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The analogy that electrons flowing in wires is like water flowing through a tube can be remarkably effective for teaching and learning about fluid flow in LC systems. 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. We also introduce several free tools that can be used to calculate pressure drops in different elements of LC systems, including connecting capillaries and packed columns. Knowing what pressure drops to expect for system components under different chromatographic conditions is very valuable in many troubleshooting situations., IN LAST MONTH'S INSTALLMENT of 'LC Troubleshooting' we discussed the conceptual similarities between the flow of current in electrical systems and the flow of fluids in LC systems, We also [...]

The analogy that electrons flowing in wires is like water flowing through a tube can be remarkably effective for teaching and learning about fluid flow in liquid chromatography (LC) systems. In this installment, we will go over the basics of this analogy, as well as explicitly discuss the phenomena observed in the two cases and the physical laws that we can use to rationalize and predict the behavior that we see. In the second installment in this series, we will then use this knowledge to demonstrate how these ideas can be used to troubleshoot problems when they arise in LC systems., IN MY INTERACTIONS with liquid chromatography (LC) users from various backgrounds and levels of training and experience, I observe that perceptions about how flow and pressure 'work' in LC systems [...]

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 MY INTERACTIONS with people learning about various aspects of liquid chromatography (LC), I find that the concept of gradient delay volume (GDV) is one of the most difficult ideas [...]

The gradient delay volume is one of the most important, yet least understood, parameters that affect how gradient elution separations in liquid chromatography (LC) work. This parameter has implications for method development and method transfer during the lifecycle of an LC method. 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., IN MY INTERACTIONS with people learning about various aspects of liquid chromatography (LC), I find that the concept of the 'gradient delay volume' (GDV) is one of the most difficult [...]

The gradient delay volume is arguably one of the most important, yet least appreciated, parameters affecting how gradient elution separations in LC work. This has implications both for method development and for method transfer during the life cycle 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., IN MY INTERACTIONS with people learning about various aspects of LC, I find that the concept of 'gradient delay volume' (GDV) is one of the most difficult ideas to grasp [...]

Two-dimensional liquid chromatography (2D-LC) is a technique that extends the separation capabilities of conventional liquid chromatography by adding a second separation step to resolve compounds that are coeluted from a first column. This approach holds tremendous potential to solve difficult separation challenges in fields ranging from pharmaceutical analysis to biofuel characterization. Currently, method development is a significant bottleneck impeding more widespread implementation of 2D-LC methods, particularly for new users who are uncertain about how to proceed. 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., TWO-DIMENSIONAL LIQUID CHROMATOGRAPHY (2D-LC) holds tremendous potential to impact many areas of science that rely on liquid chromatography (LC) to move those fields forward. For example, 2D-LC can be used [...]

Similar to Stephen Covey's 'Seven Habits of Highly Effective People,' effective troubleshooters take a systematic approach to liquid chromatography (LC) troubleshooting and rely on several key principles to help them navigate the complex task of troubleshooting. In this installment I add to this list of principles described in prior installments with three specific ideas: 1) Ask--does it make sense?; 2) Get a second opinion; and 3) Join a chromatography discussion group to help grow your chromatography network., As 2023 comes to a close, I've been reflecting a bit on the problems with instrumentation that I've encountered this year, both through messages from folks in my professional network [...]

As chromatography technologies change, our approaches to using them in ways that value reliability, as well as the ways we should approach troubleshooting and fixing problems, should also change. In [...]

As chromatography technologies change, our approaches to using them in ways that value reliability, as well as the ways we should approach troubleshooting and fixing problems, should also change. In this installment, I continue the discussion started last month about how some specific troubleshooting topics have evolved over time, this time focusing on mobile phase degassing. Understanding how approaches to degassing have evolved is helpful for troubleshooting problems with pumps and detection, designing reliability into new methods, and when considering updates to legacy methods that have been in use for decades., In recent months, I've been hearing from several readers of this column that they've appreciated the more educational, 'back-to-basics' flavor of some of the 'LC Troubleshooting' installments in the last [...]

In recent months, I've been hearing from several readers of this column that they've appreciated the more educational, 'back-to-basics' flavor of some of the 'LC Troubleshooting' installments this past year. [...]

