Reversed-phase classification

Neue, U. D., Serowik, E., Iraneta, P., Alden, B. A. and Walter, T. H., A Universal Procedure for the Assessment of the Reproducibility and the Classification of Silica-Based Reversed-Phase Packings 1. Assessment of the Reproducibility of Reversed-Phase Packings. /. Chromatogr. A, 849 87—100, 1999. 

Euerby, M. R. and Petersson, P., Chromatographic Classification and Comparison of Commercially Available Reversed-phase Liquid Chromatographic Columns Using Principal Component Analysis, /. Chromatogr. A, 994 13—36, 2003. 

Classification as low polarity is based upon elution in the last half of the reverse-phase C gradient described in the Experimental section. 

The technique used to develop the four-solvent systems was based on procedures elucidated by Lehrer (6), Rohrschneider (7), and Glajch (8). After trials with individual solvents chosen from the comers of the Snyder solvent-selectivity triangle—a system of classification of solvents by the degree to which they function as proton donors, proton acceptors, or dipole interactors—an ideal solvent system was calculated. Ethanol, acetonitrile, and tetrahydrofuran were the reverse-phase solvents used, and water was the carrier solvent. Once the ideal solvent strength of one solvent-water combination was empirically determined, that of the other combinations could be estimated by use of the following equation (9) ... 

R. Kaliszan,M. A. van Straten,M. Markuszewski, C. A. Cramers and H. A. Claessens, Molecular mechanism of retention in reversed-phase high-performance liquid chromatography and classification of modern stationary phases by using quantitative structure-retention relationships,/. Chromatogr. A 855 (1999),455-486. 

M. Euerby, A. McKeown, and R Petersson, Chromatographic classification and comparison of commercially available perfluorinated stationary phases for reversed-phase liquid chromatography using principal component analysis,/. Sep. 

A useful classification of the various LC techniques is based on the type of distribution mechanism applied in the separation (see Table 1.2). In practice, most LC separations are the result of mixed mechanisms, e.g., in partition chromatography in most cases contributions due to adsorption/desorption effects are observed. Most LC applications are done with reversed-phase LC, i.e., a nonpolar stationary phase and a polar mobile phase. Reversed-phase LC is ideally suited for the analysis of polar and ionic analytes, which are not amenable to GC analysis. Important characteristics of LC phase systems are summarized in Table 1.3. 

Stationary phases used for achiral, small molecule separations are often further separated into two major classifications normal and reversed phase. Normal-phase chromatography uses a polar stationary phase with a less polar mobile phase, while reversed-phase chromatography operates using the exact opposite conditions. Additionally, there are several functionalized stationary phases that can operate in either normal or reversed phase, e.g., cyano and amine resins. 

Reversed phase silicas are discussed in depth here as this type of packing materials is often applied in preparative chromatography. According to the classification used above, reversed phase silica is an example of a designed adsorbent. 

The previous chapter discussed the solvent and its interaction with the solute. To complete the chromatographic system the adsorbent has to be selected. As mentioned in Chapter 3.2.1 one has to distinguish between enantioselective and non-enantioselective adsorbents. Both groups of adsorbents are classified into polar, semi-polar and nonpolar adsorbents (see Tab. 4.4). This classification is based on the surface chemistry of the packing material. Interaction between mobile phase and adsorbent characterizes the phase system, which is distinguished between normal phase (NP) chromatography and reversed phase (RP) chromatography. This differentiation is historic and appointed by the ratio of the polarity of the adsorbent and the mobile phase. 

HPLC provides reliable quantitative precision and accuracy, along with a linear dynamic range (LDR) sufficient to allow for the determination of the API and related substances in the same run using a variety of detectors, and can be performed on fully automated instrumentation. HPLC provides excellent reproducibility and is applicable to a wide array of compound types by judicious choice of HPLC column chemistry. Major modes of HPLC include reversed phase and normal phase for the analysis of small (<2000 Da) organic molecules, ion chromatography for the analysis of ions, size exclusion chromatography for the separation of polymers, and chiral HPLC for the determination of enantiomeric purity. Numerous chemically different columns are available within each broad classification, to further aid method development. 

