María Celia García-Álvarez-Coque

• Professor
• University of Valencia

Reversed-phase liquid chromatography (RPLC) relies on a non-polar stationary phase and a more polar hydro-organic mobile phase, where compound retention is primarily governed by hydrophobicity, with more hydrophobic compounds being retained longer. The introduction of secondary equilibria in the chromatographic system through additives, such as ani...

In this work, the polyphenolic composition of honeys from three different floral origins (chestnut, heather, and thyme), coming from different geographical areas of Spain was investigated. First, samples were characterized in terms of total phenolic content (TPC) and antioxidant capacity, which was established by three different assays. The results...

The polyphenolic composition of Algerian date palm (Phoenix dactylifera L) leaves (in Hamray, Safray, Ghars, Horra, Deglet Nour (DN) cultivars), and pollen (in DN cultivars), were investigated for the first time in these cultivars. The polyphenolic fractions were extracted using three solvents (acetone, methanol and ethanol), and characterized in t...

The use of ionic liquids (ILs) in liquid chromatography (LC) as mobile phase additives, or immobilized on the stationary phase, has grown during the last decades. In these approaches, ILs no longer play the role of solvents, being just used as reagents able to establish hydrophobic, electrostatic and other specific interactions with the stationary...

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...

Hydrophilic interaction liquid chromatography (HILIC) is able to separate from polar to highly polar solutes, using similar eluents to those in the reversed-phase mode (RPLC) and a polar stationary phase, where water is adsorbed onto its surface. It is widely accepted that multiple modes of interaction take place in the HILIC environment, which can...

Basic compounds yield long retention times and broad and asymmetric peaks in reversed-phase liquid chromatography, due to interaction with residual silanols in the columns. The addition of the surfactant sodium dodecyl sulphate in the so called micellar liquid chromatography enhances the efficiency, but long retention is achieved, due to electrosta...

The capability of liquid chromatography with microemulsions (MEs) as mobile phases was studied for the analysis of four parabens (butylparaben, ethylparaben, methylparaben, and propylparaben) and seven β-adrenoceptor antagonists (acebutolol, atenolol, carteolol, metoprolol, oxprenolol, propranolol, and timolol). MEs were formed by mixing aqueous so...

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 t...

In reversed-phase liquid chromatography (RPLC), positively charged basic compounds yield broad and asymmetric peaks, as a result of ionic interactions with free silanols that remain on conventional silica-based columns. Diverse solutions have been proposed to mask the silanophilic activity, which is translated to an improved peak shape. In this wor...

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 experime...

The description of the profiles of chromatographic peaks has been studied extensively, with a large number of proposed mathematical functions. Among them, the accuracy achieved with modified Gaussian models that describe the deviation of an ideal Gaussian peak as a change in the peak variance or standard deviation over time, has been highlighted. T...

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 sepa...

A glycidyl methacrylate-based monolith was modified with imidazolium-based ionic liquid (IL) to be used as stationary phase for solid-phase extraction (SPE). The host monolithic support was prepared by in-situ UV polymerization in spin column format. Two approaches were developed to incorporate the IL into the polymeric monolithic matrix: generatio...

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 m...

Gradient elution in liquid chromatography reduces the analysis time, improves the efficiency and increases the peak capacity. The study of this chromatographic mode has been based mainly on kinetic dispersion models. The plate model has been applied to a lesser extent, despite being the basis for the concepts of plate height and chromatographic eff...

Modelling the retention behaviour of solutes in liquid chromatography, based on the composition of the mobile phase is a common task in the chromatographic practice. Along the development of liquid chromatography (LC), several models have been proposed to help in understanding the retention mechanisms, and especially, allow the prediction of retent...

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,...

Multi-linear gradients are a convenient solution to get separation of complex samples by modulating carefully the gradient slope, in order to accomplish the local selectivity needs for each particular solute cluster. These gradients can be designed by trial-and-error according to the chromatographer experience, but this strategy becomes quickly ina...

In reversed-phase liquid chromatography, the performance for basic compounds is affected by the interaction of the protonated (cationic) species with the anionic free silanols on the alkyl-bonded stationary phases. Using aqueous-organic mobile phases in the absence of additives, the retention may be too high, and the peaks be broad and asymmetric....

