S. Pous-Torres

Determination of the hydrophobicity of organic compounds measured as log Po/w through a new chromatographic method

A new chromatographic method to determine the octanol-water partition coefficient (logP(o/w)) of organic substances is proposed in this paper. This method is based on a previously reported model that relates the retention factor in reversed-phase liquid chromatography with solute (p), mobile phase (P(m)(N)) and stationary phase (P(s)(N)) polarity parameters: logk=(logk)(0)+p(P(m)(N)-P(s)(N)). P(m)(N) values are calculated through expressions that depend only on the organic solvent fraction in the mobile phase. (logk)(0) and P(s)(N) parameters are characteristic of the chromatographic system and are determined from the retention of a selected set of 12 compounds. Then, the p value of a solute determined in a properly characterized system is easily derived from the retention factor data. Solute p values are slightly dependent on the chromatographic system but they are linearly related to those obtained in the reference system (Spherisorb ODS-2 column and acetonitrile as organic modifier). Therefore, they can be easily transferred from any experimental system to the reference one. A Quantitative Structure-Property Relationship study reveals that the p parameter in the reference chromatographic system depends, mainly, on the hydrophobicity of the compound, expressed as the n-octanol/water partition coefficient (logP(o/w)), and five additional structural descriptors which can be easily calculated through the CODESSA program from the chemical structure of the solute. In this work the p descriptors of a wide set of structurally different organic compounds have been determined in several chromatographic systems and transferred to the reference one from these and the CODESSA structural parameters. The logP(o/w) values have been determined. The obtained values agree with those determined from classical experimental techniques and validate the new method as a useful tool to determine the hydrophobicity of a wide variety of compounds in a broad logP(o/w) range.

Combined effect of solvent content, temperature and pH on the chromatographic behaviour of ionisable compounds II: benefits of the simultaneous optimisation.

A previously reported eight-parameter mechanistic model [Part I of this work, J. Chromatogr. A 1163 (2007) 49] was applied to optimise the separation of 11 ionisable compounds (nine diuretics and two beta-blockers), considering solvent content, temperature and pH as experimental factors. The data from 21 experiments, arranged in a central composite design, were used to model the retention. Local models were used to predict efficiency and peak asymmetry. The optimisation strategy, based on the use of peak purity as chromatographic objective function and derived concepts, was able to find the most suitable experimental conditions yielding full resolution in reasonable analysis times. It also allowed a detailed inspection of the separation capability of the studied factors, and of the consequences of the shifts in the protonation constants originated by changes in solvent content and temperature. The size of the resolution structures suggested that the ranked importance of the factors was pH, organic solvent and temperature, giving rise to relatively narrow domains of full resolution. The three factors were found, however, worthwhile in the optimisation of selectivity. Predicted optimal conditions corresponding to two different optimal resolution regions were verified experimentally. In spite of the difficulties associated to the use of pH as optimisation factor, satisfactory agreement was found in both cases.

Performance of a Chromolith RP-18e column for the screening of β-blockers

The chromatographic performance of a monolithic column (Chromolith RP-18e) was comprehensively examined in the isocratic separation of ten beta-blockers, using ACN-water mobile phases, and compared with the performance of three microparticulate RP columns manufactured with different types of silica: Spherisorb ODS-2, Kromasil C18 and XTerra MS C18. The comparison considered the analysis time, selectivity, peak shape (column efficiency and asymmetry) and resolution, and was extended to a wide range of mobile phase compositions. The Chromolith column showed good performance for the analysis of beta-blockers with regard to the packed columns. In terms of selectivity and analysis time, the greatest similarity was found between the Chromolith and XTerra columns. The addition of a silanol blocking agent (0.1% triethylamine) to both Chromolith and Spherisorb columns yielded, apparently, a similar blocking degree of the silanol groups (based on the similar peak shapes), and gave rise to similar selectivity.

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, tending to inflate them and increase their volume. This decreases the linear velocity inside the column, and increases the retention at relatively low pressure (<200 bar). In contrast, frictional heating, which is an issue for microparticulate columns, seems to be less significant for the highly permeable Chromoliths. The usefulness of the retention time versus the inverse of the delivered flow rate plots to measure the deviations, whatever their origin, is shown. This allows the correction of the retention times to the ideal behaviour, where the retention factors are independent of the flow rate.

