I2 M. WINITZ 
their synthesis. Thus, the synthesis of a given amino acid by a general method may 
afford many problems unique unto itself. 
If the synthesis leads to an amino acid which contains more than one asymmetric 
center, then a diastereomevic mixture of the racemates generally results. Separation 
of the racemates must precede their resolution into the optical antipodes, and for 
this purpose two general approaches have been used, namely, differential solubility 
and partition or ion-exchange chromatography. Although certain instances are known 
wherein it is possible to separate diastereomeric amino acids on the basis of differential 
solubility, such a process more frequently requires the prior conversion of the amino 
acid into a suitable derivative. It would, of course, prove most advantageous if par- 
tition or ion-exchange chromatography could be employed to separate the diastereo- 
meric racemates, and although such procedures have been employed for the separation 
of threonine from allothreonine**, hydroxylysine from allohydroxylysine*®: 4°, and 
hydroxyproline from allohydroxyproline*””, among others, the procedures have been 
applicable only to the separation of micromolar amounts of material and hence have 
proven inadequate for preparative purposes. The problem of increasing the scale of 
such chromatographic separations to the preparative range was undertaken by 
Dr. Micu1 in our laboratory* some 2 years ago. Attempts were first made to separate 
isoleucine from alloisoleucine, and after a long and tedious process of juggling con- 
ditions in many trials, conditions were finally found that would permit the separation 
of several of these diastereomers. The conditions evolved are shown in Fig. 4 A. 
Since the use of the usual citrate or phosphate buffers as eluants here would have 



A B 



L-ISOLEUCINE-D-AL/OISOLEUCINE (3g ) DL-THREONINE-DL-ALLOTHREONINE (5g ) 

COLUMN: 7.5em «150 cm COLUMN: 7.5m «150 cm 


RESIN: Amberlite CG 120, type 2, NHg*— form RESIN: Amberlite CG 120, type 2, NHy*— form 


ELUANT: 0.2Mammonium formate in 40% ethanol ELUANT: 0.2/ammonium acetate in 60% ethanol 
pH: 3.8 
FLOW RATE: 80m! 4h 

pH: 6.3 



FLOW RATE: 85 ml h 





Wmaramamarararaterat 
RRR RSD 
20 21 
EFFLUENT (Liters) 
22 23 24 W 12 
EFFLUENT (Liters) 







L-HY DROXY PROLINE-D-ALLOHYDROXYPROLINE (20g ) 
COLUMN: 3.0 cm «150 cm 






RESIN: Amberlite CG 120, type 2, NHg *— form 
ELUANT: 0.2Mammonium acetate in 40% ethanol 
pH: 5.8 
FLOW RATE: 10-12 ml h 


revevere"e?a? 
PRI 


05 0.6 0.7 08 O09 1.0 hy] 1.2 1.3 
EFFLUENT (Liters) 

Fig. 4. Conditions for separation of diastereomers of various amino acids in macro quantities via 
ion-exchange chromatography. 
References p. 22/24 