The lack of retention of highly polar solutes in liquid chromatography with reversed phase columns (RPLC) can be solved through the use of the hydrophilic interaction liquid chromatographic (HILIC) mode. Due to the complexity of the separation mechanisms in HILIC and the different factors that may have significant influence, the selection of the ap...

Microemulsions (MEs) are stable, isotropically clear solutions consisting of an oil and water stabilized by a surfactant and a co-surfactant. Oil-in-water microemuslion liquid chromatography (MELC) is a relatively new chromatographic mode, which uses an O/W ME as mobile phase. Retention, selectivity and efficiency can be modified by changing the co...

Gradient elution may provide adequate separations within acceptably short times in a single run, by gradually increasing the elution speed. Similarly to isocratic elution, chromatograms can be predicted under any experimental condition, through strategies based on retention models. The most usual approach implies solving an integral equation (i.e.,...

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, s...

Oil-in-water microemulsions (O/W MEs) are obtained spontaneously by mixing two immiscible liquids (water and oil) in the presence of a surfactant. A co-surfactant is also often needed for ME stabilization. The surfactant provides a microstructure with a definite boundary between oil and water phases. O/W MEs are used as mobile phases in a chromatog...

Performance of chromatographic columns is of major importance in the development of more efficient separation methods. So far, a common practice is to study the column behavior in isocratic elution by modifying the flow rate and fitting the theoretical plate height values versus the mobile phase linear velocity, according to the Van Deemter equatio...

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. I...

The presence of anionic free silanols in the silica-based stationary phases gives rise to broad and asymmetrical peaks when cationic basic compounds are chromatographed using hydro-organic mobile phases. The addition to the mobile phase of a reagent with ionic character prevents the access of analytes to the free silanols, improving the peak shape....

The reversed-phase chromatographic behaviour of six tricyclic antidepressants (amitryptiline, clomipramine, doxepin, imipramine, nortryptiline and maprotiline) was examined in this work with acetonitrile-water mobile phases, in the absence and presence of the ionic liquids 1-hexyl-3-methylimidazolium chloride and 1-hexyl-3-methylimidazolium tetrafl...

Most reports in reversed-phase liquid chromatography (RPLC) with micellar mobile phases make use of the anionic sodium dodecyl sulfate. This surfactant masks efficiently the silanol groups that are the origin of the poor efficiencies and tailing peaks observed for basic compounds in conventional RPLC. However, it has the handicap of yielding excess...

The most efficient optimisation methodologies in liquid chromatography are based on the modelling and prediction of the chromatographic behaviour for each compound in the sample. However, when the column suffers some ageing after the modelling process, predictions may differ significantly from the actual separation. Repeating the modelling is espec...

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 strategie...

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,...

In reversed phase-liquid chromatography, the usual solution to the "general elution problem" is the application of gradient elution with programmed changes of organic solvent (or other properties). A correct quantification of chromatographic peaks in liquid chromatography requires well resolved signals in a proper analysis time. When the complexity...

Background: The disadvantages of the classical approach used to characterize column performance in liquid chromatography, based on the observation of Van Deemter plots, are the need of measuring the extra-column variance, the use of data from only one compound eluted with a single mobile phase, which limits the conclusions of the approach, and the...

To characterize column performance in liquid chromatography, several parameters must be obtained from experimental data. These parameters can be computed through the numerical integration of the net signal to calculate the moments after subtraction of the baseline. This requires the establishment of the peak integration limits. The whole process in...

The search of the best conditions in liquid chromatography is routinely carried out with the information provided by standards. However, there are samples with insufficient knowledge about their chemical composition. In other cases, the identity of the components is known, but there are no standards available, and in others the identity of peaks in...

Basic drugs are positively charged in the usual working pH (2–8) in reversed-phase liquid chromatography. This gives rise to a strong association with the residual ionized silanols in conventional silica-based stationary phases, which is translated in poor peak shape and high consumption of organic solvent to get appropriate retention times. Micell...

Single conventional columns in reversed-phase LC are insufficient for analysing the isoindoles of primary amino acids, due to their limited functionality. An interesting possibility for increasing the separation power is the combination of several columns of different nature, where the length is modified by coupling small segments. This approach ma...

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-...

In reversed-phase liquid chromatography, cationic basic compounds yield broad and asymmetrical peaks, as a result of their ionic interaction with the anionic free silanol groups present in the silica-based stationary phases (commonly derivatised with C18 groups). A simple way to improve the peak shape is the addition to the hydro-organic mobile pha...

