Estrategias para mejorar la calidad de las separaciones en cromatografía líquida de fase inversa

La Memoria de Tesis Doctoral propone estrategias dirigidas a mejorar la separación de mezclas que muestran una resolución cromatográfica extraordinariamente baja, en las condiciones usuales de elución, mediante cromatografía líquida de alta eficacia (HPLC, high-performance liquid chromatography). La HPLC es actualmente la técnica analítica de separación más extendida, debido a su amplio campo de aplicación, fiabilidad, robustez y sensibilidad. Lamentablemente, la eficacia que proporciona suele ser inferior a la conseguida en cromatografía de gases, electroforesis capilar y otras técnicas de electromigración, lo que representa una limitación para el análisis de muestras complejas. Los estudios incluidos en la Memoria se han centrado, principalmente, sobre aspectos teóricos fundamentales de interés general en HPLC utilizando elución isocrática y en gradiente, con columnas individuales o acopladas, relacionados con la predicción de las condiciones de elución, la modelización de picos cromatográficos, la investigación de los mecanismos de retención que existen en el interior de las columnas cromatográficas cuando se establecen equilibrios secundarios en presencia de diversos tipos de aditivos añadidos a las fases móviles (los surfactantes dodecilsulfato sódico y polioxietilen(23)lauril éter, líquidos iónicos derivados del 3-metilimidazolio y las aminas trietilamina y dimetiloctilamina), y la combinación de mecanismos de separación. En esta última serie de investigaciones, deben resaltarse contribuciones novedosas sobre la correcta conexión de columnas cromatográficas en HPLC, y el desarrollo de herramientas quimiométricas para la predicción y optimización de cromatogramas cuando se utilizan columnas acopladas en elución isocrática y de gradiente. También, con el fin de describir de una manera sencilla y global el comportamiento de mezclas de solutos en diversas condiciones, utilizando ambos tipos de elución, cabe destacar el desarrollo de gráficos que proporcionan información global sobre la retención o sobre la cinética de las interacciones soluto-fase estacionaria. Otras aportaciones se refieren a la propuesta de diversas metodologías para monitorizar la posible adsorción de un aditivo sobre la fase estacionaria, favorecer la inyección directa de muestras fisiológicas en una columna cromatográfica y realizar la correcta medida de la supresión de la actividad de los grupos silanol en las columnas de base sílice mediante la adsorción de aditivos añadidos a la fase móvil. Las investigaciones se llevaron a cabo con compuestos de diversa naturaleza (sulfonamidas, β bloqueantes, diuréticos y flavonoides), eluidos con mezclas de acetonitrilo-agua en presencia y ausencia de diversos aditivos, así como fases móviles micelares que sólo contenían un surfactante en ausencia de disolvente orgánico. Para realizar los estudios, se han utilizado columnas cromatográficas de fase inversa de diverso tipo: C18 convencionales y con grupos embebidos para modificar su polaridad, ciano y fenilo. En los diversos estudios, se han desarrollado softwares de optimización basados en la modelización tanto del tiempo de retención como del perfil de los picos cromatográficos, para elución isocrática y de gradiente, utilizando gráficos de Pareto para encontrar soluciones que proporcionen resoluciones y tiempos de análisis aceptables, y algoritmos genéticos para reducir el tiempo de cálculo.The PhD. work proposes strategies to improve the separation of mixtures with extremely low chromatographic resolution, under the usual conditions of elution, using high-performance liquid chromatography (HPLC). This is currently the analytical separation technique most widely used due to its wide scope, reliability, robustness and sensitivity. Unfortunately, the efficiency that it offers is usually lower than that achieved in gas chromatography, capillary electrophoresis and other electromigration techniques. This represents a limitation for the analysis of complex samples. The studies included in this work have been focused mainly on fundamental theoretical aspects of general interest in HPLC, using isocratic and gradient elution with single or coupled columns, related with the prediction of the elution conditions, modeling of chromatographic peaks, investigation of the retention mechanisms inside the chromatographic columns when secondary equilibria are established in the presence of several types of reagents added to the mobile phases (the surfactants sodium dodecyl sulfate and polyoxyethylene(23)lauryl ether, ionic liquids derived from 3 methylimidazolium and the amines dimethyloctylamine and triethylamine), and the combination of separation mechanisms. In the latter research, novel contributions on the correct connection of chromatographic columns in HPLC and the development of chemometric tools for the prediction and optimization of chromatograms when coupled columns are used under isocratic and gradient elution, should be highlighted. New graphics have been also developed to describe in a simple fashion the global behavior of mixtures of solutes in diverse conditions, using both elution modes. These graphics provide global information on the retention kinetics of the solute-stationary phase interactions. Other contributions concern various methods for monitoring the possible adsorption of an additive on the stationary phase, for performing direct injection of physiological samples in a chromatographic column, and for the correct measurement of the suppression of the activity of the silanol groups on silica-based columns through the use of additives in the mobile phase. The research was carried out with compounds belonging to different families (sulfonamides, β-blockers, diuretics and flavonoids), eluted with acetonitrile-water mixtures with and without additives and micellar mobile phases containing only a surfactant in the absence of organic solvent. Several types of reversed-phase chromatographic columns were used: conventional C18 columns and C18 columns with embedded groups to change its polarity, and cyano and phenyl columns. In the different studies, diverse optimization software has been developed based on the modeling of both retention and chromatographic peak profiles for isocratic and gradient elution, and assisted by Pareto plots to find solutions offering good resolution and acceptable analysis time. Genetic algorithms were also used in some cases to reduce the computation time

Prediction of Peak Shape and Characterization of Column Performance in Liquid Chromatography as a Function of Flow Rate

Traditionally, column performance in liquid chromatography has been studied using information from the elution of probe compounds at different flow rates through van Deemter plots, which relate the column plate height to the linear mobile phase velocity. A more recent approach to characterize columns is the representation of the peak widths (or the right and left peak half-widths) for a set of compounds versus their retention times, which, for isocratic elution, give rise to almost linear plots. In previous work, these plots have been shown to facilitate the prediction of peak profiles (width and asymmetry) with optimization purposes. In this work, a detailed study on the dependence of the peak widths (or half-widths) on the flow rate is reported. A new approach to quantify the deterioration of column performance for slow and fast flow rates and to characterize chromatographic columns is proposed. The approach makes use of the width (or half-widths) for a set of compounds with similar interaction kinetics and does not require knowledge of the extra-column contributions to the total variance. The chromatographic data of two sets of compounds of different natures (sulfonamides and β-blockers), eluted from Spherisorb and Chromolith columns with acetonitrile-water mixtures, are used to illustrate the approach

Program for the interpretive optimization of two-dimensional resolution

The challenge of fully optimizing LC × LC separations is horrendous. Yet, it is essential to address this challenge if sophisticated LC × LC instruments are to be utilized to their full potential in an efficient manner. Currently, lengthy method development is a major obstacle to the proliferation of the technique, especially in industry. A program was developed for the rigorous optimization of LC × LC separations, using gradient-elution in both dimensions. The program establishes two linear retention models (one for each dimension) based on just two LC × LC experiments. It predicts LC × LC chromatograms using a simple van-Deemter model to generalize band-broadening. Various objectives (analysis time, resolution, orthogonality) can be implemented in a Pareto-optimization framework to establish the optimal conditions. The program was successfully applied to a separation of a complex mixture of 54 aged, authentic synthetic dyestuffs, separated by ion-exchange chromatography and ion pair chromatography. The main limitation experienced was the retention-time stability in the first (ion-exchange) dimension. Using the PIOTR program LC × LC method development can be greatly accelerated, typically from a few months to a few days