186 Bitancourt, Nogueira, and Schwarz 



order to avoid decomposition during chromatography, chromato- 

 grams were always short (10 cm.) and were sometimes run in the 

 cold (5 to 8° C.) or in an atmosphere of nitrogen. The inconveniences 

 of this decomposition were put to a marked advantage in double 

 chromatograms, i.e., two-dimensional chromatograms in which the 

 same solvent is used in both directions (27). 



Multiple levels of pH were established in some chromatograms 

 (6) by drawing longitudinal, evenly spaced, lines with a straight- 

 edge and a capillary pipette filled with buffer solutions (Figure 2). 



Electrophoresis was initially carried out in an Elphor apparatus, 

 with a voltage of 140 volts, and later in a Pheromatic II apparatus 

 with 200 volts. Whatman No. 1 filter paper strips, 4 x 40 cm., re- 

 ceived the solution on a 5 mm. wide transversal band in the mid- 

 dle of the strij). The strip was moistened with a 0.1 M phosphate 

 buffer solution and placed in the apparatus filled with the same 

 buffer. The operation usually lasted 6 to 8 hrs. 



A combination of electrophoresis and chromatography was used 

 in several instances. For this purpose after electrophoresis, the iono- 

 gram, reduced to 3 cm. width, was machine sewed onto two strips 

 of filter paper, one of them for dipping in the chromatographic 

 solvent and the other one for running the chromatogram (Figure 3). 



Inspection of Chromatograms and lonograms and Color Reactions 



Chromatograms and ionograms were examined under long-wave 

 (366 m^) and short-wave (254 m^^) ultraviolet light and fluorescent 

 and ultraviolet-absorbing spots and zones were outlined with a pen- 

 cil. The contrast between ultraviolet-absorbing spots and the sur- 

 rounding paper was greatly enhanced by spraying the chromatogram 

 with a 0.02 per cent alcoholic solution of safranin. Under short- 

 wave ultraviolet light, absorbing spots appeared dark blue in contrast 

 to the brilliant red of safranin. Significant changes of the color or 

 intensity of fluorescence of some of the spots were produced by ex- 

 posure of the chromatograms and ionograms to ultraviolet radiation 

 for a few minutes. This also induced fluorescence in some of the 

 nonfluorescent, short-wave, ultraviolet-absorbing spots. 



14ie following reagents were ai)plied to the chromatograms and 

 ionograms: FeCl.j reagents (acjueous solutions, Salkowski, Gordon- 

 Weber); p-dimethylaminobenzaldehyde (Ehrlich) ; safranin; N HCl; 

 2,6-dichlorophenol-indophenol (DCPIP); 2,4-dinitrophenylhydrazine 

 (DNPH); benzidine (Van Eck's); ammoniacal AgNO;,. In many cases 

 more than one reagent was applied lo the same chromatogram or 

 ionogram which for that purpose was cut into several longitudinal 

 strips, one for each reagent (Figure 4). The composition of the rea- 

 gents is given elsewhere (29). 



