Aqueous normal phase chromatography
Encyclopedia
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).
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 silanol
s (-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 ).
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.
Chromatography
Chromatography is the collective term for a set of laboratory techniques for the separation of mixtures....
technique which involves the mobile phase region between reversed-phase chromatography (RP) and organic normal-phase chromatography (ONP).
Principle
In normal-phase chromatography, the stationary phase is polarChemical polarity
In chemistry, polarity refers to a separation of electric charge leading to a molecule or its chemical groups having an electric dipole or multipole moment. Polar molecules interact through dipole–dipole intermolecular forces and hydrogen bonds. Molecular polarity is dependent on the difference in...
and the mobile phase is nonpolar
Chemical polarity
In chemistry, polarity refers to a separation of electric charge leading to a molecule or its chemical groups having an electric dipole or multipole moment. Polar molecules interact through dipole–dipole intermolecular forces and hydrogen bonds. Molecular polarity is dependent on the difference in...
. 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
Cyanide
A cyanide is a chemical compound that contains the cyano group, -C≡N, which consists of a carbon atom triple-bonded to a nitrogen atom. Cyanides most commonly refer to salts of the anion CN−. Most cyanides are highly toxic....
and amino
Amine
Amines are organic compounds and functional groups that contain a basic nitrogen atom with a lone pair. Amines are derivatives of ammonia, wherein one or more hydrogen atoms have been replaced by a substituent such as an alkyl or aryl group. Important amines include amino acids, biogenic amines,...
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
Hexane
Hexane is a hydrocarbon with the chemical formula C6H14; that is, an alkane with six carbon atoms.The term may refer to any of four other structural isomers with that formula, or to a mixture of them. In the IUPAC nomenclature, however, hexane is the unbranched isomer ; the other four structures...
or heptane
Heptane
n-Heptane is the straight-chain alkane with the chemical formula H3C5CH3 or C7H16. When used as a test fuel component in anti-knock test engines, a 100% heptane fuel is the zero point of the octane rating scale...
mixed with a slightly more polar solvent such as isopropanol, ethyl acetate
Ethyl acetate
Ethyl acetate is the organic compound with the formula CH3COOCH2CH3. This colorless liquid has a characteristic sweet smell and is used in glues, nail polish removers, and cigarettes...
or chloroform
Chloroform
Chloroform is an organic compound with formula CHCl3. It is one of the four chloromethanes. The colorless, sweet-smelling, dense liquid is a trihalomethane, and is considered somewhat hazardous...
. 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
Methanol
Methanol, also known as methyl alcohol, wood alcohol, wood naphtha or wood spirits, is a chemical with the formula CH3OH . It is the simplest alcohol, and is a light, volatile, colorless, flammable liquid with a distinctive odor very similar to, but slightly sweeter than, ethanol...
, acetonitrile
Acetonitrile
Acetonitrile is the chemical compound with formula . This colourless liquid is the simplest organic nitrile. It is produced mainly as a byproduct of acrylonitrile manufacture...
or THF
Tetrahydrofuran
Tetrahydrofuran is a colorless, water-miscible organic liquid with low viscosity at standard temperature and pressure. This heterocyclic compound has the chemical formula 4O. As one of the most polar ethers with a wide liquid range, it is a useful solvent. Its main use, however, is as a precursor...
. Retention increases as the amount of the polar solvent (water) in the mobile phase increases. Normal phase chromatography, an adsorptive
Adsorption
Adsorption is the adhesion of atoms, ions, biomolecules or molecules of gas, liquid, or dissolved solids to a surface. This process creates a film of the adsorbate on the surface of the adsorbent. It differs from absorption, in which a fluid permeates or is dissolved by a liquid or solid...
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 silanol
Silanol
Silanol, also known as silyl alcohol, is a chemical with formula SiH3OH. It is the simplest silicon alcohol, and is a heavy, volatile, colorless, flammable liquid. At room temperature it is a polar liquid...
s (-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 ).
Features
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
Formic acid
Formic acid is the simplest carboxylic acid. Its chemical formula is HCOOH or HCO2H. It is an important intermediate in chemical synthesis and occurs naturally, most notably in the venom of bee and ant stings. In fact, its name comes from the Latin word for ant, formica, referring to its early...
or acetic acid, which is compatible with detector techniques that include mass spectral analysis.