Dwight R. Stoll

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?

The offerings of commercially available columns for reversed-phase liquid chromatography (LC) continue to expand. Are these columns similar or different compared to what is already available?

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.

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?

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

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?

Several new materials and columns have been introduced in recent years for size-exclusion separations of proteins. How do I know which one to choose, and which separation conditions will be the best for my protein separation?

Is a mixer needed between the injector and column in high performance liquid chromatography (HPLC)?

What happens when we inject a sample into the mobile-phase stream? Many LC practitioners are surprised to learn just how serious the effect of the injected sample solvent can be.

Many LC users are unclear what happens when we combine two (or more) flow streams in LC systems, and when mixers are needed to blend the fluids. This discussion explains why mixers are needed, and when and how you might consider using something other than the default mixer setting.

What types of mixers are needed with different types of liquid chromatography (LC) pumps? Are different mixers needed for different applications?

Much of the conventional wisdom regarding size-phase separations of proteins has been negated thanks to development of superior chemistries and advances in research. In this article, details that the authors have found to be especially beneficial in achieving effective SEC separations are examined.

Several new materials and columns have been introduced in recent years for size-exclusion separations of proteins. How do I know which one to choose, and which separation conditions will be the best for my protein separation?

Several new materials and columns have been introduced in recent years for reversed-phase separations of proteins. How do I know which one to choose, and which separation conditions will be best for my protein separation?