Aqueous normal-phase chromatography

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Aqueous normal phase chromatography
Classification chromatography
Other techniques

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).


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[1] and long-chain alkyl groups[2]. 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[citation needed]).


An interesting feature of these phases is that both polar and nonpolar compounds can be retained over some range of mobile phase composition (organic/aqueous). The retention mechanism of polar compounds has not been elucidated yet for the silica hydride phases. This property distinguishes it from a pure HILIC (hydrophilic interaction chromatography) column where separation by polar differences is obtained, 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 0.1 to 0.5% formic or acetic acid, which is compatible with detector techniques that include mass spectral analysis.


  1. ^ J.J. Pesek, M.T. Matyska, LCGC, 24 (2006) 296
  2. ^ Pesek, J. J.; Matyska, M. T.; Prabhakaran, S. J. (2005). "Synthesis and characterization of chemically bonded stationary phases on hydride surfaces by hydrosilation of alkynes and dienes". Journal of Separation Science 28 (18): 2437. doi:10.1002/jssc.200500249. PMID 16405172.  edit
  3. ^ Pesek, J. J.; Matyska, M. T.; Gangakhedkar, S.; Siddiq, R. (2006). "Synthesis and HPLC evaluation of carboxylic acid phases on a hydride surface". Journal of Separation Science 29 (6): 872. doi:10.1002/jssc.200500433. PMID 16830499.  edit
  4. ^ Hemström, P.; Irgum, K. (2006). "Hydrophilic interaction chromatography". Journal of Separation Science 29 (12): 1784–821. doi:10.1002/jssc.200600199. PMID 16970185.  edit