0000000000006022
AUTHOR
M.c. García-alvarez-coque
Retention mechanisms for basic drugs in the submicellar and micellar reversed-phase liquid chromatographic modes.
The reversed-phase liquid chromatographic (RPLC) behavior (retention, elution strength, selectivity, efficiency, and peak asymmetry) for a group of basic drugs (beta-blockers), with mobile phases containing the anionic surfactant sodium dodecyl sulfate (SDS) and acetonitrile, revealed different separation environments, depending on the concentrations of both modifiers: hydro-organic, submicellar at low surfactant concentration and high concentration of organic solvent, micellar, and submicellar at high concentration of both surfactant and organic solvent. In the surfactant-mediated modes, the anionic surfactant layer adsorbed on the stationary phase interacts strongly with the positively ch…
SENSITIZED LANTHANIDE FLUORESCENCE DETECTION OF STEROIDAL HORMONES
Several steroidal hormones with an α,β-unsaturated carbonyl group sensitized the fluorescence of terbium(III) ions: medroxyprogesterone, medroxyprogesterone acetate, methyltestosterone, nandrolone, nandrolone decanoate, progesterone, testosterone, testosterone enanthate, and testosterone propionate, but other steroids which have this chromophore did not give any signal: clostebol acetate, dehydrotestosterone, dydrogesterone, methandienone, and methenolone enanthate. Sensitized fluorescence was used to detect the steroids when eluted with micellar mobile phases of sodium dodecyl sulphate (SDS)-acetonitrile and SDS-pentanol containing 0.01 M Tb(III), with 245 nm and 545 nm as excitation and e…
A hybrid genetic algorithm with local search: I. Discrete variables: optimisation of complementary mobile phases
Abstract A hybrid genetic algorithm was developed for a combinatorial optimisation problem. The assayed hybridation modifies the reproduction pattern of the genetic algorithm through the application of a local search method, which enhances each individual in each generation. The method is applied to the optimisation of the mobile phase composition in liquid chromatography, using two or more mobile phases of complementary behaviour. Each of these phases concerns the optimal separation of certain compounds in the analysed mixture, while the others can remain overlapped. This optimisation approach may be useful in situations where full resolution with a single mobile phase is unfeasible. The o…
Retention-structure relationship studies for some steroidal hormones in micellar liquid chromatography
The retention behavior in a C18 column of fourteen steroidal hormones (clostebol acetate, dehydrotestosterone, dydrogesterone, medroxyprogesterone, medroxyprogesterone acetate, methandienone, methyltestosterone, nandrolone, nandrolone decanoate, progesterone, testosterone, testosterone enanthate, testosterone propionate and, stanozolol) with pure micellar mobile phases prepared from sodium dodecyl sulfate (SDS), and hybrid mobile phases comprising SDS-acetonitrile and SDS-pentanol, was correlated with their octanol-water partition coefficients. Similar correlations were found with retention data obtained by other authors in gas chromatography, conventional reversed-phase liquid chromatograp…
Origin and correction of the deviations in retention times at increasing flow rate with Chromolith columns.
Chromoliths can be used at flow rates beyond those feasible for conventional microparticulate packed columns. Ideally, the plots of the retention time versus the inverse of delivered flow rate should exhibit y-intercept of zero. However, significant positive deviations correlating with the solute polarity were observed for several compounds chromatographed with a Chromolith column, owing to the increased system pressure. Consequently, the dead time marker exhibits a smaller deviation, making the retention factors depend on the flow rate. Chromoliths are made of a silica-based monolith encapsulated within a PEEK tube, and should suffer larger stress with pressure than stainless steel columns…
A New Calibration Method for the Accurate Determination of Ethylene Content in Ethylene-Propylene Copolymers by CRYSTEX-IR
Summary: 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 the whole polymer sample. In this work, a new approach based on multiple linear regression (MLR) models is presented, which makes use of two independent IR absorbance signals acquired simultaneously by the detector. A compromise model to predict directly C2% is proposed, which balances good accuracy and reduced experimental effort. MLR mode…
New approaches based on modified Gaussian models for the prediction of chromatographic peaks
Abstract 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 peaks. However, it has the drawback of the uncontrolled growth of the predicted signal outside the elution region, which departs from the experimental baseline. To solve this problem, the Parabolic-Lorentzian Modified Gaussian (PLMG) model was developed. This combines a parabola that describes the variance change in the peak region, and a Lor…
Use of a three-factor interpretive optimisation strategy in the development of an isocratic chromatographic procedure for the screening of diuretics in urine samples using micellar mobile phases.
Screening of diuretics in urine is feasible through direct injection of the samples into the chromatographic system and isocratic reversed-phase liquid chromatography (RPLC) with micellar-organic mobile phases of sodium dodecyl sulfate (SDS) and 1-propanol. The surfactant coverage of the chromatographic column makes the addition of organic competing amines less necessary than in conventional aqueous-organic RPLC to achieve well-shaped peaks. Also, the range of elution strengths of micellar mobile phases required to elute mixtures of hydrophobic and hydrophilic diuretics is smaller. This allows the isocratic separation of the diuretics within adequate analysis times. An interpretive methodol…
Resolution assessment and performance of several organic modifiers in hybrid micellar liquid chromatography
The performance of four criteria that measure the elementary resolution (modified selectivity, modified RS, peak purity, and orthogonal valley-to-peak ratio) was critically assessed using as global resolution function, the product of elementary measurements. The peak purities and valley-to-peak criteria yielded the best description of the overall separation according to the shape of the resolution surfaces compared to the peak arrangements in the chromatograms, the capability of defining unambiguously the composition regions of complete resolution, and the resolution achieved in the predicted optimums. Peak purities were used to compare the effect of five organic modifiers (1-propanol, 1-bu…
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 …
Performance of amines as silanol suppressors in reversed-phase liquid chromatography
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 phase of a reagent (usually called additive) with cationic character. This associates with the stationary phase to prevent the access of analytes to the free silanol groups. Cationic additives may interact electrostatically with the anionic silanols. The hydrophobic region of the additive may also associate with the alkyl chains bound to the stationa…
Stationary phase modulation in liquid chromatography through the serial coupling of columns: A review
Liquid chromatography with single columns often does not succeed in the analysis of complex samples, in terms of resolution and analysis time. A relatively simple solution to enhance chromatographic resolution is the modulation of the stationary phase through the serial coupling of columns. This can be implemented with any type of column using compatible elution conditions and conventional instruments. This review describes the key features of column coupling and published procedures, where two or more columns were coupled in series to solve separation problems. In all reports, the authors could not resolve their samples with single columns, whereas significant enhancement in chromatographi…
EVALUATION OF THE ELUTION STRENGTH OF THE SURFACTANT AND ORGANIC SOLVENT IN HYBRID MICELLAR MOBILE PHASES
The global ability of a mixed mobile phase (with two or more modifiers) to elute a solute is often measured without distinguishing between the elution strength of each modifier. An algorithm is proposed to evaluate the elution strength of the modifiers in hybrid micellar mobile phases containing a surfactant and an organic solvent. The algorithm is based on a mechanistic retention model that takes into account the competing equilibria of solutes among aqueous-organic phase, micelle, and stationary phase. The change in the elution strength of surfactant and organic solvent with respect to the concentration of both modifiers is also examined. The results are discussed according to the values …
Optimisation of gradient elution with serially-coupled columns. Part I: single linear gradients.