Background: Micellar LC was first proposed as a 'green' mode using mobile phases of water and surfactant. However, in most procedures a small amount of organic solvent is required to decrease the retention to convenient values. Results & methodology: Mixed micellar mobile phases prepared with both cationic (sodium dodecyl sulphate) and nonionic su...

Micellar liquid chromatography (MLC) is a reversed-phase (RP) mode, which often does not require gradient elution, since the peaks of mixtures of compounds within a large range of polarities appear more evenly distributed in the chromatograms of isocratic elution, giving rise to a “gradient effect”. However, the use of a gradient of organic solvent...

Background: Current liquid chromatography instruments may yield two-way signals with full spectra collected as a function of time. These signals can be processed to recover chromatographic peaks in case of low resolution in the time order without any peak shape assumption. In Part I of this work, a two-way chromatographic objective function (COF) t...

The plate model proposed by Martin and Synge has been used for the characterization of columns up-to-date. In this approach, the column is divided into a large number (N) of identical theoretical plates. Mobile phase transference between plates takes place in infinitesimal steps with mixing of the solutions in the adjacent plates during the flow. T...

Many inorganic and organic compounds occur in ionic form or are ionised at the pH of the mobile phase, giving rise to poor performance (too short or too long retention and poor efficiency) with typical reversed-phase liquid chromatography columns. Early in the development of this technique, the addition of lipophilic anions and cations to conventio...

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...

Reversed phase liquid chromatography (RPLC) is a mode of high-performance liquid chromatography (HPLC) that employs a nonpolar stationary phase (most frequently a hydrocarbon chain chemically bonded to porous silica particles) and a polar mobile phase constituted by water and at least a water-miscible organic solvent, which performs as a modifier....

After more than three decades of experience, micellar liquid chromatography (MLC) seems to be an alternative to conventional reversed phase liquid chromatography (RPLC) with hydro-organic mobile phases, with increasing interest in ?green? chemistry. Most MLC procedures use hybrid micellar mobile phases containing a surfactant (frequently sodium dod...

Interpretive optimization strategies in liquid chromatography are based on the accurate description (modeling) of the chromatographic behavior. The first step in these strategies consists of gathering information about the compounds in the sample, usually attending only to retention and covering reasonably wide regions of the factors involved. For...

The reversed phase liquid chromatography (RPLC) mode with surfactant above the critical micellar concentration (CMC) has been called micellar liquid chromatography (MLC). In pure micellar systems, the retention behavior is explained by considering three phases or environments: surfactant-modified stationary phase, bulk aqueous solvent, and micellar...

Getting useful chemical information from samples containing many compounds is still a challenge to analysts in liquid chromatography. The highest complexity corresponds to samples for which there is no prior knowledge about their chemical composition. Computer-based methodologies are currently considered as the most efficient tools to optimise the...

The serial coupling of chromatographic columns of controlled lengths containing different stationary phases multiplies the available selectivity in reversed phase liquid chromatography, which expands the probabilities of success in the separation of complex samples. However, the full exploitation of the separation capability of coupled columns requ...

Micellar liquid chromatography (MLC) is a reversed-phase liquid chromatographic (RPLC) mode, which uses a surfactant as modifier. The anionic sodium dodecyl sulphate (SDS) is the most usual, but the addition of an organic solvent is needed to decrease the retention and increase the efficiency. Particularly, positively charged basic compounds are st...

Polyoxyethylene(23)lauryl ether (known as Brij-35) is a non-ionic surfactant, which has been considered as an alternative to the extensively used in micellar liquid chromatography anionic surfactant sodium lauryl (dodecyl) sulfate, for the analysis of drugs and other types of compounds. Brij-35 is the most suitable non-ionic surfactant for micellar...

The popularity of ionic liquids (ILs) has grown during the last decades in several analytical separation techniques. Consequently, the number of reports devoted to the applications of ILs is still increasing. This review is focused on the use of ILs (mainly imidazolium-based associated to chloride and tetrafluoroborate) as mobile phase additives in...

When a new separation problem is faced with high-performance liquid chromatography (HPLC), the analysis is addressed conventionally with a single column, trying to find out a single experimental condition aimed to resolve all compounds. However, in practice, the system selectivity may be insufficient to achieve full resolution. When a separation fa...

