Comparison of the performance of butanol and pentanol as modifiers in the micellar chromatographic determination of some phenethylamines
Abstract A procedure was developed for the determination of several phenethylamines (amphetamine, arterenol, ephedrine, phenylephrine, phenylpropanolamine, mephentermine, methoxyphenamine, pseudoephedrine and tyramine), using micellar mobile phases of sodium dodecyl sulfate (SDS), a C18 column and UV detection. The drugs were eluted at short retention times with conventional acetonitrile–water or methanol–water mobile phases. In contrast, in the micellar system, they were strongly retained due to association with the surfactant adsorbed on the stationary phase, and needed the addition of butanol or pentanol to be eluted from the column. These modifiers allowed a simple way of controlling th…
Interpretive optimisation strategy applied to the isocratic separation of phenols by reversed-phase liquid chromatography with acetonitrile–water and methanol–water mobile phases
An optimisation protocol is presented for the resolution of complex mixtures in isocratic RPLC with binary mobile phases of organic solvent and water, which is based on the prediction of peak position and shape of the individual compounds. A good description of the retention was achieved through the application of statistical weights to the widely used linear or quadratic relationships between the logarithm of the retention factor (log k) and the organic solvent concentration in the mobile phase. The maximisation of the product of peak purities for each compound is shown as a competitive resolution strategy versus the worst value of a selectivity parameter. Peak purities allow one to associ…
Micellar-organic versus aqueous-organic mobile phases for the screening of β-blockers
A comparative study of the performance of reversed-phase liquid chromatography with micellar-organic (MLC) and aqueous-organic (RPLC) mobile phases is reported for the separation of 16 -blockers (acebutolol, alprenolol, atenolol, bisoprolol, carteolol, celiprolol, esmolol, labetalol, metoprolol, nadolol, oxprenolol, pindolol, practolol, propranolol, sotalol, and timolol). MLC with hybrid mobile phases of sodium dodecyl sulfate (SDS) and propanol is revealed as a very competitive technique for the screening of these drugs. Using a conventional Spherisorb C18 column, the theoretical plates ( N) and asymmetry factors (B/A) for the optimal mobile phase compositions were in the ranges N = 2200–4…
Levels in the interpretive optimisation of selectivity in high-performance liquid chromatography: a magical mystery tour.
Interpretive approaches for selectivity optimisation, which are those supported by retention models, are able to exploit efficiently the capabilities of the chromatographic system. The resolution of a mixture is usually faced in a first trial by looking for a unique experimental condition, able to resolve all compounds in the sample. If this is not possible, the problem can be outlined with less ambitious aims, focusing on only some compounds. In an extreme case, a single analyte can be individually optimised. Current strategies that give answer to the different goals pursued in the analysis, which are classified as total, partial and specific, are reviewed. Optimisation oriented to deconvo…
Global treatment of chromatographic data with MICHROM
Abstract The program MICHROM for the general treatment of chromatographic data is presented. MICHROM takes part in all the stages of the analytical process. It allows determination of dead time, smoothing of chromatograms, measurement of peak parameters, fitting of skewed peaks, and deconvolution of overlapped peaks. Tools for the experimental design, optimization of the mobile phase composition to resolve a mixture of analytes, and simulation of chromatograms in several experimental conditions, are implemented. Routines for the graphical representation of chromatograms, resolution surfaces, contour maps, management of data series, optimization and regression analysis, are also included. Th…
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…
Prediction of the retention in reversed-phase liquid chromatography using solute-mobile phase-stationary phase polarity parameters.
Abstract A previously reported algorithm, based on the equation: log k=( log k) 0 +p(P N m −P N s ) , that relates the retention in reversed-phase liquid chromatography with solute (p), mobile phase (PmN) and stationary phase (PsN) relative polarity parameters, is improved. The retention data reported by several authors for different sets of compounds, eluted with acetonitrile–water and methanol–water mixtures, are used to test the algorithm and elaborate a database of p values. The methodology is successfully applied to predict the retention using PmN values calculated as PmN=1.00−(2.13ϕ)/(1+1.42ϕ) for acetonitrile–water and PmN=1.00−(1.33ϕ)/(1+0.47ϕ) for methanol–water, ϕ being the organi…
Considerations on the modelling and optimisation of resolution of ionisable compounds in extended pH-range columns
Abstract The problems associated to the modelling and optimisation of the chromatographic resolution of mixtures involving ionisable solutes at varying pH and acetonitrile content are discussed. Several retention models that separate the contributions of solute, column and stationary phase, were used. The retention was predicted with low errors in large pH domains (2–12), which was an essential requirement to face the optimisation of resolution. The selected mixture was particularly problematic under the viewpoint of resolution, owing to the excessively diverse acid–base behaviour of solutes. This variety led to sudden drops in retention at different pH for each solute, yielding numerous pe…
Models and objective functions for the optimisation of selectivity in reversed-phase liquid chromatography.
