HPLC

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.

This article describes a direct analysis of glyphosate, aminomethylphosphonic acid (AMPA), glufosinate, and 3-methylphosphinicopropionic acid (MPPA) in water by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) without derivatization. The chromatographic separation was performed using a hydrophilic interaction liquid chromatography (HILIC) column and typical LC–MS mobile phases. Method performance was evaluated, showing excellent results. The low limits of quantification (LLOQs) obtained meet the requirements of EU guidelines and could also be used to get an agreement in France where regulations require lower LLOQs (NOR: DEVL1703763V).

A snapshot of key trends and developments in the chromatography sector according to selected panellists from companies who exhibited at Analytica 2018.

The Column spoke to Arcadius V. Krivoshein, Assistant Professor of Chemistry at the University of Houston–Clear Lake, USA, about his work developing EPP, an experimental anticonvulsant that can help to stop convulsions during epileptic seizures, and the role of chiral HPLC in this research.

An overview and assessment of the latest HPLC and mass spectrometry instruments, modules, chromatography data systems, and related software.

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

This instalment describes high performance liquid chromatography (HPLC) and related products introduced at Pittcon 2018 in Orlando, Florida, USA, and in the year prior. It highlights new HPLC and mass spectrometry (MS) systems, modules, chromatography data systems, and other HPLC-related software and provides brief descriptions of their significant benefits and innovative features.

Chromatographic principles and best practices for obtaining highly precise retention time, peak width, and resolution predictions for the optimization of reversed-phase liquid chromatography (LC) separations using retention modelling software will be discussed. The importance of fully characterizing the LC instrumentation, how to generate accurate input data, the selection of appropriate models, and peak tracking will be addressed along with a suggested workflow. Adhesion to a few basic rules and simple precautions and the use of modern retention modelling software programmes can assist the rapid development of highly accurate retention models to enable the development of robust and optimized reversed-phase LC separations using either ultrahigh-pressure liquid chromatography (UHPLC) or high performance liquid chromatography (HPLC) conditions. Examples of retention modelling for small and large molecules will be highlighted.

Certain details related to mobile-phase preparation that can be assumed in reversed-phase LC separations can be much more consequential in HILIC.

The drug discovery process can be accelerated by chromatographic profiling of analogs by measuring their nonspecific binding to proteins and lipids and then by modelling in vivo distribution. A balanced potency and chromatographically determined membrane and protein binding ensure the selection of compounds with the highest probability to show the desired in vivo distribution behaviour for efficacy and reduced toxicity. The first part of the article will discuss the high performance liquid chromatography (HPLC)-based measurements of lipophilicity and biomimetic properties, while the second part will discuss the models derived from the measured data of known drug molecules and drug discovery compounds.

Characterization of protein modifications is an essential aspect of biopharmaceutical development. Traditionally, the characterization process of chromatographic peaks involves manual, larger-scale fractionation to obtain a sufficient amount of material for further analytical studies. This article presents a fully automated process for online peak fractionation and reduction of therapeutic antibodies with subsequent quadrupole time-of-flight mass spectrometry (QTOF-MS) characterization. This innovative technique significantly accelerates MS peak characterization compared to traditional approaches and avoids the risk of unintended modifications of the variants as a result of the isolation process, for example, deamidation during storage of isoforms. This approach considerably reduces the required sample amount and can be used for the characterization of product-related impurities during early stage development.