of serum. ]. Biol. Chem., 3: 138-140.
DuRoN, 0. S. and A. NowoTNY, 1963. Microdetermination oflongchain carboxylic acids by transesterification with boron trifluoride. Anal. Chem., 35: 370-372.
ETTRE, L. S. and F.J. KABOT, 1963. Relative response of fatty acid methyl esters on the flame ionization
detector. J. Chromatog., 11: 114-116.
NELSON, G. J. and N. K. FREEMAN, 1960. Phospholipide and phospholipide-fatty acid component of humanserum lipoprotein fraction. J. Biol. Chem., 235: 578-583.
TsuYUKI, H. and S. lToH, 1969a. Fatty acid composition offinless porpoise oil. Sci. Rep. Whales Res. Inst.,
21: 131-135.
TsuYUKI, H. and S. I TOH, l 969b. Fatty acid composition of Many toothed pilot whale oil. Sci. Rep. Whales
Res. Inst., 21: 137-141.

Sci. Rep. Whales Res. Inst.,

No. 23, 1971.