Normal-phase chromatography retention mechanism

Detailed discussion of normal-phase chromatography process, mechanism, and retention theories, as well as types and properties of used stationary phases, is given in Chapter 5. 

The mechanism of reversed phase chromatography can be understood by contrast with normal phase chromatography. Normal phase liquid chromatography (NPLC) is usually performed on a polar silica stationary phase with a nonpolar mobile phase, while reversed phase chromatography is performed on a nonpolar stationary phase with a polar mobile phase. In RPLC, solute retention is mainly due to hydrophobic interactions between the solutes and the nonpolar hydrocarbon stationary surface. The nonpolar... 

Figure 5-1. Hypothetical representation of the adsorption mechanism of retention in normal-phase chromatography. S denotes sample molecule, E denotes molecule of strong polar solvent, and X and Y are polar functional groups of the stationary phase. Prior to retention, the surface of stationary phase is covered with a monolayer of solvent molecules E. Retention in normal-phase chromatography is driven by the adsorption of S molecules upon the displacement of E molecules. The solvent molecules that cover the surface of the adsorbent may or may not interact with the adsorption sites, depending on the properties of the solvent. (Reprinted from reference 1, with permission.)...

In normal-phase chromatography, polar stationary phases are employed and solutes become less retained as the polarity of the mobile-phase system increases. Retention in normal-phase chromatography is predominately based upon an adsorption mechanism. Planar surface interactions determine successful use of NPC in separation of isomers. The nonaqueous mobile-phase system used in NPC has found numerous applications for extremely hydrophobic molecules, analytes prone to hydrolysis, carbohydrates, and sat-urated/unsaturated compounds. In the future, with the advent of new stationary phases being developed, one should expect to see increasingly more interesting applications in the pharmaceutical industry. 

Regardless of the exact retention mechanism — adsorption, liquid-liquid partition or their combination — the stationary phase in normal-phase chromatography is more... 

Strong solvents can act and modify the retention of solutes in two different ways. In some cases, they compete with the feed components for adsorption. This is the mechanism through which they reduce the adsorption of the feed components and accelerate their elution. This is what occurs in normal phase chromatography when polar solvents are used as components of the mobile phase e.g., propanol added to dichloromethane). Usually, the concentration at which these additives are used is low, often of the order of a few percent. However, not all additives act by competition for retention. 

The retention mechanism and solvent selectivity have been studied most carefully with alumina or silica as stationary phases. The knowledge of both for bonded phases used in normal-phase chromatography is much more limited. Nevertheless, it is safe to assume that similar selectivity rules for solvent strength and selectivity can be applied, especially since the results obtained for alumina and silica correlate well with each other. 

Although SPE can be done in a batch equilibration similar to that used in LLE, it is much more common to use a small tube (minicolumn) or cartridge packed with the solid particles. SPE is often referred to as LSE, bonded phase or sorbent extraction SPE is a refinement of open-column chromatography. The mechanisms of retention include reversed phase, normal phase, and ion exchange. 

Those QSRR equations, which comprise physically interpretable structural descriptors, can be discussed in terms of the molecular mechanism of the chromatographic process [90]. There is literature evidence that different structural parameters of analytes account for retention differences in gas chromatography on polar as compared to non-polar stationary phases. Also, the structural descriptors in QSRR equations, which are valid for normal-phase HPLC, are different from those valid for reversed-phase HPLC. In the case of apparently similar chromatographic systems the differences in retentive properties of stationaty pha.ses may be reflected by the magnitude of the regression coefficients for analogous descriptors [9I.92]. Comparative QSRR studies are especially valuable when new chromatographic phases are introduced. 

Retention mechanisms and mobile phase ects in normal-phase liquid chromatography... 

Although the primary restilt is the same, i.e., the retention time of the component band decreases with increasing mobile phase concentration of the strong solvent, there are important differences in the behavior of overloaded columns in normal and in reversed phase chromatography. The competitive retention mechanism, its behavior in nonlinear chromatography, and its consequences when the strong solvent is adsorbed are discussed in Qiapter 13. The solubility mechanism is often much simpler. The isotherm coefficients merely depend on the concentration of the strong solvent in the mobile phase. 

The adsorption from solutions of polycyclic aromatic hydrocarbons, alkylbenzenes and benzene derivatives is of great interest from many points of view. To understand the mechanism of their adsorption and to predict the equilibrium adsorption constant also it is possible to use the contributions of functional groups of fragments of molecules to retention in reversed-phase and normal-phase liquid chromatography [21]. The contributions of different molecular groups as well as the dependence of these contributions on mobile phase composition have been evaluated from experimental data in work [25]. 

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