The chemical analysis was done at the 

 Hawaiian Sugar Planter's Association (HSPA) 

 Experiment Station, where the paper chronnato- 

 graphic technique is regularly used. I wish to 

 thank Dr. J. H. Payne, Principal Technologist 

 at the HSPA Experiment Station, for the use of 

 laboratory facilities and materials; Dr. Chen- 

 Chuan Tu, Senior Technologist, for valuable 

 technical advice; and chemists Richard M. Oka- 

 moto and George Akatsuka for aid in running the 

 chromatograms. I am also grateful to Tamotsu 

 Nakata, Scientific Illustrator at the Honolulu 

 Biological Laboratory, for preparing the figures. 



MATERIAL 



Samples of fresh muscle tissue of adult 

 tunas captured in Hawaiian water s were obtained 

 at the Honolulu fish market and from the Bureau 

 of Commercial Fisheries research vessel Charles 

 H. Gilbert. It was not possible to collect uni- 

 formly fresh samples, since those obtained at 

 the market had been kept chilled from 1 to 10 

 days, whereas those taken on the research ves- 

 sel were frozen immediately after the completion 

 of fishing operations. Samples from the fish 

 market included yellowfin, bigeye and albacore, 

 and those from the vessel consisted of skipjack 

 (Katsuwonus pelamis) and little tunny (Euthynnus 

 yaito). Two species of frigate mackerels (Auxis 

 thazard and A. thynnoides), which were taken 

 on November 30, 1957 fronn the Bureau's re- 

 search vessel, John R. Manning, and kept fro- 

 zen for about 8 months, were also used inthis 

 study. Fresh samples of these two species were 

 not immediately available. 



San-iples of tuna larvae were obtained 

 from zooplankton collections taken by the Gilbert 

 working in Hawaiian waters, and from another 

 of the Bureau' s research ves sels, Hugh M. Smith 

 working in Hawaiian and equatorial waters. The 

 zooplankton samples were frozen aboard the ves- 

 sels immediately upon capture. At the laboratory, 

 the sannples were thawed and tuna larvae and 

 postlarvae were sorted out in a bath of ice water 

 under a dissecting microscope. The larvae were 

 refrozen for later use. Because they could be 

 readily identified visually, only yellowfin and 

 skipjack larvae were employed in this prelimi- 

 nary work. Theselarvae me asured 4. 5 to 10.2 

 mnn, in total length. 



Solvents used in the experiments were 

 some of those which had been used successfully 

 by other workers in separating amino acids. 

 They were (1) 80 percent solution of phenol in 

 water, (2) m-cresol: acetic acid: water (48:2:50), 

 (3) n-butanol:methylethylketone: 17 N ammonia: 

 water (5:3:1:1) and (4) n-butanol:acetic acid: water 



(4:1:5). A 0. 1 percent solution of ninhydrinin 

 95 percent ethanol was used as the indicator solu- 

 tion to visually detect the amino acid spots on the 

 paper. 



Whatman No. 1 filter paper was used 

 throughout the experiments. For one-dimen- 

 sional chromatograms, 7 x 22-inch sheets of 

 paper were used. For two-dimensional work, 

 18 1/2 X 22-inch sheets were used. 



ONE -DIMENSIONAL CHROMATOGRAMS 



Procedure 



In one-dimensional chromatography the 

 samples were placed about 1 inch apart in a 

 row near one end of the filter paper. They were 

 allowed to dry at room temperature and the 

 amino acids were separated in a descending 

 chromatogram by dipping the end of the paper 

 bearing the samples into a suitable solvent. In 

 order to intensify the color of the various amino 

 acids present, the paper was heated in a con- 

 vectionoven at about 80''C. for lOto 15 minutes 

 aiter applying the indicator solution. The out- 

 line of each acid spot was then marked with 

 pencil. 



The first series of chromatograms was 

 intended to define sample size and solvent appro- 

 priate to the clear separation of amino acids 

 present in adult muscle tissue. Phenol was 

 used as the solvent for the initial tests which 

 consisted of 10 samples. Two of these samples 

 consisted of snnall pieces of muscle tissue (ap- 

 proximately 8 mm. ^) pressed directly onto the 

 filter paper. Although excess tissue was re- 

 moved, considerable streaking occurred and 

 there was no separation of the acids. Samples 

 of fluid which were squeezed from muscle tissue 

 varying in size from 1 to 30 jxl. were also 

 tested. Of these, only the 1 |jl1. sample appeared 

 pron-iising. Therefore, further tests were made 

 on this sample size with a sannple from each of 

 6 species of tunas. The chromatograms showed 

 some streaking, and the separation of the amino 

 acids was indistinct. 



A second solvent, m-cresol:acetic acid: 

 water, was tried with a 1 [d. sannple from each 

 of the previous 6 species. Reasonably good 

 separations were obtained, but the spots were 

 not well defined and were of the type known as 

 "comets. " 



A third solvent, a solution of n-butanol: 

 methylethylketone: 17 N ammonia: water, also 

 was tested as before. With this solvent, excel- 

 lent separation was obtained from samples of 



