Dwight R. Stoll

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?

Taking a systematic approach to restarting liquid chromatography instrumentation following the COVID-19 shutdowns will save money and time in the long run.

COVID-19-related laboratory shutdowns are sure to cause a myriad of problems with liquid chromatography (LC) instrumentation across the globe. Taking a systematic approach to restarting these systems will save money and time in the long run by preventing problems that may otherwise appear in days or weeks following startup.

When you restart liquid chromatography (LC) instrumentation that was idle during the COVID-19 shutdown, you need to follow a systematic approach. Otherwise, problems may appear in days or weeks following startup.

There are still many methods in use that have been developed for use at “room temperature.” With such a method, can one reasonably expect to obtain the same separation in Anchorage, Alaska, as in Mumbai, India?

The hydrophobic subtraction model has been very successful. Nevertheless, the accompanying public database, which has parameters for 750 commercially available columns, is an underutilized column characterization tool. Here is some guidance on how to use both the model and the free database.

How do I know when bioinert liquid chromatography columns, systems, or components are needed for my separations of biomolecules?

In situ measurements of the mobile-phase pH before and after the column help to rationalize the effects of mismatch in pH and concentration between the mobile phase and sample buffer mismatch in reversed-phase LC separations.

If I inject a sample buffered at a pH different from that of the mobile phase, how quickly is the sample buffer neutralized inside the column?

We return to the important topic of buffers, this time focusing on what happens when there is a mismatch between the mobile-phase buffer pH and the pH that the sample is buffered at.

What concentration of aqueous buffer should I use in the mobile phase when developing a re-versed‑phase liquid chromatography (LC) method for the analysis of ionogenic compounds?

If I increase the flow rate of my separation when using UV absorbance detection, should I expect peak area to change?

A deeper theoretical understanding the relationship between peak area and flow rate will help analysts diagnose problems when using UV absorbance detection.

The sample solvent can have a big impact on peak shape in both reversed-phase and hydrophilic interaction liquid chromatography (HILIC) separations, especially when large volumes are injected. Diluting the sample with weak solvent can be an effective solution to mitigate this problem, but we have to be careful to not lose analytes of interest to precipitation or phase separation.

For decades the prevailing perception was that satisfactory re-equilibration of reversed-phase columns following gradient elution took a long time. In the early 2000s we showed that this perception was not well founded, and demonstrated that adequate re‑equilibration could be achieved in seconds. Recently, we have shown the same for HILIC columns. All of this work so far has been with small molecules. In this article, we present an overview of this work, and summarize the practical utility of it all.

Many users have questions about how long LC columns should be re-equilibrated following solvent gradient elution separations. Here we show satisfactory results with short re-equilibration periods, for both reversed-phase and HILIC separations.

Retention of peptides is strongly dependent on solvent composition in reversed-phase separations with gradient elution. In this instalment we provide tips, tricks, and suggestions for best practices to help minimize retention time variations over time.

Carefully diluting a sample with weak solvent can mitigate the impact of sample solvent on peak shape in both reversed-phase and HILIC separations, but we need to understand how the choice of sample diluent can affect analyte recovery.