The use of computational software can greatly facilitate the teaching of chemical equilibria. It allows the treatment of complex problems without the need of simplifications, and the outcome of a very large number of calculations in a short time. Two practical examples of different complexity are proposed in this work to show the application of a g...

In reversed-phase liquid chromatography in the absence of additives, cationic basic compounds give rise to broad and asymmetrical peaks as a result of ionic interactions with residual free silanols on silica-based stationary phases. Ionic liquids (ILs), added to the mobile phase, have been suggested as alternatives to amines to block the activity o...

The effect of the modifier concentration and flow rate on the chromatographic performance of a second generation Chromolith® RP-18e column, under isocratic and gradient elution with acetonitrile-water mixtures, was examined using four sulphonamides as probe compounds. The acetonitrile concentration was varied between 5 and 55% (v/v), and the flow r...

The optimisation of the resolution in high-performance liquid chromatography is traditionally performed attending only to the time information. However, even in the optimal conditions, some peak pairs may remain unresolved. Such incomplete resolution can be still accomplished by deconvolution, which can be carried out with more guarantees of succes...

Micellar liquid chromatography makes use of aqueous solutions or aqueous-organic solutions containing a surfactant, at a concentration above its critical micelle concentration. In the mobile phase, the surfactant monomers aggregate to form micelles, whereas on the surface of the non-polar alkyl-bonded stationary phases they are significantly adsorb...

An interpretive approach is reported for the simultaneous optimisation of resolution and analysis time in the separation of complex samples by reversed-phase liquid chromatography, using serially-coupled columns of different lengths and nature, and multi-linear gradient elution. The results complement those reported in JCA 1350 (2014)51 for single...

The so-called "fundamental equation for gradient elution" has been used for modelling the retention in gradient elution. In this approach, the instantaneous retention factor (k) is expressed as a function of the change in the modifier content (ϕ(ts )), ts being the time the solute has spent in the stationary phase. This approach can only be applied...

A mixture of compounds often cannot be resolved with a single chromatographic column, but the analysis can be successful using columns of different nature, serially combined through zero-dead volume junctions. In previous work (JCA 1281 (2013) 94), we developed an isocratic approach that optimised simultaneously the mobile phase composition, statio...

Method validation is being applied in the reported analytical methods for decades. Even before this protocol was defined, authors already somehow validated their methods without full awareness. They wished to assure the quality of their work. Validation is an applied approach to verify that a method is suitable and rugged enough to function as a qu...

Column selection in reversed-phase liquid chromatography (RPLC) can become a challenge if the target compounds interact with the silica-based packing. One of such interactions is the attraction of cationic solutes to the free silanols in silica-based columns, which is a slow sorption-desorption interaction process that gives rise to tailed and broa...

In micellar liquid chromatography (MLC), chromatographic peaks are more evenly distributed compared to conventional reversed-phase liquid chromatography (RPLC). This is the reason that most procedures are implemented using isocratic elution. However, gradient elution may be still useful in MLC to analyse mixtures of compounds within a wide range of...

Surfactant addition above the critical micellar concentration (CMC), in reversed-phase liquid chromatography (RPLC), was proposed as a way to modify the selectivity and analysis time, giving rise to a chromatographic mode called micellar liquid chromatography (MLC). However, solutions containing only surfactant are too weak and yield poor peak shap...

The addition of reagents to an 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, whose main features are outlined in th...

Many solvents can be used to prepare mobile phases in liquid chromatography. Also, mixtures of solvents at different ratios are used to modify the mobile phase properties. This can make solvent selection for method development a hard task, unless suitable guidelines are followed. This chapter summarizes the most common strategies used by skilled ch...

Peak profiles in chromatography are characterised by their height, position, width and asymmetry; the two latter depend on the values of the left and right peak half-widths. Simple correlations have been found between the peak half-widths and the retention times. The representation of such correlations has been called half-width plots. For isocrati...

Gel permeation chromatography (GPC), also known as size exclusion chromatography (SEC), is the technique routinely used at high temperature to analyze the molar mass distribution in polyolefins. The distribution of comonomer along the molar mass distribution in a copolymer is a key microstructural feature that determines the macroscopic properties...

The combination of the selectivity of different columns serially coupled improves the separation expectancies with regard to the separation offered by each single column. In the reported approaches, either a pre-selected isocratic mobile phase composition or gradient program, giving rise to acceptable retention, is used. In previous work (JCA 1281...

