0000000000214411
AUTHOR
J.a. Navarro-huerta
Study of the performance of a resolution criterion to characterise complex chromatograms with unknowns or without standards
The search for best conditions in liquid chromatography is routinely carried out with information provided by chemical standards. However, sometimes there are samples with insufficient knowledge about their chemical composition. In other cases, identities of the components are known, but there are no standards available, and in other cases the identities of peaks in chromatograms taken under different conditions are ambiguous. Most resolution criteria used to measure separation performance cannot be applied to these samples. In this work, a global resolution function valid for all situations was developed based on automatic measurements of peak prominences (area fraction exceeding the line …
Modelling retention and peak shape of small polar solutes analysed by nano-HPLC using methacrylate-based monolithic columns.
Abstract The development of methacrylate-based monolithic columns was studied for the separation of pharmaceutical hydrophilic compounds in nano-liquid chromatography. The selected polymerisation mixture consisted of 7.5% hexyl methacrylate, 4.5% methacrylic acid and 18.0% ethylene dimethacrylate (w/w), in a binary porogenic solvent (35:35 w/w 1-propanol/1,4-butanediol). The polymer synthesised with this mixture has a good permeability, not excessive back-pressure, and reasonable retention times for polar and non-polar solutes. Monolithic columns (12 cm total capillary length, 100 μm i.d.), prepared with this mixture, were able to produce hydrogen bonding and electrostatic interactions, giv…
Estimation of peak capacity based on peak simulation.
Peak capacity (PC) is a key concept in chromatographic analysis, nowadays of great importance for characterising complex separations as a criterion to find the most promising conditions. A theoretical expression for PC estimation can be easily deduced in isocratic elution, provided that the column plate count is assumed constant for all analytes. In gradient elution, the complex dependence of peak width with the gradient program implies that an integral equation has to be solved, which is only possible in a limited number of situations. In 2005, Uwe Neue developed a comprehensive theory for the calculation of PC in gradient elution, which is only valid for certain situations: single linear …
Interpretive search of optimal isocratic and gradient separations in micellar liquid chromatography in extended organic solvent domains
Abstract Micellar liquid chromatography (MLC) is a reversed-phase mode with mobile phases containing an organic solvent and a micellised surfactant. Most procedures developed in MLC are implemented in the isocratic mode, since the general elution problem in chromatography is less troublesome. However, gradient elution may be still useful in MLC to analyse mixtures of compounds within a wide range of polarities, in shorter times. MLC using gradients is attractive to determine by direct injection moderate to low polar compounds in physiological samples. In these analyses, the use of initial micellar conditions (isocratic or gradient) with a fixed amount of surfactant above the critical micell…
Ultra-short ion-exchange columns for fast charge variants analysis of therapeutic proteins
Abstract The purpose of this work was to study the potential of recently developed ultra-short column hardware for ion exchange chromatography (IEX). Various prototype and commercial columns having lengths of 5, 10, 15, 20 and 50 mm and packed with non-porous 3 µm particles were systematically compared. Both pH and salt gradient modes of elution were evaluated. Similarly, what has been previously reported for reversed phase liquid chromatography (RPLC) mode, an “on-off” retention mechanism was observed in IEX for therapeutic proteins and their fragments (25–150 kDa range). Because of the non-porous nature of the IEX packing material, the column porosity was relatively low (e = 0.42) and the…
Secondary chemical equilibria in reversed-phase liquid chromatography
Abstract The addition of reagents to a reversed-phase liquid chromatography (RPLC) mobile phase enables the separation of ionizable compounds, inorganic anions, and metal ions, using conventional instrumentation, silica-based materials, and hydro-organic mixtures, thanks to a variety of secondary equilibria. This gives rise to several chromatographic modes, the main features of which are outlined in this chapter. The effect of the mobile phase pH on the retention of ionizable compounds is described, together with the recommended experimental practice. The mechanism of adsorption of amphiphilic anions or cations on the stationary phase to attract analytes with opposite charge, or suppress th…
Testing experimental designs in liquid chromatography (I): Development and validation of a method for the comprehensive inspection of experimental designs.
