Peter J. Schoenmakers

Modelling retention in reversed-phase liquid chromatography as a function of pH and solvent composition

Abstract Models describing the concomitant effects of pH and organic modifier concentration on retention in reversed-phase liquid chromatography are established. Two different octadecyl-modified silica columns were used. The retention behaviour of several acidic, basic and neutral solutes were studied, using methanol as the organic modifier. The suggested models accurately describe retention as a function of pH and composition. A unified formalism for retention modelling that is applicable to all ionogenic solutes is also established. This formalism is tested with the modelling of retention for several weak bases. The resulting (general) model describes retention accurately and is applicable to all solutes studied.

Effects of pH in reversed-phase liquid chromatography

Abstract 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.

Effects of the column pressure drop in packed-column supercritical-fluid chromatography

SummaryColumns packed with small particles (i.e. 3 to 10µm) may be used for fast separations in supercritical-fluid chromatography (SFC). The analysis times required for such packed columns are typically an order of magnitude smaller than what may be achieved with contemporary open-tubular (capillary) columns (25 to 100µm i.d.).However, packed columns give rise to much higher pressure drops over the column. The maximum allowable pressure drop will determine how many plates can be achieved with packed columns. In this paper the effects are described of the pressure drop on retention (capacity factor) and efficiency (number of plates) for packed columns of various lengths and internal diameters, packed with three different octadecylsilica materials. Carbon dioxide has been used as the mobile phase under several different sets of pressure and temperature conditions.

RES, AN EXPERT SYSTEM FOR THE SET-UP AND INTERPRETATION OF A RUGGEDNESS TEST IN HPLC METHOD VALIDATION .1. THE RUGGEDNESS TEST IN HPLC METHOD VALIDATION

Abstract In method validation, an intralaboratory repeatability study and an interlaboratory reproducibility study can be performed as part of a precision test. In HPLC, an intralaboratory ruggedness test can be performed to detect problems that would otherwise be encountered in a reproducibility study. In a ruggedness test, variations in ambient factors that are expected to occur in practice, are simulated. Several steps determine the success of a ruggedness test. The complexity and lack of standard procedures for some of these steps is the main reason why ruggedness testing is still not widely accepted.

An expert system for the selection of criteria for selectivity optimization in high-pressure liquid chromatography

SummarySystematic procedures for the optimization of chromatographic selectivity require objective criteria to characterize the quality of separation in a chromatogram. Numerous criteria have been suggested. Different criteria yield different results and the choice will depend on a large number of factors. It is genuinely difficult to select the most suitable criterion in a particular situation. For these reasons, an expert system has been developed to assist chromatographers in the selection of optimization criteria. A structured representation of the required knowledge and its implementation in an expert-system shell are presented in this paper.

Thermodynamic model for supercritical fluid chromatography

Abstract The possibility to apply a thermodynamic model to supercritical fluid chromatography (SFC) is investigated. Thermodynamics can be used to characterize the (mobile) phases used in SFC, as well as for the description and ultimately the prediction of retention behaviour. The solubility parameter, as obtained from a thermodynamic equation of state, has been used for characterization purposes. An equation which describes retention in terms of thermodynamic functions is derived. This equation enables the prediction of the effect of pressure or density on the retention behaviour. Since the interactions of solutes with the stationary phase are not taken into account, the retention data can be calculated only relative to some arbitrary pressure. Close agreement between experimental and calculated data is observed, and illustrated with representative examples. Some practical conclusions are drawn.

Correction of the resolution function for non-ideal peaks

Abstract The resolution function is commonly used to describe the extent of separation between successive peaks in a chromatogram. However, the resolution is usually defined in such a way that it is applicable only to symmetrical (Gaussian) peaks. Moreover, the resolution does not provide a realistic estimate of the extent of separation between two peaks with greatly different areas. Nevertheless, the main advantage of the resolution is that its value can be predicted from retention and efficiency data for the individual peaks. Simple methods are described to correct the resolution function for (i) large variations in peak areas and (ii) peak asymmetry. The corrections are derived as modifications of the resolution equation. An important consequence of these modifications is that the resolution for a pair of peaks has two different values, one for each peak. The new resolution equations were evaluated using computer-generated (exponentially modified Gaussian) peak profiles. The effects of varying degrees of peak asymmetry and varying peak-area ratios were studied.

Calculation of pressure, density and temperature profiles in packed-column supercritical fluid chromatography

Abstract The simultaneous calculation of pressure, density and temperature profiles for packed columns used in supercritical fluid chromatography (SFC) is described. The profiles are obtained by solving the heat balance equation numerically at a large number of locations in the column. The compressibility of the mobile phase(carbon dioxide) and the variations of the isochoric heat capacity, thermal conductivity and viscosity with temperature and pressure are taken into account. The results show that pressure profies over packed columns in SFC are approximately linear. At high mobile phase densities, a supercritical fluid behaves like a liquid, but the viscosity is much lower. The temperature increases slightly along the column length. At lower densities the compressibility of the fluid becomes more apparent. The density decreases along the column length and so does the temperature of the eluent. Both the variations in density and in temperature are enhanced by using smaller particles or higher flow-rates. Temperature effects are reduced by using columns with smaller inner diameters. Temperature effects do not appear to be a major source for concern in packed-column SFC.

Interpretation of infrared spectra with modular neural-network systems

Abstract The interpretation of infrared (IR) spectra is not straightforward and requires much time and expertise. In this study the interpretation of IR spectra using artificial neural networks is addressed. The conventional approach is to design a single neural network to cover the problem domain. In this study a different approach is taken by tackling specific sub-problems with small, dedicated neural networks. Such networks are intended to form the modules of a larger, structured system for spectrum interpretation. The problem domain chosen in this preliminary work concerned the decision on the presence or absence of various functional groups (i.e., alcohols and carbonyls). Several modules were created, as well as a large, ‘flat’ neural network which covered the entire problem domain. The performance of the specialized modules was compared with that of the flat network and with that of a genuine expert.

Comparison of stationary phases for packed-column supercritical fluid chromatography

Abstract A study of the applicability of different stationary phases for packed-column supercritical fluid chromatography is described. The compatibility of these phases with carbon dioxide and with a number of test solutes was established qualitatively from the observed peak shapes. Retention and selectivity differences between the different columns were studied quantitatively. The stationary phases studied include chemically modified silicas, polysiloxane-coated silicas, modified and unmodified porous graphitic carbon and modified and unmodified poly(styrene-divinylbenzene) copolymers. It is concluded that differences in selectivity between different stationary phases are often due to interactions between the solute molecules and active sites on the surface. Such interactions lead to poor peak shapes and are therefore undesirable. For most of the solutes studied, with the notable exception of polyaromatics, a poly (ethylene glycol)-coated carbon phase provided the best results. Owing to the very high retentivity of unmodified carbon, this phase turned out to be very stable. The coating of different types of polymers on solid surfaces may be used to create stationary phases without residual active adsorption sites, but with considerable differences in selectivity.