Reversed-phase stationary phases characterization

R. Vervoort, E. Ruyter, A. Debets, H. Claessens, C. Cramers, and G. De Jong, Characterization of reversed-phase stationary phases for the liquid chromatographic analysis of basic pharmaceuticals by thermodynamic data, /. Chromatogr. A 964 (2002), 67-76. 

Reversed-phase stationary phases are more or less hydrophobic, and the degree of this property is characterized by their hydrophobicity H. As a general rule, retention times are longer the more C atoms the bonded stationary phase contains. (The reason is that the volume taken up by the bonded nonpolar groups, i.e. that required by the actual stationary phase, is greater with long chains than it is with shorter chains retention is directly proportional to the volume ratio between the stationary and mobile phases see Section 2.3.) Figure 10.7 demonstrates this effect. 

Buszewski, B., Gadza-la-Kopciuch, R. M., Markuszewski, M. L, Kaliszan, R. Chemically bonded silica stationary phases synthesis, physicochemical characterization, and molecular mechanism of reversed-phase HPLC retention. Anal. Chem. 1997, 69, 3277-3284. 

Advances in understanding solute interachons in liquid-liquid systems in a nonequilibrium environment brought reversed-phase (RP)-HPLC into the forefront of lipophilicity determinahon. The development and manufacturing of rigid, reproducible and well-characterized stationary phases and columns, as well as the accessibility and high level of automation of modern HPLC systems, have made RP-HPLC the method of choice for many laboratories. 

Doyle, C. A. and Dorsey, J. G., Reversed-Phase HPLC Preparation and Characterization of Reversed-Phase Stationary Phases, In Handbook of HPLC, Katz, E., Eksteen, R., Schoenmakers, P. and Miller, N., Eds., Marcel Dekker, New York, pp. 293-323, 1998. 

The effect of temperature on the acid base chemistry of the stationary phase can also play a role in separation. Free silanol groups on the stationary phase may exhibit changes in acid base chemistry with temperature [28]. Also, reverse phase columns with amine, amide, or acidic functional groups will be affected by the interaction of the temperature, the ionization state of the stationary phase, the mobile phase acidity, and the ionization state of the solute. Most non-linear van t Hoff plots can be rationalized in these terms, but it is difficult to predict a priori what the effects will be on a given system. Thus, it is important to characterize the system under study if a simple change in temperature produces unexpected effects. 

Kibbey, C. E. and Meyerhoff, M. E. (1993) Preparation and Characterization of Covalently Bound Tetraphenylporphyrin-silica Gel Stationary Phases for Reversed-phase and Anion-exchange Chromatography. Anal. Chem. 65, 2189-2196. 

Valko et al. have developed chromatographic methods which are based on established reversed phase methods with acetonitrile water gradients. The lipophilicity is characterized as a so-called chromatographic hydrophobicity index (CHI), which approximates the percentage of acetonitrile necessary for equal distribution between mobile and stationary... 

The k" measures the magnitude of retention in CEC due to reversible binding of the analyte to the stationary phase. Inspection of Eq. (11) shows that for all components (neutral or charged), k" is always positive, as a chromatographic retention factor should be. Further, while the retention factor in HPLC and the velocity factor in CZE are able to characterize the respective differential migration processes alone, both of them are required to characterize CEC. 

The peak shapes of metal chelating analytes are often poor because metal impurities in the stationary phase behave as active sites characterized by slowo desorption kinetics and higher interaction energies compared to reversed phase ligand sites. This phaiomaion is typical of silica-based stationary phases [31] ultrapure silicas were made commercially available to reduce it. However, styrene-divinylbenzene-based chromatogripliic packings suffer from the same problem and it was hypothesized that metals may be present in the matrix at trace conditions because they were used as additives in the polymerization process they may have been c tured via Lewis acid-base interactions between the aromatic ring n electrons and impurities in the mobile phase [32]. 

J. Layne, Characterization and comparison of the chromatographic performance of conventional, polar-embedded, and polar-endcapped reversed-phase liquid chromatography stationary phases, /. Chromatogr. A 957, (2002), 149-164. 

M. H. Abraham, M. Roses, C. F. Poole, and S. K. Poole, Hydrogen bonding. 42. Characterization of reversed phase high-performance hquid chromatographic C18 stationary phases,/. Phys. Org. Chem. 10 (1997), 358-368. 

A general rule is that QSRR equations are characterized by two kinds of structural descriptors one which accounts for bulkiness of analyte and one which encodes its polar properties [85]. Bulkiness descriptors are always significant in GC on non-polar phases and in reversed-phase HPLC whereas the significance of polar descriptors increases as the polarity of both the stationary phases and the analytes increases [7,12],... 

QSRR Eqs. (11.15) and (11.16) clearly demonstrate that the organic modifier of binary aqueous eluents used in reversed-phase liquid chromatography also modifies the stationary phase. The hydrocarbon brush on the silica matrix adsorbs the modifier and gets to some extent its properties 117]. QSRR enables differences in the mechanism of reversed-phase retention in individual HPLC. systems employing the. same stationary phase material, are characterized in a numerical manner. 

The predominant mode of HPLC, reversed phase, involves the separation of material based on the partitioning between a relatively polar mobile phase and a nonpolar stationary phase. Normal phase HPLC—nonpolar mobile phase and polar stationary phase—is considered an orthogonal technique to reversed-phase HPLC when qualifying reference standards. In fact it is common for the elution order to be entirely reversed when switching an analysis from reversed to normal phase. Therefore, highly nonpolar impurities can be easily characterized by normal phase separations. 

In the following, the synthesis of the most often employed stationary phase is discussed spherical silica with an n-octadecyl modification. The synthesis route has been chosen because all synthesis steps are well characterized and documented in standard operation procedure (SOP) protocols. The objective of this work was to develop a manufacturing process for a reversed phase C18-bonded silica column for HPLC according to standardized and validated procedures and to perform certification of the column, the tests and the mobile phases (du Fresne von Hohenesche et al., 2004). Figure 3.13 shows a scheme of the whole manufacturing process, and Table 3.7 summarizes the main steps. 

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