ISOLATION OF PORPHYRINS 85 



lating ground levels. While Hellstrom used absorption spectra measured at 

 room temperature, Hausser and co-workers used the spectra obtained at the 

 temperature of liquid air. According to Rabinowitch [JlflSa) the al)sorp- 

 tion bands of porphyrins in the visible part of the spectrum are <lue to a 

 common electronic transition with superimposed vibrational changes, but 

 different electronic transitions are responsible for the i^oret band, and for 

 the bands in the red and infrared of dihydro- and tetrahydroporphyrin 

 derivatives. 



7. METHODS OF ISOLATION AND ESTIMATION 

 OF PORPHYRINS 



7.1. Isolation 



The ether-soluble porphyrins can be directly extractefl from urine 

 with ether after acidification, or from feces, bile, or serum with mix- 

 tures of ether and acetic acid. The excess of acetic acid is partly 

 removed by washing the ether solution, and the porphyrins are 

 extracted by 5% hydrochloric acid from ether and, after neutraliza- 

 tion of this extract with sodium acetate, again taken back into 

 ether. The above procedure is particularly necessary and must be 

 repeated for the separation of the porphyrins of feces and bile from 

 accompanying bile pigments. The ether must be peroxide-free, and 

 precipitates appearing in the interfaces should be redissolved in 

 acetic acid and, if they contain porphyrins, worked up again. The 

 easy contamination of porphyrins by traces of metal from glassware 

 or reagents (particularly copper and zinc) had already been observed 

 by Willstatter and needs careful watching. The porphyrin solutions 

 should not be unduly exposed to light. As an alternative to the con- 

 centration of porphyrins by solvent extraction, flocculation and 

 adsorption have been used. Garrod (980) used freshly precipitated 

 calcium phosphate as adsorbent (cf. also 599). It is better, however, 

 to filter through a short column of aluminium oxide, or to shake with 

 talc (1056,2906). Flocculation at the isoelectric point, pH 3 to 4, is 

 only effective if the urine is extremely rich in porphyrin, and is only 

 to be recommended for concentrated and previously purified solutions. 



For the separation of the ether-soluble porphyrins, Zeile (3170) 

 recommends the following procedure. Four extractions of the ether 

 solution with one fifth of the volume of 0.54% hydrochloric acid 

 removes coproporphyrin, deuteroporphyrin, and mesoporphyrin. 

 This leaves protoporphyrin in the ether, which is now extracted with 

 one fifth of the volume of 8% hydrochloric acid. The solution in 