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 those of the underlying bonded phase. The chromatographic behaviour (in terms of selectivity, analysis time and peak shape) of eight C18 columns in the analysis of weakly acidic phenols and basic β-blockers was examined with hydro-organic and micellar-organic mobile phases. The behaviour of the columns differed significantly when the cationic basic drugs were eluted with hydro-organic mobile phases. With micellar-organic mobile phases, the adsorption of surfactant, instead of making the columns similar, gave rise to a greater diversity of behaviours (especially in terms of selectivity and analysis time), for both groups of phenols and β-blockers, which should be explained by the residual effect of the underlying bonded stationary phase and the different amount of surfactant covering the packing. Therefore, the implementation of a micellar-organic procedure in RPLC will depend significantly on the selected type of C18 column.

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 from different linear relationships that can be established between the peak variance, standard deviation, or half-widths with the retention time. Some of them describe exclusively the column contribution to the peak broadening, whereas others consider the extra-column effects also. The estimation of peak skewness was also possible for the approaches based on the half-widths. The proposed approaches were applied to the characterisation of different columns (Spherisorb, Zorbax SB, Zorbax Eclipse, Kromasil, Chromolith, X-Terra and Inertsil), using the chromatographic data obtained for several diuretics and basic drugs (beta-blockers).

Interpretive optimisation of organic solvent content and flow-rate in the separation of β-blockers with a Chromolith RP-18e column

The chromatographic performance of a Chromolith RP-18e column was comprehensively examined for a group of basic drugs (beta-blockers), eluted with isocratic ACN-water mixtures at increasing flow-rate up to 6 mL/min. As the flow-rate increases at fixed mobile phase composition, peak distribution (selectivity) is maintained, but the relative peak widths increase. This reduces the resolution below satisfactory values for closely eluting compounds. With the monolithic column, flow-rate becomes thus an important factor to be optimised, in addition to the mobile phase composition. Since, theoretically, retention factors (k) are independent of the flow-rate, the classical quadratic model relating log k with the solvent content allows the prediction of the retention at any combination of organic solvent content and flow-rate. The small deviations found for the most retained compounds were corrected by including, in the quadratic model, an additional term correlating linearly log k with the flow-rate. Peak shape and resolution changes were predicted by taking advantage of the approximated linear relationships between peak half-widths and retention times, which offered similar coefficients for peaks eluting at different organic solvent contents and flow-rates. The accuracy of the predictions in critical conditions was experimentally verified to be satisfactory.

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. The sequential elimination of the most retained compounds attenuates this problem. This means that the homologous series should contain at least four compounds with low retention. Otherwise, overestimations of dead time are yielded.

Correction of the deviations in the retention times with Chromolith columns associated to the flow rate: Implications in the modelling of the retention behaviour

In a previous work (J. Sep. Sci. 2009, 32, 2793-2803), we reported an interpretive optimisation approach to achieve maximal resolution in minimal analysis time, based on models describing the retention and peak shape as a function of mobile phase composition and flow rate. The method was applied to the separation of a group of basic drugs in a Chromolith column. In that work, we found that the retention factors were sensitive to the flow rate. The reason of the observed deviations in retention times is the increase in the column volume at the applied pressure, which decreases the linear velocity inside the column. This behaviour forced to include a correction term in the model that described the retention. We show here how the deviations in retention times can be evaluated, allowing retention models that do not include the flow rate as a variable, similar to isocratic chromatography at fixed flow rate. The logarithm of the deviations in the retention times with flow rate is shown to correlate with the solute polarity. This correlation is compared with similar correlations for the retention factor at fixed mobile phase composition and the extrapolated retention factor in water at fixed flow rate.

Optimal experimental designs in RPLC at variable solvent content and pH based on prediction error surfaces

AbstractWhen pH is used as factor in reversed-phase liquid chromatographic (RPLC) separations, the need for providing quality and informative data with the minimal experimental effort becomes imperative. The most rational way to achieve this is by means of experimental designs. The interest in finding optimal designs involving solvent content and pH in RPLC is considerable, since these factors allow large variations in selectivity when ionisable compounds are involved. Unfortunately, the equations that describe the retention of these compounds with pH are nonlinear. As a consequence, factorial and other designs based on geometrical considerations are not well suited, whereas D-optimal and related designs can only be applied in an iterative fashion. In this work, an extension of G-optimal designs, aimed to enhance the quality of the predictions, is examined for problems involving solvent content and pH. The study was carried out with a set of probe ionisable compounds, for which information on retention behaviour was accurately known. A stepwise strategy was used to obtain a rapid estimation of the best design with a given number of experiments. The objective of the study was to investigate the distribution and number of points in the ideal design for compounds of different acid–base behaviour, and the possibility of finding common designs for groups of compounds. A further goal was to derive design construction rules containing the information requirements, without needing any further mathematical treatment. FigureThree-dimensional view of an error surface