202 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES 



erately soluble in chloroform, slightly soluble in benzene and in carbon 

 tetrachloride. It is soluble in water to the extent of 5 mg./ml. 



It is inactivated by oxidation and by evaporation at 40° to 45° C. in 

 acid and in alkaline solutions, although it is fairly stable at fH 5 to 6. 

 If the solutions are adjusted to ^H 6.8, it retains its potency for 

 3 months. The crude penicillin does not dialyze through a collodion 

 membrane and resists heating at 60° to 90° C. for short periods; it 

 remains active when heated at 100° C. for 5 minutes but not for 10 min- 

 utes. 



The methods of isolation of penicillin from the culture media can be 

 classified under the extraction and adsorption procedures. 



Fleming first reported that penicillin is insoluble in ether. This was 

 found (146) to be due to the alkaline reaction of the filtrate; for at ^H 

 2.0 ether removes completely the antibacterial substance. The ether 

 extract is evaporated with some water in vacuo at 40° to 45° C, the 

 residual water containing the active substance, which is extremely labile. 



For practical purposes, penicillin is extracted from the acidified cul- 

 ture by means of different organic solvents, such as ether or amyl ace- 

 tate (6). It is then removed from the solvent by shaking with phos- 

 phate buffer or with water at ^H 6.7. Since penicillin is rapidly de- 

 stroyed at a high acidity, the first extraction must be carried out very 

 quickly and at a low temperature. In the presence of the solvents, peni- 

 cillin is stable for several days. The aqueous extract may be partly de- 

 colorized by shaking with charcoal and filtering. The solution is cooled, 

 acidified, and extracted several times with ether or amyl acetate; the 

 extracts are passed through an adsorption alumina column, or through 

 a 2.5 per cent precipitate of an alkaline earth carbonate on silica gel. 

 Water may often contain a pyrogenic or heat-producing substance that 

 must be removed from the penicillin. 



The following four main zones were recognized in the chromato- 

 grams, beginning from the top : 



1. A dark brownish-orange layer, the depth of which is inversely propor- 



tional to the amount of charcoal used for the decolorization ; this zone 

 contains some penicillin 



2. A light yellow layer containing most of the penicillin but none of the 



pyrogen 



