ESTIMATION OF PORPHYRINS 87 



adsorbs the porphyrin on a column of ahiminium oxide, removes 

 other pigments by washing the column first with 20% acetic acid and 

 then with glacial acetic acid, and finally elutes uroporphyrin with 

 dilute ammonia. Grinstein et al. {1056) use talc as adsorbent and 

 esterify the adsorbate directly with methyl alcoholic hydrochloric 

 acid. From bones or shells uroporphyrin may be directly extracted 

 as ester by treatment with methyl alcoholic hydrochloric acid after 

 pre-extraction with petroleum ether or ether + alcohol. It is then 

 brought into chloroform. 



Final purification. For the final purification and identification the 

 porphjrins are usually transformed into their methyl esters and 

 identified by their melting points and mixed melting points. For 

 further purification the methyl esters may be subjected to fractional 

 crystallization, or to chromatography on alumina, calcium carbonate, 

 or talc {835, 8 Jf 5, 1056, 29 11). 



7.2. Estimation 



For the estimation of porphyrins extraction methods similar to 

 those described above are used. For the estimation of total por- 

 phyrins in urine the ether solution is extracted with 5% hydrochloric 

 acid (Fischer). 



Spectrocolorimetric and fluorimetric methods are particularly suit- 

 able for quantitative porphyrin estimations. Whenever sufficient 

 material is available, the spectrocolorimetic method is preferable. 

 The fluorimetric methods are far more sensitive and must be applied 

 for porphyrin estimations in urine of normal or nearly normal por- 

 phyrin content. There is, however, greater need for purification of 

 the porphyrin solutions, since impurities interfere with the porphyrin 

 fluorescence, and this purification is difficult to carry out without 

 losses. 



Spectrocolorimetric method. The main absorption band of a porphyrin solution 

 in dilute hydrochloric acid is sharp and excellently suited for comparison with 

 that of a standard porphyrin solution. If pure coproporphyrin is not available, 

 mesoporphyrin in 0.5% hydrochloric acid can be used for the standard, which 

 keeps for many weeks in the dark. The method was first used by Weiss {3025), 

 who emploj-ed amyl alcohol for extraction and extracted the porphyrins from this 

 by phosphoric acid. It is preferable to use the method of Fischer as recommended 

 by Schreus, in which porphyrin is concentrated first by ether extraction and then 

 by extraction with hydrochloric acid {2Jf68,2986). Losses of porphyrin during 

 the removal of excess acetic acid by washing {2830) can be minimized by buffering 

 the washwater with sodium acetate. 



