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 chromatographic performance was obtained when the columns were combined. Particularly interesting is the reduction in the analysis time in the isocratic mode, which alleviates the "general elution problem" of liquid chromatography, and may represent a stimulus for the proposal of new procedures, especially in combination with mass spectrometric, electrochemical and refractometric detection. Developments proposed to make the serial coupling of columns useful in routine and research laboratories are outlined, including optimisation strategies that facilitate the selection of the appropriate column combination and elution conditions (solvent content, flow rate or temperature) in both isocratic and gradient modes. The availability of zero dead volume couplers, able to connect standard columns, and the commercialisation of short columns with multiple lengths, have expanded the possibilities of success.

A chromatographic objective function to characterise chromatograms with unknown compounds or without standards available.

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 this work, a new COF based on the automatic measurement of the protruding part of the chromatographic peaks (or peak prominences) that indicates the number of perceptible peaks and global resolution, without the need of standards, is developed. The proposed COF was found satisfactory with regard to the peak purity criterion when applied to artificial peaks and simulated chromatograms of mixtures built using the information of standards. The approach was applied to mixtures of drugs containing unknown impurities and degradation products and to extracts of medicinal herbs, eluted with acetonitrile-water mixtures using isocratic and gradient elution.

Serial versus parallel columns using isocratic elution: a comparison of multi-column approaches in mono-dimensional liquid chromatography.

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 mono-dimensional HPLC with serial or parallel columns. In this work, the separation performance offered by the serial coupling of columns of different nature and length, operated at varying mobile phase composition in isocratic elution, is compared with the results offered by parallel columns. The resolution capability of both approaches is characterised through the limiting peak purities. It is demonstrated that serial columns of different lengths perform as new columns that increase enormously the probabilities of success. The potential of the approach is illustrated through the separation of 15 sulphonamides. In spite of the poor individual performance of the four selected columns (phenyl, cyano and two C18 columns, with nearly null resolution for the cyano column), it was found that the serial coupling of the phenyl and cyano columns of appropriate lengths succeeded in the full resolution of the 15 compounds in 20-25min, and the serial coupling of the two C18 columns yielded acceptable resolution in less than 20min.

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 and tetrafluoroborates of 1-ethyl-, 1-butyl- and 1-hexyl-3-methylimidazolium) as modifiers of the chromatographic behaviour of a group of 10 ß-blockers is performed, and compared with triethylamine and dimethyloctylamine. In order to gain more insight in the behaviour of ILs in RPLC, the changes in the nature of the chromatographic system, at increasing concentration of the additives, were followed based on retention and peak shape modelling. The multiple interactions that amines and ILs experience inside the chromatographic system suggest that the suppressing potency should be measured based on the shape of chromatographic peaks and not on the changes in retention. The ILs 1-hexyl-3-methyl-imidazolium chloride and tetrafluoroborate offered the most interesting features for the separation of the basic drugs.

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 sensitive to situations where the components that coelute in a mixture show some spectral differences. Therefore, they are useful to find out experimental conditions where the spectrochromatograms can be recovered by deconvolution. Two-way multivariate selectivity yielded the best performance and was applied to the separation using diode-array detection of a mixture of 25 phenolic compounds, which remained unresolved in the chromatographic order using linear and multi-linear gradients of acetonitrile-water. Peak deconvolution was carried out using the combination of orthogonal projection approach and alternating least squares.

Adsorption of the anionic surfactant sodium dodecyl sulfate on a C18 column 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, especially for basic compounds. However, the risk of full desorption of surfactant is the main limitation in the high submicellar mode. This study examines the adsorption of the anionic surfactant sodium dodecyl sulfate under micellar and high submicellar conditions on a C18 column, applying two methods. One of them uses a refractive index detector to obtain direct measurements of the adsorbed amount of sodium dodecyl sulfate, whereas the second method is based on the retention and peak shape for a set of cationic basic compounds that indirectly reveal the presence of adsorbed monomers of surfactant on the stationary phase.

Optimisation of gradient elution with serially-coupled columns Part II: Multi-linear gradients.

An interpretive approach is reported for the simultaneous optimisation of resolution and analysis time in the separation of complex samples by reversed-phase liquid chromatography, using serially-coupled columns of different lengths and nature, and multi-linear gradient elution. The results complement those reported in JCA 1350 (2014) 51 for single linear gradients, using combinations of five columns: three C18 columns of different characteristics, a cyano column and a phenyl column. The relationships between the logarithm of the retention factor (lnk) and the organic solvent content (φ), and between the peak half-widths and the retention times, for each analyte, were determined using isocratic retention times. From the established correlations, the gradient retention time was predicted using the fundamental equation for gradient elution (modified to be used for serially-coupled columns), and with this information, the peak profile. Two chromatographic goals (maximal peak purity and minimal analysis time) were simultaneously evaluated using the Pareto optimality concept. The rigorous predictive system can be easily applied to columns from different manufacturers and succeeds at different levels: single and combined columns, using isocratic and gradient elution. The computation time for gradient elution optimisation was significantly reduced to practical values with the help of genetic algorithms (GAs).

