A. Pappa-Louisi

Retention models for isocratic and gradient elution in reversed-phase liquid chromatography.

One- and multi-variable retention models proposed for isocratic and/or gradient elution in reversed-phase liquid chromatography are critically reviewed. The thermodynamic, exo-thermodynamic or empirical arguments adopted for their derivation are presented and discussed. Their connection to the retention mechanism is also indicated and the assumptions and approximations involved in their derivation are stressed. Special attention is devoted to the fitting performance of the various models and its impact on the final predicted error between experimental and calculated retention times. The possibility of using exo-thermodynamic retention models for prediction under gradient elution is considered from a practical point of view. Finally, the use of statistical weights in the fitting procedure of a retention model and its effect on the calculated elution times as well as the transferability of retention data among isocratic and gradient elution modes are also examined and discussed.

On the equations describing chromatographic peaks and the problem of the deconvolution of overlapped peaks

The problem of the appropriate choice of the function that describes a chromatographic peak is examined in combination with the deconvolution of overlapped peaks by means of the non-linear least-squares method. It is shown that the majority of the functions proposed in the literature to describe chromatographic peaks are not suitable for this purpose. Only the polynomial modified Gaussian function can describe almost every peak but it is mathematically incorrect unless it is redefined properly. Two new functions are proposed and discussed. It is also shown that the deconvolution of an overlapping peak can be done with high accuracy using a non-linear least-squares procedure, like Microsoft Solver, but this target is attained only if we use as fitted parameters the position of the peak maximum and the peak area (or height) of every component in the unresolved chromatographic peak. In case we use as fitted parameters all the parameters that describe each single peak enclosed in the multi-component peak, then Solver leads to better fits, which though do not correspond to the best deconvolution of the peak. Finally, it is found that Solver gives much better results than those of modern methods, like the immune and genetic algorithms.

Study on the electrochemical detection of the macrolide antibiotics clarithromycin and roxithromycin in reversed-phase high-performance liquid chromatography

The optimal conditions of the amperometric detection of the macrolide antibiotics clarithromycin and roxithromycin were found by cyclic voltammetric studies and HPLC-electrochemical detection responses obtained in different temperatures (25.5-60 degrees C) and different but almost isoelutropic binary, ternary and quaternary mixtures of aqueous buffer (pH 7), methanol, acetonitrile and isopropanol. These conditions were also proved to be applicable for the quantitative detection of clarithromycin in human plasma using roxithromycin as an internal standard and vice versa. It was demonstrated that increased attention has to be paid to eluent composition and column temperature to ensure sensitive and reproducible electrochemical responses as well as regularly shaped peaks for both macrolides tested.

Modeling the combined effect of temperature and organic modifier content on reversed-phase chromatographic retention - Effectiveness of derived models in isocratic and isothermal mode retention prediction

Models considering simultaneously mobile phase organic content and column temperature were developed in this study by an extension of different equations describing the influence of temperature on solute retention. This extension was achieved by two methods: a semi-thermodynamic and a direct combination of equations expressed separately the dependence of the retention upon each of these factors. The above approaches gave a great number of expressions for the logarithm of the solute retention factor in terms of both temperature and organic content in the mobile phase, ln k(T,phi), determined from the dependence of the standard enthalpy of the retention process on T. From the final expressions of ln k(T,phi) we tested only those with the minimum number of adjustable parameters, i.e. those that correspond to a constant standard enthalpy of the retention process. For this test we examined the retention behaviour of a sample of alkylbenzenes in aqueous acetonitrile eluents. These compounds exhibit ln k versus 1/T plots with a very small curvature. We found that a new equation for ln k(T,phi) based on the adsorption model for retention performs better than all the others. The average percentage prediction error ranges from 0.7 to 1.4%.

On the use of genetic algorithms for response surface modeling in high-performance liquid chromatography and their combination with the Microsoft Solver

Four genetic algorithms--the classical, Haupts, Brunettis and a modification of the classical algorithm suggested in the present paper--are examined when they are used for the modeling of response surfaces in high-performance liquid chromatography (HPLC). We found that the best results are obtained from our modification and the worst by Haupts algorithm. The classical genetic algorithm gives satisfactory results, better than those of Brunettis algorithm. We also ascertained that all genetic algorithms may get stuck in a local minimum other than the global one, except for our modification, which can be considered to approach a global method. Finally, the time needed for the optimization of a genetic algorithm and the combination of a genetic algorithm with a non-linear least-squares routine are considered and discussed.