Interpretive methodologies are the most efficient tools for finding the optimal conditions in chromatography. These methodologies are supported by models or algorithms able to infer the system behaviour upon changes in the experimental factors. Once the models are built with data obtained from sets of carefully designed experiments, molecular modelling or other approaches, they can be applied to predict the performance of new conditions. The different elements involved in these methodologies, for both isocratic and gradient elution, are given. Special attention is devoted to the description of retention, owing to its major impact on the prediction of chromatographic resolution. Several mode…
Enhancement in the computation of gradient retention times in liquid chromatography using root-finding methods.
Abstract 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 fundamental equation of gradient elution), which has an analytical solution only for certain combinations of retention model and gradient programme. This limitation can be overcome by using numerical integration, which is a universal approach although at the cost of longer computation times. In this work, several alternatives to impr…
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…
Performance of a Chromolith RP-18e column for the screening of β-blockers
The chromatographic performance of a monolithic column (Chromolith RP-18e) was comprehensively examined in the isocratic separation of ten beta-blockers, using ACN-water mobile phases, and compared with the performance of three microparticulate RP columns manufactured with different types of silica: Spherisorb ODS-2, Kromasil C18 and XTerra MS C18. The comparison considered the analysis time, selectivity, peak shape (column efficiency and asymmetry) and resolution, and was extended to a wide range of mobile phase compositions. The Chromolith column showed good performance for the analysis of beta-blockers with regard to the packed columns. In terms of selectivity and analysis time, the grea…
Updating chromatographic predictions by accounting ageing for single and tandem columns.
The most efficient optimization methodologies in liquid chromatography are based on the modeling and prediction of the chromatographic behavior for each compound in the sample. However, when the column suffers some ageing after the modeling process, predictions may differ significantly from the actual separation. Repeating the modeling is especially troublesome when several columns are involved, as is the case of coupled columns. We propose a shortcut to correct the time and peak profiles in these situations, after evaluating the effects of ageing. The original models are corrected by introducing parameters accounting for column ageing, obtained using the data of a small subset of compounds…
Resolution of overlapped non-absorbing and absorbing solutes using either an absorption null-balance detection window or multivariate deconvolution applied to capillary electrophoresis of anionic surfactants.
Abstract Non-absorbing alkyl ether sulfates (AES) can be separated using anthraquinone-2-carboxylic acid (AQCA) as a probe; however, absorbing alkyl benzene sulfonates (ABS), if present, interfere indirect detection of most AES oligomers. Overcoming of this interference, as well as the simultaneous characterisation and evaluation of AES, fatty acids and ABS, was accomplished by using a diode-array detector and the procedures here discussed. First, it was shown that ABS can be made undetectable by using a 9 nm wide and 227 nm centred charge-absorptivity null-balance detection window (NBDW), where its contribution to the absorbance cancels the dilution effects that its presence induces on the…
Effects of pH and the presence of micelles on the resolution of diuretics by reversed-phase liquid chromatography
A comparative study on the performance of two RPLC modes on the separation of 18 diuretics with diverse acid-base behaviour (acetazolamide, althiazide, amiloride, bendroflumethiazide, benzthiazide, bumetanide, canrenoic acid, chlorothiazide, chlorthalidone, ethacrynic acid, furosemide, hydrochlorothiazide, piretanide, probenecid, spironolactone, triamterene, trichloromethiazide and xipamide) was carried out. A conventional octadecylsilane column and acidic acetonitrile-water mobile phases, in the absence and presence of micelles of the anionic surfactant sodium dodecyl sulphate (SDS), were used. The effects of pH and the modifiers acetonitrile and SDS on peak asymmetry, efficiency, selectiv…
Performance of short-chain alcohols versus acetonitrile in the surfactant-mediated reversed-phase liquid chromatographic separation of β-blockers
Organic solvents are traditionally added to micellar mobile phases to achieve adequate retention times and peak profiles, in a chromatographic mode which has been called micellar liquid chromatography (MLC). The organic solvent content is limited to preserve the formation of micelles. However, at increasing organic solvent contents, the transition to a situation where micelles do not exist is gradual. Also, there is no reason to neglect the potentiality of mobile phases containing only surfactant monomers instead of micelles (high submicellar chromatography, HSC). This is demonstrated here for the analysis of β-blockers. The performance of four organic solvents (methanol, ethanol, 1-propano…
Interpretive optimisation of organic solvent content and flow-rate in the separation of β-blockers with a Chromolith RP-18e column
The chromatographic performance of a Chromolith RP-18e column was comprehensively examined for a group of basic drugs (beta-blockers), eluted with isocratic ACN-water mixtures at increasing flow-rate up to 6 mL/min. As the flow-rate increases at fixed mobile phase composition, peak distribution (selectivity) is maintained, but the relative peak widths increase. This reduces the resolution below satisfactory values for closely eluting compounds. With the monolithic column, flow-rate becomes thus an important factor to be optimised, in addition to the mobile phase composition. Since, theoretically, retention factors (k) are independent of the flow-rate, the classical quadratic model relating …
Correction of the deviations in the retention times with Chromolith columns associated to the flow rate: Implications in the modelling of the retention behaviour
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 Chromolith column. In that work, we found that the retention factors were sensitive to the flow rate. The reason of the observed deviations in retention times is the increase in the column volume at the applied pressure, which decreases the linear velocity inside the column. This behaviour forced to include a correction term in the model that describe…