M. WINITZ 
oO 
y-hydroxyglutamic acid, which possesses the same empirical formula, could result. 
As authentic samples of y-hydroxyglutamic acid were available, a chromatographic 
comparison of this material with the unknown synthetic material was made in three 
different solvent systems. The Ry values were identical in every instance. However, 
it was noted that in contradistinction to the usual rapid purple color given by y- 
hydroxyglutamic acid with the ninhydrin reagent, the unknown synthetic product 
exhibited some unique color reactions. Thus, when a solution of this material was 
spotted on filter paper and the paper sprayed with a solution of ninhydrin in acetone 
and subsequently heated, a bright yellow spot appeared. The yellow color gradually 
changed to gray-brown after 4-8 h, and finally to purple after 18-24 h. If, however, 
NH2—CH-COOH 
CH2 
0 ee HO-CHCOOH 
NH 2CH2COOH + gta Gogh OH> y—Hydroxyglutamic Acid 
Glycine Pyruvic Acid NH2—CH-COOH 
HO-ECOOH 
CH3 
B-Hydroxy—B-methylaspartic Acid 
Fig. 2. Potential products arising from copper-catalyzed condensation of pyruvic acid and glycine. 
the paper was sprayed with a basic ninhydrin solution and heated, the yellow spot 
appeared but almost immediately turned purple. Such behavior indicated that the 
new material was other than the suspected y-hydroxyglutamic acid. Had it, however, 
responded to ninhydrin in the usual manner, it might have been all too tempting, on 
the basis of its mobility behavior alone, to assign it the erroneous structure. 
From what has already been said, it becomes readily apparent that visual obser- 
vation of the color-staining process on paper will sometimes permit distinctions 
between amino acids that cannot be made on the basis of movement analysis alone. 
A few instances of the specific color reactions revealed by the different amino acids 
after development with various color-inducing reagents are listed in Table I. 
Thus, it is commonly known that whereas most amino acids exhibit a blue or reddish- 
purple color after treatment with the ninhydrin reagent, proline displays a yellow 
color, tyrosine gives a dull greenish-purple, hydroxyproline yields a brown-yellow 
color (which serves to distinguish it from the nearby alanine spot?*), glycine shows a 
rather grayish-purple color, asparagine reveals an orange-brown color (which serves 
to distinguish it from the nearby glycine spot!’), and aspartic acid is characterized 
by a rather bright blue. Treatment of the paper chromatogram with weak alkali 
intensifies the blue color of the ninhydrin spot of phenylalanine!’, whereas treatment 
of the paper with cyclohexylamine prior to spraying with ninhydrin leads to a blue 
color for aspartic acid, orange for cystine, grayish-green for histidine, bluish-gray for 
phenylalanine, yellow for proline, carmine for hydroxyproline, grayish-purple for 
threonine, gray for tyrosine, and reddish-brown for glycine, with all of the other protein- 
derived amino acids yielding the more usual purple color!’. In addition, a variety of 
other staining reagents are available which are more or less specific for certain amino 
References p. 22/24 
