Normal-phase chromatography separation modes

Normal Phase Chromatography—Separations mode run on nonbonded, anhydrous porous silica using a nonpolar mobile phase. See Adsorption Chromatography.)... 

Reverse-Phase Chromatography—Separation mode on bonded phase columns in which the solvent/column polarities are the opposite of normal-phase separations. Polar compounds elute before nonpolar compounds, Nonpolar columns require polar solvents. 

Normal-phase chromatography is still widely used for the determination of nonpolar additives in a variety of commercial products and pharmaceutical formulations, e.g. the separation of nonpolar components in the nonionic surfactant Triton X-100. Most of the NPLC analyses of polymer additives have been performed in isocratic mode [576]. However, isocratic HPLC methods are incapable of separating a substantial number of industrially used additives [605,608,612-616], Normal-phase chromatography of Irgafos 168, Irganox 1010/1076/3114 was shown [240]. NPLC-UV has been used for quantitative analysis of additives in PP/(Irganox 1010/1076, Irgafos 168) after Soxhlet extraction (88%... 

Adsorption Chromatography—Separation mode resulting from compounds that have different adhesion rates for the packing surface. (See Normal-Phase Chromatography.)... 

Normal phase (NP) separations are comparatively rarely used in environmental analysis. Again, the reasons lie in the range of analytes amenable to this mode of separation, and in the limited compatibility of typical normal phase HPLC (NP-HPLC) mobile phases with mass spectrometric detection (this also applies to IC). Not only for this reason has interest recently grown in hydrophilic-lipophilic interaction chromatography (HILIC), which represents a viable alternative to the separation of very polar compounds with mobile phases that have a much better compatibility with MS detection, for example, acetonitrile/water with a low water content, typically below 10%, 32 Nonetheless, NP chromato-graphy retains its important role in sample preparation, particularly for the cleanup of complex environmental samples. In the off-line approach, fractions are collected and the relevant one is injected into the reversed phase HPLC (RP-HPLC) system, often after solvent exchange. 

A less obvious example of normal-phase chromatography is the separation of saccharides and oligosaccharides in foods910 and in biological mixtures,1112 using a mobile phase consisting of acetonitrile/water or acetonitrile/dilute phosphate buffer. Although the separation mode has occasionally been misidentified as reversed phase, it is normal phase by virtue of the fact that increased aqueous levels of the mobile phase reduce carbohydrate retention, and elution order follows carbohydrate polarity.1... 

Reversed-phase HPLC is the dominant method used for the majority of pharmaceutical applications (>95%). Normal-phase chromatography may be required for separations that are not compatible with reversed-phase mode. 

The first step in method development is selecting an adequate HPLC mode for the particular sample. This choice depends on the character of the sample compounds, which can be either neutral (hydrophilic or lipophilic) or ionic, low-molecular (up to 2000 Da) or macromolecular (biopolymers or synthetic polymers). Many neutral compounds can be separated either by reversed-phase or by normal-phase chromatography, but a reversed-phase system without ionic additives to the aqueous-organic mobile phase is usually the best first choice. Strongly lipophilic samples often can be separated either by non-aqueous reversed-pha.se chromatography or by normal-phase chromatography. Positional isomers are usually better separated by normal-phase than by reversed-phase chromatography and the separation of optical isomers (enantiomers) requires either special chiral columns or addition of a chiral selector to the mobile phase. 

There are also stationary phases that effectively partition solutes in either reversed-phase or normal-phase mode. These stationary phases are typically silica particles derivatized with cyano, diol, or amino functional groups. Particles with a cyano-functionality separate based on polarity utilizing nitrile interactions between the stationary phase and the solute. The amino group of typical amino stationary phases interacts primarily with anionic and organic acid portions of the solute. Diols utilize hydroxyl interactions similar to underivatized silica but offer a slightly different selectivity. These and other bonded-silica phases offer alternatives to underivatized silica, but they are used much less frequently. The mobile phases employed with these stationary phases are the same as used in standard reversed-phase or normal-phase chromatography. 

Figure 1.3. Schematic diagrams depicting separation modes of (a) normal-phase chromatography (NPC) and (b) reversed-phase chromatography (RPC).

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. 

Both reversed phase and normal phase chromatography have been used for the separation of the androstanes. In general normal phase chromatography using unmodified silica as the stationary phase is the preferred mode of separation since better resolution of all the epimeric steroids except the 17-epimers can be achieved (Hunter et al.,... 

In the reversed-phase mode, higher separation selectivity than in normal-phase chromatography can be usually achieved for compounds with even minor differences in the size of the molecules. This is the main reason for the predominant role of reversed-phase separations in contemporary HPLC. On the other hand, NPLC usually offers significantly better separation of isomers than RPLC. 

Due to the complex stmcture of these antibiotics, most of them function equally well in reversed, normal and modified polar ionic phases. All three solvent modes generally show different selectivities with different analytes. Sometimes, equivalent separations are obtained in both the normal and the reversed phase mode. This ability to operate in two different solvent modes is an advantage in determining the best preparative methodology where sample solubility is a key issue. In normal phase chromatography, the most commonly used solvents are typically hexane, ethanol, methanol and so on. The optimization of chiral resolution is achieved by adding some other organic solvent, such as acetic acid, tetrahydrofuran (THF), diethylamine (DBA) or triethylamine (TEA) [50, 51]. 

Combining different separation techniques governed by different mechanisms to a multidimensional method is suitable to increase the potential of the individual techniques by an order of magnitude (31,32). HPLC is one of the most powerful separation techniques available today for nonvolatile substances. For reasons mentioned above, HPLC most often employs the reverse phase separation mode. On-line coupling of HPLC with AMD using normal phase chromatography results in separation numbers around 500. 

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