Determination of Nucleosides and Purine and Pyrimidine Bases by Ion Exchange Chromatography

Determination of Nucleic Acid Related Substances by Ion Exchange Chromatography Part III

Abstract

Nucleosides and purine and pyrimidine bases were separated using both of anion and cation exchange chromatography. Anion exchange chromatography was carried out using a column of φ9 mm×220mm packed with Hitachi model 2630 anion exchanger(strongly basic type). Nucleosides and purine and pyrimidine bases were eluted at an early stage by 0.1M ammonium acetate-acetic acid(pH 4.6)separating from nucleotides. The nucleosides and the purine and pyrimidine basesobtained by anion exchange chromatography were moreover separated by cation exchange chromatography. The determination was carried out using a column of φ9mm×400mm packed with Hitachi model 2613 cation exchanger(strongly acidic type), by the stepwise eluting method. Urd, Ura, Thy, Ino and Xan could be separated by 0.05M monopotassium phosphate-phosphoric acid (pH 2.4)as the first eluting solution, Hyp and Guo by 0.4M monopotassium phosphate-phosphoric acid(pH 3.4)as the second eluting solution, and Ado, Cyd, Gua, Ade, and Cyo by 0.4M monopotassium phosphate-potassium hydroxide (pH 5.0) as the third eluting solution. The Nucleosides and the purine and pyrimidine bases in dried purple lavers were determined by the method. The very small amounts of Ade, Gua, Ado, Cyd, and Urd were observed in these samples.