Many high-performance liquid chromatography (HPLC) users are confronted with questions about how to improve upon the performance of an existing or recently developed method. These days, we have many technological options to consider, but how do we choose one (or a few) to try? The important variables that are most important for improving the speed of a separation are the maximum pressure available to drive the separation, and column temperature. Understanding the relationship between these variables and analysis time, and their effects on other choices made during method development, is helpful for developing methods that are both effective and time efficient., In the January, 2023 installment of 'LC Troubleshooting,' I kicked off a series of articles aimed at addressing the general question, 'How do I improve my separation?' Given the diversity [...]

With so many options for method parameters to adjust during method development, identifying a starting point can be intimidating. Starting with scouting gradients can simplify the process, and yield rich results that inform subsequent method development steps. In this installment, we discuss how to design scouting gradients, how to use the resulting data to make decisions about next steps (including whether to use isocratic or gradient elution), and how to improve the separation once an elution mode has been chosen., One of the characteristics of the development process for chromatographic methods that I think keeps many chromatographers so engaged in the work is that there are many 'knobs to turn.' [...]

Many high performance liquid chromatography (HPLC) users are confronted with questions about how to improve upon the performance of an existing or recently developed method. These days, we have many [...]

Some liquid chromatography (LC) troubleshooting topics never get old, because there are some problems that persist in the practice of LC, even as instrument technology improves over time. There are [...]

When high performance liquid chromatography (HPLC) users are confronted with questions about how to improve upon the performance of an existing or recently developed method, there are many technological options [...]

In many applications of liquid chromatography (LC), excellent peak shapes are observed with simple, easy-to-use mobile phases and operating conditions. However, some specific combinations of analyte and LC column chemistry can lead to terrible peak shapes and even total adsorption of analytes to components of the LC system such that no peaks are observed at all. In this installment of 'LC Troubleshooting', I discuss two specific cases where an understanding of specific interactions between particular analyte functional groups and LC instruments or column materials is critical to achieving successful separations., For many applications, liquid chromatography (LC) is a fantastically successful analytical technique. I often share with my students that LC instruments are found in so many laboratories because LC can [...]

In this latest installment of our series on recurring challenges in liquid chromatography (LC), we turn our attention to peak widths that are wider than expected. Estimating the expected peak widths can help determine when a column and LC system are not working quite right, and to decide when troubleshooting should be initiated., Writing this 'LC Troubleshooting' column and thinking about topics each month is interesting in the sense that there are some topics that just never get old. Whereas in the chromatography [...]

The analysis of peptides using liquid chromatography--mass spectrometry (LC--MS) for proteomics applications is powerful and can yield tremendously rich data sets. However, the exquisite sensitivity and global applicability of MS detection also makes it prone to contaminants that can easily and seriously compromise the quality of a data set. A short list of tips and tricks can increase the likelihood of consistently producing good LC--MS data in this context and streamline the troubleshooting process when problems do eventually occur., Some applications of liquid chromatography (LC) involve highly specific 'tips and tricks' that are acquired by analysts over time, and they can make the difference between producing data that is [...]

In reversed-phase liquid chromatography (RPLC), mobile-phase additives are used for pH control because mobile-phase pH can strongly affect retention, peak shape, and method robustness for ionizable compounds. For instance, an [...]

Some liquid chromatography (LC) troubleshooting topics never get old because there are some problems that persist in the practice of LC, even as instrument technology improves over time. There are many ways for things to go wrong in an LC system that ultimately manifest as detector baselines that do not look right. Developing a short list of the likely causes of these results can help streamline our troubleshooting experience when baseline-related problems occur., Writing this 'LC Troubleshooting' column and thinking about topics each month is interesting in the sense that there are some topics that just never get old. Whereas in the chromatography [...]

Verifying system performance is important when using chromatographic instrumentation for analyzing both known and unknown samples. Typically, system suitability tests (SSTs) are used to verify performance, which involves running an established method and comparing the results to pre-established acceptance criteria. As two-dimensional liquid chromatography (2D-LC) becomes used more widely and in regulated laboratory environments, development and implementation of SSTs will be critical for successful routine use of the 2D-LC technique., Although I have not written in the past about system suitability tests (SSTs ) for the 'LC Troubleshooting' column, they were mentioned several times in the past by John Dolan, [...]