Characterization of chromatographic supports for the analysis of basic compounds (original) (raw)
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Chromatographia, 2001
Reversed-phase liquid chromatography (RP-HPLC) has become a powerful and widely employed technique in the separation and analysis of a great variety of compounds with different functionalities. The most common type of stationary phase for RP-H PLC consists of nonpolar, hydrophobic organic species (e.g., octyl, octadecyl) attached by siloxane bonds to the surface of a silica support. In the first part of this article, a description of the many beneficial properties that make porous silica the most employed support in RP-HPLC will be presented, starting from the synthesis of silica. It is noteworthy that the chromatographic properties of the final column are strictly correlated to the preparation type. A silica surface possesses a number of attractive properties, but also some drawbacks. Unreacted or residual silanols interact with basic compounds and can induce peak tailing, which means a loss in chromatographic performance. This problem has lead many manufacturers to produce stationary phases with reduced silanol activity which improve dramatically the peak shape of basic compounds. In the second part of this review, different approaches are proposed to obtain less reactive stationary phases.
Orthogonality and similarity within silica-based reversed-phased chromatographic systems
Journal of Chromatography A, 2005
The starting point of this study was a current set of 32 chromatographic systems used to select initial conditions for method development to determine the impurity profile of a drug. The system exhibiting the best selectivity is then selected for further method development. In this current set eight silica-based phases are applied in conjunction with four mobile phases at different pH. In order to save time and resources, the possibilities for a meaningful subset selection were investigated. The most differing systems in terms of selectivity, in other words only the most orthogonal systems, need to be selected. Since the stationary phases are all silica-based, the selectivity differences are examined within a more homogeneous group than if, for instance, also zirconia-or polymer-based columns would be involved. To select the subset of systems also the best overall separation performances are taken into account. The selection is based both on the HPLC-DAD data of a generic set of 68 drugs, and on the LC-MS-DAD results for a mixture of 15 drugs, less different in structure. The orthogonality is evaluated using weighted-average-linkage dendrograms and color maps, both created from the Pearson-correlation coefficients r between normalized retention times τ. The Derringer's desirability functions are applied to define the systems with the best overall separation performances. Proposals for different representative subsets of the initial 32 systems are made.
Simplification of a chromatographic test methodology for evaluation of base deactivated supports
Chromatographia, 2002
Today reversed-phase liquid chromatography (RP-LC) is the most employed chromatographic technique for analysis of basic compounds. Unfortunately, the strong ionic interactions of basic compounds with residual silanols result in asymmetric peaks and non-reproducible retention. This problem and the continuous increase in the use of RP-LC techniques have furthered the need for a new generation of"base deactivated" stationary phases. Selecting the appropriate stationary phase for a specific separation is an important parameter in the development of LC methods. To characterize and evaluate the relative chromatographic performance of stationary phases, a series of tests are proposed in the literature. In this work, a chromatographic test previously developed is discussed with the aim of simplifying its methodology. The number of test compounds was reduced (seven instead of fourteen), as well as that of the mobile phases (one or l",,vo instead of three) and columns per support (three instead of five). This simplification allows a faster and easier method to evaluate new chromatographic supports.
Journal of Chromatography A, 2004
To select appropriate stationary phases from the continuously expanding supply of potentially suitable HPLC columns, the properties of 28 frequently applied stationary phases were determined by measuring several chromatographic parameters. From these results, based on chromatographic expertise, eight stationary phases with different properties and selectivities were selected. The aim of this study is to apply chemometric tools to evaluate the initially selected set of columns, i.e. a more systematic approach for making such a selection is examined. Starting from the information obtained on the 28 stationary phases, the re-evaluation was performed independently based on the chemometric techniques Pareto-optimality, principal component analysis (PCA), and Derringer's desirability functions. The aim was to select a set of efficient columns exhibiting large selectivity differences. The chemometrically selected stationary phases were divided in groups based on hydrophobicity, a critical retention-determining property in reversed-phase chromatography. This allowed to further reducing the selection to three columns. It is demonstrated that the selection by the chemometric approaches in general is fairly comparable with the initial selection.
