Evolving neural network optimization of cholesteryl ester separation by reversed-phase HPLC

Cholesteryl esters have antimicrobial activity and likely contribute to the innate immunity system. Improved separation techniques are needed to characterize these compounds. In this study, optimization of the reversed-phase high-performance liquid chromatography separation of six analyte standards (four cholesteryl esters plus cholesterol and tri-palmitin) was accomplished by modeling with an artificial neural network–genetic algorithm (ANN-GA) approach. A fractional factorial design was employed to examine the significance of four experimental factors: organic component in the mobile phase (ethanol and methanol), column temperature, and flow rate. Three separation parameters were then merged into geometric means using Derringer’s desirability function and used as input sources for model training and testing. The use of genetic operators proved valuable for the determination of an effective neural network structure. Implementation of the optimized method resulted in complete separation of all six analytes, including the resolution of two previously co-eluting peaks. Model validation was performed with experimental responses in good agreement with model-predicted responses. Improved separation was also realized in a complex biological fluid, human milk. Thus, the first known use of ANN-GA modeling for improving the chromatographic separation of cholesteryl esters in biological fluids is presented and will likely prove valuable for future investigators involved in studying complex biological samples

Hydrophilic interaction liquid chromatography (HILIC)—a powerful separation technique

Hydrophilic interaction liquid chromatography (HILIC) provides an alternative approach to effectively separate small polar compounds on polar stationary phases. The purpose of this work was to review the options for the characterization of HILIC stationary phases and their applications for separations of polar compounds in complex matrices. The characteristics of the hydrophilic stationary phase may affect and in some cases limit the choices of mobile phase composition, ion strength or buffer pH value available, since mechanisms other than hydrophilic partitioning could potentially occur. Enhancing our understanding of retention behavior in HILIC increases the scope of possible applications of liquid chromatography. One interesting option may also be to use HILIC in orthogonal and/or two-dimensional separations. Bioapplications of HILIC systems are also presented

A study of the relative importance of lipophilic, π-π and dipole-dipole interactions on cyanopropyl, phenyl and alkyl LC phases bonded onto the same base silica

Cyano (CN), butyl (C4), phenyl and octadecyl (C18) phases prepared from the same base silica gel were chromatographically characterized in order to assess the relative importance of lipophilic, π-π and dipole-dipole interactions in governing retention on these differing phases. Dipole interactions of analytes (possessing dipole moments and low lipophilicity) with CN phases were primarily responsible for the elution order. However, as the analytes' lipophilicity increased, the lipophilic interaction predominated over the dipole interaction. In comparison, retention on the phenyl phase appeared to be complex, being controlled by a mixture of lipophilic, π-π and dipole-dipole interactions. Retention on the C4 and C18 phases was dictated by the analyte's lipophilicity and its accessibility into the phase

Possibilities and Limitations of Computer-Assisted Method Development in HILIC: A Case Study

peer reviewedIn the present study, we investigated the possibilities and limitations of computer-assisted method development (CAMD) for the HILIC separation optimization of a mixture of 13 isomeric hydroxy- and aminobenzoic acids on a ZIC-HILIC column. The isocratically obtained Neue and Kuss retention parameters enabled the accurate gradient retention modeling for peaks eluting well within the gradient (mean error of 2.7 %). The prediction errors for peaks eluting at the end of the gradient could be reduced from 8.8 to 6.1 % by implementing the isocratic regime after the gradient into the expression for the gradient retention factor. The prediction of the corresponding peak widths improved significantly for certain compounds and gradient profiles using individual gradient N values for each compound compared to employing a single N value for all compounds and gradient profiles. Two gradient optimization strategies (constructing the Rs map based on individual retention modeling and predictive elution stretching and shifting, PEWS2) resulted in a reasonable separation of the challenging mixture of 13 isomeric hydroxy- and aminobenzoic acids on the ZIC-HILIC column. Overall, the optimization was limited by the steep decrease in N (dropping to the isocratic N value) and corresponding increase in peak width when increasing the gradient time. The discrimination factors d0 were used to assess the resolution between peaks varying widely in height. The best separation was found to be obtained via the PEWS2 approach. Both the individual retention modeling and PEWS2 strategies corresponded to a total instrument time less than 12 h (including equilibration). Finally, it was found that the salt concentration had a significant effect on both the retention and the peak shape of the compounds, resulting in a small “solution domain” at 10 mM. Coupled columns with higher efficiencies are suggested to improve the resolution and robustness of the separation. © 2016 Springer-Verlag Berlin Heidelber