Mobile and stationary phases for SFC: Effects of using modifiers

The effects of modifiers on the mobile-phase and the stationary-phase properties in packed-column supercritical-fluid chromatography were studied. Modifiers, may affect retention and improve peak shapes and efficiencies, because of (i) increased mobile-phase polarity, (ii) increased mobile-phase density, (iii) stationary-phase deactivation, and (iv) increased solvation or swelling of the stationary phase. In this paper these four parameters are evaluated and evidence to establish their relative importance is considered.The introduction of a modifier can lead to a substantial increase in the density of the mobile phase. The reliability of several methods for calculating the critical properties and densities of binary fluids is investigated. The Chueh and Prausnitz method gives the most accurate results for the calculation of the critical properties. The Lee and Kesler equation of state yields accurate density estimates.Adsorption isotherms have been measured on several stationary phases for packed-column SFC. These data suggest that stationary-phase deactivation is the most important effect of adding modifiers. Adsorption data on different stationary phases can also be used to judge their applicability. Relevant indicators are the maximum amount of modifier that can be adsorbed on the surface (saturation level) and the initial steepness of the isotherm (surface activity). The latter parameter can be related more closely to the peak shape observed in the absence of modifiers.All silica-based materials studied so far give rise to broad and highly asymmetrical peaks for certain classes of polar solutes. Different silica-based materials appear to differ in degree of activity, but the kind of interactions are the same. On a polystyrene-divinylbenzene stationary phase the mobile-phase modication effect could be studied without the interference of active sites.     

An overview of contemporary method development in liquid chromatography

Summary This paper provides an overview of method development in liquid chromatography. It is explained why every practicing chromatographer needs to understand the process of method development. Thus, this paper is directed towards chromatographers who develop new separation methods, as well as to those who apply published methods. The method-development process is first explained in broad terms, after which the different aspects of the process are described in more detail.First, attention is paid to the initial stages of method development, which consist of the accurate definition of the separation problem and the selection of the most suitable chromatographic method. Method-development expert systems are discussed with regards to their applicability in this area.Selectivity optimization is shown to rely on three main factors,i. e. the selection of the parameter space, the experimental design and the optimization criterion. Selectivity-optimization procedures are described very briefly.System optimization concerns those factors which do not affect the chemistry of the system (mobile and stationary phase), but are essentially determining the analysis time and the sensitivity.The method-validation stage can be divided into three main parts: qualitative method validation (i.e. peak integrity and identity), calibration and quantitative method validation. The requirements of calibration procedures are briefly reviewed and an inventory of method-validation procedures is presented.     

Application of the reversed-phase liquid chromatographic model to describe the retention behaviour of polydisperse macromolecules in gradient and isocratic liquid chromatography

This paper illustrates how conventional models of chromatographic behaviour can be used to predict the separation behaviour of polydisperse macromolecules. Using polystyrene and polymethylmethacrylate homo- and co-polymeric standards, the models were validated by comparing experimental retention behaviour with that predicted by the chromatographic model. The experimental retention time of each of the samples was entered into a spreadsheet application, which calculated the parameters that best described retention (for a given model). When a correlation between the relevant parameters and molecular mass was established, that correlation was used to predict the change in retention behaviour over the molecular-mass range. An expression introduced in a previous paper, to calculate the critical mobile-phase composition of a homopolymer was validated using polystyrene homopolymers. A second expression, which can predict the elution behaviour of copolymers, was also validated. This expression can predict the retention of a copolymer, based solely onthe retention of the homopolymeric units that make up the copolymer.     

