Normal-phase adsorption

Dugo, P, Favoino, O., Luppino, R., Dugo, G., Mondello, L. (2004). Comprehensive two-dimensional normal-phase (adsorption)-reversed-phase liquid chromatography. Anal. Chem. 76, 2525-2530. 

To eliminate these effects, a mobile phase is chosen that is a good solvent for the polymer and whose solubility parameter, 8, is close to that of the packing. Thus the polymer and packing are well solvated and potential adsorptive sites on both are "deactivated". As demonstrated by Dawkins (21,22) and Mori (10,23), if > solvent normal-phase adsorption... 

The nature of the stationary phase within an LC column is of critical importance for determining the separation process. Normal-phase adsorption, normal-... 

In normal-phase adsorption chromatography, the stationary phase is composed of strongly polar silica particles whereas the mobile phase is composed of nonpolar solvents such as n-hexane. Analytes are separated due to their varying degree of adsorption onto the solid surface. Polar samples are thus retained on the polar surface of the column packing longer than less polar materials. 

Normal-phase adsorption on silica gel with a relatively less polar mobile phase is the most widely used mode in conventional TLC. To improve separations, silica gel may be impregnated with various solvents, buffers, and selective reagents. Other commercial precoated layers include alumina, florisil, polyamide, cellulose, and ion exchangers. 

Figure 3.17 Normal-phase adsorption chromatography on silica.

Hydrophobic Effect. The primary retention force in normal phase adsorption is the attraction of solute polar moieties to the polar stationary phase. In contrast, the retention force in RPLC is repulsion from the mobile phase. The stationary phase is a relatively passive surface, the solute attraction for the hydrocarbon stationary phase being weak and non-selective. How does this come about, and what are the resultant selectivity characteristics ... 

The compatibility of electrochemical detection with the various modes of liquid chromatography is limited. For all practical purposes, electrochemical detection is not suitable for use with normal phase adsorption or partition chromatography due to the solvents of low dielectric constant used as the mobile phase. On the other hand, reverse-phase adsorption and partition (including ion-exchange or ion-pairing systems) are highly com-... 

In this section peptide separation by gel permeation, normal-phase adsorption, ion-exchange, and liquid-solid adsorption HPLC will be considered. Compared to reversed-phase HPLC, these elution modes have attracted considerably less interest for peptide separations. This is due to a number of factors arising from the diverse characteristics of the solutes... 

From the literature there is evidence that in GC on polar phases and in normal-phase (adsorption) liquid chromatography (HPLC and TLC) the chemically specific, molecular size-independent intermolecular interactions play the main retention-determining role. For example, the HPLC retention parameters determined for substituted benzenes on porous graphite are described by QSRR equations comprising polarity descriptors but containing no bulk descriptors [93-95]. Because, in general, it is difficult to quantify the polarity properties precisely, the QSRR for GC on polar phases and for normal-phase HPLC are usually of lower quality than in the case of GC on non-polar phases and in the case of reversed-phase liquid chromatography. 

However, in normal phase adsorption systems (or liquid-solid chromatography) the interaction of the mobile phase solvent with the solute is often less Important than the competing Interactions of the mobile phase solvent and the solute with the stationary phase adsorption sites. Solute retention is based upon a displacement mechanism. Multicomponent mobile phases and their combination to optimize separations in liquid-solid chromatography have been studied in detail (31-35). Here, solvents are classified as to their interaction with the adsorption surface (Reference 32, in particular) ... 

A secondary goal of these preliminary studies was to characterize supercritical fluid normal phase adsorption chromatography ... 

As already discussed, RP-HPLC is suitable for the majority of natural product isolations, though the hydrophobicity of the desired matabolite/s might dictate the use of normal phase operation. An example of the application of this mode of operation to natural products work is the isolation of tocopherols from soya bean oil and wheat germ (6). This example also emphasizes the power of normal phase (adsorption) chromatography for the separation of positional isomers. Baseline resolution of a, p, y, and 8-tocopherol (Fig. 16) was achieved under semipreparative HPLC conditions utilizing a lO-pm silica stationary phase (Alltech Econosil). It is notable that P- and y-tocopherols were completely resolved even though they differ only in the position of one methyl substituent on the benzopyran nucleus. 

Solvent strengths are also measured in terms of polarity, and dielectric constants are generally used to quantify relative strengths (Table 1). A high dielectric constant indicates a polar solvent with a strong power of elution, and a low dielectric constant indicates a nonpolar solvent with a lower ability to elute a component from a sorbent. This elution strength applies to normal phase adsorption chromatography. 

It has been shown that there are many approaches to the separation of heterocyclic bases by chromatographic techniques. Normal-phase adsorption, reversed-phase, ionpairing, and ion-exchange chromatographic methods have been reported extensively in the cited literature. 

A later study (Aguas 2006) adopted a somewhat different approach to the analysis of acrylamide and extended it to the case of cocoa, which is at least as complex a matrix as coffee. The initial extraction of acrylamide and its C3-intemal standard with water was essentially unchanged, but the aqueous extract was then defatted by hquid-liquid extraction with dichloromethane. The subsequent clean-up strategy was very different from the earlier methods (Tareke 2002 Andrzejewski 2004) in that a normal phase SPE sorbent was used to selectively elute the amide from more polar impurities. The SPE phase chosen for this purpose was of the aminopropyl type that has weak anion exchange properties in addition to the characteristic normal phase adsorption properties. However, such an approach requires that the cocoa extract must be in a relatively nonpolar solvent so that acrylamide is retained on... 

FIGURE 7.12 Comprehensive normal-phase (adsorption) liquid chromatography (LC) x reversed-phase LC separation of the oxygen heterocyclic fraction of a lemon oil sample (for peak identi cation, see Ref. [133]). (From Dugo, P. et al., Anal. Chem., 73, 2525, 2004. With permission.)... 

In normal phase LLC the stationary phase is polar and the mobile phase consists of nonpolar solvents. This mode is suited for more polar, water-soluble analytes. The elution order is similar to that in normal phase adsorption chromatography because nonpolar solutes prefer the moving phase and elute with low k values. Solutes preferring the polar stationary phase elute later with high k. ... 

Normal-phase adsorption HPLC has been employed, as well as ion-exchange chromatography, bounded-NH2 partition chromatography, reversed-phase chromatography, and ion pair reversed-phase chromatography. Cjg columns have been the most employed. RP-8, NH2, and silica columns have been also employed. UV detection has been the most used technique. However, electrochemical detection, as well as fluorescence after derivatization, can also be used. 

---