Time of RJN ■ 24 'ft HOURS 



Figure 2. --One -dimensional chr omatogranns of 

 adult and larval samples of two species of tunas 

 developed in n-butanol:methylethylketone:am- 

 monia:water . 



of the adults and 2 to 7 larvae of each of the lat- 

 ter two species. 



Results 



The results of the two-dimensional chro- 

 matograms were not always satisfactory and 

 varied considerably from one experiment to 

 another. In many instances the variations were 

 extreme, from to 1 1 or 12 spots, even among 

 samples taken from fish of the same species. 

 Part of these variations were probably due to 

 the inexact manner in which the samples had 

 been prepared. The comparisons discussed 

 here are based only on those chr omatograms 

 which yielded the maximunn spots per species, 

 and may be somewhat arbitrary. Nevertheless, 

 they give us sonne idea whether this method is 

 applicable in species identification of the tunas. 



Figures 3-7 show five of the better traces 

 obtained with samples fron-i adult tunas. Some 

 differences were evident between little tunny, 

 albacore, bigeye and skipjack, but differences 

 between bigeye and yellowfin could not be deter- 

 nnined, since the trace of the latter species 

 showed some streaking and the separation of 

 the spots was not sufficiently clear. 



TWO-DIMENSIONAL CHROMATOGRAMS 



Procedure 



In two-dimensional chr omatograms the 

 sample to be analyzed was placed in one corner 

 of a large (18 x 22 inches) sheet of filter paper. 

 The end of the paper nearest the sample was 

 placed in the fir st solvent until the solventfront 

 had descended to the opposite edge. After dry- 

 ing, an edge of the paper adjacent to the sample 

 was dipped into a second solvent and allowed to 

 develop as in the first instance. The paper was 

 then dried and treated with the indicator solution. 



Because successful separation of the 

 amino acids was obtained with n-butanol:methy- 

 lethylketone: 17 N ammonia:water and n-butanok 

 acetic acid:water in one -dimensional chromatog- 

 raphy, these solvents were used in the order 

 mentioned. The samples of adults were prepared 

 by squeezing out the muscle fluid by hand. Lar- 

 val samples consisted of whole tuna larvae which 

 were squeezed directly on the paper with a glass 

 rod. Samples from adults of 5 species of tunas 

 (yellowfin, bigeye, albacore, skipjack and little 

 tunny) and the larvae of 2 species of tunas (yellow- 

 fin and skipjack) were developed for 2 1 to 24 

 hours in each solvent. The amount of sample 

 usedwas 10 fil. of muscle tissue fluid fromeach 



Figure 3. --Two-dimensional chroinatogr am of 

 adult little tunny. 



