358 
PACIFIC SCIENCE, Vol. XIII, October, 1959 
drop of hydrochloric acid was added to pre- 
vent degeneration. The material was returned 
to the laboratory where it was refrigerated or 
frozen for long-term storage. Spectrophoto- 
metric curves and chromatographic separa- 
tions were made using 10 per cent hydrochloric 
acid as diluent. Spectrophotometric curves 
were run on a Beckman Model DU spectro- 
photometer. 
The writer used two methods of analysis. 
Chromatographic columns were prepared, us- 
ing corn starch packed in a column two feet 
deep, in four-foot sections of soft glass tubing 
(Vs in., outside diam.). A very small piece of 
glass wool at each end of the column held 
the corn starch in place. 
The tube was placed in a stopper which in 
turn fitted into a suction flask. Approxi- 
mately one half cc. of aplysia purple was 
placed in the top of this column. As soon as 
the substance had penetrated the column, the 
top of the tube was filled with 10 per cent 
hydrochloric acid and suction was applied to 
the base of the column. About 8 hours later, 
when pigment had reached the bottom, the 
tube was removed and cut into sections cor- 
responding with the color components. The 
starch was removed and the color components 
were eluted with dilute hydrochloric acid. 
Instead of cutting up the column, suction 
can be continued and each fraction can be 
caught as it drips from the bottom of the 
column. This method is very slow, requiring 
several days for one complete separation. 
It was found that quicker determinations, 
especially where it was unnecessary to collect 
the fractions, could be made by paper chro- 
matography. Several alterations in the meth- 
ods were used, depending upon the nature 
and purpose of the particular investigation 
being carried on. Square sections of Whatman 
No. 1 filter paper measuring 18 X 18 cm. 
were spotted or lined with aplysia purple at a 
point V/i cm. from the margin which would 
become the base. The liquid was applied with 
either a fine pipette or with an Esterbrook 
No. 2 drawlet pen. Each paper was then 
folded twice parallel to the line of travel of 
the components, after which the folds were 
relaxed enough so that the papers would 
stand without any two parts of the paper 
being in mutual contact. The papers were 
stood in petri dishes containing about cm. 
of dilute hydrochloric acid and each paper 
and its petri dish was then covered with a bell 
jar. Separations made by this method were 
complete in one hour. Great care was neces- 
sary, however, in making the original spot or 
line. Thin lines and small spots produced the 
best results, especially where the distance of 
upward migration was limited. Strips 3 feet 
long were used with large initial wide lines of 
pigment. These resulted in excellent separa- 
tions but required from 8 to 12 hours. The 
papers were dried and stored until the time of 
elution. Lining rather than spotting simpli- 
fied the separation and subsequent elution. 
The papers were cut into strips parallel with 
the original pigment line so that each strip 
contained only one color component. Elution 
was then made by "siphoning” dilute hydro- 
chloric acid lengthwise through the strips 
thus formed. 
EXPERIMENTAL DATA 
To determine the spectrophotometric dif- 
ferences between the purple secretion of 
young specimens whose purple appears bluish 
and that of adult specimens which is of a 
reddish hue, studies were made upon diluted 
aplysia purple. The most striking differences, 
as shown in Figure 1, were: (1) the differences 
in relative height of the two peaks, and (2) 
the variation of 15 m^u in the location of the 
broad peak in the high 500 region. 
The components of the purple of A. cali- 
fornica were separated in quantity by column 
chromatography. Though no blue region was 
distinct as a separate band on the starch col- 
umn, the collected fraction from the extreme 
trailing region of the lavender zone gave high 
concentrations showing a deep blue gross 
color, which produced a different absorption 
curve than that of the earlier lavender collec- 
