Anthony J. Alexander

Modifying conventional high-performance liquid chromatography systems to achieve fast separations with Fused-Core columns: A case study

The theoretical increase in performance from the use of high efficiency columns with conventional HPLC equipment is generally not observed due to the design limitations of such equipment, particularly with respect to extra-column dispersion (ECD). This study examines the impact of ECD from a Waters Alliance 2695 system on the performance of 2.7 μm HALO C(18) Fused-Core superficially porous particle columns of various dimensions. The Alliance system was re-configured in different ways to reduce extra-column volume (ECV) and the ECD determined in each case as a function of flow rate up to a maximum of 2 mL/min. The results obtained showed a progressive decrease in ECD as the ECV was reduced, irrespective of the flow rate employed. However, this decrease in ECD was less than theoretically expected for the lower ECV configurations. The inability to reduce the actual extra-column dispersion further was attributed to additional dispersion associated with the design/volume of the auto-injector. This was confirmed by making sample injections with a low dispersion manual injection valve, instead of auto-injection, for the two lowest ECV configurations studied. In each case, the measured and predicted ECD values were in good agreement. The auto-injector module is an integral part of the Alliance 2695 instrument and cannot be easily modified. However, even with autosampler injection, for a 3mm ID × 100 mm Fused-Core column approximately 70% of the maximum plate count (∼84% of the resolution or more) could still be obtained in isocratic separations for solutes with k ≥ ∼4.5 when using the lowest ECV configuration. This study also highlights some of the problems inherent in trying to measure accurately the true extra-column dispersion of a chromatographic system and compares the results obtained to those theoretically predicted. Using this same lowest volume instrument configuration, two real-world pharmaceutical methods were scaled to separations that are ∼3-3.5-fold faster, while still maintaining comparable data quality (resolution and signal-to-noise ratios).

Comparison of supercritical fluid chromatography and reverse phase liquid chromatography for the impurity profiling of the antiretroviral drugs lamivudine/BMS-986001/efavirenz in a combination tablet

Dual and triple combinations of antiretroviral drugs are a cornerstone of human immunodeficiency virus type 1 (HIV-1) treatment. Supercritical fluid chromatography (SFC) and reverse phase liquid chromatography (RPLC) methods have been developed for the impurity profiling of a prototype combination tablet containing three such drugs: lamivudine, BMS-986001 and efavirenz. Separation by SFC was achieved using a Princeton 2-ethyl pyridine stationary phase and a mobile phase B consisting of methanol with 10 mM ammonium acetate and 0.1% isopropyl amine. This combination of mobile phase additives was required for both the separation of minor components and to minimize peak tailing of the active pharmaceutical ingredients (APIs). Separation by RPLC was achieved using a Discovery HSF5 stationary phase and a mobile phase consisting of 10 mM ammonium acetate, pH 5.5 and methanol. Mobile phase gradient elution was employed in each case to elute components with a wide range of polarities. Both these methods were found to have advantages and disadvantages. Out of the three APIs and 13 possible impurity/degradation products selected, all were resolved by RPLC. By SFC, 15 peaks were resolved with one co-eluting pair and a high degree of orthogonality was achieved relative to RPLC. A more even distribution of peaks across the separation space, a non-sloping baseline and fewer system peaks were significant advantages associated with the SFC method. Particular attention had to be paid to optimizing the reverse phase diluent strength/initial mobile phase composition to avoid distortion of the peak shapes for early eluting components. This was not an issue with SFC, as the diluent of choice (methanol) was also the solvent of choice (in combination with ≤20% water) for the dissolution of the triple combination tablet. As with RPLC, SFC was found to exhibit the required sensitivity for successful quantitation of potential impurities/degradation products at the 0.05-0.1 area% level.

Investigation of polyethylene membranes as potential sample support for linking SDS-PAGE with MALDI-TOF MS for the mass measurement of proteins

Publisher Summary Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a classic technique used for the separation and molecular weight (MW) determination of biomolecules; however, the technique gives only a rough estimation of MW, and can be subject to systematic errors if the species under investigation has different electrophoretic migration behavior then the MW markers. While, matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) routinely gives MW values with greater accuracy, and has become increasingly popular for the mass measurement of biopolymers. This chapter discusses preliminary investigations into the application of a novel membrane material, a porous polyethylene (PE), for use as a surface to link SDS-PAGE with MALDI-TOF MS. The PE membrane provides access to higher molecular weights than the more common transfer membrane materials, which permits the mass analysis of the large proteins for which MALDI-TOF MS is ideally suited. Furthermore, the use of PE reduces the severe ion suppression effects typically observed in the MALDI analysis of high mass mixtures. This also permits more accurate mass measurements to be made via the use of internal calibration. While, it remains to be shown that proteins can be desorbed from PE membranes following the electro transfer of bands from SDS-PAGE gels, results to date are very encouraging.