Separation of porphyrins by counter-current distribution.

The recent development by Craig (l-3) of an all-glass machine for liquidliquid counter-current fractionation makes possible the application of his method to the separation of porphyrins which cannot readily be separated by the sensitive Willstatter “HCl number” method. In this paper we have studied the application of the Craig method to the separation of a mixture of three known porphyrins, using the Willstiitter system of aqueous HCl and ether. The principle of separating porphyrins by liquid-liquid partition was first used in 1906 by Willstatter and Mieg (4). In their method, the porphyrin mixture was dissolved in ether and the ether solution was extracted successively with equal volumes of increasingly higher concentrations of aqueous HCl. They defined a partition value called the HCl number as that per cent of HCl (in gm. per 100 cc.) in the aqueous phase which would extract two-thirds of the porphyrin from the ether phase when the volume of the aqueous phase saturated with ether was equal to the volume of the ether phase saturated with water. In 1937 Zeile and Rau (5) attempted to extend the method of Willstatter and Mieg to the separation of a mixture of two porphyrins that differed only slightly in their HCl numbers, namely deuteroporphyrin (HCl number = 0.36) and mesoporphyrin (HCl number = 0.60) (Fig. 1). They calculated that, starting with a 1: 1 mixture of these porphyrins, it should be possible to obtain one-third of the mesoporphyrin admixed with only 1.3 per cent of deuteroporphyrin after 60 successive extractions. The Craig method, as we shall see below, permits a complete separation of these two porphyrins, uncontaminated with each other, and at the same time has made possible the recognition of unsuspected porphyrin impurities in the original crystalline materials.