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PARTITION CHROMATOGRAPHIC SEPARATION OF ADENINE AND GUANINE ( partition-chromatographic-separation-adenine-and-guanine )

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PARTITION CHROMATOGRAPHIC SEPARATION OF ADENINE AND GUANINE BY PEHR EDMAN, EINAR HAMMARSTEN, BENGT LOW, AND PETER REICHARD (From the Biochemical Department, Karolinska Inatitutet, Stockholm, Sweden) (Received for publication, June 22,1948) In the course of an investigation into the metabolic pathways of nucleo- tide purines by means of tracer elements, it became necessary to prepare from biological sources pure specimens of adenine and guanine in sticient quantities for analyses in the mass spectrometer. In general, for prepar- ative purposes, use has been made of the insolubility of adenine and gua- nine under certain strictly specified conditions. For small quantities of pu- rines, however, these methods are unsatisfactory because of the difficulties in maintaining the specified conditions in small volumes. The employ- ment of the liquid-liquid distribution principle in the form of parti- tion chromatography seemed more promising as a separatory procedure for small amounts of purine, especially since Vischer and Chargaff (1) have shown that it is possible to separate adenine and guanine on paper strip chromatograms in the system quinoline-collidine-water. After the conclusion of our work a paper by Tinker and Brown (2) was brought to our attention. They employed the system butanol-1 M phosphate buffer, pH 6.5, for the separation of the purines in the Craig apparatus. In our work partition chromatography on starch (3, 4) has effected the desired separation of adenine and guanine. For two reasons our choice of solvents has been restricted. In the first place we prefer not to have foreign nitrogen in the system. Secondly, it was desirable to follow the development of the chromatogram directly in the effluent. This could be conveniently done by taking advantage of the fact that the purines absorb strongly in the ultraviolet region and there- fore the absorption of the solvent should be negligible. In preliminary ex- periments, we first chose n-butanol as a solvent, but the purines proved to be insufficiently soluble in this medium. The addition, however, of a small amount of the monomethyl’ ether’ of ethylene glycol to the butanol in- creased the solubility of the purines, and this solvent mixture was found to serve as an excellent developing agent in the chromatography. In our first experiments the purines were put on the column as hydro- chlorides. At that time the separation was never complete, because the 896 Downloaded from http://www.jbc.org/ by guest on June 7, 2018



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