Clam Poison:i. The Paper Chromatographic Purification of Clam Poison Dihydrochloride

Abstract

A semimicro (10–25 mg) paper chromatographic method for purification of clam poison has been developed. This procedure when applied to a partially purified (toxicity 4150 MU/mg) sample of the toxin gave a 74% recovery of clam poison dihydrochloride of purity identical with that of the best material obtained by Schantz etal. (2) via the alumina chromatographic method. The upper limit of purity of toxin resulting from paper chromatography is estimated to be 98% because of traces of impurity introduced by the paper. The paper chromatographic procedure has been found capable of enriching low-grade toxic residues (35%) from the alumina process to a purity of 75% with an over-all recovery of 70%.

Clam Poison: Iii. Paper Electrophoresis of Clam Poison

“…The substance was recovered by aqueous elution and the toxicity (5700 RlIU/mg) and specific rotation (+12G0) were identical, within experimental error, with those of the starting material. The infrared spectrum was also identical with that reported previously for clam poison (2,8 011 Whatman 3Mi\/I paper in 1 N forinic acid a t 900 v for 1.75 hours revealed the presence of Weber-positive toxin a t an apparent mobility of 18-19 cm, ninhydrinpositive spots a t 21 and 30 cm, and ultraviolet-fluorescent spots a t 6.5 and 8 cm. These results indicated that electropl~oresis is capable of separating components in the tailings toxin which are not separable by paper chromatography.…”

“…The recovered toxin exhibited physical, chemical, and biological characteristics which agreed in all respects with those reported previously for pure clan1 poisoil (1,2), except that the specific rotation (+98&4") was approximately 257, below normal. Since paper cl~romatography proved incapable of producing further improvement in the toxicity and rotation it was uncertain whether the low rotation referred t o above was caused by the presence of impurities or whether the toxin recovered from the tailings differed fro111 clain poison in some subtle manner.…”

“…sheets of 3MM paper which had been chrolnatographically pretreated were conditioned further, by elution with 1 i V formic acid and prolonged electropl~oretic treatinent as described in the experimental section, before the quantities of extractable iinpurities were determined. Results of these experiments are given in Table 11, fro111 which it is clear that untreated paper yields substantial amounts of impurities which call be reduced appreciably by chromatographic pretreatment (2,3). A further reduction can be effected by prolonged washing of the pretreated papers with 1 N formic acid, followed by electrophoretic conditioning.…”

“…The purification of clam poison by ion exchange followed by chromatography on acidwashed alumina, by chromatography on Norit A followed by crystallization as the helianthate, by countercurrent distribution (I), or by paper cl~romatography (2), provided material concluded to be chemically homogeneous because of the close correspondence in chemical, physical, and biological properties of the resultant specimens. Nevertl~eless, the fact that this exceedingly toxic substance of low molecular weight has not been obtained in a crystalline form possessiilg a definite melting point (2) makes it desirable t o examine its purity by methods other than those mentioned above.…”

“…Nevertl~eless, the fact that this exceedingly toxic substance of low molecular weight has not been obtained in a crystalline form possessiilg a definite melting point (2) makes it desirable t o examine its purity by methods other than those mentioned above. Recently, we described a heavy-paper technique by means of which the toxin in clam poisoil tailings was enriched approximately fourfold in toxicity to 5000-5800 mouse units (MU)/mg in 67% yield.…”