Normal-phase chromatography applications

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. 

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 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. 

There are many applications of normal-phase chromatography to separate all eight vitamers using silica or bonded silica phases such as diol or amino (Table 1.5 Figure 1.4). The separation of 3- and y-tocopherols and tocotrienols is the most difficult task, but it can be achieved with normal-phase HPLC. In addition to its... 

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. 

There are a number of modes of HPLC available to the analyst. The main difference between them is the type of column used. Two have been mentioned already - reversed-phase and normal phase chromatographies. Five are be discussed briefly here, each having their own applications and suitabilities for certain types of compound. An example of an LC instrument is shown in Figure 3.15. 

Reverse phase supports will bind from aqueous solution most analytes that contain a hydrophobic moiety or domain. Since the majority of secondary metabolites do possess some degree of hydrophobicity, such supports are of good general applicability to natural products work. In addition, most crude natural product extracts are, by their nature, highly heterogeneous and tend to exhibit better solubility in the water/water-miscible organic-solvent mixtures used in reverse phase HPLC than in the less polar solvents employed for normal phase chromatography. 

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 can also be influenced by other components of the mobile phase present in small concentrations. The user may not be aware of the presence of these components. In such a case, troubleshooting can be quite difiicult. A common example of this problem is water in normal-phase chromatography. Water is always present in all solvents (see Table 9.3) and can shift retention substantially. However, this is not the only example. For instance, a contamination of methanol with amines that influences the retention of basic analytes in reversed-phase chromatography has been observed. To avoid this situation, the use of HPLC-grade solvents is generally recommended for HPLC applications. 

Hydrophilic interaction chromatography can he viewed as an extension of normal-phase chromatography to the retdm of very polar analytes and aqueous mobile phases. Suitable stationary phases are the same as used in normal-phase chromatography. The most important application is the separation of sugars, oligosaccharides, and complex carbohyttotes. 

There are a few techniques related to normal phase chromatography that are worth mentioning. Silica can be coated with the salts of heavy metals. This results in unique selectivities for analytes that form complexes with the metal ions. An example of this is argentation chromatography (20). The silica is coated with silver nitrate, which gives it a spedal sel ivity for compounds with al atic double bonds. The technique has acquired a broad range of applications. However, while Ag -coated TLC plates are commercially available, HPLC columns have to be prepared by the user. Other metal salts can be used in a similar manner for different applications. 

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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