Normal phase chromatography fundamentals

In extraction techniques (LLC, SPE, etc.), normal-phase liquid chromatography (NPLC), and thin-layer chromatography, aliphatic hydrocarbons (e.g., -hexane, -heptane) are usually used. The elution strength of these solvents is often modified by addition of more polar solvents. The fundamental problem with the eluents in NPLC is dissolved water and trace amounts of olefins. These contaminations can induce a change in the wavelength cut-off values (UV detection, spectrophotometry), baseline perturbation, and poor reproducibility of retention data. Halogenated solvents such as dichloromethane can react with some organic solvents (e.g., acetonitrile) to form crystalline products. 

The best operative conditions to separate the 20 natural amino acids by using a wide variety of commercially available stationary phases used both in normal and in reversed-phase chromatography and by two-dimensional (2D) chromatography technique are described. Resolution of amino acids derivatives, which play a fundamental role in the peptide and protein sequence structures, is also reported. 

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 differences between achiral and enantioselective HPLC are smaller than is generally assumed. Enantioselective HPLC does not require special units or detectors, and all physico-chemical fundamentals of the chromatography are identical. However, it should be noted that many of the described enantioselective HPLC separations are performed imder normal-phase conditions. Applications of the popular water/mefhanol or water/acetonitrile gradients are in the minority, since many CSPs are not compatible with these gradient systems or do not show any retention. 

Equation (5) is regarded as a fundamental equation of column chromatography as it relates the retention volume of a solute to its distribution ratio. Planar separations (PC and TLC). Separations are normally halted before the mobile phase has travelled completely across the surface, and solutes are characterized by the distance they have migrated relative to the leading edge of the mobile phase (solvent front). A solute retardation factor, Rf, is defined as... 

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