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, stationary phase nature and column length. In this work, we take the challenge of implementing optimal linear gradients for serial columns to decrease the analysis time for compounds covering a wide polarity range. For this purpose, five ACE columns of different selectivity (three C18 columns of different characteristics, a cyano and a phenyl column) we…
Comparison of surfactant-mediated liquid chromatographic modes with sodium dodecyl sulphate for the analysis of basic drugs
In reversed-phase liquid chromatography (RPLC), basic drugs are positively charged at the usual working pH range and interact with free anionic silanols present in conventional silica-based stationary phases. This translates into stronger retention and tailed and broadened peaks. This problem can be resolved by the addition of reagents to the mobile phase that are adsorbed on the stationary phase, avoiding the access of solutes to silanols. Among these additives, surfactants under micellar conditions have provided good silanol suppressing potency through the technique known as micellar liquid chromatography (MLC). The most common example of this is anionic sodium dodecyl sulphate (SDS). Whe…
Micellar versus hydro-organic mobile phases for retention-hydrophobicity relationship studies with ionizable diuretics and an anionic surfactant
Abstract Logarithm of retention factors (log k ) of a group of 14 ionizable diuretics were correlated with the molecular (log P o/w ) and apparent (log P app ) octanol–water partition coefficients. The compounds were chromatographed using aqueous–organic (reversed-phase liquid chromatography, RPLC) and micellar–organic mobile phases (micellar liquid chromatography, MLC) with the anionic surfactant sodium dodecyl sulfate (SDS), in the pH range 3–7, and a conventional octadecylsilane column. Acetonitrile was used as the organic modifier in both modes. The quality of the correlations obtained for log P app at varying ionization degree confirms that this correction is required in the aqueou…
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…
Description of the retention behaviour in micellar liquid chromatography as a function of pH, surfactant and modifier concentration
Abstract Micellar liquid chromatography permits the elution of solutes of diverse polarity. One of the most outstanding advantages of the technique is its capability of predicting the retention with high accuracy, as a function of different experimental variables. The separation of a group of compounds is usually optimized by varying the concentrations of surfactant and modifier in the mobile phase. The pH is, however, for many solutes, a variable that should be considered in the description of their elution behaviour. A global model that takes into account, simultaneously, the concentrations of surfactant and modifier, and the pH as chromatographic variables, is proposed for ionizable solu…
Comparative study of solvation parameter models accounting the effects of mobile phase composition in reversed-phase liquid chromatography
Solvation parameter models relate linearly compound properties with five fundamental solute descriptors (excess molar refraction, dipolarity/polarizability, effective hydrogen-bond acidity and basicity, and McGowan volume). These models are widely used, due to the availability of protocols to obtain the descriptors, good performance, and general applicability. Several approaches to predict retention in reversed-phase liquid chromatography (RPLC) as a function of these descriptors and mobile phase composition are compared, assaying the performance with a set of 146 organic compounds of diverse nature, eluted with acetonitrile and methanol. The approaches are classified in two groups: those t…
Approaches to model the retention and peak profile in linear gradient reversed-phase liquid chromatography.
Abstract 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 concentration ranges of modifier. However, more complex models may not allow an analytical integration of the general equation for gradient elution. Alternative approaches for modelling the retention in linear gradient elution are here proposed. Those based on the quadratic logarithmic model and a model proposed for normal liquid chromatography yie…
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 …
Characterization of Chemical Composition along the Molar Mass Distribution in Polyolefin Copolymers by GPC Using a Modern Filter-Based IR Detector
Summary 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 of the material, and thus, its range of possible applications and performance. The direct coupling of a modern filter-based infrared (IR) detector to a high temperature GPC instrument, by means of a heated flow-through cell, is here described. The analyses are carried out by recording the continuous IR absorbance chromatograms at selected …
Robust interpretive optimisation in high-performance liquid chromatography considering uncertainties in peak position.
In the context of interpretive chromatographic optimisation, robustness is usually calculated by introducing deliberated shifts in the nominal optimal conditions and evaluating their effects on the monitored objective function, mimicking thus the experimental procedures used in method validation. However, such strategy ignores a major source of error: the uncertainties associated to the modelling step, that may give rise to deceiving results when conditions that were expected to yield baseline separation are reproduced in the chromatograph. Two approaches, based on the peak purity concept, are here proposed to evaluate the robustness of the objective function under the perspective of measur…
On the Measurement of Dead Time in Micellar Liquid Chromatography
Abstract Modelling of the retention of solutes in micellar liquid chromatography allows the optimization of the resolution of a mixture of solutes and the determination of physico-chemical retention parameters. Both tasks imply the calculation of capacity factors, which are severely affected by the value of dead time. However, the determination of the dead time is not easy when a micellar mobile phase is used owing to the wide and variable perturbations that appear at the heads of the chromatograms. Four different criteria of determination of a reference time in the chromatograms are proposed and compared. The criteria are applied to mobile phases containing a varying concentration of surfa…
Submicellar and micellar reversed-phase liquid chromatographic modes applied to the separation of beta-blockers.
The behaviour of a reversed-phase liquid chromatographic (RPLC) system (i.e. elution order, resolution and analysis time), used in the analysis of β-blockers with acetonitrile-water mobile phases, changes drastically upon addition of an anionic surfactant (sodium dodecyl sulphate, SDS). Surfactant monomers cover the alkyl-bonded phase in different extent depending on the concentration of both modifiers, in the ranges 1 × 10-3-0.15 M SDS and 5-50% acetonitrile. Meanwhile, the surfactant is dissolved in the mobile phase as free monomers, associated in small clusters or forming micelles. Four characteristic RPLC modes are yielded, with transition regions between them: hydro-organic, micellar, …
Enhanced spectrophotometric determination of nicotinic acid in a sodium dodecyl sulphate micellar medium.
The spectrophotometric determination of pyridine and pyridine derivatives by means of the König reaction was studied in micellar media of sodium dodecyl sulphate (SDS), N-cetylpyridinium chloride and Triton X-100. The sensitivity was largely increased in SDS micellar medium. The attack of the pyridine ring with cyanogen bromide to produce a glutaconic aldehyde was not affected by the presence of SDS, but the yield of the coupling reaction with an arylamine to produce a polymethine dye was largely increased. In the SDS micellar medium, aniline was superior to other coupling reagents. The limits of detection (LODs) were 6 x 10(-7), 1 x 10(-6) and 5 x 10(-7)M for pyridine, pyrrol-ylmethylpyrid…
Approaches to estimate the time and height at the peak maximum in liquid chromatography based on a modified Gaussian model
The time and height at the peak maximum are key parameters to describe a chromatographic peak with prediction or optimization purposes, or in the qualitative/quantitative analysis of samples. Three different approaches to estimate these parameters, using the experimental points in the peak maximum region, are here described and compared. The approaches are based on the reliable description of the peak profile using a modified Gaussian model with a parabolic variance (PVMG). In the first approach, non-linear fitting of the chromatographic data to the PVMG model is carried out to obtain the time and height at the peak maximum (Approach I). In the other two approaches, the PVMG model is linear…
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…
Implementation of gradients of organic solvent in micellar liquid chromatography using DryLab®: Separation of basic compounds in urine samples
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 polarities, decreasing the analysis time. Also, it benefits the determination of moderately to low polar compounds in physiological fluids performing direct injection: an initial micellar eluent with a low organic solvent content, or a pure micellar (without surfactant) solution, will provide better protection of the column against the proteins i…
Combined effect of solvent content, temperature and pH on the chromatographic behaviour of ionisable compounds. III: Considerations about robustness
Abstract We previously reported a model able to predict the retention time of ionisable compounds as a function of the solvent content, temperature and pH [J. Chromatogr. A 1163 (2007) 49]. The model was applied further, developing an optimisation of the resolution based on the peak purity concept [J. Chromatogr. A 1193 (2008) 117]. However, we left aside an important issue: we did not consider incidental overlaps caused by shifts in the predicted peak positions, owing either to uncertainties in the source data, modelling errors, or the practical implementation in the chromatograph of the optimal mobile phase (or any other). These shifts can ruin the predicted separation, since they can eas…
Are analysts doing method validation in liquid chromatography?
International audience; 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 quality control tool in different locations and times. The performance parameters and statistical protocols followed throughout a validation study vary with the source of guidelines. Before single laboratory validation, an analytical method should be fully developed and optimized. The purpose of the validation is to confirm p…
A chromatographic objective function to characterise chromatograms with unknown compounds or without standards available
Abstract 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 chromatographic resolution, and further finding the optimal separation conditions. However, most chromatographic objective functions (COFs) described in the literature to measure the resolution are based on mathematical models fitted with the information obtained from standards, and cannot be applied to samples with unknown compounds. In …
Peak deconvolution in one-dimensional chromatography using a two-way data approach.
A deconvolution methodology for overlapped chromatographic signals is proposed. Several single-wavelength chromatograms of binary mixtures, obtained in different runs at diverse concentration ratios of the individual components, were simultaneously processed (multi-batch approach), after being arranged as two-way data. The chromatograms were modelled as linear combinations of forced peak profiles according to a polynomially modified Gaussian equation. The fitting was performed with a previously reported hybrid genetic algorithm with local search, leaving all model parameters free. The approach yielded more accurate solutions than those found when each experimental chromatogram was fitted in…
Effect of buffer nature and concentration on the chromatographic performance of basic compounds in the absence and presence of 1-hexyl-3-methylimidazolium chloride
Abstract 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 performance is improved by addition to the mobile phase of ionic liquids, from which 1-hexyl-3-methylimidazolium chloride ([C6MIm][Cl]) has especially good characteristics. A recent report has also revealed that the use of the phosphate system as buffer, at varying concentration and pH, may have a significant role in the chromatograph…
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…
Determination of inorganic species by thermal lens spectrometry
Abstract The application of thermal lens spectrometry (TLS) to the determination of inorganic species is reviewed. The requirements of a chromogenic reaction to be advantageously adapted to TLS detection, and the necessary conditions for reaching very low limits of detection are discussed. Methods for the determination of metals and P, Si, S and N compounds in several samples are examined. Flow-injection and extraction methods, associated photoinduced reactions and surface phenomena, speciation and equilibrium studies are discussed. Gas phase NO 2 methods are also commented upon. Trends are given.
