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…

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…

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…

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…

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…

Isocratic and gradient elution in micellar liquid chromatography with Brij-35

Polyoxyethylene(23)lauryl ether (known as Brij-35) is a nonionic surfactant, which has been considered as an alternative to the extensively used in micellar liquid chromatography anionic surfactant sodium lauryl (dodecyl) sulfate, for the analysis of drugs and other types of compounds. Brij-35 is the most suitable nonionic surfactant for micellar liquid chromatography, owing to its commercial availability, low cost, low toxicity, high cloud temperature, and low background absorbance. However, it has had minor use. In this work, we gather and discuss some results obtained in our laboratory with several β-blockers, sulfonamides, and flavonoids, concerning the use of Brij-35 as mobile phase mo…

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 …

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…

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…

Modeling of Retention in Reversed Phase Liquid Chromatography

Description of the Retention and Peak Profile for Chromolith Columns in Isocratic and Gradient Elution Using Mobile Phase Composition and Flow Rate as Factors

The effect of the modifier concentration and flow rate on the chromatographic performance of a second generation Chromolith® RP-18e column, under isocratic and gradient elution with acetonitrile-water mixtures, was examined using four sulphonamides as probe compounds. The acetonitrile concentration was varied between 5 and 55% (v/v), and the flow rate between 0.1 and 5.0 mL/min, keeping the other factors constant. The changes in both retention and peak profile were modelled, and used to build simple plots, where the logarithm of the retention factor was represented against the modifier concentration (in gradient elution, against the initial modifier concentration), and the half-widths or wi…

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…

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…

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…

Prediction of Peak Shape and Characterization of Column Performance in Liquid Chromatography as a Function of Flow Rate

Traditionally, column performance in liquid chromatography has been studied using information from the elution of probe compounds at different flow rates through van Deemter plots, which relate the column plate height to the linear mobile phase velocity. A more recent approach to characterize columns is the representation of the peak widths (or the right and left peak half-widths) for a set of compounds versus their retention times, which, for isocratic elution, give rise to almost linear plots. In previous work, these plots have been shown to facilitate the prediction of peak profiles (width and asymmetry) with optimization purposes. In this work, a detailed study on the dependence of the …

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…

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…