Characterization of bonded phases by solid-state NMR spectroscopy

A pragmatic test procedure for comparison and evaluation of reversed phases is described. The differences in retention between RP8 and RP18 can be compensated for by adjustment of the eluent composition. A 10% increase in the water content doubles the k′ values, as does the exchange of an RP8 with an RP18 column. It is possible to differentiate between the two columns with the pair ethylbenzoate and toluene. Under stand-ard conditions (methanol-water, 55–45, v/v; 49–51, w/w) with RP8 ethylbenzoate is always eluted together or after toluene, whereas with RP18 it is always eluted in front of toluene.

Comparison and characterization of reversed phases

Fused silica capillaries of different diameters were slurry-packed with an identical sorption material and evaluated chromatographically. The extent of band broadening was evaluated fluorometrically at different column lengths, together with porosity measurements obtained from a fluorescence-quenching marker. Kinetic analysis of the plate-height vs velocity data reveals that the inner diameter is a very important variable in the preparation of such packed columns. Extremely high efficiencies were obtained with columns of the smallest diameter (44 ..mu..m).

Separation efficiency of slurry-packed liquid chromatography microcolumns with very small inner diameters.

This review is in support of the development of selective, reproducible and validated capillary electrophoretis (CE) methods. Focusing on pharmaceutical and biological applications, the successful use of CE is demonstrated by more than 800 references, mainly from 1994 until 1998. Approximately 80 recent reviews have been catalogued. These articles sum up the existing strategies for method development in CE, especially in the search for generally accepted concepts, but also looking for new, promising reagents and ideas. General strategies for method development were derived not only with regard to selectivity and efficiency, but also with regard to precision, short analysis time, limit of detection, sample pretreatment requirements and validation. Standard buffer recipes, surfactants used in micellar electrokinetic capillary chromatography (MEKC), chiral selectors, useful buffer additives, polymeric separation media, electroosmotic flow (EOF) modifiers, dynamic and permanent coatings, actions to deal with complex matrices and aspects of validation are collected in 20 tables. Detailed schemes for the development of MEKC methods and chiral separations, for optimizing separation efficiency, means of troubleshooting, and other important information for key decisions during method development are given in 19 diagrams. Method development for peptide and protein separations, possibilities to influence the EOF and how to stabilize it, as well as indirect detection are considered in special sections.

Strategies for capillary electrophoresis: Method development and validation for pharmaceutical and biological applications

The measurement and meaning of void volumes in reversed-phase liquid chromatography.

The effect of the properties of the silica gel matrix, and of the octadecylsilane used, on the chromatographic selectivity of C18 bonded phases is described. Preparation of bonded phases with monochlorodimethyloctadecylsilane is easier because absolute water exclusion is not required in this case. The surface coverage (μmol/m2) is smaller than with dichloromethyl- or trichlorooctadecylsilanes, but in these cases a secondary silanization with HMDS is essential to remove newly formed silanol groups. Bonded phases on Si 60 give the largest amount of hydrocarbon per unit column volume, large k′ values and good selectivity, however, surface coverage and efficiency of these packed columns is less than with Si 100 silica. The chemical properties of the silica surface also influence bonded phase selectivity. Bonded phases should not be compared with only a single eluent, because properties of bonded phases differ with test solutes and test conditions, i.e. eluent composition.

Properties and diversity of C18 bonded phases

The problems in defining a true dead volume in chromatography on silica and reversed phase columns are discussed and demonstrated. Also discussed are the influence of pore size distribution of the stationary phase, the size of marker molecule, as well as of negative adsorption and ionic interactions on dead volume determination. Some practical suggestions for suitable Vm markers are given.

B. Dreyer

Papers

Properties and diversity of C18 bonded phases

Is there a “true” dead volume for HPLC columns?