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…
Protocol to compare column performance applied to hydrophilic interaction liquid chromatography
Abstract 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 appropriate stationary phase plays a fundamental role in the development of analytical procedures to obtain good performance. In this work, the chromatographic performance of bare silica and six polar stationary phases with different functionalized groups (with neutral, cationic, anionic and zwitterionic character) are investigated. The beh…
Analysis of tricyclic antidepressants in pharmaceuticals by microemulsion liquid chromatography
Abstract 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 electrostatic interaction between the cationic species of basic compounds and the anionic sulphate group of the surfactant. This forces the addition of a strong organic solvent to get appropriate times. An alternative is the use of a microemulsion (ME), formed by mixing surfactant, oil and an alcohol as co-surfactant. Association of hydrophobic com…
Finding the best separation in situations of extremely low chromatographic resolution.
Abstract Samples with a large number of compounds or similarities in their structure and polarity may yield insufficient chromatographic resolution. In such cases, however, finding conditions where the largest number of compounds appears sufficiently resolved can be still worthwhile. A strategy is here reported that optimises the resolution level of chromatograms in cases where conventional global criteria, such as the worst resolved peak pair or the product of elementary resolutions, are not able to detect any separation, even when most peaks are baseline resolved. The strategy applies a function based on the number of “well resolved” peaks, which are those that exceed a given threshold of…
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…
1-Hexyl-3-methyl imidazolium tetrafluoroborate: an efficient column enhancer for the separation of basic drugs by reversed-phase liquid chromatography.
Abstract 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 shorter retention times the most noticeable features. However, other characteristics as selectivity or resolution are not usually considered, or are only examined for selected chromatographic conditions. In this work, the effect of the addition of the ionic liquid 1-hexyl-3-methyl imidazolium tetrafluoroborate to acetonitrile–water mixtures i…
Chromatographic Efficiency in Micellar Liquid Chromatography: Should it Be Still a Topic of Concern?
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 as a handicap for MLC, in spite of the fact that the hybrid mode often offers similar or even improved efficiencies (for basic compounds) relative to that attained in the hydro-organic mode. Only the efficiencies for apolar non-ionizable compounds are still clearly inferior. This work describes the type of interactions and polarity changes with org…
Performance of different C18 columns in reversed-phase liquid chromatography with hydro-organic and micellar-organic mobile phases
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 broad peaks. The effect of silanols is minimised by the addition of a competing agent in the mobile phase, such as the anionic surfactant sodium dodecyl sulphate (SDS). In micellar-organic RPLC, the adsorption of an approximately fixed amount of SDS monomers gives rise to a stable modified stationary phase, with properties remarkably different from th…
Reversed-phase liquid chromatography with mixed micellar mobile phases of Brij-35 and sodium dodecyl sulphate: a method for the analysis of basic compounds
Micellar liquid chromatography (MLC) is a reversed-phase liquid chromatographic (RPLC) mode, which uses a surfactant as a modifier, with significant changes in retention and selectivity with regard to the classical RPLC mode that employs mixtures of water and organic solvent. The anionic sodium dodecyl sulphate (SDS) is the most usual surfactant in MLC, but it also requires the addition of an organic solvent to decrease the retention times and increase the efficiency. In particular, positively charged basic compounds are strongly retained by the stationary phase modified by adsorption of SDS monomers and require the addition of a strong solvent, such as propanol or pentanol. The non-ionic s…
Description of the retention behaviour of solutes in micellar liquid chromatography with organic modifiers: Comparison of two methods
Two methods for the description of the retention behaviour of solutes in micellar liquid chromatography are compared. One of them divides the parameter space into triangular subspaces, fitting a different equation in each subspace. The second method makes use of a unique equation, valid in the whole parameter space. In both cases, equations of the type log k=f (μ, ϕ), and 1/k=f (μ, ϕ), (μ and ϕ are the concentration of surfactant and alcohol, respectively), were used to describe the retention. The use of the hyperbolic function, 1/k=c0+c1μ+c3μϕ, to describe the whole parameter space yielded the best prediction. When a small portion of the parameter space was modelled, a simpler hyperbolic f…
RAPID LIQUID CHROMATOGRAPHIC DETERMINATION OF TETRACYCLINES IN ANIMAL FEEDS USING A SURFACTANT SOLUTION AS MOBILE PHASE
ABSTRACT A chromatographic procedure was developed for the determination of oxytetracycline (OTC), tetracycline (TC), chlortetracycline (CTC), doxycycline (DC) and minocycline (MINO) in animal feeds. Clear analyte-rich extracts were obtained using a 1 : 1 acetonitrile/water mixture buffered at pH 3. The extracts were injected into a conventional unprotected C18 chromatographic column and eluted with a mobile phase of 0.05 M sodium dodecyl sulfate/5% 1-butanol/0.01 M oxalic acid at pH 3. Good resolution was achieved for the five compounds, whereas OTC and TC coeluted with an optimized aqueous-organic mobile phase of methanol/acetonitrile/0.01 M oxalic acid at pH 3. Mean recoveries from spike…
Resolution of mixtures of steroidal hormones with micellar eluents of sodium dodecyl sulphate and acetonitrile or pentanol
An optimization strategy was applied to explore the capability of hybrid micellar eluents of sodium dodecyl sulphate (SDS), using acetonitrile or pentanol as modifiers, to resolve mixtures of eleven steroids showing a wide range of hydrophobicity (clostebol acetate, dehydrotestosterone, dydrogesterone, medroxyprogesterone, medroxyprogesterone acetate, methandienone, methyltestosterone, progesterone, testosterone, testosterone enanthate and testosterone propionate). The accurate prediction of the retention behaviour of the steroids, with relative errors in the 0.8–1.7% and 0.4–2.9% ranges for SDS-acetonitrile and SDS-pentanol eluents, respectively, demonstrated the reliability of the methodo…
Micellar liquid chromatographic separation of amino acids using pre- and post-column o -phthalaldehyde/ N -acetylcysteine derivatization
Abstract The isocratic analysis of primary proteic amino acids using a C18 column, micellar mobile phases of sodium dodecyl sulphate (SDS) and 1-propanol at varying pH, and pre- and post-column formation of the o-phthalaldehyde (OPA)/N-acetyl- l -cysteine (NAC) isoindoles, is examined. An interpretive optimization strategy was applied to find the best separation conditions. For this purpose, empirical polynomial models were used to describe the elution behavior of the isoindoles and free amino acids, using as separation factors the concentrations of surfactant and modifier (two-factor problem), or these two factors and the pH of the mobile phase (three-factor problem). The separation of the…
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…
Effect of short-chain alcohols on surfactant-mediated reversed-phase liquid chromatographic systems.
The behaviour of β-blockers in a reversed-phase liquid chromatographic (RPLC) column with mobile phases containing a short-chain alcohol (methanol, ethanol or 1-propanol), with and without the surfactant sodium dodecyl sulphate (SDS), was explored. Two surfactant-mediated RPLC modes were studied, where the mobile phases contained either micelles or only surfactant monomers at high concentration. Acetonitrile was also considered for comparison purposes. A correlation was found between the effects of the organic solvent on micelle formation (monitored by the drop weight procedure) and on the nature of the chromatographic system (as revealed by the retention, elution strength and peak shape of…
Silanol suppressing potency of alkyl-imidazolium ionic liquids on C18 stationary phases
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 manufacturer, give rise to different amounts of residual silanols in the packings, and consequently, different chromatographic performance. One of the most efficient and widespread strategies to reduce or even eliminate the different performance among columns is the addition of a reagent to the mobile phase to block the silanol sites. However, the i…
Comparison of two serially coupled column systems and optimization software in isocratic liquid chromatography for resolving complex mixtures.
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 interactions), and then optimizes continuous factors as the mobile phase composition, pH, temperature and flow-rate. However, the optimization of the stationary phase nature and column length (which are discrete factors) may be interesting. In this regard, the optimization of the coupling of individual columns may yield separations that are not possible wi…
Chromatographic determination of diuretics in urine samples using hybrid micellar mobile phases with fluorimetric detection
Abstract A rapid and simple chromatographic procedure, that uses hybrid micellar mobile phases of sodium dodecyl sulphate (SDS), propanol and phosphate buffer at pH 3, and fluorimetric detection ( λ exc =270 nm, λ em =430 nm), is reported for the determination of mixtures of seven diuretics of diverse efficacy (amiloride, bendroflumethiazide, bumetanide, furosemide, hydroflumethiazide, piretanide and triamterene), in urine samples. The application of a sequential optimization procedure for the resolution of the mixtures of the diuretics was problematic. However, their separation was achieved through the use of an interpretive optimization procedure based on the accurate description of the r…
A complementary mobile phase approach based on the peak count concept oriented to the full resolution of complex mixtures
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 resolution of such samples in reversed-phase liquid chromatography is approached using two or more isocratic mobile phases with a complementary resolution behaviour (complementary mobile phases, CMPs). Each mobile phase is dedicated to the separation of a group of compounds. The CMPs are selected in such a way that, when the separation is cons…
Towards unsupervised analysis of second-order chromatographic data: automated selection of number of components in multivariate curve-resolution methods.
