Chromatographic modes hydrophobic interaction

A common feature of protein retention in reversed-phase and other interactive chromatographic modes such as ion exchange and hydrophobic interaction... 

The mechanisms described above form the basis for the chromatographic modes described in Chapter 2, namely, normal-phase, reversed-phase, size-exclusion, ion-exchange, and affinity chromatographies. However, other modes that are variations of those mentioned above, such as hydrophobic-interaction chromatography (HIC), chiral, ion-exclusion, and ion-pair chromatographies are also used and will be mentioned. 

There are five major chromatographic modes that can be applied to the analysis of solutes in solution normal phase, reversed phase, ion exchange, size exclusion, and affinity. In addition, a variety of submodes exist, such as hydrophobic interaction, chiral separations, ion suppression, and ion pairing. 

The ultimate challenge for any chromatographic technique is to separate neutral, ionic, acidic, and basic compounds found in real samples in the same run. Toward this goal, a series of papers published simultaneous separations of acidic, neutral, and basic compounds. Klamfl et al. used a mixed-mode (C6/ SAX) silica-based packing to combine reverse EOF, hydrophobic interactions, as well as electrophoretic migration for the separation of a lab-made sample... 

In common with other application areas of chromatographic separation, a considerable amount of effort has been expended recently on the development of different elution conditions and types of stationary phases for peptide separations in attempts to maximize column selectivities without adversely affecting column efficiences. Peptide retention will invariably be mediated by the participation of electrostatic, hydrogen bonding, and hydrophobic interactions in the distribution phenomenon. The nature of the predominant distribution mechanism will be dependent on the physical and chemical characteristics of the stationary phase as well as the nature of the molecular forces which hold the solute molecules within the mobile and stationary zones. The retention of the solute in all HPLC modes can be described by the equation... 

In RPLC, the influence of pressure on the chromatographic behavior is related to the hydrophobic interactions involved in the retention mechanism and to the change upon adsorption in the numbers of acetonitrile and water molecules in the solvent shells of the protein molecule and of the bonded layer. The importance of the changes in the retention factor and the saturation capacity with a change in the average column pressure will thus depend on the retention mode used and will vary with the hydrophobicity of the molecule [128]. In RPLC, it is larger with polymeric than with monomeric bonded phases [126]. 

RPC-5 and other mixed mode chromatographic techniques are based on ionic as well as on hydrophobic interactions. RPC-5 is the earliest of these techniques having been introduced more than 20 years ago [26]. The resin applied originally consists of a charged reversed-phase matrix with a quaternary ammonium derivative (such as methyltrialkyl (Cg-Cjo) ammonium chloride) being adsorbed on a non-porous polymer support such as Plaskon or Teflon. In contrast to other HPLC-resins, the surface-forming groups of RPC-5 are physically adsorbed to the polymeric support and are not covalently bound therefore RPC-5, in principle, can be called a... 

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