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Highlights: [•] UHPLC pressure at constant flow rate and temperature reduces analyte molar volume. [•] For proteins, UHPLC pressure at constant temperature facilitates α-helix folding. [•] The degree of protein folding at certain conditions can be controlled by pressure. [•] Protein folding by pressure manifests as change in retention time. [•] Pressure variations in UHPLC can be used to study protein secondary structure. [Copyright &y& Elsevier]

The goal of this study was to investigate the effect of pressure modulation on the retention, selectivity, and peak efficiency of proteins and small basic and neutral molecules using reversed-phase liquid chromatography. A backpressure restrictor was used in the experiments to study pressure variation at a constant flow rate. Experiments were conducted in isocratic mode at constant temperature using mobile phase additives namely sodium perchlorate and potassium hexafluorophosphate. Upon increase of pressure at constant flow rate, an increase of the retention factor of analytes was observed depending on the type of analyte and molecular weight of the compound. However, retention factors of all analytes decreased with an increase in flow rate at constant pressure. Pressure alone can have a significant effect on retention of polar and ionized analytes; however, the effects are much more pronounced with larger analytes such as proteins in the presence of liophilic mobile phase additives. Selectivity changes could be obtained for a mixture of analytes by modulating the level of backpressure applied and this could be an effective tool in HPLC method development. The effects of pressure should be considered when transferring methods from conventional HPLC applications to fast LC applications at high pressures, especially for proteins and large peptides. In the case of ionized large molecules and proteins, strong liophilic additives are recommended for use in the mobile phase to streamline the transfer of a method originally developed on a fast LC system to a conventional LC system or vice versa. [ABSTRACT FROM AUTHOR]

The pharmaceutical industry today is driven to create new, more efficient ways to discover, develop, deliver and monitor drugs. Pharmaceutical companies are being faced with major challenges in reducing drug discovery and development timelines. Automation and the introduction of new analytical technologies that increase speed of analysis are integral in the analytical laboratory. The development of rapid chromatographic methods in preformulation and formulation development is playing an increasing role to support this drive in efficiency and productivity. The introduction of ultra fast-HPLC systems that can operate at pressures of up to 15,000 psi with columns packed with sub-2-µm particles have allowed for high speed and efficient separations. The consequent reduction of time, solvent, and waste disposal, and the analysis of more samples per unit time makes ultra-fast HPLC a very attractive technology. Faster method development and decision making can be achieved during late-phase preformulation/formulation development for the analysis of both singly charged and multiply charged basic compound analysis. The use of ultra fast-HPLC (UHPLC) for pH scouting experiments and the determination of the analyte's ionogenic nature was shown to be an effective tool for rapid and systematic method development. The implementation of this technology was also evaluated for the analysis of drug product formulations and excipient compatibility studies. Increased speed of analysis and significant gains in resolution per unit time were obtained compared to separations performed using conventional HPLC systems (operating pressures of <5,500 psi). Also, the use of liophilic ions as mobile phase additives with operation under high pressures led to enhanced separation selectivity, retention, and peak symmetry of multiply charged basic compounds. [ABSTRACT FROM AUTHOR]

The separation of basic compounds can be challenging and the use of inorganic mobile phase additives have been successfully used in chromatographic methods development. About fifteen years ago the role of these additives as ion-interaction agents for selectively adjusting retention of ionic analytes was discovered and the theory of chaotropicity was applied to reversed phase chromatography. In the last ten years the studies of the influence of these counterions on the retention of ionizable analytes and the interaction with the stationary phase in various hydro-organic eluents has expanded our knowledge of this phenomenon. The general view and understanding of the process have been significantly updated and the use of these ionic additives (liophilic ions) in the mobile phase has become regular practice in the pharmaceutical industry for optimization and fine tuning of complex separations. This paper reviews the latest developments in the field and discusses the modification and expansion of our theoretical understanding of the process. The paper also describes their application in practical separations for a wide variety of analytes, from small molecules to peptides and even chiral separations. [ABSTRACT FROM AUTHOR]

In recent years, the use of halogen-containing molecules has proliferated in the pharmaceutical industry, where the incorporation of halogens, especially fluorine, has become vitally important for blocking metabolism and enhancing the biological activity of pharmaceuticals. The chromatographic separation of halogen-containing pharmaceuticals from associated isomers or dehalogenation impurities can sometimes be quite difficult. In an attempt to identify the best current tools available for addressing this important problem, a survey of the suitability of four chromatographic method development platforms (ultra high-performance liquid chromatography (UHPLC), core shell HPLC, achiral supercritical fluid chromatography (SFC) and chiral SFC) for separating closely related mixtures of halogen-containing pharmaceuticals and their dehalogenated isosteres is described. Of the 132 column and mobile phase combinations examined for each mixture, a small subset of conditions were found to afford the best overall performance, with a single UHPLC method (2.1 × 50 mm, 1.9 μm Hypersil Gold PFP, acetonitrile/methanol based aqueous eluents containing either phosphoric or perchloric acid with 150 mM sodium perchlorate) affording excellent separation for all samples. Similarly, a survey of several families of closely related halogen-containing small molecules representing the diversity of impurities that can sometimes be found in purchased starting materials for synthesis revealed chiral SFC (Chiralcel OJ-3 and Chiralpak IB, isopropanol or ethanol with 25 mM isobutylamine/carbon dioxide) as well as the UHPLC (2.1 × 50 mm, 1.8 μm ZORBAX RRHD Eclipse Plus C18 and the Gold PFP, acetonitrile/methanol based aqueous eluents containing phosphoric acid) as preferred methods. [ABSTRACT FROM AUTHOR]