L ite r a tu r e C ited (1) Berl and K ullm ann, Ber., 60B, 814 (1927).
(2) Dowzard and Russo, I n d . E n g . C h e m ., Anal. Ed., 8 , 74 (1 9 3 0 ).
(3) E astm an K odak Co., “ S ynthetic Organic Chemicals,” Vol. X , No. 2, p. 4, April, 1937.
(4) Franklin, “ Nitrogen System of C om pounds,” American Chemical Society M onograph, p. 321, New York, Reinhold Publishing Corp., 1935.
(5) Friedel, Biochem. Z., 209, 65 (1929).
(6) Monsch, Helv. Chini. Acla, 13, 509 (1930).
(7) Thiele, Z. angew. Chem., 15, 780 (1902).
(8) Walsh, I n d . E n o . C h e m . , Anal. E d . , 6, 468 (1934).
Re c e iv e d A p ril 30, 1937.
Q uantitative D eterm ination o f Oil in Fish Flesh
MAURICE E. STANSBY A N D JA M ES M . LEMON
U. S. B u rea u o f F isheries, T echnological L ab o rato ry , College P a rk , M d.
F is h o ils , b e in g u n u s u a lly s u s c e p tib le to o x id a tio n a n d p o ly m e r iz a tio n , are d iffic u lt to e x tr a c t q u a n tita tiv e ly fr o m fish flesh e x c e p t a fte r d r y in g t h e fle sh in a v a c u u m d e s ic c a to r fo r a p erio d o f a w eek or m o re.
A m e t h o d is d e sc r ib e d in w h ic h t h e d ry in g a n d o il- e x tr a c tio n p r o c esse s are c o m b in e d b y e x tr a c tin g t h e fish w it h a c e to n e . T h e cr u d e o il t h u s o b ta in e d is p u rified b y tr e a t
m e n t w it h e t h y l e th e r . A rap id s e m
i-q u a n t ita tiv e m e t h o d is a lso d e sc r ib e d in w h ic h t h e o il is e x tr a c ted b y a g it a t in g th e m o is t fish flesh w it h a n h y d r o u s s o d iu m s u l
fa te a n d e t h y l e th e r .
D a ta are p r e se n te d t o sh o w t h a t th e w id ely q u o te d v a lu e o f 7.1 p er c e n t fo r t h e oil c o n te n t o f th e c o m m o n m a c k e r e l is in error, a v a lu e o f 12 t o 15 p er c e n t b e in g clo ser to t h e average o il c o n t e n t o f th is sp e c ie s.
O
R D IN A R Y m ethods of determ ining oil in flesh products such as m e at are n o t entirely satisfactory for fish, because of th e tendency of fish oil to decompose very readily when heated w ith th e flesh. Such decomposition prevents a com plete extraction of th e oil from th e fish. In the methods ordinarily used for such products th e substance is first dried thoroughly, in order to prevent th e extraction of water soluble m aterial and to ensure a complete recovery of the oil.
The dried m aterial is then extracted for ab o u t 15 hours in a continuous extractor, using ethyl ether or petroleum ether, more frequently th e former. T he solvent is then removed by evaporation and th e oil is dried and weighed.
In fish flesh, th e greatest decomposition of the oil usually occurs during th e drying process; in order to minimize these changes, it is custom ary to employ a vacuum desiccator for this step, drying the fish over sulfuric acid. Since fish con
tains large quantities of gluelike substances, it has a tendency to dry to a hornlike mass, enclosing m oisture which is very difficult to remove. By mixing th e fish flesh w ith sand or cotton, this difficulty is lessened, and a fairly thorough dry
ing is attain ed in from 10 days to 2 weeks. This procedure m ay result in a loss of oil either during the mixing of th e flesh w ith th e sand or during the subsequent transfer of the dried m aterial to th e extraction apparatus. M oreover, such a lengthy procedure is undesirable and in endeavoring to avoid it, the following m ethods were developed. T he first m ethod described is a rapid sem iquantitative one. T he second m ethod is believed to give a t least as great precision as any existing m ethod and w ith a considerable saving in tim e.
This m ethod, in conformance w ith general usage, assumes th a t the oil content of a substance is equivalent to th e con
te n t of substances extracted by ethyl ether from th e dried material.
S e m iq u a n t ita tiv e M e th o d
A brief description by Sebelin (4) has been given of a m ethod, devised by Bull, for determ ining th e oil content of fish. This m ethod consists of mixing a weighed portion of finely ground fish w ith anhydrous sodium sulfate an d then shaking w ith a measured q u an tity of benzene. An aliquot
342 INDUSTRIAL AND E N G IN E E R IN G CHEM ISTRY VOL. 9, NO. 7 th e percentage of oil in the fish. T he principle of this m ethod, th e extraction of oil by shaking w ith an oil solvent and de
rubber stoppers are unsuitable. T he following procedure was adopted:
Twenty grams of the finely ground flesh, free of skin and bones, are weighed into a shaking bottle. Twenty-five grams of anhydrous sodium sulfate and exactly 100 ml. of ethyl ether are added, and the bottle and contents are shaken for 60 minutes.