A method to apply multivariate curve-resolution unattendedly is presented. The algorithm is suitable to perform deconvolution of two-way data (e.g. retrieving the individual elution profiles and spectra of co-eluting compounds from signals obtained from a chromatograph equipped with multiple-channel detection: LC-DAD or GC-MS). The method is especially adequate to achieve the advantages of deconvolution approaches when huge amounts of data are present and manual application of multivariate techniques is too time-consuming. The philosophy of the algorithm is to mimic the reactions of an expert user when applying the orthogonal projection approach--multivariate curve-resolution techniques. Ba…
Error analysis and performance of different retention models in the transference of data from/to isocratic/gradient elution.
The transferability of retention data among isocratic and gradient RPLC elution modes is studied. For this purpose, 16 beta-blockers were chromatographed under both isocratic and gradient elution with acetonitrile-water mobile phases. Taking into account the elution mode where the experimental data come from, and the mode where the retention should be predicted, the following combinations are possible: isocratic predictions from (i) isocratic or (ii) gradient experimental designs; and gradient predictions from (iii) isocratic or (iv) gradient data. Each of these possibilities was checked using three retention models that relate the logarithm of the retention factor: (a) linearly and (b) qua…
Estimation of significant solvent concentration ranges and its application to the enhancement of the accuracy of gradient predictions.
Abstract The solvent concentration range actually useful for gradient predictions is significantly narrower than the total range scanned in a gradient run. This range, called “solvent informative range” (SIR), if known with the highest accuracy, allows to predict gradient retention times ( t g ) with minimal error. The small size of the SIR supports the application of the linear solvent strength theory (LSST). Furthermore, LSST allows a closed-form solution to the integral required to predict gradient retention times, which eliminates numerical integration, needed with other retention models. A methodology that calculates the SIR by applying error analysis, and uses it to improve the accura…
Optimisation of chromatographic resolution using objective functions including both time and spectral information.
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 success by including spectral information. In this work, two-way chromatographic objective functions (COFs) that incorporate both time and spectral information were tested, based on the peak purity (analyte peak fraction free of overlapping) and the multivariate selectivity (figure of merit derived from the net analyte signal) concepts. These COFs are s…
Complementary mobile-phase optimisation for resolution enhancement in high-performance liquid chromatography.
An optimisation methodology in high-performance liquid chromatography (HPLC) is presented for the selection of two or more mobile phases having an optimal complementary resolution. The complementary mobile phases (CMPs) are selected in such a way that each one resolves optimally only some compounds in the mixture, while the remainder, resolved by the other mobile phase(s), can overlap among them. The methodology is based on the computation of a peak purity measurement for each solute, using an asymmetrical peak model for peak simulation. Two global resolution criteria (product of elementary resolutions and worst elementary resolution) and two methods for solving the problem (a systematic ex…
Micellar liquid chromatography: suitable technique for screening analysis.
The screening capability of micellar liquid chromatography (MLC) is discussed using the reported chromatographic data of several sets of compounds (amino acids, beta-blockers, diuretics, phenethylamines, phenols, polynuclear aromatic hydrocarbons, steroids and sulfonamides) and new results (sulfonamides and steroids). The chromatographic data are treated with an interpretive optimisation resolution procedure to obtain the best separation conditions. Usually, the pH and the concentration of surfactant (sodium dodecyl sulfate, SDS, or cetyltrimethylammonium bromide) for the optimal mobile phase were 2.5-3 and0.12 M, respectively. The nature and concentration of organic solvent depended on the…
Prediction of peak shape as a function of retention in reversed-phase liquid chromatography
Optimisation of the resolution of multicomponent samples in HPLC is usually carried out by changing the elution conditions and considering the variation in retention of the analytes, to which a standard peak shape is assigned. However, the change in peak shape with the composition of the mobile phase can ruin the optimisation process, yielding unexpected overlaps in the experimental chromatograms for the predicted optimum, especially for complex mixtures. The possibility of modelling peak shape, in addition to peak position, is therefore attractive. A simple modified-Gaussian model with a parabolic variance, which is a function of conventional experimental parameters: retention time (tR), p…
Evaluation of several global resolution functions for liquid chromatography
Abstract An interpretative approach, that makes use of the overlapped fraction of each chromatographic peak as elementary resolution criterion, was applied to the separation of mixtures of compounds. The elementary resolution measurements for all peaks in the chromatogram were reduced to a single numerical value using several functions: normalised by the mean resolution product, unnormalised product, geometrical mean of the unnormalised product, and worst elementary resolution value. The descriptive capability of these reduction functions was evaluated through the observation of global resolution diagrams and the change in the shape of the chromatograms in the selected factor space. michrom…
Improvement of peak shape and separation performance of beta-blockers in conventional reversed-phase columns using solvent modifiers.
A comparative study of peak shape, elution behavior, and resolution of 16 beta-blockers (acebutolol, alprenolol, atenolol, bisoprolol, carteolol, celiprolol, esmolol, labetalol, metoprolol, nadolol, oxprenolol, pindolol, practolol, propranolol, sotalol, and timolol) chromatographed with hybrid mobile phases of triethylamine (TEA)-acetonitrile and sodium dodecyl sulfate (SDS)-propanol is performed using conventional reversed-phase columns and isocratic elution. Both solvent modifiers (TEA and SDS) prevent the interaction of the basic drugs with the alkyl-bonded phase. However, the protection mechanisms of silanols on the packing are different. Whereas TEA associates with the silanol sites (b…
Chromatographic Determination of Thiols After Pre‐column Derivatization witho‐Phthalaldehyde and Isoleucine
Abstract The reaction of primary amines with excess o‐phthalaldehyde (OPA) and thiol yields unique isoindole derivatives that are readily separated by reversed‐phase liquid chromatography. In a previous work, a spectrophotometric procedure was proposed for the assay of N‐acetylcysteine by derivatization with OPA and isoleucine at pH 9.5, with satisfactory results. The chromatographic determination of this and other low molecular‐weight thiols, after isoindole formation with isoleucine, using mobile phases of acetonitrile–water at pH 3 and spectrophotometric detection, is now examined. From the assayed thiols (thioglycolic acid, 3‐mercaptopropionic acid, tiopronin, N‐acetylcysteine, N‐acetyl…
A three-factor optimisation strategy for micellar liquid chromatography
An interpretive optimisation methodology for micellar liquid chromatography (MLC) is shown, taking into account pH, surfactant (sodium dodecyl sulphate) and organic modifier (propanol) concentration. Two objectives are considered: to develop a highly practical straightforward three-factor optimisation for practical MLC, and, in order to avoid unecessary experiments, to link two and three-factor optimisations through a step-wise construction of the experimental design at different pH levels. The whole pH range for an ODS column (from 3 to 7) is covered. The proposed strategy was thoroughly evaluated using the chromatographic data from 81 experimental mobile phases, applied to the separation …
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…
Spectrophotometric Determination of the Antihistamines, Carbinoxamine Maleate and Doxylamine Succinate, in anN-Cetylpyridinium Chloride Micellar Medium
Abstract The pyridine antihistamines carbinoxamine maleate and doxylamine succinate were derivatized by hydrolysis with cyanogen bromide and coupling with aniline to form a polymethine dye, and determined by colorimetry. In a micellar cationic medium of N-cetylpyridinium chloride (NCPC), the reaction times were reduced and the sensitivities improved. In 0.1 M NCPC the apparent molar absorptivities of the derivatives were 5,900 and 3,500 M−1 cm−1, and the limits of detection (as 3s) were 8.6x10−7 M and 1.7x106 M, for carbinoxamine and doxylamine, respectively. The sensitivity was enhanced in a three- to six-fold factor with respect to a non-micellar medium. The reproducibility was 0.8% for 1…
Towards the optimization of complementary systems in reversed-phase liquid chromatography
Previously reported optimization methodology, which seeks complementary mobile phases (CMP) in isocratic chromatography, has been extended to include more than one system simultaneously (i.e. more than one organic solvent and/or column). In the literature the benefits of complementarity are not usually fully exploited—few working conditions giving rise to interactions as different as possible are examined, without developing a fully linked optimization. The proposed approach is compared critically with use of a single mobile phase or CMP which consider one system only. The strategy greatly expands the capability of isocratic chromatography in the analysis of complex samples that cannot be r…
Thermal lens spectrometry in biochemical analysis.
The photothermal spectroscopic techniques, with special emphasis on the thermal lens spectrometry (TLS), are introduced to the non-specialist in laser spectroscopy. The following topics are treated on an elementary basis: fundamentals and analytical characteristics, instrumentation, selectivity and multi-wavelength capability, the models describing the signal-concentration relationship, the sensitivity, background noise and limits of detection, the influence of light scattering and flow. Applications related to the fields of clinical and biochemical analysis and organic pollution are given. The thermal lens circular dichroism and the infrared TLS are also briefly outlined.
