Normal-phase chromatography polar adsorbent

Bonded phases may be used in both normal and reverse phase chromatography. When normal phase chromatography is done on bonded phase packings, the packing is more polar than the mobile phase. Polar bonded phases such as the cyanopropyl and aminopropyl functionalities are popular for this use. These bonded phases are less subject to changing retention times of compounds because water is adsorbed from the mobile phase onto the stationary phase, a frequent concern when using bare silica packings for normal-phase separations. 

The silanol group (Si-OH), present at least at low concentrations in all reversed-phase columns, is capable of adsorbing a layer of a polar solvent such as methanol or water. A column conditioning procedure based on this adsorption is an essential step in normal-phase chromatography on silica columns. This process is probably also important in reversed-phase... 

Unlike the more popular reversed-phase chromatographic mode, normal-phase chromatography employs polar stationary phases, and retention is modulated mainly with nonpolar eluents. The stationary phase is either (a) an inorganic adsorbent like silica or alumina or (b) a polar bonded phase containing cyano, diol, or amino functional groups on a silica support. Tlie mobile phase is usually a nonaqueous mixture of organic solvents. As the polarity of the mobile phase decreases, retention in normal-phase chromatography... 

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 order to accomplish the desired separation, the selection of appropriate stationary phase and eluent system is imperative. The most commonly used stationary phases in normal-phase chromatography are either (a) inorganic adsorbents such as silica and alumina or (b) moderately polar chemically bonded phases having functional groups such as aminopropyl, cyanopropyl, nitrophenyl, and diol that are chemically bonded on the silica gel support [16]. Other phases that are designed for particular types of analytes have also... 

The main use of unmodified silica in HPLC is as an adsorbent in normal phase chromatography where the mobile phase is less polar than the stationary phase, and in this mode a non-aqueous mobile phase is commonly selected. Due to the high polarity of water and its strong affinity for free silanols on the silica surface, the presence of even very... 

The principle of adsorption chromatography (normal-phase chromatography) is known from classical column and thin-layer chromatography. A relatively polar material with a high specific surface area is used as the stationary phase, silica being the most popular, but alumina and magnesium oxide are also often used. The mobile phase is relatively nonpolar (heptane to tetrahydrofuran). The different extents to which the various types of molecules in the mixture are adsorbed on the stationary phase provide the separation effect. A nonpolar solvent such as hexane elutes more slowly than a medium-polar solvent such as ether. 

Solvents commonly used in normal phase chromatography are aliphatic hydrocarbons, such as hexane and heptane, halogenated hydrocarbons (e.g., chloroform and dichloromethane), and oxygenated solvents such as diethyl ether, ethyl acetate, and butyl acetate. More polar mobile phase additives such as isopropanol, acetone, and methanol are frequently used see Table 2). The technique is particularly suited to analytes that are very hydrophobic, e.g., fat-soluble vitamins such as tocopherols (6J and other hydrocarbon-rich metabolites that exhibit poor solubility in the water-miscible solvents employed in other separation modes. In addition, since the geometry of the polar adsorbent surface is fixed, the technique is useful for the separation of positional isomers the proximity of functional groups to the adsorbent surface, and hence the strength of interaction, may well differ between isomers. 

Before the development of reversed-phase bonded phases, normal-phase chromatography was the most popular separation technique. It relies on the interaction of analytes with polar functional groups on the surfooe of the stationary phase, which is strongest when nonpolar solvents are used as mobile phase. Previously, it was also called adsorption chromatography. However, the technique has expanded from the exclusive application of metal oxide adsorbents such as silica and alumina as stationary phases to the use of polar bonded phases. Thus the name adsorption chromatography has become too narrow. 

Retention volumes in normal-phase chromatography depend much on the polarity and polarizability of the analyte molecules, and little on their molecular masses [42]. Thus, all alkyl benzenes, or all alkyl esters, will be eluted in a narrow range of retention volumes. On the other hand, structural changes affecting the polarity of a molecule are easily seen. The amount of water adsorbed by the stationary phase controls its activity, hence the resolution between analytes, and this is in turn a sensitive function of water dissolved in the mobile phase. Thus, control of the water content of the eluent is a primary concern. The influence of the composition of the mobile phase on retention has been discussed by Snyder, who introduced the concept of eluotropic strength [42],... 

Normal Phase Chromatography. Normal phase mode involves the polar interaction of sample molecules with a polar stationary phase, commonly silica-based packings with an aminopropyl bonded phase. Alternatively, pure silica gel modified in situ by equilibration (and simultaneous regeneration) with a mobile phase containing a polyfunctional amine that is adsorbed could be used (47). 

Normal-phase chromatography can also be applied to concentrate polar analytes that are dissolved in a nonpolar solvent, such as hexane. The hexane can be passed through a polar solid phase such as silica, Florisil, alumina, or a diol to adsorb the polar analyte. The polar analyte can be eluted with a smaller volume of a more polar solvent (e.g., ethyl acetate). Normal-phase SPE is not used very much for trace enrichment in environmental analysis, but is useful for matrix isolation and cleanup techniques. 

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