Effects of pH in reversed-phase liquid chromatography

The effects of concomitant variations in pH and organic modifier concentration on retention, efficiency and peak symmetry are considered for reversed-phase liquid chromatography (RPLC) on octadecyl-modified silica (ODS) columns. A number of factors are discussed, which make the systematic exploitation of pH effects in RPLC more complicated than the optimization of solvent composition. If the pH is varied, a second factor (usually the concentration of organic modifier) will need to be varied simultaneously to maintain retention (capacity factors) in the optimum range. When pH is considered as a parameter in RPLC, not only its effects on retention, but also the variations in efficiency (plate count) and peak shape (asymmetry) need to be considered. These parameters turn out to vary drastically between individual solutes and between different experimental conditions. The results of a study involving a number of acidic, basic and neutral solutes, two different ODS columns and mixtures of either methanol or acetonitrile with aqueous buffers are reported. In the earlier part of the study, using methanol as the organic modifier, reproducible data for retention, peak width and peak symmetry were obtained and these data are reported. In the later part of the study, using acetonitrile, a gradual change in retention as a function of time was observed, this effect coinciding with a decrease in column efficiency. It is concluded that ODS columns are subject to considerable degradation during studies in which the pH is varied. Although this effect can be described mathematically, the preferred solution is thought to be the use of pH-stable columns.     

Application of Combinatorial Procedures in the Search for Serine-Protease-Like Activity with Focus on the Acyl Transfer Step

Recursive deconvolution of a 729-membered peptide library has identified three active sequences, in which both Ser and His are present in one of the two tripeptidic chains generated on a steroidal scaffold (see structural formula), for the cleavage of an activated p-nitrophenyl ester. This combinatorial approach aims at searching for serine-protease-like activity.     

Criteria for judging the quality of separation in chromatograms containing solvent peaks

Summary Formal procedures used for optimizing chromatographic selectivity require objective, numerical functions to assess the quality of each chromatogram obtained during the process. Many of such optimization criteria have been suggested for chromatograms in which all or a number of well-shaped peaks need to be separated. This paper describes a method by which resolution can be measured in chromatograms in which very large, highly non-symmetrical background peaks,e.g. typical solvent peaks, are present. Using this method, various optimization criteria can be calculated. The method is evaluated using a simulation program which constructs chromatograms from the recorded profiles of individual experimental peaks. It is also demonstrated for use in an experimental optimization procedure in reversed-phase liquid chromatography.     

Optimizing separations in reversed-phase liquid chromatography by varying pH and solvent composition

Summary An interpretive optimization procedure in which pH can be one of the variables is presented with the emphasis on optimizing separations. When varying the pH in reversed-phase liquid chromatography the retention of ionogenic solutes will change. Thus, the selectivity between ionogenic and neutral solutes or between ionogenic solutes mutually can be optimized. However, pH also greatly affects the efficiency (plate count) and peak shape (asymmetry). Optimum selectivity (i.e. large differences in retention times) may be observed under conditions where peaks are broad and asymmetrical. Thus, it is essential to simultaneously consider retention, peak width and peak shape and their effects on separation (effective resolution) in pH-optimization studies. A procedure in which this is done is presented and applied to optimizing the separation of a synthetic mixture of selected pharmaceuticals. After initial experiments to establish the parameter space (boundaries for pH and binary methanol — water composition), twelve experiments are performed according to a 3×4 experimental design. At each loaction the retention, peak height, peak area and peak symmetry are recorded for each solute. These data are then used to build models for each of the four characteristics and for each solute. From this set of models the response surface, describing the quality of separation as a function of pH and composition, can be calculated. A variety of optimization criteria (quantifying quality of separation) can be used. The optimum corresponds to the highest point on the response surface.     

Novel system for classifying chromatographic applications, exemplified by comprehensive two-dimensional gas chromatography and multivariate analysis

For practical chromatographers it is extremely difficult to judge the merits and limitations of new technological developments. On the other hand, it is nearly impossible for those at the forefront of technology to judge the implications of their efforts for all specific applications of chromatography. Both chromatographers and researchers can be aided by a classification of the numerous specific applications into a few well-defined categories. In this paper, we propose such a classification of all chemical analysis by chromatography into three generic types of applications, viz. target-compound analysis, group-type separation, and fingerprinting. The requirements for each type are discussed in general terms. The classification scheme is applied to assess the benefits and limitations of comprehensive two-dimensional gas chromatography (GC x GC) and the possible additional benefits of using multivariate-analysis (MVA) techniques for each type of application. The conclusions pertaining to the generic types of applications are indicative for the implications of new developments for specific chemical analysis by chromatography.