Chapter 2 HPLC of Transfer RNAS Using Ionic-Hydrophobic Mixed-Mode Chromatography and Hydrophobic-Interaction Chromatography

Abstract

Publisher Summary This chapter focuses on high-performance liquid chromatography (HPLC) of transfer RNAs (tRNAs), using mixed-mode or ionic-hydrophobic chromatography. Mixed-mode chromatography is performed using a stationary phase consisting of a C 18 -bound silica matrix (ODS-Hypersil) that was noncovalently coated with the ion-pairing reagent methyltrioctylammonium chloride. This material provides sites for hydrophobic and electrostatic interactions and results in high resolution of tRNAs. When evaluated with a variety of tRNAs (baker's yeast) differing in hydrophobic and ionic properties, the matrix appears to favor a separation mechanism based predominately upon hydrophobic interactions. The retention mechanism is markedly different from either hydrophobic interaction or reversed-phase chromatography. The described support is successfully employed to isolate specific tRNAs in the microgram and milligram range. It was also capable of resolving isoaccepting tRNA species. Hydrophobic-interaction chromatography (HIC) is effective for resolution of tRNAs on weakly hydrophobic stationary phases that are prepared by either reacting free silanol groups on the surface of silicagel with C 1 -C 4 alkyltri-chlorosilanes or by acylating part of the primary amino groups of an aminopropyl bonded-phase silica support (APS-Hypersil) with aliphatic organic acids of C 2 -C 8 chain lengths.