The optimisation of the experimental conditions in gradient reversed-phase liquid chromatography requires reliable algorithms for the description of the retention and peak profile. As in isocratic elution, the linear relationship between the logarithm of the retention factor and the solvent contents is only acceptable in relatively small concentrat...

Although there is a great deal of stationary phases having different selectivities (even practically orthogonal selectivities), these very rarely are taken as a factor to be optimized during method development. The chromatographer selects the stationary phase usually in a trial-and-error fashion (or based on the solute nature and expected interacti...

The description of skewed chromatographic peaks has been discussed extensively and many functions have been proposed. Among these, the Polynomially Modified Gaussian (PMG) models interpret the deviations from ideality as a change in the standard deviation with time. This approach has shown a high accuracy in the fitting to tailing and fronting peak...

Achievement of the highest separation efficiency and quick delivery of results are key requirements in liquid chromatography for enhancing productivity and reducing analysis cost, especially in the pharmaceutical industry. This work concerns two of the most popular current solutions to get fast separations: the use of a silica-based monolithic colu...

Two approaches are proposed to measure the effect of different experimental factors (such as the modifier concentration and temperature) on the elution strength and peak shape in reversed-phase liquid chromatography, which quantify the percentage change in the retention factor and peak width (referred to the weakest conditions) per unit change in t...

Ionic liquids are dual modifiers composed by a large anion and a large cation, which interact with both the hydrophobic alkyl-bonded phase and the anionic residual silanols in C18 columns. The deactivation of the silanol groups has important implications on the chromatographic analysis of basic drugs, being the improvement of peak profiles and shor...

Infrared detection has been shown to be very appropriate for high temperature analysis of polyolefins. After some early reports in which dispersive or single-band filter-based detectors were applied, Fourier transform detectors have been described for this application, in order to improve the method sensitivity. Modern simple filter-based detectors...

The chromatographic behavior of seven tricyclic antidepressants (amitryptiline, clomipramine, doxepin, imipramine, maprotiline, nortryptiline, and trimipramine) was examined with micellar mobile phases containing the nonionic surfactant Brij-35. Acetonitrile-water mixtures were also used for comparison purposes. Tricyclic antidepressants are modera...

A general systematic approach including ionic strength effects is proposed for the numerical calculation of concentrations of chemical species in multiequilibrium problems. This approach extends the versatility of the approach presented in a previous article and is applied using the Solver option of the Excel spreadsheet to solve real problems such...

Chromatographic problems are usually addressed trying to find out a single experimental condition aimed to resolve all compounds in the sample. However, very often, the chromatographic system is not able to provide full resolution. When a separation fails, the usual choice is introducing a drastic change in the chromatographic system (e.g. column,...

CRYSTEX is a fully automated instrument for the determination of Xylene Solubles in polypropylene and ethylene-propylene copolymers, providing an excellent correlation with gravimetric methods. The instrument can be equipped with a dual band infrared (IR) detector to quantify the ethylene weight percentage (C2%) in the amorphous fraction, and in th...

Micellar liquid chromatography (MLC) was first proposed as an attractive alternative to avoid the use of organic solvents. It was soon apparent that pure micellar solutions yield poor efficiencies. This problem was remediated by the addition of a small amount of an organic solvent. However, the general opinion of the poor peak shape has prevailed a...

Residual silanols on C18 columns yield undesirable slow-kinetics ion-exchange interactions with positively charged basic compounds that result in asymmetrical peaks, low efficiencies and long retention times. The purity of the silica employed as supporting material, and the technique used to form the bonded phase, which varies with the brand and ma...

Situations of minimal resolution are often found in liquid chromatography, when samples that contain a large number of compounds, or highly similar in terms of structure and/or polarity, are analysed. This makes full resolution with a single separation condition (e.g., mobile phase, gradient or column) unfeasible. In this work, the optimisation of...

The chromatographic elution has been studied from different perspectives. However, in spite of the simplicity and evident deficiencies of the plate model proposed by Martin and Synge, it has served as a basis for the characterization of columns up-to-date. This approach envisions the chromatographic column as an arbitrary number of theoretical plat...

In a previous work (J. Sep. Sci. 2009, 32, 2793-2803), we reported an interpretive optimisation approach to achieve maximal resolution in minimal analysis time, based on models describing the retention and peak shape as a function of mobile phase composition and flow rate. The method was applied to the separation of a group of basic drugs in a Chro...