In this review, hydrophilic interaction liquid chromatographic (HILIC) applications for pharmaceutical analysis are discussed. The HILIC technique uses an aqueous/organic modifier mobile phase with a high organic modifier fraction and a hydrophilic stationary phase (SP). In general, the SPs are silica or polymer based. The silica-based columns are most often applied and are divided into bare-silica particle, chemically bonded silica particle, and monolithic phases. The bare-silica columns are most frequently used. The much less applied polymer-based columns are also divided into particle and monolithic phases. In this review, the applications are grouped and discussed according to the SP type used. Whenever possible, the considered HILIC application is compared with other HILIC methods, e.g. on other SPs, and/or with other separation modes, for that application. The advantages and drawbacks of HILIC are also discussed, as well as the method validation issues, when executed.
Analysis of basic compounds at high pH values by reversed-phase liquid chromatography
Journal of Separation Science, 2004
Reversed phase high performance liquid chromatography (RPLC) is currently the method of choice for the analysis of basic compounds. However, with traditional silica materials, secondary interactions between the analyte and residual silanols produce peak tailing which can negatively affect resolution, sensitivity, and reproducibility. In order to reduce these secondary interactions, which comprise ion exchange, hydrogen bonding, and London forces interactions, chromatographic analyses can be carried out at low or high pH values where silanol groups and basic compounds are mostly uncharged. The chromatographic behaviour of a particular bidentate stationary phase, Zorbax Extend C 18 , was studied with a set of basic and neutral compounds. Thanks to a higher chemical stability than traditional silica based supports, analyses were carried out with a high pH mobile phase, which represents a good alternative to the acidic mobile phases generally used to reduce ion exchange interactions. The performance of this bidentate stationary phase was also compared with that of other supports and it was proved that it is advantageous to work with high pH mobile phases when analyzing basic compounds.
Journal of Separation Science, 2003
Capillary liquid chromatography as a tool for separation of hydrophobic basic drugs. Relation between tests for column characterization and real analysis Two capillary columns for reversed phase (RP) capillary liquid chromatography (CLC), viz. Nucleosil 100 -5 C18 and LiChrosorb RP-select B, were characterized by the Walters test, i.e. the chromatographic test proposed for RP stationary phases. Hydrophobicity indices were determined not only in acetonitrile/water mobile phase, as proposed in the test, but they were also measured in buffered systems. This approach was used to quantify the influence of mobile phase composition on the modification of the surface of the stationary phases. In the next step, small basic compounds differing in their hydrophobicity and basicity were selected and their retention on the stationary phases in mobile phases of the same composition as used for column testing was examined. Furthermore, the retention of newly synthesized drugs, chemotherapeutics derived from thioacridine and pyridoquinoline, differing in their structures, basicity, and hydrophobicity, was also studied. The composition of the mobile phases had to be shifted to higher contents of organic modifiers -acetonitrile or methanol -in order to elute these hydrophobic compounds from the columns. The question we wanted to answer was: How is the method for testing of reversed phases related to retention, separation efficiency, and peak symmetry of various analytes?
Silanol effects in reversed-phase liquid chromatography
Journal of Chromatography A, 1990
A large number of butyl-, hexyl-, octyl-and octadecylsilica stationary phases were synthesized by the usual low-temperature and also novel high-temperature silylation methods. The phases were characterized by measuring their silanol concentrations, organic functional group concentrations and wettabilities and, additionally, by reversed-phase liquid chromatographic retention measurements. Unusual retention data, not expected on the basis of the solvophobic retention theory, were observed and are explained. Separation selectivity and peak asymmetry are interpreted by taking into account the surface concentrations of both the silanol groups and the organic functional groups. The peak shape is better, but the separation selectivitiy is lower, when access to all silanol groups of a stationary phase is equally easy or equally difficult.
Journal of Pharmaceutical and Biomedical Analysis, 2007
The chromatographic performance of several base-deactivated stationary phases was evaluated with a specific chromatographic test. Seven basic test compounds, possessing different physico-chemical properties were injected on different supports with two mobile phases: one at pH 7.0 (acetonitrile-phosphate buffer, 40:60, v/v), and the other at pH 3.0 (acetonitrile-phosphate buffer, 15:85, v/v). Chromatographic parameters obtained under these conditions were treated by principal component analysis (PCA) to separate base deactivated supports according to their silanol activity (pH 7.0 mobile phase) and hydrophobic properties (pH 3.0 mobile phase). The information given by the specific test column evaluation was improved with complementary chemometric tools such as hierarchical cluster analysis. The same base deactivated supports were also tested following a general test procedure issued from the literature and obtained fundamental properties (in particular silanol activity and hydrophobicity) were compared with column evaluation obtained with the specific test: results were in good agreement, although the use of the specific test offered a better differentiation between numerous base-deactivated supports.