General Chromatographic Considerations

The aim of any chromatographic separation may be defined as the achievement of an optimal combination of speed of elution, sample size, and resolution of the solutes. Good resolution can only be obtained if there is adequate control over the differential migration rates of a group of solutes as they move down a column (column selectivity) and over the extent of zone dispersion for each of the solutes (column efiiciency). Historically, the various modes of liquid chromatography have been considered as separate and independent phenomena. It is now clear that they all have a common theoretical basis. Column selectivity in HPLC, irrespective of the mode, arises due to differences in the distribution equilibria 

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 steric exclusion (gel permeation) separations, the retention of partially retained solutes is described by 

For icNi-exchange HPLC, the retention of a cationic peptidic solute of charge z can be related to the ionic strength of the eluent and the pH such that 

With polar liquid-liquid adsorption chromatography, based on chemically bonded normal-phase systems, the distribution coefficient can be equated with the solubility parameter 8j of a solute such that retention is given by 

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The sample preparation in LC analysis is as important as the chromatographic separation itself. The procedure will often require considerable skill copied with a basic understanding of chromatographic methodology. The analyst will need to have some familiarity with micro techniques including general micro-manipulation, microfiltration, centrifugation and derivatization. 

Some general considerations governing the nature of selective enantiomeric interactions for both gas and liquid chromatographic phases (at least of the bonded monomeric ligand type) have been forthcoming [721,742,754,756,781,782,790). It is generally assumed that three points of simultaneous interaction at least one of which must be stereochemically controlled, are required to distinguish the chirality of a molecule. These... 

Most publications dealing with chromatographic reactors focus on theoretical issues of this very complex system. Models of different complexity were derived and used to predict the behavior of chromatographic reactors. Such models typically take into consideration different types of mass transfer, adsorption isotherms, flow profiles, and reactions. A general scheme of these models, not including the reaction, is presented in Fig. 4. There are also several review papers... 

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