Multi-scale optimisation vs. genetic algorithms in the gradient separation of diuretics by reversed-phase liquid chromatography.

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 finding the best gradient is the use of global search methods, such as genetic algorithms (GAs). Recently, a new global approach where the level of detail is increased along the search has been proposed, namely Multi-scale optimisation (MSO). In this strategy, cubic splines are applied to build intermediate curves to define any arbitrary solvent variation function. Subdivision schemes are used to generate the cubic splines and control their level of detail. The search was subjected to a number of restrictions, such as avoiding long elution and favouring a balanced peak distribution. The aim of this work is evaluating and comparing the results of GAs and MSO. Both approaches were tested with a set of 14 diuretics and probenecid, eluted with acetonitrile-water mixtures using a C18 column. Satisfactory baseline resolution was obtained with an analysis time of 15-16 min. We found that GAs optimisation offered results equivalent to those provided by MSO, when the penalisation parameters were included in the cost function.

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 gradient elution similar to those in isocratic elution. The approach was applied to Zorbax Eclipse XDB C18 and Chromolith SpeedROD C18 columns, using four sulphonamides as probe compounds. The results are compared with those obtained in isocratic elution, and gradient elution where the gradient program is kept constant while the flow is varied, giving rise to changes in the mean retention factor at each flow rate. The mean compression factor obtained experimentally was higher than expected, with mean values of 0.98 and 1.02 for the Zorbax and Chromolith columns, whereas the predicted values were 0.87 and 0.92, respectively. Better efficiencies were obtained at lower flow and higher gradient slope. The resolution shows similar values in isocratic and gradient elution when the mean retention factor is kept constant. When the gradient slope is constant, the resolution is usually smaller, although it improves at higher flows.

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.

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 extracts) on the number of significant peaks found in the fingerprints of a medicinal herb (a green tea sample), using Plackett­Burman designs. Acetonitrile, ethanol and methanol were used for the extraction, and a linear gradient for chromatographic analysis, where the acetonitrile content was increased from 5.0% to 42.5% (v/v) in 45 min. The maximal number of significant peaks in the fingerprints was obtained using a methanol­water mixture as extraction solvent, high ultrasonication time and high temperature. The reported approach can be generalised to other complex samples and situations.

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.