178 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES 



with ether, then with water, sodium hydroxide being used to adjust the 

 fH. Since penicillin is destroyed readily in alkaline solution, care must 

 be taken in adding the alkali. The "nonpyrogenic" or "therapeutic" 

 fraction, which contains about 8o per cent of the penicillin, is extracted 

 with pyrogen-free water. It is a deep reddish-orange liquid, yellow in 

 dilute solution, with a characteristic smell and bitter taste. 



By means of adsorption, distribution between solvents, and reduc- 

 tion, a barium salt or penicillin was finally obtained (3, 7, 8) which was 

 homogeneous by chromatographic analysis and gave 450 to 500 Oxford 

 units per milligram of dry material. The active substance was found to 

 be a salt of a strong dibasic acid with ^H values approximately 2.3 and 

 2.5y having a formula of C24H;..OioNoBa. The molecule contained one 

 carboxyl, one latent carboxylic, two acetylatable, at least five C-Me 

 groups, and no easily reducible double bond. The penicillin thus pre- 

 pared was more sensitive to oxidizing agents than to reducing agents j it 

 was unstable toward dilute acids and alkalies, and to heat (loss of COo), 

 primary alcohols, and various heavy metal ions. Tentative suggestions 

 were made concerning its chemical nature as follows: (a) a polysubsti- 

 tuted hydroaromatic ring structure j (b) the acidic groups (carboxyl) 

 not conjugated with the chromophore responsible for the absorption j 

 (c) the possible presence of a trisubstituted a-unsaturated ketone 

 grouping. 



Another method for obtaining penicillin has been suggested (598). 

 In this method, the culture medium was adjusted to -pH 3 to 4, satu- 

 rated with ammonium sulfate and extracted with chloroform. The con- 

 centrated chloroform extract was treated with phosphate buffer at fH 

 7.2 to remove the active substance. This process was repeated, the less 

 active substance being separated from the active fraction by extraction 

 with chloroform at different ranges. By precipitating the concentrated 

 extracts from petroleum ether, the free acid form of penicillin was ob- 

 tained. By saturating the chloroform-benzol solution with dry am- 

 monia gas, an ammonium salt was obtained which gave a dark yellow 

 microcrystalline powder. The salt was more stable than the acid form. 

 By acetylating or benzoylating the ammonium salt a further increase in 

 stability was obtained. The analysis of the penicillin prepared by this 

 method was found to fit best the formula Ci4Hic)NO,( or C]4Hi-N05 -f- 



