B54: G. ROUSER, B. JELINEK, A. J. SAMUELS, K. KINUGASA 
Aliquots for platelets and white cells are expressed on a wet weight basis assuming 
1 ml of packed cells (1300 x g) to be equivalent to I gram. Erythrocyte aliquots are 
expressed as milliliters of packed cells (1300 x g). 
Filter paper chromatography was carried out using water-saturated phenol in an 
ammonia atmosphere in the first dimension, followed by 2,4-lutidine saturated with 
water in the second dimension. The chromatograms were sprayed with ninhydrin 
dissolved in 7-butanol (1 g/l) while still sightly moist with lutidine and allowed to 
develop at room temperature before they were photographed. Lutidine increases the 
color obtained with ninhydrin, and in its presence the proline spot is blue rather than 
yellow when fully developed. 
Identification of compounds 
The tentative identifications of compounds based on migrations in phenol and lutidine, 
behavior after hydrogen peroxide treatment, and stability to 6 N hydrochloric acid 
in a sealed tube at 100° for 24 h was confirmed as follows. Several two-dimensional 
chromatograms were prepared in the usual manner. The dry papers were heated at 
105° for 30-60 min and amino acid spots visualized by their fluorescence under 
ultraviolet light. The spots were marked and eluted with water or a dilute sodium 
carbonate solution. When such eluates are evaporated to dryness in a small beaker, 
crystals can usually be obtained in the center of the beaker that are largely free of 
coloration from lutidine. A comparison of these crystals with those obtained from 
authentic compounds obtained in a similar manner can give valuable information as 
to the nature of the material. 
Dinitroflucrobenzene derivatives were prepared from the compounds eluted from 
paper and, after removal of excess dinitrofluorobenzene by extraction with diethyl 
ether, the derivatives were applied to washed strips of Whatman No. 1 paper along- 
side authentic compounds treated in the same manner. The paper strips were washed 
by capillary descent in 2 N acetic acid prior to use. Chromatographic comparisons 
were carried out in: (1) dioxane-concentrated ammonia (4 : I, v/v); (2) 2,4-lutidine— 
water (II: 1, v/v); (3) isobutyric acid saturated with water; (4) 1 N aqueous 
hydrochloric acid; (5) 5°% potassium dihydrogen phosphate in water layered with 
an equal volume of isoamyl alcohol; (6) 5° potassium dihydrogen phosphate in 
water adjusted to pH 7.0 with concentrated ammonia and layered with an equal 
volume of isoamyl alcohol; and (7) 5% disodium hydrogen phosphate in water 
layered with an equal volume of isoamyl alcohol. Solvents 5 to 7 are used only for 
ascending paper chromatography. 
The differentiation of glutathione, cysteine and cystine (as cysteic acid), and 
cysteinylglycine in leukocyte samples was achieved as follows: With some batches 
of filter paper the spot applied to paper could be warmed with air from a hair dryer to 
oxidize cysteine and cysteinylglycine without oxidizing glutathione. With other batches 
of paper it was necessary to treat with 30% hydrogen peroxide to obtain oxidation. 
The differentiation of cysteic acid and cysteinylglycine (probably as the sulfonic 
acid diketopiperazine) was facilitated by observing the color changes of the spots 
after spraying with ninhydrin. Cysteinylglycine was first yellow, then blue-green, 
and finally purple. The cysteic acid spot was purple at all times. Authentic cysteinyl- 
glycine was prepared by the hydrolysis of glutathione (250 mg) in 1 N hydrochloric 
acid (10 ml) in a boiling water bath for 1 h. 
References p. 447/448 
