ABSTRACT
This chapter describes four primary modes of separation. These modes may be classified as normal-phase chromatography (NPC), reverse-phase chromatography (RPC), ion-exchange chromatography (IEC), and size-exclusion chromatography (SEC). Hydrophilic interaction chromatography (HILIC), a variation of NPC, is also included. Method development strategies that include column selection, identifying the optimal pH, selection of mobile phase and modifiers, and setting column temperatures, are described. A variety of column packing types is outlined. Silica (SiO2) support material in column packing is discussed as a primary packing phase. Typical bonded phases used in RPC, NPC, and IEC are reviewed. Detectors interfaced with high-performance liquid chromatography (HPLC) and selection criteria are discussed. Detectors described include UV/Vis, refractive index, fluorescence, conductivity, mass spectrometry, and evaporative light scattering detector (ELSD). Method development and compatibility with LC-NMR are discussed. The convention of using the reversed-phase HPLC mode with mobile phases (acetonitrile/water or methanol) employed in LC-NMR is included. Integration of CE and NMR and the process of transitioning from analytical to preparative chromatography and general considerations with respect to equipment integration and specialized expertise are described.
