ABSTRACT
High-performance liquid chromatography (HPLC) has become the dominant analytical technique in pharmaceutical, chemical, and food industries, as well as in environmental laboratories, in clinical chemistry for therapeutic drug monitoring, and in bioanalysis [1]. The rise of HPLC to the most widely used instrumental analytical systems originates in part from the broad variety of selectivities introduced by the enormous number of stationary phases available and the easy adjustment of selectivity by changing the composition of the mobile phase. The classical separation systems based on pure silica or alumina-now called normal phase chromatography-with nonpolar mobile phases would not have provided the variety and the simplicity of separation methods, and the reproducibility, now state-of-the-art in HPLC. The availability of the so-called reversed phases (RPs) based on chemically modied silica, where adsorption is the highest from aqueous solutions, opened for HPLC direct access to aqueous, and hence, bioanalytical systems. These phases are the workhorses in HPLC. Their diversity allows us to select appropriate columns for a wide variety of applications ranging from separations of aromatic hydrocarbons, pharmaceuticals, and pesticides to applications
2.1 Introduction ............................................................................................................................ 47 2.2 The Base Material: Silica ....................................................................................................... 49
2.2.1 Physical Parameters .................................................................................................... 49 2.2.2 Measurement of Physical Parameters .........................................................................50 2.2.3 Chemical Properties of Silica .....................................................................................50 2.2.4 Bonding Technology ................................................................................................... 51 2.2.5 Bonded Phases with Functional Groups ..................................................................... 53 2.2.6 RP with Shielding Groups (Polar-Embedded RP) ......................................................54 2.2.7 RP with Other Functional Groups .............................................................................. 55 2.2.8 Alternative Bonding Technologies ............................................................................. 57 2.2.9 Other Inorganic Carriers for Bonded Phases.............................................................. 58 2.2.10 Polymers as Stationary Phases ................................................................................... 58 2.2.11 Porous Carbon ............................................................................................................ 58
2.3 Retention with RP ................................................................................................................... 58 2.3.1 Hydrophobic Properties of RP .................................................................................... 59 2.3.2 RP with Shape Selectivity ..........................................................................................60 2.3.3 Inuence of Analyte Structure on Retention .............................................................. 62 2.3.4 Inuence of Eluent Composition ................................................................................64 2.3.5 Acetonitrile or Methanol ............................................................................................64 2.3.6 Inuence of Temperature ............................................................................................68 2.3.7 Characterization and Comparison of RP ....................................................................69
