The purification and identification of flavin nucleotides by high-performance liquid chromatography

✍ Scribed by Barrie Entsch; Robin G. Sim

• Publisher: Elsevier Science
• Year: 1983
• Tongue: English
• Weight: 653 KB
• Volume: 133
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(83)90101-x

✦ Synopsis

Many preparations of flavin nucleotides contain nucleotide isomers of the natural compounds which are difficult to remove or separate. The method of dynamic complex-exchange (or pairedion) chromatography has been used with high-performance liquid chromatography to achieve resolution and purification of isomers. A solution of nucleotide in water was chromatographed isocratically on a Cur-substituted silica column with a mobile phase of methanol, water, and tetrabutylammonium phosphate at neutral pH. Commercial preparations of FMN and FAD contained multiple components. The purified isomers were subjected to ionexchange chromatography directly on a quatemary nitrogen-substituted silica column to remove methanol and tetrabutylammonium cation, and thus obtain pure nucleotide in aqueous buffer suitable for use with proteins. With analytical equipment, a milligram of pure FMN or FAD was produced in 1 day. The same procedure was useful for the rapid identification and quantitation of flavin nucleotides in proteins. After exposure of a protein solution to heat treatment, the supematant was subjected to dynamic complex-exchange chromatography, as described above.