Principle
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 increases as the amount of nonpolar 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 surfaces" distinguish 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. Mobile phases for ANPC are based on an organic solvent (such as methanol or acetonitrile) with a small amount of water; thus, the mobile phase is both "aqueous" (water is present) and "normal" (less polar than the stationary phase). 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.
Typically the amount of the nonpolar component in the mobile phase must be 60% or greater with the exact point of increased retention depending on the solute and the organic component of the mobile phase. 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. Thus a continuum of solvents can be used from 100% aqueous to pure organic. ANP retention has been demonstrated for a variety of polar compounds on the hydride based stationary phases (J.J. Pesek, M.T. Matyska, J. Sep. Sci., in press).
Read more about this topic: Aqueous Normal-phase Chromatography
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