Liquid Chromatography (LC/HPLC)

We’ll see how to find the “sweet spot” in terms of retention for a liquid chromatographic separation as well as how much retention change is to be expected for a selected change in mobile-phase percent organic or column temperature.

This installment describes HPLC and related products introduced at Pittcon 2017 Chicago and in the year prior. It highlights new HPLC systems, modules, and software, with innovative features and significant benefits to the users.

When considering column efficiency, more is not always better. We look at some ways to quickly estimate the effects of changes in column length and particle diameter rather than trying the experiments in the laboratory.

Rudolf Krska, from the University of Natural Resources and Life Sciences in Vienna, Austria, talks about the latest analytical techniques and challenges facing analysts involved in the evolving field of mycotoxin analysis.

Delivering samples to the analytical column in ‘clean’ mobile phase is important for robust methods and high quality results.

The carotenoid test allows one to build a simple classification map of stationary phases used in reversed-phase liquid chromatography, on the basis of the shape recognition(plotted on the x axis) the polar surface activity(plotted on the y axis) and the phase hydrophobicity (related by the bubble size).

There is increasing demand to analyze samples with a wide range of polarities, in fields such as environmental analysis and proteomics.

We explore the careers and achievements of the winners of LCGC’s 10th annual awards: Pat Sandra and Deirdre Cabooter.

A reader’s problem of a method that fails the repeatability of the system suitability test serves as an example of how to approach LC method troubleshooting.

Part II of this series describes additional features of the HPLC Teaching Assistant software, including the possibility to simulate the impact of the mobile phase temperature on HPLC separations; understand the chromatographic behavior of a mixture of diverse compounds in both isocratic and gradient elution modes; show the influence of instrumentation (injected volume and tubing geometry) on the kinetic performance and sensitivity in HPLC; and demonstrate the impact of analyte molecular weight on thermodynamic (retention and selectivity) and kinetic (efficiency) performance.

What could be causing a peak to be eluted before the column dead time? In last month’s “LC Troubleshooting” (1) we looked at problems two readers had with ghost peaks in gradient runs. This month, we’ll continue looking at submitted questions and examine one submitted by another reader of this column.

Here we propose an exemplary workflow for the analysis of phenolic extracts (i.e. wine) enabling confident differential analysis using high performance liquid chromatography in combination with low-field drift tube ion mobility quadrupole time-of-flight mass spectrometry (HPLC×IMS-QTOFMS). In this workflow, single-field collisional cross section values from low-field drift-tube IMS using nitrogen as drift gas (DTCCSN2) are readily extracted in addition to a retention time and a high resolution mass spectrum for each compound. “Alternating frames” experiments utilizing post-drift tube fragmentation also allow drift time-aligned MS/MS spectra to be obtained. Molecular feature extraction was highly repeatable with average precision values of 0.28% for retention time, 0.18% for drift time, and 1.5 ppm m/z determined for 233 molecular features found in all six technical replicates. The improved selectivity of this strategy increases confidence in intersample molecular feature alignment (i.e. compound identity confirmation), including the resolution of co-eluting isomeric compounds.

Interfering peaks or high baseline background can compromise the results of gradient LC separations.

This article describes the development of a HPLC method for the assay of green fluorescent protein (GFPuv) in-process samples from our model therapeutic protein production process. Specificity of the method is evaluated by demonstrating a suitable HPLC method to separate and detect closely related protein degradation species.

Cartenoid compounds can be used as probes for studying the stationary bonded phases devoted for reversed-phase liquid chromatography, that is, C18, phenyl-hexyl, and cholester. From one analysis achieved in supercritical fluid chromatography (SFC) that favors the chromatographic behaviors due to the stationary phase properties, bonding density, ligand type (monomeric or polymeric), and endcapping treatment, two separation factors are calculated allowing us to build a bi-dimentional map. These two axes are related either to the shape selectivity or the polar surface activity (residual silalnos). Each point on the map corresponds to a column. The retention factor of beta-carotene, which describes the phase hydrophobicity, is indicated by the size of the point. More than 200 stationary phases were studied, including small particle sizes and superficially porous ones. Moreover, the results are now available on a website, allowing you to check and compare, by selecting the required tabs, columns, manufacturer brands, and ligand nature.

Fluorescence detection can be a strong alternative to ultraviolet or other detectors for some compounds.

Reversed-phase liquid chromatographic columns can be compared quantitatively for differences in selectivity by means of the hydrophobic-subtraction model. This allows selection of columns that are either equivalent or different in selectivity. The present paper both presents a summary of this approach and shows in detail how to use it in practice.

A practical step-by-step guide to setting up an HPLC instrument with hints and tips to avoid common pitfalls.

Detectors based on ultraviolet absorbance are the most common detectors in use for liquid chromatography.

There can be significant benefits by standardizing HPLC columns in a pharmaceutical development laboratory. Here is a story of how one organization attempted to encourage its staff to develop HPLC methods using fewer column brands and dimensions to reduce waste and efforts in method transfers downstream.

A reliable autosampler is one key requirement for unattended operation of a liquid chromatograph.

A simple, rapid, and robust ultrahigh-performance liquid chromatography (UHPLC) method has been developed for the simultaneous determination of natural and artificial vanilla flavouring substances as well as some precursors has been developed using an automated method scouting or method optimization workflow. The most suitable mobile phase and stationary phase combination was identified in a scouting run. These conditions were used to create a two-dimensional model in computer simulation software. Temperature and gradient time were varied to establish the optimum fast and robust separation conditions. This approach resulted in a 5.5 min gradient method that allowed for fast screening of 11 compounds of interest.

Put yourself in their spot: How would you tackle analyzing a bag of gummy bears that showed up on your lab bench? Here, we offer some insights from the very capable finalists at The Conference on Small Molecule Science (CoSMoS), which was held in August 2015 in San Diego, California.

Structural, bioanalytical, characterization, and quality control studies are critical for successful drug development. These studies must be as accurate, sensitive, and selective as possible, and liquid chromatography coupled to tandem mass spectrometry (LC–MS–MS) has been the technique of choice for many areas of small molecule analysis for the past 30 years. During that time, rapid improvements in analytical technologies have supported the development of more sensitive and robust methods. However, the pharma and biopharma industry continues to need more powerful instruments and more diverse methods, particularly as therapeutics have expanded to include large molecules. This work follows on from an earlier article that explored the limitations of LC–MS–MS for bioanalysis of biologics. This article considers some of the current issues for analysis of small and large molecules, and emerging trends in method development.

Appropriate analytical methods are required to evaluate the presence, metabolism, degradation, and removal of specific compounds in complex mixtures. There is an increasing demand to analyze samples with a wide range of polarities in a variety of applications, including environmental analysis, biomarker discovery, and proteomics. Multiple analyses on complementary columns are often needed to cover the separation of all compounds with a large difference in polarity. This article describes a generic method involving an ultrahigh-pressure liquid chromatography (UHPLC) system equipped with two external switching valves to connect hydrophilic interaction liquid chromatography (HILIC) and reversed-phase LC columns in series for the sequential analysis of polar and apolar compounds. The method was successfully applied to separate 32 pharmaceutical compounds with a wide range of polarities, which could be useful for analyzing pharmaceutical compounds in the environment.