Columns | Column: LC Troubleshooting

Liquid chromatography (LC) is an incredibly powerful analytical tool that can be used to characterize materials across an astonishingly wide array of applications, ranging from 1 megaDalton polystyrenes to small genotoxic impurities of concern in pharmaceutical manufacturing. However, with this power also comes with a lot of detail related to successful operation of an LC instrument and analysis of the data it produces. This can be challenging when onboarding new LC users—how do we train someone new, where do we start, and which resources are best? In this installment, I will highlight ten high priority topics that every new user should be familiar with, no matter the training mechanism.

This article explores advanced oligonucleotide analysis techniques, including chromatography methods for therapeutic RNA, siRNA, and mRNA, enhancing nucleic acid research.

In this installment, we explore the key relationships between efficiency and mobile phase flow rate and particle size, as well as secondary determinants of efficiency, including analyte retention, size, and chemistry, and instrumental effects.

In the latest edition of "LC Troubleshooting", Dwight Stoll explores troubleshooting tips for liquid chromatography, particularly focusing on peak width challenges in large molecule separations and effective solutions.

In this installment of “LC Troubleshooting,” Dwight Stoll touches on highlights from Pittcon 2025 talks, as well as troubleshooting advice distilled from a lifetime of work in separation science by LCGC Award winner Christopher Pohl.

In this installment, I primarily discuss considerations to keep in mind when choosing buffering additives that will be used for LC methods involving UV absorbance detection.

In this installment of "LC Troubleshooting," Dwight Stoll discusses several essential principles related to when and why buffers are important, as well as practical factors, such as commonly used buffering agents, that are recommended for use with different types of detectors.

It has been more than five years since the last update in this column on the evolution of the Hydrophobic Subtraction Model (HSM) of reversed-phase selectivity and characteristics of new stationary phases recently characterized using the model. In this installment, Dwight Stoll discusses the continuing evolution of the model.

In this installment of “LC Troubleshooting,” we describe an artifact that can arise because of compound degradation during the transfer of fractions of the first dimension (1D) column effluent to the 2D separation.

In this installment of “LC Troubleshooting,” we discuss strategies that can be used to minimize the likelihood of compound degradation in the isolation process.

In this month’s column, I highlight some of the primary considerations we face in method development and point to resources that can help users overcome uncertainty and develop highly effective 2D-LC methods.

In this installment, we apply the concepts developed in last month’s installment by demonstrating how they can be used to help troubleshoot problems in LC involving pressure and flow.

The analogy that electrons flowing in wires is like water flowing through a tube can be remarkably effective. In this installment, the basics of that analogy are discussed.

The concept of gradient delay volume (GDV) in liquid chromatography (LC) poses challenges for both beginners and experienced practitioners. The GDV, which affects the arrival time of mobile phase composition changes at the column inlet, can have a significant impact on method throughput, influencing the time required for mobile phase changes at both the beginning and end of the LC method. Different pump designs and column characteristics affect efficient use of the available analysis time, as well as overall throughput. Notably, achieving repeatable equilibration, rather than full equilibration of LC columns following mobile phase gradients, is often sufficient for many LC applications, which can also be leveraged to increase method throughput.

In this installment, I illustrate the impact of different gradient delay volumes when transferring a method between instruments and discuss some strategies that can be used to mitigate these challenges.

The gradient delay volume is arguably one of the most important, yet least appreciated, parameters that affect how gradient elution separations in LC work. This has implications both for method development and for method transfer during the lifecycle of a LC method. In this installment, I will review the concept of gradient delay volume, its physical connection to the LC instrument, and how it can impact method development and separation quality.

There are three important habits and practices that can improve the effectiveness and efficiency of any troubleshooter.

Troubleshooting continues to evolve constantly, changing how we overcome issues such as chemical causes of peak tailing.

Mobile phase degassing has become very useful for troubleshooting problems with pumps and detection.

The author outlines multiple protective and troubleshooting methods for liquid chromatography and how these approaches have changed over time.

We present two cases where understanding the specific interactions between particular analyte functional groups and LC instruments and columns is critical for successful separations.

Several factors strongly influence separation speed, including the pressure available to drive the separation, column temperature, particle size, and column length. Developing efficient methods to improve speed while not sacrificing accuracy require an understanding of the abovementioned variables and analysis time.

Scouting gradients can simplify LC method development. Here’s what you need to get started using them.

The variables that are most important for improving the separation of complex samples are quite different from those for simpler samples. Considering these differences can save time and resources.

When can analyte retention deviate from what is expected or normal? We explain three subtle causes.

Even relatively simple mixtures are not always easy to separate. What are the options for adjusting selectivity in reversed-phase LC separations?

When considering different ways to improve an established LC method, it helps to start by reviewing some essential concepts.

Estimating expected peak widths helps us determine when a column and LC system is not performing optimally.

Knowing the tips and tricks of producing quality LC–MS data for peptide analysis can help streamline troubleshooting when problems occur.

There are various physical and chemical causes of low detection sensitivity. Here, we address some of these causes, and how to troubleshoot them.