HPLC

In this instalment of the LCGC Blog, Tony Taylor discusses noisy baselines in high performance liquid chromatography (HPLC).

Many analysts have strongly held beliefs about buffer preparation methods, but these positions are not always supported by experimental evidence.

The effect of dwell volume on chromatographic selectivity can be successfully modelled using retention prediction software. Hence, the robustness of reversed-phase LC gradient methodologies, with respect to dwell volume, can be conveniently assessed.

Ultrahigh efficiency separations based on the presence of one deuterium in benzene, toluene, and naphthalene were achieved by recycle chromatography using C18 silica columns. The discrimination mechanism of H/D isotopic species is discussed based on the dispersion interactions of a CH/CD group of the solute with the stationary phase as well as the mobile phase.

The impact of ionic strength, buffer capacity, and pH-response on the retention behavior and peak shape of mAb species characterization is evaluated for IEX-MS. The aim of the present study was to understand the impact of ionic strength, buffer capacity, and pH-response on the retention behavior and peak shape of mAb species.

An in-line mixer between the sample injector and column may resolve problems with peak shape caused during sample dilution.

Legacy LC methods seem like they come from a different planet. This month, we look at which conditions to keep, and which ones to let go.

Ultrahigh-efficiency separations based on the presence of one deuterium in benzene, toluene, and naphthalene were achieved by recycle chromatography using C18 silica columns. Larger isotopic separation factors, α(H/D), were observed in methanol–water than in acetonitrile–water, when the mobile phases provided similar retention factors (k), or similar methylene selectivity, α(CH2). Isotopic resolutions between nondeuterated and perdeuterated aromatic hydrocarbons at long separation times were estimated by using the plate counts obtainable by recycle operation as a function of a cycle time, along with the retention factors and the separation factors experimentally observed.

This article gives a brief overview of the advantages and limitations of recently introduced mathematical procedures such as the Fourier deconvolution of extracolumn effects, iterative curve fitting, multivariate curve resolution, modified power law, and use of first and second derivatives in enhancing resolution. High-throughput analyses in gas chromatography (GC), LC, and supercritical fluid chromatography (SFC) could benefit from these simple and effective approaches in many challenging separations applications.

The design of a user-friendly vacuum-jacketed column (VJC) is described for improved LC–MS performance, which does not require a large vacuum chamber with multiple vacuum pumps. Using this configuration, the experimental peak capacities measured for a 2.1 mm × 100 mm column packed with sub-2-μm particles and placed in the VJC-MS probe are doubled with respect to standard LC–MS systems.

Re-equilibrating a reversed-phase stationary phase following aqueous gradient elution can be achieved much faster than you think.

Our annual review of new liquid chromatography (LC) columns and accessories, introduced at Pittcon and other events.

How long does it take to re-equilibrate reversed-phase stationary phases following gradient elution, especially when starting with a highly aqueous eluent?

How long does it take to re-equilibrate reversed-phase stationary phases following gradient elution, especially when starting with a highly aqueous eluent?

How many new columns truly offer new selectivity?

In reversed-phase separations, retention generally increases as the fraction of water in the eluent increases. When we encounter situations where retention is too low for an analyte of interest, we tend to use eluents with higher and higher levels of water. But how much water is too much?

The challenges we face in troubleshooting problems with liquid chromatography (LC) separations are highly diverse. This month we take a closer look at topics that have garnered more attention recently.

Is an ultraviolet (UV) detector signal good for anything if the analyst is using a mass spectrometer?

Within the broad scope of analytical techniques required to characterize a protein, chromatographic methods have shifted towards high-flow analyses that can drop development time significantly. However, fast analytical methods for charge heterogeneity have lagged in development because current column technologies are ultrahigh-pressure liquid chromatography (UHPLC)-incompatible. This article will demonstrate the development of a high-flow method for charge variant analysis made possible through a bioinert titanium column flow path.

The use of ultrahigh-pressure liquid chromatography (UHPLC) is now commonplace among pharmaceutical laboratories. However, until depreciation cycles replace traditional high performance liquid chromatography (HPLC) systems that operate at a maximum pressure of 400 bar, the advantages of UHPLC cannot be realized worldwide. Thus, product methods developed using UHPLC capabilities cannot directly transfer these methods to receiving laboratories without qualified UHPLC availability.

The drive behind multipath liquid chromatography (LC), a new concept developed by Kevin Schug and his group, is presented.

These ten propositions are widely acknowledged, but frequently neglected, by practitioners of high performance liquid chromatography (HPLC).

Two-dimensional liquid chromatography (2D-LC) is drawn out of the chromatographic toolbox if resolution for compounds of interest is insufficient. Recently, several studies have started to highlight 2D-LC as a tool of choice to streamline analytical workflows to increase automation making them less time-consuming. This article highlights two proven cases where 2D-LC does more than simply increase peak capacity.

Many workers in pharmaceutical laboratories are unable to change any aspect of their methods, although they often encounter severe problems and create many out-of-specification (OoS) results. They are particularly afraid to investigate these problems from a chromatographic perspective in case they generate new unforeseen problems. In the literature, however, there are numerous examples showing that it is worthwhile trying to understand the reasons for “unexplainable” behaviour in ultrahigh-pressure liquid chromatography (UHPLC) using modelling. By using modelling, problems can be recognized and often eliminated with legal operations according to the allowed tolerance limits mentioned in pharmacopoeia descriptions. The following article aims to show that “visual chromatographic modelling” can be a useful aid.

Comprehensive two-dimensional liquid chromatography (2D-LC) was used for detailed profiling of various nonionic ethoxylated surfactants applied in pharmaceutical formulations. Hydrophilic-interaction chromatography (HILIC) and reversed-phase liquid chromatography (LC) were used as the first and second dimensions, respectively. Detection was performed with evaporative light-scattering detection (ELSD) for general profiling and with single-quadrupole mass spectrometry (MS) for structure elucidation of individual peaks and for class-type confirmation of peak-groups.