Correlation between hydrophobicity of amino acids and retention data in reversed-phase liquid chromatography with micellar eluents
Hydrophobic character is usually expressed in terms of the partition coefficient in 1-octanol-water (log PO/W). However, measurement of this coefficient is often problematic. Retention in micellar liquid chromatography is mainly due to hydrophobic interactions and can also be used as an index of hydrophobicity. A hydrophobicity scale was established with retention data foro-phthalaldehyde (OPA)-N-acetyl-L-cysteine (NAC) amino acid derivatives, using the glycine derivative as reference. Since the OPA-NAC derivatives only differ in the nature of R1 in the amino acid (R1CH(COOH)NH2), in the absence of electrostatic interactions the hydrophobic character of the substituent was responsible for r…
Effect of sodium dodecyl sulphate and Brij-35 on the analysis of sulphonamides in physiological samples using direct injection and acetonitrile gradients
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 may still be convenient to shorten the total analysis time. This work compares the separation of 15 sulphonamides using conventional hydro-organic RPLC and MLC with the surfactants sodium dodecyl sulphate (SDS) and Brij-35, and both isocratic and gradient elution with acetonitrile. The observed behaviour is rationalized attending to the interacti…
A theoretical plate model accounting for slow kinetics in chromatographic elution.
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 plates, each of them consisting of identical repeating portions of mobile phase and stationary phase. Solutes partition between both phases, reaching the equilibrium. Mobile phase transference between the theoretical plates is assumed to be infinitesimally stepwise (or continuous), giving rise to the mixing of the solutions in adjacent plates. This yi…
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…
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…
Peak capacity estimation in isocratic elution.
Peak capacity (i.e. maximal number of resolved peaks that fit in a chromatographic window) is a theoretical concept with growing interest, but based on a situation rarely met in practice. Real chromatograms tend to have uneven distributions, with overlapped peaks and large gaps. The number of resolved compounds should, therefore, be known from estimations. Several equations have been reported for this purpose based on three perspectives, namely, the intuitive approach (peak capacity as the size of the retention time window measured in peak width units), which assumes peaks with the same width, and the outlines of Giddings and Grushka, which consider changes in peak width with retention time…
MICELLAR CHROMATOGRAPHIC PROCEDURE WITH DIRECT INJECTION FOR THE DETERMINATION OF SULFONAMIDES IN MILK AND HONEY SAMPLES
The capability of liquid chromatography with micellar mobile phases of sodium dodecyl sulfate (SDS), of allowing the direct injection of biological fluids into reversed-phase columns, was applied to the determination of sulfonamides in milk and honey samples. The chromatographic behavior of a group of 15 sulfonamides was studied at pH 3.0 where the drugs showed a greater separation space. Acetonitrile was added to the mobile phase to decrease the retention of the most hydrophobic drugs and increase the efficiencies, which yielded a higher resolution. The samples were diluted with 0.10 M SDS to facilitate the solubilization of the matrix compounds and release the protein-bound drugs. The pro…
Suitability of 1-hexyl-3-methylimidazolium ionic liquids for the analysis of pharmaceutical formulations containing tricyclic antidepressants
Abstract 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 tetrafluoroborate, which have interesting features for the separation of basic compounds, in terms of peak shape combined with reduced retention. Tricyclic antidepressants are low polarity drugs that strongly associate to the alkyl chains of conventional stationary phases. They are also positively charged in the usual working pH range (2–8) in r…
An approach to evaluate the information in chromatographic fingerprints: Application to the optimisation of the extraction and conservation conditions of medicinal herbs.
A new approach is reported for high-performance liquid chromatography to measure the level of information in fingerprints. For this purpose, the concept of peak prominence, which is the protruding part of each visible peak with regard to the valleys that delimit it, was used. The peaks in the fingerprints are ranked according to the areas of the peak prominences, and a threshold is established to discriminate between the significant peaks and those that are irreproducible. The approach was applied to evaluate the impact of several extraction conditions (solvent nature and composition, time and temperature of the treatment, amount of sample, and time and temperature of conservation of the ex…
SINGLE-PEAK RESOLUTION CRITERIA FOR OPTIMIZATION OF MOBILE PHASE COMPOSITION IN LIQUID CHROMATOGRAPHY
Three criteria that evaluate the single peak resolution (peak purity) in chromatography, the free height fraction, free area fraction, and valley ratio, are examined. The main advantages of these criteria against other criteria based on peak pair separation, are that the measurements are not affected by the identity of neighboring peaks and are normalized, which make them very intuitive. The methodology is illustrated through the isocratic separation of mixtures of several sulphonamides (sulphacetamide, sulphadiazine, sulphadimethoxine, sulphaguanidine, sulphamerazine, sulphamethazine, sulphamethoxazole, sulphamethizole, sulphamonomethoxine, sulphanilamide, sulphapyridine, sulphaquinoxaline…
Gaining insight in the behaviour of imidazolium-based ionic liquids as additives in reversed-phase liquid chromatography for the analysis of basic compounds.
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 of silanols. However, the dual character of ILs should be considered: both cation and anion may be adsorbed on the stationary phase, thereby creating a double asymmetrical layer positively or negatively charged, depending on the relative adsorption of both ions. In this work, a study of the performance of six imidazolium-based ILs (the chlorides an…
MICELLAR LIQUID CHROMATOGRAPHIC DETERMINATION OF ANTI-CONVULSANT DRUGS IN PILLS AND CAPSULES
A simple chromatographic procedure is reported for the determination of several anti-convulsant drugs in pharmaceuticals: carbamazepine, and the benzodiazepines bentazepam, halazepam, oxazepam, pinazepam, and tetrazepam. The procedure utilizes a C18 column, a hybrid micellar mobile phase of 0.1 M SDS-3% butanol-0.1% triethylamine-0.01 M phosphate buffer (pH 3), and UV detection (230 nm). The drugs eluted in less than 13 min, in accordance to their relative polarities, as indicated by their octanol-water partition coefficients. The limits of detection (μg/mL), and intra and inter-day repeatabilities (%), for 4 μg/mL were: carbamazepine (0.03, 1.0, 4.1), bentazepam (0.05, 1.3, 1.6), halazepam…
Parabolic-Lorentzian modified Gaussian model for describing and deconvolving chromatographic peaks.
Abstract A new mathematical model for characterising skewed chromatographic peaks, which improves the previously reported polynomially modified Gaussian (PMG) model, is proposed. The model is a Gaussian based equation whose variance is a combined parabolic-Lorentzian function. The parabola accounts for the non-Gaussian shaped peak, whereas the Lorentzian function cancels the variance growth out of the elution region, which gives rise to a problematic baseline increase in the PMG model. The proposed parabolic-Lorentzian modified Gaussian (PLMG) model makes a correct description of peaks showing a wide range of asymmetry with positive and/or negative skewness. The new model is shown to give b…
Limits of multi-linear gradient optimisation in reversed-phase liquid chromatography
Abstract The concept of limiting peak purity was applied to quantify the degree of completion of the separation capability of a chromatographic system using multi-linear gradients. The objective was to check whether the complexity of a gradient program deserves be increased to enhance resolution by inserting more linear segments, or on the contrary, no significant improvements can be expected under more complex gradients. A set of 19 isoindole derivatives of primary amino acids was selected to test the performance of isocratic, single linear and multi-linear gradients. Accurate simulated chromatograms were obtained via numerical integration of the general equation of gradient elution, using…
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…
Peak half-width plots to study the effect of organic solvents on the peak performance of basic drugs in micellar liquid chromatography.
The addition of the anionic surfactant sodium dodecyl sulphate (SDS) to hydro-organic mixtures of methanol, ethanol, propanol or acetonitrile with water yielded enhanced peak shape (i.e. increased efficiencies and symmetrical peaks) for a group of basic drugs (β-blockers) chromatographed with a Kromasil C18 column. The effect can be explained by the thin layer of surfactant associated to the hydrocarbon chain on the stationary phase in the presence of the organic solvents, which covers the free silanols on the siliceous support avoiding their interaction with the cationic basic drugs. These instead interact with the anionic head of the surfactant increasing their retention and allowing a mo…
Adsorption of the anionic surfactant sodium dodecyl sulfate on a C18column under micellar and high submicellar conditions in reversed-phase liquid chromatography
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 nonpolar alkyl-bonded stationary phases they are significantly adsorbed. If the mobile phase contains a high concentration of organic solvent, micelles break down, and the amount of surfactant adsorbed on the stationary phase is reduced, giving rise to another chromatographic mode named high submicellar liquid chromatography. The presence of a thinner coating of surfactant enhances the selectivity and peak shape, es…
Combined effect of solvent content, temperature and pH on the chromatographic behaviour of ionisable compounds.
