IDENTIFICATION OF THE ELUSIVE AMINO ACID 13 
resulted in the ultimate isolation of large amounts of buffer salts that would have 
interfered with the isolation of the desired components, volatile acetate and formate 
buffers patterned after those used by Moore and STEIN, were employed. The buffer 
used here was 0.2 WM with respect to ammonium ion and the resin was Amberlite 
CG 120 in the ammonium form. After equilibration of the column with buffer, 3 g 
of a diastereomeric mixture containing approximately equal parts of L-isoleucine and 
p-alloisoleucine, dissolved in the appropriate buffer, was poured onto the column. As 
is indicated by the graph (Fig. 4 A), the amino acid emerged in two distinctly separate 
fractions after the passage of 171. Evaporation of each separate fraction 7 vacuo 
resulted in a residual mass which was subjected to sublimation under high vacuum 
at 50° in order to remove contaminating buffer salts. The residual amino acid was 
recovered in approx. 95°% over-all yield, was found to be analytically and chromatog- 
raphically pure, and required no further purification or recrystallization. 
Fig. 4 B illustrates the conditions employed in the separation of threonine from 
allothreonine. The starting material here was a 50 : 50 mixture of threonine and 
allothreonine, the buffer ammonium acetate, and the resin again Amberlite CG 120. 
It was observed that separation of the threonines appeared to be appreciably more 
sensitive to alcohol concentration than to pH. Thus, although successful separation 
of several hundred milligrams of the diastereomeric mixture could be obtained in 40%, 
alcohol in the pH range of 5.8—6.6, it was only after the alcohol concentration was in- 
creased to 60% that the resolution became great enough to permit the separation of 
several grams of material. Recovery of the amino acid from the separate fractions was 
effected in over 90%, yield and, as in the case of the isoleucines, the material was 
found to be analytically and chromatographically pure. 
In Fig. 4 C, the conditions employed for the separation of L-hydroxyproline from 
p-allohydroxyproline are given. Extremely large quantities of a mixture of the two 
diastereomers could be separated under these conditions, as is evidenced by the fact 
that 20 g of a 50 : 50 mixture of hydroxyproline and allohydroxyproline could be 
separated on a column only 3 cm in dia. and 150 cm long, in contrast to the 7.5 x 
150-cm column which was employed in the separation of the isoleucines and of the 
threonines. Recovery of analytically and chromatographically pure amino acids 
from the separate fractions proceeded in high yield as before. In any event, the data 
showed that the use of ion-exchange chromatography would permit the separation 
of diastereomeric amino acids on a scale sufficiently large for preparative purposes. 
The next step in establishing the identity of a naturally occurring amino acid 
necessitates resolution of the synthetic racemic material into its optical antipodes by 
any one of a number of chemical or biological procedures. A distinct advantage which 
accrues from the use of biological procedures over purely chemical procedures is that 
they yield isomers of known optical configuration by virtue of the known optical speci- 
ficity of the biological agent itself. In addition, biological procedures permit a more gen- 
eral approach and a more uniform resolution procedure, and do not require the tedious 
and time-consuming manipulations so generally encountered with chemical proce- 
dures. Probably the most satisfactory of all presently available biological resolution 
procedures is that developed by the late Dr. J. P. GREENSTEIN, who employed as the 
biological agents two separate enzyme systems isolated from hog kidney. One enzyme 
system, known as acylase I, is capable of acting asymmetrically only on the L-isomers 
of N-acylated pL-amino acids, while the other enzyme system, known as amidase, 
References p. 22/24 