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) were combined, aimed to resolve a mixture of 15 sulphonamides using acetonitrile-water gradients. A gradient predictive system, based on numerical integration, was built to simulate chromatograms under linear gradient profiles. Two approaches were compared: the optimisation of the combination of columns pre-selecting the gradient profile, developed by De Beer et al. (Anal. Chem. 82 (2010) 1733), and the optimisation of the gradient program after pre-selecting the column combination using isocratic elution, developed for this work. Several refinements concerning the gradient delays along the solute migration and peak half-width modelling were included to improve the realism of the predictions. Pareto plots (expressed as analysis time versus predicted global resolution) assisted in the selection of the best separation conditions. The massive computation time in the gradient optimisation, once the column combination was optimised, was reduced to ca. 3min by using genetic algorithms.

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 in the physiological fluid, and once the proteins are swept away, the elution strength can be increased using a positive linear gradient of organic solvent to reduce the analysis time. This work aims to encourage analysts to implement gradients of organic solvent in MLC, which is rather simple and allows rapid analytical procedures without pre-treatment or the need of re-equilibration. The implementation of gradient elution is illustrated through the separation of eight basic compounds (ß-blockers) in urine samples directly injected into the chromatograph, the most hydrophobic showing large retention in both conventional RPLC and MLC. The use of the DryLab(®) software to optimise gradients of organic solvent with eluents containing a fixed amount of surfactant above the critical micellar concentration is shown to provide satisfactory predictions, and can facilitate greatly the implementation of gradient protocols.

Simultaneous optimization of mobile phase composition, column nature and length to analyse complex samples using serially coupled columns.

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 reduction in the number of different column lengths needed without loss of performance. The massive computation needed to develop the approach was cropped by restricting the maximal analysis time, total pressure and combined column length. Guidelines to model peak position and half-widths with low errors are given, which increase the reliability of the optimizations. Pareto plots, expressed as analysis time versus predicted resolution, assisted in the selection of the best separation conditions. Five ACE columns of different selectivity (C18, C18-HL, AQ, CN and Phenyl), available at two or three different lengths, were used to demonstrate the approach. Isocratic experiments with acetonitrile-water in the 10-20% (v/v) range were developed to model each single column, aimed to analyse a mixture of 15 sulphonamides. No single column was able to succeed in the full separation. In contrast, the comprehensive optimization of mobile phase composition, together with column nature and length, allowed baseline resolution of the mixture in approximately 20 min.

Approaches to model the retention and peak profile in linear gradient reversed-phase liquid chromatography.

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 yielded accurate predictions of the retention time for a wide range of initial concentrations of organic modifier and gradient slopes, with errors usually below 1-2%. Based on the half-width changes of chromatographic peaks along one or more gradients, an approach is also reported to predict the peak profile with low errors (usually below 2-3%). The proposed approaches were applied to two sets of probe compounds (diuretics and flavonoids), eluted with acetonitrile-water gradients. The changes in retention and peak shape in isocratic and gradient elution are illustrated through diagrams that define triangular regions including all possible values of retention factors or peak half-widths (or widths) inside the selected working ranges.

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 with a single column, based on the combined selectivities and the potential increase in plate count. The idea is highly attractive, but there are only few reports in the literature using this approach. The theory behind the use of serially coupled columns is indeed rather simple, but its implementation may be troublesome. The most problematic factor is the connection of the serial columns, which ideally should not modify the result of the sum of behaviors of the columns. A proper serial connection of columns needs a zero-dead volume (ZDV) union and a system to link tightly the columns to each other. In this work, two different approaches to solve this problem are compared using isocratic elution, one consisting in the use of holders, which are screwed to maintain attached the columns linked by ZDV junctions, and the other using ZDV fingertight column couplers, which are screwed directly to the columns maintaining them tightly attached without the need of column holders. The advantages and problems associated to these approaches are described, and information on the accuracy in the prediction of retention times, peak widths and asymmetries are given for the combined columns. Guidelines to prepare software to make reliable predictions are also presented. A set of 15 sulphonamides were used to probe the systems.

New approaches based on modified Gaussian models for the prediction of chromatographic peaks.

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 Lorentzian function that decreases the variance growth out of the peak region. The PLMG model has, however, the drawback of its high flexibility that makes the optimisation process difficult when the initial values of the model parameters are far from the optimal ones. Based on the fitting of experimental peaks of diverse origin and asymmetry degree, several semiempirical approaches that make use of the halfwidths at 60.65% and 10% peak height are here reported, which allow the use of the PLMG model for prediction purposes with small errors (below 2-3%). The incorporation of several restrictions in the algorithm avoids the indeterminations that arise frequently with this model, when applied to highly skewed peaks.

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 some compounds in the sample, while the other compounds will be resolved using a second (or subsequent) condition(s). Complementary separations, being a simple and attractive idea, present, however, challenges in terms of computation volume and complexity of the required algorithms. This work describes in detail different approaches that have been developed up-to-date for this purpose, and introduces a new approach based on the peak count concept that is benefited of the best features of the previous approaches: high reliability in finding the solution, accessibility to analysts without specialised programming skills and short computation time.