The organic solvent content and the pH in the mobile phase are the usual main factors in reversed-phase liquid chromatographic separations, owing to their strong effects on retention and/or selectivity. Temperature is often neglected. However, even in cases where the impact of this factor on selectivity is minor, the reduction in analysis time is still an interesting reason to consider it. In addition, ionisable compounds may exhibit selectivity changes, owing to the interaction of organic solvent and/or temperature with pH. The separation of ionisable compounds (nine diuretics: bendroflumethiazide, benzthiazide, bumetanide, chlorthalidone, furosemide, piretanide, probenecid, trichloromethi…
Approaches to find complementary separation conditions for resolving complex mixtures by high-performance liquid chromatography
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, solvent, pH). There are, however, other possibilities that take advantage of the gathered information in the failed separation, without the need of new experiments, based on the concept of complementary separations (e.g. isocratic mobile phases, gradients, columns, chromatographic modes). One separation condition will focus on the resolution of so…
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…
Effect of ionization and the nature of the mobile phase in quantitative structure-retention relationship studies.
Abstract The octanol–water distribution constant, commonly called partition coefficient, Po/w, is a parameter often retained as a measure of the hydrophobicity of a molecule. log Po/w, for a given molecule, can be conveniently evaluated constructing correlation lines between standard retention factor logarithms (log k) in reversed-phase liquid chromatography (RPLC) and standard log Po/w values. Many compounds of pharmaceutical interest can be quite hydrophobic and have, simultaneously, basic nitrogen atoms or acidic sulfur containing groups in their structure. This renders them ionizable. The hydrophobicity of the molecular drug form (Po/w value) is completely different from its ionic form …
HYDRO-ORGANIC AND MICELLAR-ORGANIC REVERSED-PHASE LIQUID CHROMATOGRAPHIC PROCEDURES FOR THE EVALUATION OF SULPHONAMIDES IN PHARMACEUTICALS
Two reversed-phase liquid chromatographic procedures were developed for the analysis of ten pharmaceutical formulations used in medicine and veterinary, which contained one of the following sulphonamides: sulphacetamide, sulphadiazine, sulphaguanidine, sulphamethazine, sulphamethizole, sulphamethoxazole, or sulphathiazole. In both chromatographic modes, the same C18 column was utilized and the mobile phase contained a low amount of acetonitrile and citric buffer at pH 3.0. In one of the procedures, the surfactant sodium dodecyl sul phate was added to the mobile phase. The presence of micelles allowed a decrease in the amount of organic solvent from 11% to 6%, and facilitated sample dissolut…
Analysis of basic drugs by liquid chromatography with environmentally friendly mobile phases in pharmaceutical formulations
Abstract 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. Micellar mobile phases containing surfactants give rise to modified stationary phases, where silanols are masked, improving the peak shape. However, mobile phases containing the anionic surfactant sodium dodecyl sulfate (SDS) require a small amount of organic solvent to conveniently decrease the retention of cationic analytes. An alternative is…
CHROMATOGRAPHIC DETECTION OF SESQUITERPENE LACTONES IN PARTHENIUM PLANTS FROM NORTHWEST ARGENTINA
Several chloroformic extracts containing some sesquiterpene lactones from autochtonous plants of Parthenium hysterophorus, collected in the province of Salta (Argentina), were separated and identified by high performance reversed-phase liquid chromatography with C18 column and propanol-water mobile phases, using gradient elution and concentrations of propanol larger than 10% (v/v). Lactones previously purified and characterized by H1 RMN, C13 RMN, IR, and mass spectrometry, were used as standards. The objective of the study was to check if the nature of the lactones was modified according to the altitude of the region where the plants were grown. Among the lactones, five were already descri…
Correlation between hydrophobicity and retention data of several antihistamines in reversed-phase liquid chromatography with aqueous-organic and micellar-organic mobile phases
Abstract The correlation between the retention of 12 antihistamines (carbinoxamine, chlorpheniramine, cyclizine, cyproheptadine, dexbrompheniramine, dexchlorpheniramine, diphenhydramine, doxylamine, pheniramine, phenyltoloxamine, pyrilamine and tripelennamine), in reversed-phase liquid chromatography (RPLC) with aqueous-organic (methanol-water) and micellar-organic (sodium dodecyl sulphate (SDS)-pentanol) mobile phases of varying composition, and their octanol-water partition coefficients expressed as log P o/w (ranging between 2.02 for pheniramine and 4.92 for cyproheptadine), was examined. For this study, the retention of the drugs was measured in six mobile phases of methanol-water, and…
Determination of furosemide in urine samples by direct injection in a micellar liquid chromatographic system.
A sensitive, selective and efficient micellar liquid chromatographic (MLC) procedure was developed for the determination of furosemide (4-chloro-N-furfuryl-5-sulfamoylanthranilic acid) in urine samples by direct injection and UV detection. The procedure makes use of a C18 reversed-phase column and a micellar mobile phase of 0.05 mol l(-1) sodium dodecyl sulfate-6% v/v propanol and phosphate buffer at pH 3 to resolve furosemide from its photochemical degradation products. The importance of protecting the standards and urine samples to be analysed from light in the assay of furosemide, avoiding its degradation, was verified. The limit of quantification was 0.15 microg ml(-1) and the relative …
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…
Separation of Proteic Primary Amino Acids under Several Reversed‐Phase Liquid Chromatographic Conditions
Abstract The reversed‐phase liquid chromatographic (RPLC) analysis of proteic primary amino acids with acetonitrile‐water, using pre‐column derivatisation with o‐phthalaldehyde (OPA) and N‐acetylcysteine (NAC), was compared with RPLC modes using trifluoroacetic acid or pentadecafluorooctanoic acid and evaporative light‐scattering detection, or sodium dodecyl sulphate micelles with pre‐ and post‐column derivatisation. The importance of column lifetime, risk of potential damages in the instrumentation, retention and resolution, was considered. Among the assayed approaches, the best is still aqueous‐organic RPLC with pre‐column derivatisation. It not only yields the most reliable results, but …
Gradient design for liquid chromatography using multi-scale optimization.
Abstract 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 of the sample is high, the gradient program should be accommodated to the local resolution needs of each analyte. This makes the optimization of such situations rather troublesome, since enhancing the resolution for a given analyte may imply a collateral worsening of the resolution of other analytes. The aim of this work is to design mul…
Half-width plots, a simple tool to predict peak shape, reveal column kinetics and characterise chromatographic columns in liquid chromatography: State of the art and new results
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 isocratic elution, the plots are parabolic, although often, the parabolas can be approximated to straight-lines. The plots can be obtained with the half-widths/retention time data for a set of solutes experiencing the same kinetics, eluted with a mobile phase at fixed or varying composition. When the analysed solutes experience different resistance to mas…
Multi-scale optimisation vs. genetic algorithms in the gradient separation of diuretics by reversed-phase liquid chromatography
Abstract 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 inappropriate for complex separations. More evolved solutions imply the sequential construction of multi-segmented gradients. However, this strategy discards part of the search space in each step of the construction and, again, cannot deal properly with very complex samples. When the complexity is too large, the only valid alternative for fi…
Net analyte signal as a deconvolution-oriented resolution criterion in the optimisation of chromatographic techniques
The performance of two multivariate calibration measurements, multivariate selectivity (SEL(s)) and scalar net analyte signal (scalar NAS), as chromatographic objective functions (COFs), was investigated. Since both assessments are straightforwardly related to the quantification of analytes in the presence of interferents, they were expected to confer new features in the optimisation of compound resolution, not present in conventional assessments. These capabilities are especially interesting in situations of low resolution, where peak deconvolution becomes an attractive alternative. For comparison purposes, chromatographic resolution (R(s)) and peak purity (p(s)) were used as reference COF…
Performance and modelling of retention in microemulsion liquid chromatography
Abstract 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 solutions of the anionic surfactant sodium dodecyl sulphate, the alcohol 1-butanol that played the role of co-surfactant, and octane as oil. In order to guarantee the formation of stable MEs, a preliminary study was carried out to determine the appropriate ranges of concentrations of the three components. For this purpose, mixtures of varia…
Determination of Pyrrole Derivatives and Hydroxyproline with 4-(Dimethylamino)benzaldehyde in a Micellar Medium
Abstract The spectrophotometric determination of pyrrole derivatives and hydroxyproline with 4-(dimethylamino)benzaldehyde (Ehrlich's reagent) in a sodium dodecyl sulphate micellar medium is studied. Pyrrole and pyrrole-2-carboxylic acid were used as model compounds for the optimization of the procedure. In the micellar medium with 0.2 M HCl at 90° C, pyrrole reacts in 15 min, whereas a HCl concentration larger than 5 M is required in the absence of the surfactant. The sensitivity increases from 1.3 to 5.6-fold with respect to the use of a non-micellar medium, for some pyrrole derivatives. The procedure is applied to the determination of hydroxyproline in meat samples.
