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

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 paired-ion) 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 C18-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 ion-exchange chromatography directly on a quaternary 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 supernatant was subjected to dynamic complex-exchange chromatography, as described above.