cherry-red in color as in the case of the water extracto Alcohol 

 served as both an extr acting oiid a preserving agent; refrigeration of 

 the extract was not necessaryp although it was used occasionallyo 



More connplex procedures were used in attennpts to 

 isolate and identify the attractive substancefs) in extracts of tuna 

 flesh and viscerao These included fractionation of the extract into two 

 or several components to determinen on pond testingo which still re- 

 tained the attractive propertieSo In some preparations „ a first sepa- 

 ration into "protein" and "non-protein" factions was achieved by 

 precipitation of the proteins <ind similar complex substanceSo Pre- 

 cipitation was accomplished in various waySj eogoo heatingo boilingj 

 saturation with sodium chloride or "salting out", adding tannic and/or 

 phosphotungstic acidp adding the salts of heavy metals such as lead, 

 barium^ etCoa as well as adding other chemicals to remove those used 

 in precipitationo In other preparations the extract was separated into 

 two fractions by dialysis to determine whether the attractant was com- 

 prised of a small molecule which would pass through a dialyzing mem- 

 branes, or a large molecule which would not pass through a membraneo 

 In still others the extract was divided into a "fatty" and a "non-fatty" 

 fraction, and each was tested in the pond to determine if the attract- 

 ant was a "fatty" or a "non«fatty" substanceo Fractionation was 

 accomplished by rennoving the fatty acids and similar compounds by 

 extraction with petroleum ether and/or acetone,. Other procedures 

 were aimed at determining whether the attractive substance consisted 

 of a positive or negative lono In these, fractionation was achieved by 

 passing the extract through anion and/or cation exchange resin columns 

 gjving two portions 5 that adsorbed by the column (subsequently removed 

 by elution or washing) and that not adsorbed by the column (the fil^- 

 trate)o In addition, columns of activated charcoal and activated alumina 

 were used to see if they would adsorb the attractive substanceo 



In many of the preparations, several of the foregoing 

 fractionation procedures were used in succession, each successive 

 portion being tested and being either accepted or rejected for further 

 fractionation depending on whether pond tests indicated that it did or 

 did not include the attractive substanceo Several of the procedures 

 were used even though they did not remove the attractive substance; 

 they did remove non^active substances and thus constituted a step in 

 the purification of the attractanto The attractant, when purified as 

 completely as possible by the successive removal of non attractive 

 substances, was then subject to basic chemical tests in an attempt to 

 determine its basic components and chemical structures 



Miscellaneous chemical compounds were prepared for 

 testing bv dissolving in tap water or in weak alcohol solutions; or in 



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