After allowing most of the fine solid particles to settle out, a 20-ml. aliquot of the solution is pipetted through a filter and into a weighed beaker. The filter is washed with three 3- to 5-ml. place. In such cases, a further aliquot of the ether solution is used for these tests.
W hen less th a n ab o u t 10 per cent of oil is present in the fish flesh this m ethod is n o t a t all accurate and a more thor
ough extraction using fresh solvent is required. In such cases th e following modified procedure is u sed :
Tw enty grams of the ground fish flesh and 25 grams of an
hydrous sodium sulfate are stirred electrically for 5 minutes in a 250-ml. centrifuge bottle with about 50 ml. of ethyl ether. The bottle is centrifuged and the solution decanted through a sintered- glass crucible. This process is then repeated five times and the filter washed. The combined filtrates are then transferred to a weighed dish, the ether is evaporated, and the oil is dried and weighed as in the previous method.
This m ethod is m ore accurate th a n th e shaking m ethod, b u t it is n o t suitable for running m any samples a t once, and hence is often of less value. F or fish of low oil content, how
ever, it is th e only available rap id m ethod. T his modifica
tion is also suitable for th e determ ination of th e oil content of fish livers. solvent with fresh acetone after 2 hours. The solutions are then heated on a steam bath until all of the acetone and most of the water is removed. They are then put in a vacuum desiccator over freshly boiled sulfuric acid and the desiccator is evacuated.
When practically all of the water has been removed, as indicated by the cessation of foaming and bubbling or after about 3 hours, the oils are removed, and 35 ml. of ethyl ether are added. After thorough shaking, the solution is poured through a filter, prefer
ably one of the sintered-glass type. The residue is washed with several small portions of ether until the wash liquid is colorless, pouring the solution in each case through the filter. The oil solution is washed into a weighed beaker and the ether removed in front of an electric fan. When the odor of ether is no longer apparent, the beaker is placed in an oven a t from 100° to 105° C.
for 45 minutes. The beaker and oil are then cooled and weighed.
Ta b l e III. Ef f e c t o f Ti m e o f Ac e t o n e Ex t r a c t i o n o n
JULY 15, 1937 ANALYTICAL ED ITION 343 Using this method a number of precautions must be observed in
order to obtain the best results. considerable oil. Fish flesh loses w ater by evaporation very readily, and for this reason it is im p o rtan t to complete the initial steps, to th e point where th e flesh has been weighed out, as rapidly as possible.
E x t r a c t i o n . F or this w ork th e F ire Underwriters model extraction ap paratus was used, although undoubtedly any type of continuous extractor would be suitable. Extractions longer th a n 16 hours give very little additional oil. A few lower values obtained w ith shorter extraction periods.
Some of these results are shown in T able I I I .
The fact th a t a 16-hour heating w'ith acetone does not de
crease th e solubility of oil in ether was established by reflux- ing a pure sample of fish oil with acetone for this period, re
moving th e acetone, and dissolving in ether. T he oil was then com pletely soluble.
R e m o v a l o f A c e t o n e an d W a te r . M ost of the water as well as th e acetone should be removed on a steam bath, since otherwise a considerable period of tim e will be required to remove wrate r in th e desiccator. On th e other hand, it was solution is filtered through paper, considerable difficulty is encountered in washing th e oil from the paper. This can be avoided by th e use of a fine grade of sintered-glass cru
cibles. T he residue rem aining after treatm ent with ether should be com pletely soluble in water.
D r y in g o f O il. This step is one of the m ost unsatis
factory of any, since even a short heating tends to decompose the oil. How'ever, a certain am ount of heating is required to remove water. Experience has shown that when a 45-minute period is used, th e error will be small unless considerable m onths. This difficulty cannot be overcome until some other solvents are found which will dissolve both fresh and decom
posed fish oils. These considerations apply to fish meal to an even greater extent. m ethod used involved drying the fish flesh mixed with sand in a vacuum desiccator over sulfuric acid. R esults in T able IV compare these m ethods. T he acetone-extraction m ethod com
pares very well with th e ordinary ether-extraction m ethod.
The cold-shaking m ethod gives approxim ate results on fairly oily fish. On samples having a low oil content, th e cold- centrifuge m ethod gives m uch b etter results.
Ta b l e V . Va r i a t i o n i n Oi l Co n t e n t o f Ma c k e r e l (Scomber
The acetone m ethod has been applied to the determ ination of oil in th e common mackerel (Scomber scombrus), w'hich is is rarely encountered. Table V shows th e seasonal variation in th e oil content of mackerel. I t should be pointed out