Hydrophobic and cation exchange mechanisms in the retention of basic compounds in a polymeric column.
A cation exchange retention mechanism concomitant with the well-known hydrophobic partition mechanism in a polymeric column has been observed and investigated. This exchange process is attributed to ionization of some acidic sites present in the polymer column at basic mobile phase pH values. Several drugs of different basicity have been chromatographed on a polymeric PLRP-S column with methanol-water and acetonitrile-water mobile phases. The cation exchange between the protonated basic drug and the buffer cations (Na+, K+ and BuNH4+) is observed at the pH range where the protonated drug and the ionized sites of the column coexist. This process produces a shift of the retention versus pH pl…
Study of the column efficiency using gradient elution based on Van Deemter plots.
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 equation. In this work, an approach is presented to extend the measurement of efficiency to linear gradient elution, where the mean retention factor is kept constant at each assayed flow. This avoids a possible source of uncertainty due to the change in the distribution equilibria profile, and makes the mean interactions with the stationary phase in grad…
A comparative study of the performance of acetonitrile and methanol in the multi-linear gradient separation of proteic primary amino acids
The performance of the separation of proteic primary amino acids using multi-linear gradients of acetonitrile and methanol was studied under an experimental-design basis, using an Inertsil ODS-3 column and pre-column derivatization with o-phthaldialdehyde (OPA) and N-acetyl-l-cysteine (NAC). Elution strength, peak properties, resolution, and analysis time, were examined. The optimal separation was established through modeling, using information obtained from isocratic data. By optimizing the separation with gradients of increasing complexity, acceptable resolution was possible, being glycine/threonine the critical pair. Multi-criteria decision-making (Derringer desirabilities) was applied t…
The role of the dual nature of ionic liquids in the reversed-phase liquid chromatographic separation of basic drugs.
The cationic nature of basic drugs gives rise to broad asymmetrical chromatographic peaks with conventional C18 columns and hydro-organic mixtures, due to the ionic interaction of the positively charged solutes with the free silanol groups on the alkyl-bonded reversed-phase packing. Ionic liquids (ILs) have recently attracted some attention to reduce this undesirable silanol activity. ILs are dual modifiers (with a cationic and anionic character), which means that both cation and anion can be adsorbed on the stationary phase, giving rise to interesting interactions with the anionic free silanols and the cationic basic drugs. A comparative study of the performance of four imidazolium-based I…
Peak dispersion in gradient elution: An insight based on the plate model.
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 efficiency. In this work, a general equation describing peak dispersion in HPLC gradient elution is derived from the plate model. This equation is studied and validated for three types of gradients: (i) a reference gradient without ramp in which the retention factor varies with time identically throughout the column, (ii) a gradient of stationary pha…
Filter-based infrared detectors for high temperature size exclusion chromatography analysis of polyolefins: calibration with a small number of standards and error analysis.
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 prove, however, comparable sensitivity while providing a number of practical advantages when coupled to chromatographic systems: reduced cell volume, simplified hardware, continuous generation of absorbance chromatograms, as well as simpler data collection and processing. A practical method for calibration, using multiple-band signals obtained wi…
Simultaneous optimization of mobile phase composition, column nature and length to analyse complex samples using serially coupled columns
Abstract 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 (2013) 94), we showed that the approach succeeds with conventional columns, assembled through zero-dead volume couplers. In this work, the simultaneous interpretive optimization of mobile phase composition and column nature and length, based on a limited number of experimental data, is demonstrated. This approach allows an impressive redu…
Extent of the influence of phosphate buffer and ionic liquids on the reduction of the silanol effect in a C18 stationary phase
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 silanol activity can be affected by the buffer concentration and mobile phase pH, factors that are not always considered sufficiently in the literature. In this work, the chromatographic behaviour of three basic β-adrenoceptor antagonists (acebutolol, nadolol and timolol), using mobile phases containing acetonitrile, was examined at different…
Enhancement of retention predictions in reversed-phase liquid chromatography using reference compounds
Abstract The use of reference compounds to correct errors associated with the preparation of mobile phase is studied, in order to enhance modelling of retention for optimisation purposes in reversed-phase liquid chromatography. The method fits individual retention models for a set of reference compounds, which are present in all injections. In second step, mobile-phase composition is corrected for each chromatogram, according to the fitted models of the reference compounds. The enhanced precision provided by the large number of replicates for the reference compounds is partially propagated to the retention models of the analytes. The approach is assayed in the separation of amino acids, aft…
Enhanced calculation of optimal gradient programs in reversed-phase liquid chromatography
Abstract The resolution of a mixture of 16 β-blockers under gradient elution was optimised using both isocratic and gradient training sets, with a reversed-phase column and acetonitrile–water eluents. Error theory was applied to measure the information extracted from different gradient experimental designs. This allows checking the expected accuracy when gradient predictions exceed the initial solvent concentrations tested in the training set. This work applies the results on modelling found in a previous study [J. Chromatogr. A 1018 (2003) 169] where the performance of several retention models was compared. Enhanced retention predictions were applied to the optimisation of gradient program…
A hybrid genetic algorithm with local search
Abstract A hybrid genetic algorithm with internal local search was developed for optimisations involving continuous variables. The reproduction probabilities were enhanced using the fitness values obtained when a local method was applied to each individual in the population. These estimations are more realistic, since consider not the apparent but the hidden, latent quality of each individual. The information gathered in the local search was also used to build an auxiliary population recording the successfully enhanced individuals, which allowed to detect the convergence and self-adapt the search limits. The size of this auxiliary population was kept constant by a cluster analysis strategy.…
Micellar versus hydro-organic reversed-phase liquid chromatography: a solvation parameter-based perspective.
The performance of the solvation parameter model is examined for micellar liquid chromatography. The results are compared with those offered with hydro-organic eluents, intending to reveal the properties that influence the retention and distinguish the particular behaviour of micellar systems. The retention data of several series of non-ionisable and ionisable compounds (mainly steroids, polyaromatic hydrocarbons, phenols, sulfonamides, beta-blockers, phenethylamines, antihistamines, and diuretics) were used as probe compounds. The micellar mobile phases contained an anionic (sodium dodecyl sulphate), non-ionic (Brij-35), or cationic (cetyltrimethylamonium bromide) surfactant, with or witho…
Performance of Markers and the Homologous Series Method for Dead Time Estimation in Reversed-Phase Liquid Chromatography
Abstract Two methods for dead time estimation (the use of markers and the homologous series mathematical method) are revised. Out of twelve assayed common markers, only KBr, KI, tartrazine, thiourea, uracil, and urea yielded retention times independent of the mobile phase composition in the range 10–90% acetonitrile, using a Zorbax Eclipse XDB−C18 column. On the other hand, the quality of the estimations provided by the homologous series method was limited by the mathematical approach and the data quality. With this method, the estimated dead time is an extrapolated value, which is severely affected by the data of the most retained compounds that act as leverage points, biasing the result. …
Modulation of retention and selectivity in oil-in-water microemulsion liquid chromatography: A review
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 concentration of the ME components and the ratio between the aqueous and oil phases. This work makes a critical survey on the information found in the literature about the mobile phase compositions that lead to the creation of successful O/W ME mobile phases, as well as the effect of pH for ionizable compounds and temperature. The viability of perfo…
Approaches to characterise chromatographic column performance based on global parameters accounting for peak broadening and skewness.
Peak broadening and skewness are fundamental parameters in chromatography, since they affect the resolution capability of a chromatographic column. A common practice to characterise chromatographic columns is to estimate the efficiency and asymmetry factor for the peaks of one or more solutes eluted at selected experimental conditions. This has the drawback that the extra-column contributions to the peak variance and skewness make the peak shape parameters depend on the retention time. We propose and discuss here the use of several approaches that allow the estimation of global parameters (non-dependent on the retention time) to describe the column performance. The global parameters arise f…
Prediction of peak shape in hydro-organic and micellar-organic liquid chromatography as a function of mobile phase composition
A simple model is proposed that relates the parameters describing the peak width with the retention time, which can be easily predicted as a function of mobile phase composition. This allows the further prediction of peak shape with global errors below 5%, using a modified Gaussian model with a parabolic variance. The model is useful in the optimisation of chromatographic resolution to assess an eventual overlapping of close peaks. The dependence of peak shape with mobile phase composition was studied for mobile phases containing acetonitrile in the presence and absence of micellised surfactant (micellar-organic and hydro-organic reversed-phase liquid chromatography, RPLC). In micellar RPLC…
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…
Furosemide assay in pharmaceuticals by Micellar liquid chromatography: study of the stability of the drug.
