0000000000342134
AUTHOR
V. Concha-herrera
Robust interpretive optimisation in high-performance liquid chromatography considering uncertainties in peak position.
In the context of interpretive chromatographic optimisation, robustness is usually calculated by introducing deliberated shifts in the nominal optimal conditions and evaluating their effects on the monitored objective function, mimicking thus the experimental procedures used in method validation. However, such strategy ignores a major source of error: the uncertainties associated to the modelling step, that may give rise to deceiving results when conditions that were expected to yield baseline separation are reproduced in the chromatograph. Two approaches, based on the peak purity concept, are here proposed to evaluate the robustness of the objective function under the perspective of measur…
Chromatographic Determination of Thiols After Pre‐column Derivatization witho‐Phthalaldehyde and Isoleucine
Abstract The reaction of primary amines with excess o‐phthalaldehyde (OPA) and thiol yields unique isoindole derivatives that are readily separated by reversed‐phase liquid chromatography. In a previous work, a spectrophotometric procedure was proposed for the assay of N‐acetylcysteine by derivatization with OPA and isoleucine at pH 9.5, with satisfactory results. The chromatographic determination of this and other low molecular‐weight thiols, after isoindole formation with isoleucine, using mobile phases of acetonitrile–water at pH 3 and spectrophotometric detection, is now examined. From the assayed thiols (thioglycolic acid, 3‐mercaptopropionic acid, tiopronin, N‐acetylcysteine, N‐acetyl…
Limits of multi-linear gradient optimisation in reversed-phase liquid chromatography
Abstract The concept of limiting peak purity was applied to quantify the degree of completion of the separation capability of a chromatographic system using multi-linear gradients. The objective was to check whether the complexity of a gradient program deserves be increased to enhance resolution by inserting more linear segments, or on the contrary, no significant improvements can be expected under more complex gradients. A set of 19 isoindole derivatives of primary amino acids was selected to test the performance of isocratic, single linear and multi-linear gradients. Accurate simulated chromatograms were obtained via numerical integration of the general equation of gradient elution, using…
Separation of Proteic Primary Amino Acids under Several Reversed‐Phase Liquid Chromatographic Conditions
Abstract The reversed‐phase liquid chromatographic (RPLC) analysis of proteic primary amino acids with acetonitrile‐water, using pre‐column derivatisation with o‐phthalaldehyde (OPA) and N‐acetylcysteine (NAC), was compared with RPLC modes using trifluoroacetic acid or pentadecafluorooctanoic acid and evaporative light‐scattering detection, or sodium dodecyl sulphate micelles with pre‐ and post‐column derivatisation. The importance of column lifetime, risk of potential damages in the instrumentation, retention and resolution, was considered. Among the assayed approaches, the best is still aqueous‐organic RPLC with pre‐column derivatisation. It not only yields the most reliable results, but …
A comparative study of the performance of acetonitrile and methanol in the multi-linear gradient separation of proteic primary amino acids
The performance of the separation of proteic primary amino acids using multi-linear gradients of acetonitrile and methanol was studied under an experimental-design basis, using an Inertsil ODS-3 column and pre-column derivatization with o-phthaldialdehyde (OPA) and N-acetyl-l-cysteine (NAC). Elution strength, peak properties, resolution, and analysis time, were examined. The optimal separation was established through modeling, using information obtained from isocratic data. By optimizing the separation with gradients of increasing complexity, acceptable resolution was possible, being glycine/threonine the critical pair. Multi-criteria decision-making (Derringer desirabilities) was applied t…
Enhancement of retention predictions in reversed-phase liquid chromatography using reference compounds
Abstract The use of reference compounds to correct errors associated with the preparation of mobile phase is studied, in order to enhance modelling of retention for optimisation purposes in reversed-phase liquid chromatography. The method fits individual retention models for a set of reference compounds, which are present in all injections. In second step, mobile-phase composition is corrected for each chromatogram, according to the fitted models of the reference compounds. The enhanced precision provided by the large number of replicates for the reference compounds is partially propagated to the retention models of the analytes. The approach is assayed in the separation of amino acids, aft…