Dwight R. Stoll

What leads to an asymmetric peak shape? Physiochemical phenomena can help chromatographers identify whether the cause of asymmetry has a chemical or physical origin.

Understanding when the effect is likely to be large enough to affect resolution is valuable for troubleshooting unexpected results that arise during both method development and the execution of established methods.

As the field moves toward routine use of pressures well above 400 bar, the effect of pressure on retention should not be overlooked.

Several physical phenomena can lead to asymmetric peak shapes, including heterogeneity of the particle density inside the column, rearrangement of the particles over time, and accumulation of debris at the column inlet frit. This month’s instalment will focus on these potential physical causes.

In a continuing series on peak shapes, we focus on potential physical causes of asymmetry, including column packing, changes in the packed particle bed, and accumulation of debris in the column.

Tailing peaks—the most common type of asymmetric peak shape—can negatively affect both the qualitative and quantitative performance of LC methods. This instalment will discuss basic concepts in peak shape, and the potential for poor fluidic connections to cause peak tailing in a separation where the peak shape would otherwise be excellent.

In this first installment in a series on the causes of peak asymmetry, we discuss basic concepts in peak shape, explore poor fluidic connections as a common cause of peak tailing, and explain what to do about it.

In recent articles, the authors reviewed the basic concepts of extracolumn dispersion and how this phenomenon can impact the quality of an LC separation. We now specifically discuss the effects of dispersion that can occur due to tubing and detectors.

In the final article of this series on extracolumn dispersion, we look at elution mode, post-column flow splitting, and a free calculator to use during method development.

Dispersion of analyte peaks outside of chromatography columns can seriously erode the resolution provided by good columns. Here, we focus on the contribution of the sample injection step to the total level of extracolumn dispersion in an LC system.

In recent articles, we reviewed the basic concepts of extracolumn dispersion and how this phenomenon can impact the quality of an LC separation. We now specifically discuss the effects of dispersion that can occur due to tubing and detectors.

This instalment focuses on basic concepts in extracolumn dispersion (ECD) that occurs in high performance liquid chromatography (HPLC) systems, and the impact of this dispersion on the performance of columns of different dimensions and efficiencies.

Dispersion of analyte peaks outside of chromatography columns can seriously erode the resolution provided by good columns. Here, we focus on the contribution of the sample injection step to the total level of extracolumn dispersion in an LC system.

Dispersion of analyte zones outside of the column often compromises the quality of an LC separation—particularly in smaller columns with smaller particles. We explain basic concepts in extra-column dispersion from the point of view of an entire instrument.

How do the characteristics of the mobile phase waves and retention properties of an analyte of interest impact retention precision?

Charged aerosol detection (CAD) is a powerful complement to ultraviolet (UV) absorbance and mass spectrometric (MS) detection for liquid chromatography (LC), particularly for analytes that have no UV chromophore, or do not ionize well by electrospray ionization. This article explores how to successfully use this technique.

Liquid chromatography (LC) pumps produce mobile-phase streams with short-term variations in mobile-phase composition. We explain the impact of these waves on retention time in reversed-phase LC and what to do about it.

A review of the operating principles of modern liquid chromatography (LC) pumps based on low- and high-pressure mixing designs, and a look at how these pumps produce mobile phase streams with small short-term variations in mobile phase composition, with a focus on the effect of these mobile-phase composition “waves” on detector baselines.

Liquid chromatography (LC) pumps produce mobile-phase streams with small short-term variations in mobile phase composition. We explain the origin of these variations and their effects on chromatographic performance.

Sometimes our approach to troubleshooting specific problems has to change in response to changes in high performance liquid chromatography (HPLC) technology over time. In this installment, we discuss changes in technologies for mobile-phase degassing, silica-based stationary phases, and models for reversed-phase selectivity.

Sometimes our approach to troubleshooting specific problems has to change in response to changes in high performance liquid chromatography (HPLC) technology over time. In this installment, we discuss changes in technologies for mobile-phase degassing, silica-based stationary phases, and models for reversed-phase selectivity.

Charged aerosol detection is a powerful complement to UV and MS, but successful implementation requires understanding a few key factors, including response dependencies on temperature, nebulization process, analyte volatility, and mobile-phase composition.

A common problem encountered in the development of 2D-LC methods is that the first dimension mobile phase properties can negatively affect the quality of subsequent second dimension separations. This installment reviews the origin of this problem and discusses potential solutions.

In 2D-LC, properties of the mobile phase used in first step can negatively affect the second step. We explain why this problem happens and how to avoid it.

When we are focused on resolving a particular problem, it can be easy to lose sight of important steps in troubleshooting problems with liquid chromatography (LC) instruments. Taking a systematic and disciplined approach to troubleshooting can improve both the efficiency and effectiveness of our troubleshooting efforts.

When we are focused on resolving a particular problem, it can be easy to lose sight of important steps in troubleshooting problems with liquid chromatography (LC) instruments. Taking a systematic and disciplined approach to troubleshooting can improve both the efficiency and effectiveness of our troubleshooting efforts.

A critical part of troubleshooting is understanding how the system should behave so that irregular behavior can be spotted. The more rules we know, the easier troubleshooting becomes. Learning these six rules is a great place to start.

A handful of approximate rules about the behaviour of reversed-phase liquid chromatography (LC) can facilitate more efficient work, both during method development and in troubleshooting problems that arise with LC systems.

If you are analyzing metal-sensitive biomolecules, and a bioinert instrument is unavailable, or insufficient, passivation or mobile-phase additives may help. Here’s how to use those solutions, with tips for avoiding potential pitfalls.

Bioinert and biocompatible liquid chromatography (LC) systems are becoming more commonplace in laboratories, but the majority of biomolecule separations still use LC systems composed primarily of stainless steel parts. Can passivation or mobile phase additives improve separations on these systems for metal-sensitive biomolecules?