86 III. PORPHYRIN CHEMISTRY 



0.54% hydrochloric acid is brought to a concentration of 0.1 to 0.15% 

 hydrochloric acid by partial neutralization with alkali and is extracted 

 with chloroform. This removes deuteroporphyrin and mesoporphyrin 

 and leaves coproporphyrin in the aqueous phase. Mesoporphyrin is 

 separated from deuteroporphyrin by extracting it from 0.67% hydro- 

 chloric acid with ether. A little of the mesoporphyrin remains in the 

 protoporphyrin fraction. This method is better than the method of 

 Schumm used by Dobriner {599), in which protoporphyrin is extracted 

 from the 5% hydrochloric acid solution by chloroform. For the sep- 

 aration of fecal porphyrins, Dobriner {600,608) has developed a 

 scheme which makes use of the insolubility of the sodium salts of 

 some porphyrins. This scheme, slightly shortened, is given in 

 Table IX. The preliminary saponification of natural porphyrin 

 ester was found to be necessary {598). 



TABLE IX 

 Separation and Identification of Fecal Porphyrin* 



{1) Extraction of total crude porphyrins with ether-acetic acid and purification by 



ether-HCl procedure. 

 {2) Saponification of natural esters with 20% NaOH followed by separation of soluble 



and insoluble N'a salts. 



(a) Coproporphyrin, soluble. 



ib) Deuteroporphyrin and protoporphyrin, insoluble. 

 (.i) Extract ether solution with 0.6% HCl. 



(a) Coproporphyrin extracted from ether by 0.2% HCl. Extraction of this 

 solution with chloroform-petroleum ether leaves coproporphyrin in 0.2% HCl. 



(6) Deuteroporphyrin extracted. Reduce HCl cone, to 0.2% and extract deutero- 

 porphyrin with chloroform. 



(c) Protoporphyrin not extracted. Extract once rnore with 1% HCl. Proto- 

 porphyrin extracted from ether by 5% HCl and from this by chloroform. 

 Finally, the methyl esters are prepared and recrystallized from chloroform-methyl 

 alcohol and identified. Coproporphyrin I and HI esters are separated as described in 

 the text. 



"According to Dobriner {600,603). 



For the extraction of protoporphyrin from blood {226,229,231)^ 

 blood or erythrocytes are homogenized in a mixture of three parts 

 ethyl acetate with one part acetic acid and are shaken for a few 

 minutes. After washing the filtrate with water, protoporphyrin is 

 extracted with 5% hydrochloric acid, brought into ether after buffer- 

 ing, and re-extracted with acid. 



Isolation of uroporphyrins. For the isolation of uroporphyrin from 

 urine, acetic acid is added to 1-2% and the ether-soluble porphyrins 

 are first removed by ether extraction. Waldenstrom {2906) then 