The basis of interpretive optimisation in liquid chromatography is the prediction of resolution, from appropriate solute retention models. The reliability of the process depends critically on the quality of the experimental design. This work develops, validates and applies a general methodology aimed to evaluate the quality of any training experimental design, which will be applied in Part II to design optimisation. The methodology is based on the systematic evaluation of the uncertainties associated to the prediction of retention times in comprehensive scans of both isocratic and gradient experimental conditions. It is able to evaluate comprehensively experimental designs of arbitrary comp…
Classification of olive leaves and pulp extracts by comprehensive two-dimensional liquid chromatography of polyphenolic fingerprints.
Abstract The development of a new comprehensive two-dimensional liquid chromatographic method is described, to obtain the profiles of polyphenolic compounds present in olive (Olea europaea L.) leaves and pulps from different genetic origin. Optimisation of the stationary phase nature, particle size, column length and internal diameter, as well as other separation conditions, was performed. Along the study, three stationary phases (C18, PFP and phenyl) in the first dimension (1D), and five (C18, amide, cyano, phenyl and PFP) in the second dimension (2D) were combined to obtain the maximal number of resolved peaks. The optimised method successfully characterised the presence of 26 and 29 comm…
Solvent selection in liquid chromatography
Many solvents and additives are used to prepare mobile phases in liquid chromatography (LC). Also, mixtures of solvents at different ratios are used to modify the mobile-phase properties. This can make solvent selection for method development a puzzling task, unless suitable guidelines are followed. This chapter summarizes the most common strategies used by skilled chromatographers in reversed-phase, normal-phase, and hydrophilic interaction LC. These are based on considerations about the global polarity of solutes, stationary phase, and mobile phase, which determine the elution strength, and on the particular profile of the contributions of intermolecular interactions to the global polarit…
Assisted baseline subtraction in complex chromatograms using the BEADS algorithm.
The data processing step of complex signals in high-performance liquid chromatography may constitute a bottleneck to obtain significant information from chromatograms. Data pre-processing should be preferably done with little (or no) user supervision, for a maximal benefit and highest speed. In this work, a tool for the configuration of a state-of-the-art baseline subtraction algorithm, called BEADS (Baseline Estimation And Denoising using Sparsity) is developed and verified. A quality criterion based on the measurement of the autocorrelation level was designed to select the most suitable working parameters to obtain the best baseline. The use of a log transformation of the signal attenuate…
Benefits of solvent concentration pulses in retention time modelling of liquid chromatography
The advantages and disadvantages of the use of isocratic experimental designs including transient increments of organic solvent (i.e., pulses) in the mobile phase(s) of lowest elution strength are explored with modelling purposes. For retained solutes, this type of mixed design offers similar or better predictive capability than gradient designs, shorter measurement time than pure isocratic designs, and retention model parameters that agree with those derived from pure isocratic experiments, with similar uncertainties. The predicted retention times are comparable to those offered by models adjusted from pure isocratic designs, and the solvent waste is appreciably lower. Under a practical st…
Global retention models and their application to the prediction of chromatographic fingerprints
Abstract The resolution of samples containing unknown compounds of different nature, or without standards available, as is the case of chromatographic fingerprints, is still a challenge. Possibly, the most problematic aspect that prevents systematic method development is finding models that describe without bias the retention behaviour of the compounds in the samples. In this work, the use of global models (able to describe the whole sample) is proposed as an alternative to the use of individual models for each solute. Global models contain parameters that are specific for each solute, while other parameters ‒related to the column and solvent‒ are common for all solutes. A special regressio…
Testing experimental designs in liquid chromatography (II): Influence of the design geometry on the prediction performance of retention models.
Abstract In liquid chromatography, the reliability of predictions carried out with retention models depends critically on the quality of the training experimental design. The search of the best design is more complex when gradient runs are used instead of isocratic experiments. In Part I of this work (JCA 1624 (2020) 461180), a general methodology based on the error propagation theory was developed and validated for assessing the quality of training designs involving gradients. The treatment relates the mathematical properties of a retention model with the geometry of the training designs and their subsequent predictions. In that work, only five usual designs were considered. Part II invest…