Hugo A.H. Billiet

Influence of organic modifiers on the rentention behaviour in reversed-phase liquid chromatography and its consequences for gradient elution

Abstract In a previous paper it was shown that the key to the description and understanding of retention behaviour under gradient conditions in reversed-phase liquid chromatography lies in the relationship between the isocratic capacity factor ( K ) and the volume fraciton of organic modifier (ϕ). Hence, the presentation of extensive data on this relationship in different organic modifier systems offers possibilities for evaluating and refining existing gradient elution models, such as the one of Snyder and co-workers. An extensive study of 32 aromatic compounds in aqueous system containing 10–100% of methanol, acetonitrile and tetrahydrofuran as organic modifiers under isocratic conditions is described. Two major conclusions are drawn that have consequences for gradient elution. Firstly, the relationship between in k and (ϕ) is generally non-linear, in accordance with previous statements. Secondly, the rate of change of in k and (ϕ) can be solute dependent. Systematic changes in the slope of the in k versus (ϕ) curves with absolute retention are observed for methanol and tetrahydro- furan as organic modifiers. It is shown that this conclusion leads to optimal gradients, which are convex rather than linear.

Gradient selection in reversed-phase liquid chromatography

Summary The rational selection of an optimal gradient in reversed-phase liquid chromatography is discussed from two viewpoints. Firstly, a general expression is derived for the numerical calculation of retention data for the case when the relationship between the capacity factor,k, and the mobile phase composition, φ, is known. For a linear gradient a graphical solution is demonstrated and analytical solutions are presented for simple but widely applicablek φ relationships. Retention data thus calculated agree well with experimental results. Secondly, the multi-component solubility parameter theory is used to predict thek φ relationship. Modification of the stationary chemically bonded phase under the influence of the organic modifier is introduced to improve the accuracy of the predictions. Empirical correlations using solubility parameters are presented as an alternative to the theoretical expressions.

Systematic study of ternary solvent behaviour in reversed-phase liquid chromatography☆

Abstract An extensive experimental survey of the retention behaviour of 32 solutes in the system methanol-tetrahydrofuran-water and 49 solutes in the system methanol-acetonitrile-water is presented. The retention data are fitted to a second order six-parameter equation, describing the capacity factor as a function of mobile phase composition. Iso-eluotropic lines, i.e., lines that connect solvents of equal eluotropic strength, are constructed in the ternary diagrams for the two systems and compared with theoretical lines, predicted from solubility parameter theory. Specific effects are defined as variations in retention for a particular solute, using solvents of equal eluotropic strength. Such effects appear to be larger between different binary mixtures than within the ternary triangle. Ternary solvents thus provide a smooth transition between two limiting binary mixtures.

Use of the solubility parameter for predicting selectivity and retention in chromatography

Abstract The theoretical and practical aspects of the solubility parameter (“polarity”) model for predicting retention behaviour and partition coefficients have been investigated. A critical discussion of the theoretical basis shows that sufficiently accurate data for the total solubility parameter can be obtained from generalized thermodynamic functions, rather than from the usual vapour pressure data. Partial polarities are obtained from theoretical and semi-empirical relations with simple physical parameters such as refractive index, dielectric constant and dipole moment. Internal pressure, calorimetric and spectroscopic experimental data are used for estimating the contributions of charge and proton transfer (acid-base interaction). Tables of partial and total polarities are presented which replace older, less accurate data. The multicomponent model is tested with chromatographic and partition experiments and is proposed as an alternative method for selecting a proper phase system (in both chromatography and partition methods where, for example the Hansch method is frequently used).

Use of gradient elution for rapid selection of isocratic conditions in reversed-phase high-performance liquid chormatography

Abstract It is shown that an initial scan of an unknown sample with a gradient elution programme offers a very rapid and easy procedure for deciding whether the sample is suitable for isocratic elution and, if so, for establishing the appropriate binary composition for isocratic separation. The resulting chromatogram is a good first-order approximation for the best possible separation in the give binary mobile phase system. In this way the selection of optimal binary compositions ca be greatly accelerated and simplified. It is shown that the transfer from one organic modifier to another can readily be mad using simple trasfer rules. In this way the selectivity can be varied, while the retention remains approximately constant.

