160 Investigations on the Life-History 



to the food, it was thought that their investigation in the case of the 

 salmon would be of some interest. In this animal the pigments do not 

 seem to have been previously studied. 



METHODS OF SEPARATION. 



If the red flesh be pounded up in a mortar with sand, and then 

 extracted with ether, it yields to the ether practically all its pigment. 

 The ether becomes a deep golden-yellow colour, and leaves merely a 

 greyish mass behind. On the evaporation of this extract a mass of 

 pinkish pigment mixed with other substances is obtained. The pink 

 pigment when treated with concentrated sulphuric or nitric acid gives 

 a pure blue colour, which fades very rapidly, especially if water be 

 added. 



This reaction shows that the muscle contains one of the class of pig- 

 ments called lipochromes, which are characterised by their solubility in 

 ether, chloroform, benzol, petroleum ether, alcohol, etc. ; by their colour, 

 which varies from yellow to red ; and by the fact that they give a blue 

 colour when treated with concentrated acid in the dry state. In order 

 to obtain the pigment pure, for further study, the method of sa.poni- 

 tication was resorted to. 



Two methods of saponification are available : (1) by means of 

 metallic sodium in ethereal solution ; (2) by means of caustic soda in 

 alcoholic solution ; both methods were employed. 



1. Small pieces of metallic sodium were added to a golden -yellow 

 extract of the flesh in ether ; on standing, the soap separated out at 

 the base of the flask and was of a reddish colour, while the ether 

 remained clear yellow. The ether was poured off and the soap washed 

 with fresh ether, which did not extract the pigment. The soap was 

 then dissolved in water, which became a pure pink colour. The addition 

 of a little acetic acid to this solution gave a pink precipitate, which was 

 filtered off. This pink precipitate dissolved readily in ether, to form, 

 when dilute, a yellow solution, and in alcohol to form a pink solution. 

 It also dissolved in petroleum ether, benzol, chloroform, etc., and gave 

 a beautiful blue colour with concentrated sulphuric or nitric acid. 



The yellow ether obtained by this method of saponification, after 

 iiltering off the soap, left on evaporation a pure yellow pigment which 

 did not give a blue colour with concentrated sulphuric or nitric acids. 



The ether of saponification is never anhydrous, and therefore when 

 evaporated it usually leaves some drops of caustic soda solution behind ; 

 the yellow pigment readily dissolves in this. 



2. In this method an alcoholic extract of the muscle was boiled with 

 caustic soda. A slight precipitate of red pigment was obtained from the 

 solution, but the mass of the pigment remained in solution with the 

 soap. 



To obtain the red lipochrome from this solution, two methods may be 

 employed. The soap and the pigment may be precipitated by the addi- 

 tion of common salt, or the caustic solution may be shaken with ether in 

 a separation funnel. 



If excess of common salt be added to the caustic solution, a. bright red 

 soap comes down, leaving the solution a clear yellow colour. The soap 

 may be washed with alcohol and then treated with dilute acid, after 

 which the pigment is readily removed by alcohol, in which it forms a 

 pink solution. 



A simpler method is to shake the caustic solution after removal of the 

 alcohol with ether, when the ether becomes deep yellow, leaving the 

 caustic solution also yellow. The evaporation of the ethereal solution 

 leaves a red pigment, which gives as before the lipochrome reactions. 



