Aqueous normal-phase chromatography

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Here we discuss a basic thermodynamic description of the effect of the type and composition of the mobile phase. In most of the HPLC separations binary eluents are employed. One of the solvents in the eluent is usually inert binary mobile phase to the surface interactions. And it does not compete with the analyte for the adsorption sutes. Another binary mobile phase of any binary eluent is an active one.

It usually called a "modifier" because it can interact with the adsorbent surface and compete with analyte molecules for binary mobile phase adsorption sites. Increasing of the concentration of the "modifier" in the eluent leads to the decreasing of the analytes retention. As we discussed before, capacity factor binary mobile phase proportional to the thermodynamic equilibrium constant, and the last is an exponent of the free Gibbs energy of the system.

In the formula 23 we have a difference of the free energy of analyte and eluent interactions with the surface. For the binary eluent system we can assume that only the "modifier" can interact with the surface. As a rough approximation we can assume that the total free Gibbs energy of that adsorption system could be considered as a difference of analyte adsorption energy and a product of modifier adsorption energy and its mole fraction.

Thus, component retention volume could be roughly estimated by using the binary mobile phase. Points are experimental values and a curves was calculated using the above equation. Retention dependencies of alkylbenzenes vs. This type of binary mobile phase influence of the mobile phase composition and its thermodynamic explanation are true for the chromatographic system with only hydrophobic interactions dispersive binary mobile phase.

In case of the presence of any specific adsorption sites, the analyte behavior may significantly differ from that described above. Any specific interactions of the analyte molecules with the eluent molecules also may introduce significant deviations to the analyte retention dependence.

Ionizible components usually show a specific behavior. Organic acids are binary mobile phase solvated with the water molecules, which block possible interaction of the hydrophobic binary mobile phase of the molecule with the adsorbent surface, and lead to the very early elution of these compounds. Organic basis usually shows a low retention also due to solvation, but in case of presense of strong acidic accessible adsorption sites on the adsorbent they show a strong retention.

A general approach to the separation of the mixtures containing an ionisible components is to suppress their ionization. Suppression of the ionization decreases a power of the molecular solvation and exposes the hydrophobic organic part of the binary mobile phase to the surface interaction.

Ionization suppression is usually made by the adding a buffer into the solvent, which shift a pH to the certain value. In the absence of buffer, easy ionizible components are eluted from the column as very broad peaks. According to the Le Chatelier principle, dissolved ionizible component is present in the solution as a mixture of ions and nonionized molecules.

But, the chromatographic behavior of ions and neutral molecules are different. Let us assume that neutral molecules will be retained, so during the run ions will move faster, and at the first moment they will be separated from the neutral molecules. But, according to the above equilibrium, in the absence of the neutral molecules ions will tend to form them, and this new neutral molecule will also be absorbed, and so on.

This process will lead to the spreading of the component along the column and causes the appearance of the broad peak.

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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 [1] 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.

Principle [ edit ] In normal-phase chromatography, the stationary phase is polar and the mobile phase is nonpolar. Journal of Separation Science. Chem B, A, Retrieved from " https: Views Read Edit View history. This page was last edited on 28 January , at By using this site, you agree to the Terms of Use and Privacy Policy. Hydrophilic interaction liquid chromatography Ion exchange chromatography.