Dwight R. Stoll

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.

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.

The high levels of non-volatile salts typically used in ion-exchange separations are incompatible with mass spectrometry. But the desire to directly couple these two powerful techniques has prompted researchers find solutions to this problem. Here are three approaches.

Direct coupling of ion-exchange separations to mass spectrometric (MS) detection is increasingly being used for analyses of molecules ranging from organic acids to proteins. These approaches leverage both the exquisite selectivity of the ion-exchange mode for charge-based separation, and the tremendous power of mass spectrometry for identification of unknowns and trace-level quantitation.

For liquid chromatography (LC) methods where the buffer pH and composition have an influence on retention, which buffer preparation method will provide the most repeatable results?

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

When can we use completely aqueous eluents with reversed-phase stationary phases, and what happens if we make a mistake?

Adding an inline mixer between the sample injector and column in a liquid chromatography (LC) system can be an effective way to resolve problems with peak shape caused by the sample diluent.

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.

Sometimes, the conditions specified in legacy liquid chromatography (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 of.

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

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?