New equations describing the combined effect of pH and organic modifier concentration on the retention in reversed-phase liquid chromatography

Six equations that express the combined effect of mobile phase pH and organic modifier content on sample retention in reversed-phase liquid chromatography (RPLC) are developed based on either the adsorption or the partition model for retention. The equations are tested against five retention data sets taken from literature. In the tests two pH scales are used, w(w)pH and s(s)pH. It is shown that a new seven-parameter equation works more satisfactorily, because it exhibits good numerical behavior, gives low values of the sum of squares of residuals and represents the experimental retention surfaces successfully. In addition, the danger of overfitting, which leads to the prediction of physically meaningless retention surfaces, is minimized by using the proposed new seven-parameter equation. Finally, the possibility of obtaining reliable pK values of weak acids or bases chromatographically by means of the derived equations is also considered and discussed.

New Approaches to Linear Gradient Elution Used for Optimization in Reversed-Phase Liquid Chromatography

Abstract The various approaches developed for prediction and optimization in linear gradient elution in reversed-phase liquid chromatography are critically reviewed. These approaches concern both single-mode and multi-mode gradient elution, the latter involving either gradients related exclusively to the mobile phase composition or combined gradients of the mobile phase composition with flow rate and/or column temperature or combined gradients of flow rate and column temperature. The advantages and disadvantages of each method are discussed and special attention is devoted to the factors that affect the quality of the prediction and their impact to the optimisation. Finally, the fitting techniques and optimisation methods adopted in linear gradient elution are presented and the most effective algorithms used for this purpose are indicated and discussed.

Theory and application of the two-mode gradient elution in liquid chromatography involving simultaneous changes in temperature and mobile-phase composition.

The theory of the dual-mode gradient elution in liquid chromatography involving any type of simultaneous changes in column temperature and mobile-phase composition is developed following Drakes approach. The theory was tested in the retention prediction of six alkylbenzenes in aqueous eluting systems modified by acetonitrile. Significant delay phenomena, i.e., a lag between the programmed gradient temperature and the temperature in the oven, and a lag between the oven temperature and the effective temperature the analyte feels in the column, were detected. When these phenomena were taken into account, the retention prediction obtained for all solutes under all dual-mode gradient conditions was excellent. The average percentage error between experimental and predicted retention times is below to 2%.

The role of mobile-phase pH in the determination of catecholamine-related compounds by reversed-phase liquid chromatography with amperometric detection

SummaryA study was conducted for the optimization of the reversed-phase high performance liquid chromatographic separation of different types (acidic, basic, amphoteric and neutral) of catechol-related compounds by varying the pH. For pH to be used as the optimizing parameter, its effects on retention, peak width and peak shape had to be investigated. Therefore, the pH dependence of the amperometric detector response, as measured by the oxidation peak height or peak area, was taken into account.

Direct RP‐HPLC determination of underivatized amino acids with online dual UV absorbance, fluorescence, and multiple electrochemical detection

The combined use of a dual-UV detector, a fluorimetric one and of a multiple electrochemical (EC) detector equipped with a dual electrode, consisting of a conventional size 3 mm diameter glassy carbon electrode (GCE) and of a pair of 30 mum thick carbon microfibers, is proposed for the determination of 15 amino acids, two dipeptides and creatinine. This online coupling of the above detection modes could partially replace amino acid analysis by derivatization methods, since it solves problems concerning the direct detection of selected underivatized amino acids. Additionally, it was proved that the use of multiple-detection allows positive peak identification in a single chromatographic run, yields more information for free amino acids and solves in some cases the problem of chromatographic resolution. In order to optimize the detection conditions of the underivatized amino acids and related compounds by different detectors, their detection characteristics were determined by adequate preliminary experiments. The electro-oxidation characteristics of the underivatized compounds of interest were determined by hydrodynamic voltammetry using a flow cell with a macrodisc GCE and by ex-situ voltammetry using both a GCE of conventional size and a carbon fiber disk microelectrode. Important practical advantages of microfiber and microdisk electrodes with respect to macroelectrodes were demonstrated.