A simplified high-performance liquid chromatographic procedure is described for the determination of furosemide (4-chloro-N-furfuryl-5-sulphamoylanthranillic acid), which makes use of UV detection, a C18, reversed-phase column, and micellar mobile phases of sodium dodecyl sulphate (SDS) and 1-propanol at pH 3 buffered with phosphate system. The most adequate experimental conditions to handle furosemide solutions in the analytical laboratory are studied. The mixture of furosemide and its degradation products which are formed upon light exposition was resolved with a mobile phase of 0.04 M SDS-2% propanol. Separation of furosemide from its common impurities and the hydrolytic product, 4-chlor…
Characterization of chromatographic peaks using the linearly modified Gaussian model. Comparison with the bi-Gaussian and the Foley and Dorsey approaches
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 introduces significant uncertainty. For this reason, several alternative procedures have been proposed to measure the area, mean time and variance, based on the assumption that the chromatographic peak can be described with a mathematical function. This allows the calculation of the peak position and variance making use of the values of the experime…
Optimised procedures for the reversed-phase liquid chromatographic analysis of formulations containing tricyclic antidepressants.
The chromatographic behaviour (retention, selectivity, peak shape and resolution) of seven tricyclic antidepressants (TCAs), amitryptiline, clomipramine, doxepin, imipramine, maprotiline, nortryptiline and trimipramine, was examined. Conventional unendcapped Cs and C18 columns and an endcapped XTerra MS C18 column recommended for the analysis of basic compounds were used together with acetonitrile-water and micellar sodium dodecylsulfate (SDS)-pentanol mobile phases. The two best combinations were XTerra C18/acetonitrile, which yielded the largest efficiencies and resolution, and C8/SDS-pentanol, which eliminated the peak tails that were still observed with the XTerra C18 column. Both the s…
Serial versus parallel columns using isocratic elution: a comparison of multi-column approaches in mono-dimensional liquid chromatography.
Abstract 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 fails, the usual practice consists of introducing drastic changes in the chromatographic system (e.g. use of another column, solvent or pH). An alternative solution is taking benefit of the combined separation capability of two or more columns, which can be attained in multiple ways, such as diverse modalities of two-dimensional HPLC, or mo…
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…
DETERMINATION OF STEROIDAL HORMONES IN URINE SAMPLES BY MICELLAR LIQUID CHROMATOGRAPHY FOLLOWING SOLID-PHASE EXTRACTION
Steroidal hormones were determined in spiked urine samples using micellar mobile phases of sodium dodecyl sulphate (SDS)-pentanol, solid-phase extraction (SPE), and detection in the UV region. In the optimized procedure, a 10 mL aliquot of urine sample is loaded into a C18 cartridge and washed with 2 mL of 50:50 (v/v) methanol-water, followed by 200 μL of 70:30 (v/v) methanol-water. The retained steroids are eluted with 2 mL of methanol and the eluate evaporated to dryness under nitrogen at 50°C. The residue is redissolved with 200 μL of the micellar mobile phase used in the chromatographic separation and injected into the chromatograph. The performance of the procedure was checked for 13 s…
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…
Determination of sulphonamides in human urine by azo dye precolumn derivatization and micellar liquid chromatography
Abstract A high-performance liquid chromatographic method for the determination of sulphonamides in urine is reported. The drugs (sulphadiazine, sulphaguanidine, sulphamethizole, sulphamethoxazole, and sulphathiazole) were diazotized with nitrite and coupled with N-(1-naphthyl)ethylenediamine dihydrochloride in a sodium dodecyl sulphate (SDS) micellar medium. Separation of the sulphonamide azo dyes was performed on a C18 column with a 0.05 M SDS-2.4% pentanol mobile phase, which permitted the direct injection of the urine samples. The limits of detection were in the 0.1–0.3 μg/ml range.
Extension of the linear solvent strength retention model including a parameter that describes the elution strength changes in liquid chromatography.
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 retention times with optimisation purposes. Particular models are used for different LC modes, such as normal phase (NPLC), reversed phase (RPLC), hydrophilic interaction (HILIC), and micellar (MLC). In this work, a general equation is proposed that includes a parameter (the elution degree, g), which characterises the way the elution strength varies wit…
High-performance micellar liquid chromatography determination of sulphonamides in pharmaceuticals after azodye precolumn derivatization
Abstract A chromatographic procedure with precolumn derivatization to form the N-(1-naphthyl)ethylenediamine dihydrochloride azodyes is proposed for the analysis of several sulphonamides (sodium sulphacetamide, sulphadiazine, sulphaguanidine, sulphamerazine, sulphamethizole, sulphamethoxazole, sulphanilamide and sulphathiazole) in pharmaceutical preparations (tablets, pills, capsules, suspensions and drops). The separation is performed with a 0.05 M sodium dodecyl sulphate/2.4% pentanol eluent at pH 7. The precolumn derivatization improved the resolution in the chromatograms and increased the selectivity in the determination of mixtures of sulphonamides and in preparations where other drugs…
Micellar modified spectrophotometric determination of nitrobenzenes based upon reduction with tin(II), diazotisation and coupling with the Bratton–Marshall reagent
Abstract Nitrobenzenes, such as the antibiotic chloramphenicol, the vasodilator nicardipine, and the herbicides dinitramin, dinobuton, fenitrothion, methylparathion, oxyfluorfen, parathion, pendimethalin, quintozene, and trifluralin, were determined by using a spectrophotometric method in the visible region (540 nm). The method was based on the reduction of the nitrobenzenes to arylamines with tin(II) chloride, diazotisation of the arylamines and coupling of the diazonium ions with the Bratton–Marshall reagent. The two latter reactions were performed in a micellar medium of sodium dodecyl sulphate. The linear calibration range was 2×10 −6 to 7×10 −5 M ( r >0.999), with limits of detection i…
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…
Modified Gaussian models applied to the description and deconvolution of peaks in chiral liquid chromatography.
Abstract 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. These models are, in fact, a family of functions of different complexity with great flexibility to adjust chromatographic peaks over a wide range of asymmetries and shapes. However, an uncontrolled behaviour of the signal may occur outside the region being fitted, forcing the use of different strategies to overcome this problem. In this wo…
Micellar liquid Chromatographic determination of diuretics by diazotization and coupling with the Bratton-Marshall reagent
Abstract A Chromatographic procedure that uses micellar mobile phases of sodium dodecyl sulphate (SDS) and propanol at pH 3 is reported for the determination, in urine samples, of thiazide diuretics (althiazide, bendroflumethiazide, chlorothiazide, hydrochlorothiazide, hydroflumethiazide, and trichloromethiazide), and other diuretics having a sulphonamide substituent in the benzene ring. Precolumn derivatization with sodium nitrite and N -(1-naphthyl)ethylenediamine, to form the coloured azo dyes of the hydrolyzed diuretics, decreased the polarity of the compounds and, consequently, their retention in the micellar eluents, and permitted detection in the visible region, eliminating thus the …
Chromatographic analysis of phenethylamine-antihistamine combinations using C8, C18 or cyano columns and micellar sodium dodecyl sulfate-pentanol mixtures.
The chromatographic behaviour of binary and ternary mixtures of several phenethylamines (phenylephrine, phenylpropanolamine, ephedrine, pseudoephedrine and methoxyphenamine) and antihistamines (pheniramine, carbinoxamine, doxylamine, chlorpheniramine, dexchlorpheniramine, dexbrompheniramine, diphenhydramine, tripolidine, azatadine and phenyltoloxamine), found in cough-cold pharmaceutical preparations, was studied using C8, C18 and cyano columns, micellar mobile phases of sodium dodecyl sulfate (SDS) and pentanol and UV detection. Using a C8 column and mobile phases of 0.05 mol l-1 SDS-6% v/v pentanol or 0.15 mol l-1 SDS-2% v/v pentanol at pH 7, more than 30 different phenethylamine-antihist…
Some observations on the prediction of retention in reversed-phase liquid chromatography using the pH as main factor
Abstract When solutes show acid–base behaviour inside the working pH of a reversed-phase liquid chromatographic (RPLC) column, besides the type and percentage of organic modifier, pH can be included to improve the possibilities of separation. Optimisation based on a retention model is an economical and reliable way to achieve the best resolution. However, the difficulties found in knowing the exact pH values and the dependence of acid−base constants of solutes with mobile phase composition makes the accurate modelling of the retention behaviour as a function of these variables rather difficult. In this work, it is shown that in fact, there is no need to have this information. Based on the e…
Modelling and prediction of retention in high-performance liquid chromatography by using neural networks
Multi-layer feed-forward neural networks trained with an error back-propagation algorithm have been used to model retention behaviour of liquid chromatography as a function of the composition of the mobile phases. Conventional hydro-organic and micellar mobile phases were considered. Accurate retention modelling and prediction have been achieved using mobile phases defined by two, three and four parameters. With micellar mobile phases, the parameters involved included the concentrations of surfactant and organic modifier, pH and temperature. It is shown that neural networks provide a competitive tool to model varied inherent nonlinear relationships of retention behaviour with respect to the…