[25] Purification of reduced nicotinamide adenine dinucleotide by ion-exchange and high-performance liquid chromatography

This chapter focuses on the purification of reduced nicotinamide adenine dinucleotide (NADH) by ion-exchange and high-performance liquid chromatography (HPLC). Forty milligrams of NADH are dissolved in 0.5 M ammonium bicarbonate (pH 9.0) and applied to a 1.1 × 30-cm AG MP-1 anion-exchange column, equilibrated previously with this same buffer at 4° in the dark. The column is eluted in the dark at 4° with starting buffer until the absorbance at 340 nm returns to baseline levels. Fractions with absorbance ratios of 2.26 ± .05 are combined and saved. For HPLC, the product from the AG MP-1 column if pumped onto a semipreparative Bondapak Cl8/Porasil B reversed-phase column, equilibrated previously with 1 M ammonium bicarbonate at pH 9.0 and 25°. Because moisture accelerates the decomposition of NADH, the purified NADH is stored in 1,2-propanediol over molecular sieves to maintain dryness. Water is removed from the 95% ethanol fraction by stirring with anhydrous sodium sulfate. The fraction is filtered, its volume reduced by rotary evaporation, and the resultant slurry suspended in 1,2- propanediol is dried over sodium sulfate and distilled.