Most chiral HPLC analyses are performed on CSPs. General classification of CSPs and rules for which columns may be most appropriate for a given separation, based on solute structure, have been described in detail elsewhere. Nominally, CSPs fall into four primary categories (there are additional lesser used approaches) donor-acceptor (Pirkle) type, polymer-based carbohydrates, inclusion complexation type, and protein based. Examples of each CSP type, along with the proposed chiral recognition mechanism, analyte requirement(s), and mode of operation, are given in Table 3. Normal-phase operation indicates that solute elution is promoted by the addition of polar solvent, whereas in reversed-phase operation elution is promoted by a decrease in mobile-phase polarity. 

Within the last several years HPLC separations have been optimized in terms of the most appropriate mobile phase composition for a particular set of solutes by exploring the whole plane of solvent selectivities using this solvent classification scheme with a minimal number of measurements in statistically-designed experiments. For reversed phase HPLC systems, the selectivity triangle is often defined by methanol, acetonitrile, and tetrahydrofuran with water as the diluent (37). 

The availability of reliable measurements or estimates of water solubility, octanol-water partition coefficient, bioconcentration factor, rate constants and the like allows one to make qualitative judgements or, through the use of mathematical simulation models such as EPA s EXAMS (19), quantitative calculations of environmental distribution and persistence. In the qualitative use, Swann and coworkers (20) classified chemical mobility in soil based upon reversed-phase HPLC retention data which in turn is related to S. The approximate water solubility equivalents in this first-estimate classification, with chemical examples, are in Table II. This classification holds for chemicals whose primary adsorption in soil is to organic matter, and excludes those chemicals (such as paraquat) which bind ionically to the soil mineral fraction. A recent tabulation of pesticides found in groundwater had 11 entries, 8 of which represented compounds with water solubilities in excess of 200 ppm with the remaining three falling in the range of 3.5 to 52 ppm (21). 

The response of heterogeneous systems to a stress field allows them to be placed in two categories (i) those in which stress induces irreversible changes e.g., precipitation, denaturation of protein, crystallization, etc.) and (ii) those in which the changes are reversible. The classification is not perfect, as the type and magnitude of stress field can be cmcial, but it provides a guide in most cases, miscibility in systems (i) is reduced by stress, while in systems (ii) it is increased. In other words, if a system can be irreversibly modified by rheological means, its solubility will be reduced. An excellent review on phase transition in shear flow was recently published [Onuki, 1997]. 

One of the most powerful features of HPLC stems from the fact that the eluent is not merely a transport medium, it rather contributes significantly to the mechanism of separation. Retention as well as selectivity arise from the combined action of mobile and stationary phase on the solutes. Because of its distinct physicochemical characteristics, a given solvent interacts in a specific manner with the solutes. Its capacity to donate or accept protons or to induce a dipole moment defines the nature of its interaction with the solutes in solution. Slight differences in these interactions complemented by stationary-phase action are often enough to provide the desired selectivity in HPLC. Solvent classification based on their elution strength or polarity represents a classic area of investigation into the fundamentals of retention mechanisms in HPLC (2S-27), and the chapter is not yet closed on its development and refinement (28-30). Here we will present a practical and comprehensive way to exploit the effect that the nature of the solvent has on retention and selectivity in reversed phase HPLC. 

The mobile phase is always liquid. The stationary phase is liquid or solid. Table 1 shows in detail this classification. There are two modifications of LSC normal phase (NP) and reversed-phase (RP), depending on the relative polarity of the two phases. The same is valid for LLC, but this subdivision is rarely... 

Like other states of matter, thermotropic mesophases are indefinitely stable at defined temperatures and pressures. Moreover, a thermotropic liquid-crystal-line material exhibits reversible phase transitions at well-defined temperatures. For example, the liquid crystal 4-n-pentyl-4 -cyanobiphenyl (5CB) melts from the solid to a nematic liquid crystal at 22.5°C and then from the nematic phase to the liquid at 35.0°C. As a consequence, the characterization and classification of thermotropic phases by microscopy also requires the use of an accurately controlled oven. 

Based on the above-mentioned six key properties of reversed phases, the stationary phases can be characterized. A wide variety of literature exists on this subject [9-15]. Of course, the synthesized stationary phases can be subjected to a full physicochemical examination (nitrogen adsorption measurements to determine the specific surface area, the pore volume and the pore size, CHN analysis to determine the surface coverage of the stationary phase, particle size measurements, etc.). However, all these characterizations are not really to the point, because in the end only the chromatographic separation counts. As a result, chromatographic tests for the characterization and classification of reversed phases have established themselves, from which a representative few, without any claim to being exhaustive, are presented here (Figures 4.1—4.3). 

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