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Hydrophilic interaction liquid chromatography. Aqueous normal-phase chromatography ANP is a chromatographic technique which involves the mobile phase region between reversed-phase chromatography RP and organic normal-phase chromatography ONP. In normal-phase chromatography, the stationary phase is polar and the mobile phase is nonpolar. In reversed phase we have just the opposite; the stationary phase is nonpolar and the mobile phase is polar.
Typical stationary phases for normal-phase chromatography are silica or organic moieties with cyano and amino functional groups. For reversed phase, alkyl hydrocarbons are the preferred stationary phase; octadecyl C18 is the most common stationary phase, but octyl C8 and butyl C4 are also used in some applications.
The designations for the reversed phase materials refer to the length of the hydrocarbon chain. In normal-phase chromatography, the least polar compounds elute first and the most polar compounds elute last.
The mobile phase consists of a nonpolar solvent such as hexane or heptane mixed with a slightly more polar solvent such as isopropanol , ethyl acetate or chloroform. Retention decreases as the amount of polar solvent in the mobile phase increases. In reversed phase chromatography, the most polar compounds elute first with the most nonpolar compounds eluting last. The mobile phase is generally a binary mixture of water and a miscible polar organic solvent like methanol , acetonitrile or THF.
Retention increases as the amount of the polar solvent water in the mobile phase increases. Normal phase chromatography, an adsorptive mechanism, is used for the analysis of solutes readily soluble in organic solvents, based on their polar differences such as amines, acids, metal complexes, etc..
Reversed-phase chromatography, a partition mechanism, is typically used for separations by non-polar differences. The "hydride surface" distinguishes the support material from other silica materials; most silica materials used for chromatography have a surface composed primarily of silanols -Si-OH. In a "hydride surface" the terminal groups are primarily -Si-H.
The hydride surface can also be functionalized with carboxylic acids  and long-chain alkyl groups. Thus, polar solutes such as acids and amines are most strongly retained, with retention decreasing as the amount of water in the mobile phase increases. A true ANP stationary phase will be able to function in both the reversed phase and normal phase modes with only the amount of water in the eluent varying.
ANP retention has been demonstrated for a variety of polar compounds on the hydride based stationary phases. Recent investigations have demonstrated that silica hydride materials have a very thin water layer about 0. The retention mechanism of polar compounds has recently been shown to be the result of the formation of a hydroxide layer on the surface of the silica hydride.
This property distinguishes it from a pure HILIC hydrophilic interaction chromatography columns where separation by polar differences is obtained through partitioning into a water-rich layer on the surface, or a pure RP stationary phase on which separation by nonpolar differences in solutes is obtained with very limited secondary mechanisms operating. Another important feature of the hydride-based phases is that for many analyses it is usually not necessary to use a high pH mobile phase to analyze polar compounds such as bases.
The aqueous component of the mobile phase usually contains from 0. From Wikipedia, the free encyclopedia. Aqueous normal phase chromatography Classification chromatography Other techniques Related Hydrophilic interaction liquid chromatography Ion exchange chromatography Aqueous normal-phase chromatography ANP is a chromatographic technique which involves the mobile phase region between reversed-phase chromatography RP and organic normal-phase chromatography ONP.