An improved optimization procedure for the selection of mixed mobile phases in reversed phase liquid chromatography

SummaryThe stepwise procedure described previously [1] for the selection of optimal mobile phase compositions in reversed phase liquid chromatography (RPLC) is improved in two ways. The optimization criterion is modified to account for variations in analysis time. The iterative approach to the optimal solvent composition makes a more efficient use of previous data. An example illustrates the resulting gain in speed and accuracy.

Description of solute retention over the full range of mobile phase compositions in reversed-phase liquid chromatography

Abstract In this paper we investigate the relationship between solute retention and mobile phase composition in reversed-phase liquid chromatography over the entire range of composition, with emphasis on mobile phases with a high water content. It will be shown that a quadratic relationship between the logarithm of the capacity factor and the volume fraction of organic modifier is generally valid for mobile phases containing less than 90% water. When more water is added to the mobile phase, a quadratic equation turns out to be insufficient. An experimental study of ten solutes and three organic modifiers is used to show that an extension of the quadratic equation by a term proportional to the square root of the volume fraction leads to a description of all experimental retention data within approximately 10%.

New approaches for fabrication of microfluidic capillary electrophoresis devices with on-chip conductivity detection.

In practice, microfluidic systems are based on the principles of capillary electrophoresis (CE), for a large part due to the simplicity of electroosmotic pumping. In this contribution, a universal conductivity detector is presented that allows detection of charged species down to the μM level. Additionally, powderblasting is presented as a novel technique for direct etching of microfluidic networks. This method allows creation of features down to 50 μm with a total processing time (design to device) of less than one day. The performance of powderblasted devices with integrated conductivity detection is illustrated by the separation of lithium, sodium, and potassium ions and that of fumaric, malic, and citric acid.

Capillary electrophoresis with on‐chip four‐electrode capacitively coupled conductivity detection for application in bioanalysis

Microchip capillary electrophoresis (CE) with integrated four‐electrode capacitively coupled conductivity detection is presented. Conductivity detection is a universal detection technique that is relatively independent on the detection pathlength and, especially important for chip‐based analysis, is compatible with miniaturization and on‐chip integration. The glass microchip structure consists of a 6 cm etched channel (20 νm×70 νm cross section) with silicon nitride covered walls. In the channel, a 30 nm thick silicon carbide layer covers the electrodes to enable capacitive coupling with the liquid inside the channel as well as to prevent interference of the applied separation field. The detector response was found to be linear over the concentration range from 20 νM up to 2 mM. Detection limits were at the low νM level. Separation of two short peptides with a pI of respectively 5.38 and 4.87 at the 1 mM level demonstrates the applicability for biochemical analysis. At a relatively low separation field strength (50 V/cm) plate numbers in the order of 3500 were achieved. Results obtained with the microdevice compared well with those obtained in a bench scale CE instrument using UV detection under similar conditions.

A simple procedure for the rapid optimization of reversed-phase separations with ternary mobile phase mixtures

SummaryA simple rapid procedure is described for estimating optimum compositions of ternary mobile phase mixtures for the separation of samples by reversed-phase liquid chromatography (RPLC). Retention data in two iso-eluotropic binary mobile phase mixtures (mixtures with equal retention times) are required to initiate the procedure. The logarithm of the capacity factor is assumed to vary linearly with the composition of isoeluotropic ternary mixtures formed by mixing the two limiting binaries. Using the product of resolution factors of adjacent peaks as the criterion, an optimum ternary composition is then calculated. After a chromatogram has been obtained with the predicted optimum ternary mobile phase, the procedure is repeated until no further improvement can be achieved. Examples of the application of the present procedure are described to illustrate its effectiveness.