A new method for isolation of hydroxy-L-proline and L-proline from gelatin.

Several investigators have reported that commercial L-proline is contaminated with small amounts of hydroxy-n-proline (l-3). During the course of investigations in this laboratory all of five commercial samples proved to contain up to 3.5 per cent hydroxyproline when tested by the method of Neuman and Logan (1). The preparation of n-proline from gelatin by the formation of complex salts is tedious and gives relatively low yields (4-8) of the contaminated product. Attempts to prepare n-proline and hydroxy-n-proline by the treatment of gelatin hydrolysates with nitrous acid also gave low yields (9) or impure products (10). Since a simple efficient method for the separation of n-proline and hydroxy-n-proline should make possible their preparation via the nitrosoamines as well as from the impure commercial products, the use of large columns was investigated. Attempts to modify the method of Rietz et al. (11) from an analytical to a large scale preparative method were unsuccessful. Direct chromatography on cellulose columns as carried out by Radhakrishnan and Meister (2) always resulted in extensive overlaps of the proline and hydroxyproline peaks. Moore and Stein (12) obtained good separation of proline and hydroxyproline in analytical amounts on a Dowex 50 column wool. The precipitate was washed lvith 95 per cent alcohol; the washings were added to the filtrate and the concentration was continued until the volume was less than 1 1. The cooled mixture was extracted three times with l-l. portions of ether, filtered to remove solid NaCl, and extracted two more times with 1 1. of ether. The ether extracts were discarded and the aqueous solution was concentrated to a syrup. The residue was taken up in 300 ml. of hot 95 per cent alcohol, filtered, and concentrated to a syrup under vacuum. One hundred ml. of 6 N HCl were added, the concentration was repeated, and a second addition and removal of HCl was effected. The amino acid hydrochlorides mere taken up in water and analyzed for proline by the method of Chinard (14) and for hydroxyproline by the method of Neuman and Logan (1). The solution contained 9.5 gm. of proline and 9.4 gm. of hydroxyproline per 100 gm. of gelatin. Separation of Proline and Hydroxyprolinc-A column of Dowex 50-X8 (200 to 400 mesh), 9.5 cm. in diameter and 80 cm. long was prepared from 11 pounds of resin which had been treated with 6 N HCl and washed with distilled water until the pH of the eluate rose above 4.5. The solution obtained from the gelatin hydrolysate was applied to the top of the column and washed in with a citrate buffer eluant. Since a more easily removable eluant was desirable for preparative purposes, dilute hydrochloric 140 acid, as recommended earlier by the above authors (13), was 9 used in the present work. Gradient elution, startjng with 0.5 120 N HCl, was found to reduce the volume of eluant necessary to obtain the two pure amino acids. HYDROXYPROLINE