Normal phase chromatography defined

The strength of the solvent is defined by the solvent strength parameter, e°, as listed in Table 2.2. A solvent with a low e° is chosen, and quantities of a second solvent with a greater s° are added until the desired separation is achieved. If the desired separation does not result from altering the concentration of the second solvent, either the nature of the second solvent can be changed or another additive can be introduced. Readers are directed to Refs. 1, 6, and 7 for in-depth discussions on the development of mobile phases for normal-phase chromatography. 

Gel filtration/permeation chromatography (also known as molecular exclusion chromatography) is a form of partition chromatography in which the solute molecules are partitioned between solvent and a stationary phase of defined porosity without an attractive interaction between the two phases. Gel filtration generally refers to aqueous systems while gel permeation is used in nonaqueous systems. The technique is normally used for the separation of biomacromolecules on the basis of size. Solutes are eluted in the order of decreasing molecular size. Gel filtration chromatography is not used as the first step in... 

A nonpolar mobile phase passing through a packed column that contains a polar stationary phase defines normal-phase HPLC (NP-HPLC). For example, if -hexane comprises the mobile-phase and silica gel is used for the stationary phase, separations of nonpolar organic analytes as shown in Fig. 4.1 is accomplished. With respect to neutral organic compounds, the polar and ionic domains cannot be reached by NP- HPLC. NP-HPLC was the first high-pressure form of liquid chromatography to be developed. If the stationary phase could be made hydrophobic by chemical treatment and the mobile phase made more polar, a reversal of mobile/stationary-phase polarities could be achieved. Like it or not, we are stuck with this nomenclature RP-HPLC has certainly extended the range of analyte polarity that... 

Polarity is a key word in many chromatographic separations since a polar mobile phase will give rise to a low solute retention in normal phase LC (liquid-solid chromatography, LSC, of adsorption chromatography), or to a high solute retention in reversed-phase LC (RPLC). Nevertheless, it is often unclear exactly what this term means. One way to define the concept of polarity is to consider the Hildebrand solubility parameter another is to consider the Snyder solvent parameter. 

The fundamental characteristic of ion-pair chromatography is that the addition of the counter ion enhances the retention of the solute, without which the solute would either move with the solvent, in the case of reversed-phase support, or be completely retained, in the case of normal-phase support (or at least experience severe tailing and poor resolution). The enhanced retention is a consequence of the partitioning of the ion pair into the stationary phase subsequent to partitioning of the ions into the stationary phase. Thus, the equilibrium constants defined in Fig. 2.22, which are unique for each particular solute, counter ion, and stationary and mobile phase, are the factors that define the retention of a particular solute, the column efficiency, and hence the efficiency of the separation. 

The normal-phase (NP) mode is not often used in high-performance liquid chromatography, probably because many analysts suppose that it is too complicated or even unreliable. This is by no means true rather, it is a valuable tool for the separation of a large group of analytes. Sihca, as the prototype of an NP stationary phase, is superior for the separation of isomers ds/trans, positional isomers, and diastereomers). An example is shown in Fig. 1. The silica surface is rigid (in contrast to the flexible hydrocarbon chains of the usual reversed-phase materials) and the analytes interact in a stericaUy defined maimer with the polar groups that... 

Triglycerides in vegetable oils are separated according to their equivalent carbon number by reversed-phase liquid chromatography and detected by differential refractometry. Quantitation is by area normalization. Elution order is determined by calculating the equivalent carbon numbers, ECNs, often defined (as we said before) as CN — 2n, where CN is the carbon number and n is the number of double bonds. To calculate ECNs more accurately, the origin of the double bond is taken into account ... 

Micellar electrokinetic chromatography (MEKC) is a particular EKC mode where the secondary phase is composed by micellized surfactant (MEKC is discussed in detail in Chapter 3 by Terabe). Solute differential retention occurs as a result of a partition mechanism between a dispersed phase defined by the total volume of micelles and the remaining aqueous phase. MEKC modes of elution comprise normal, restricted, and reversed MEKC, based on the relative migration of the analyte and secondary phase apparent velocity. 

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