Normal-phase chromatography alumina

Alumina has been widely used in normal phase chromatography. Its surface exhibits amphoteric properties (available as basic, acidic, and neutral alumina) and can react as a cation or an anion exchanger. The surface area is in the usual range around lOOm /g, however, the pore structure is not very favorable as a relatively large portion of narrow pores (<2nm) is present. 

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... 

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 solvent-strength parameters for the common solvents used in normal phase chromatography on carbon are quite different from those of silica or alumina. Thus carbon offers quite different selectivities than alumina and silica for normal-phase chromatography. However, the lack of a reproducible commercial source for carbon was for many years a significant limitation to its widespread application. In addition the sensitivity of carbon to changes in solvent strength is much less than that of silica or alumina. 

Despite the limitations of unmodified silica as a stationary phase for normal phase chromatography, it remains the most common adsorbent in this field, although there has been a recent renewal of interest in the use of alumina. 

Non-polar supports like polystyrene/divinylbenzene copolymers or carbon are also used as column materials. Alumina is polar and acidic while TiOi, and zirconia are much more neutral. They all have good aqueous stability compared to silica. Normal phase chromatography is restricted to the separation of stereochemical isomers, diastereomers, low molecular weight aromatic compounds and functionalized long chain aliphatic compounds. 

Retention in normal-phase chromatography increases as the polarity of the mobile phase decreases. The selectivity of the analytes may arise from the differences in solvent strengths (eq), acidity, basicity, and dipolar nature of the mobile phase. Furthermore, solvent localization of the mobile phase plays a major role in the retention of the analytes [15,16]. These solvent strengths have been shown to be different when used with varied stationary-phase packings such as alumina, diol, and silica [3,17]. 

In normal-phase chromatography the native, non-modified adsorbent is employed with organic solvent mixtures as eluents. Normal phase chromatography was the classical chromatography mode performed with native silica or alumina, i.e. the adsorbent s surface is hydrophilic and the interaction with the solutes takes place via the hydroxy] groups on the surface. As an example the surface of silica consists of... 

In general, the eluting strength of commonly used solvents for normal phase chromatography is stationary phase = silica gel neutral alumina increasing order proceeds as follows petroleum ethers < hexanes < cyclohexane < toluene < diethyl ether < dichloromethane < chloroform < ethyl acetate < acetone < ethanol < methanol < acetic acid. 

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. 

Normal-phase sorbents such as silica and Florisil are used to isolate low to moderate polarity species from nonaqueous solutions. Examples of applications include lipid classification, plant pigment separations, and separations of fat-soluble vitamins from lipid extracts, as well as the clean-up of organic solvent concentrates obtained from a previous SPE method or liquid-liquid extraction. Alumina is used to remove polar species from nonaqueous solutions. Examples include vitamins in feeds and food and antibiotics and other additives from feed. Normal-phase chromatography has been used for a number of years, and most applications for normal-phase column chromatography may be easily transferred over to normal-phase SPE. 

Normal phase chromatography, where the stationary phase is polar in nature e.g., silica or alumina) and the mobile phase is non polar e.g., hexane). In this mode, polar samples are retained more strongly by the column therefore allowing elution of non polar compounds first. 

Normal-phase, bonded-phase columns are likely underutilized for separations where they should be the method of choice. This is due both to the ease of use of reversed-phase, bonded-phase columns, discussed next, and also to the many problems inherent in the use of bare silica and alumina. Very straightforward method development in normal-phase chromatography can be performed by combining the solvent and stationary-phase selectivity triangles. The three columns, each used with the three recommended modifiers, should provide the maximum difference in selectivity available. These nine experiments, used in conjunction with chemometric optimization schemes, should then provide a ratio-... 

Normal phase chromatography is used where very nonpolar molecules take too long to elute from a reversed-phase column. It, too, is based on the principle that Uke dissolves Uke and uses polar columns, e.g. silica or alumina, and nonpolar mobile phases, e.g. toluene or hexane. Molecules distribute themselves between the stationary and mobile phases to different extents depending on factors such as polarity, size and pKa. Large nonpolar molecules will elute first. 

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. 

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. 

Diol phases arc about as retentive as silica or aminopropyl bonded phases (17). Compared to silica and alumina, diol phases are much less sensitive to the water content of the mobile phase. They arc also less reactive than aminopropyl phases. Furthermore, they can be washed with water without difficulty. Thus they are the best choice among the stationary phases for normal-phase chromatography. Although the stationary phase is readUy available from many sources, it is most commonly found as a packing for aqueous size-exclusion dromatography. 

Equilibration times vary widely with the type of chromatography and the history of the column. A colunm that had previously been equilibrated and was stored in the mobile phase should be up and running within a few, maybe 10, column volumes, provided the new mobile phase is really identical to the old mobile phase. If the column was stored in a solvent other than the mobile phase, equilibration times vary with the chromatographic mode. Normal-phase chromatography using silica or alumina columns may take a long time for equilibration several days at a flow rate of 1 column volume per minute have bran observed. The equilibration with normal-phase bonded phases is faster. 

In contrast to reversed-phase, the stationary phase in normal-phase chromatography is polar, usually silica or alumina, and uses nonpolar solvents, e.g., hexane and ethylacetate, that are not compatible with the API processes nsed in LC-MS. In normal-phase chromatography compounds elute progressively from the least to the most polar. The technique is not applicable to the highly polar compounds encountered... 

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