Columns | Column: LC Troubleshooting

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.

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.

When esterification occurs in your LC mobile phase, knowing how baseline quality, retention, and selectivity are affected will help you mitigate the effects.

Knowing the likely causes of baseline-related problems will help you solve them.

As two-dimensional liquid chromatography (2D-LC) becomes more widely used, system suitability tests (SSTs) become even more important.

We explain several of the more frequent causes of bad peak shapes in liquid chromatography and provide tips on how to remedy them.

Retention time fluctuations are challenging to deal with, especially in two-dimensional liquid chromatography (2D-LC) separations. Here, two approaches are presented that can help navigate this challenge.

Developing a short list of likely causes of retention-time problems in reversed-phase LC makes troubleshooting easier.

With kinetic plots, you can make better-informed column choices. Here’s how.

A kinetic plot is a powerful tool, but how do you construct one—from either experimental data or data from other sources? We explain.

Kinetic plots can help us understand how different combinations of parameters will perform in relation the time needed to acquire a particular column efficiency—and thus resolution.

Deviations from the expected pressure in modern LC systems (too low, too high, or fluctuating) can be diagnosed and more quickly resolved using the streamlined troubleshooting practices shown here.

What leads to an asymmetric peak shape? Physiochemical phenomena can help chromatographers identify whether the cause of asymmetry has a chemical or physical origin.

As the field moves toward routine use of pressures well above 400 bar, the effect of pressure on retention should not be overlooked.

In a continuing series on peak shapes, we focus on potential physical causes of asymmetry, including column packing, changes in the packed particle bed, and accumulation of debris in the column.

In this first installment in a series on the causes of peak asymmetry, we discuss basic concepts in peak shape, explore poor fluidic connections as a common cause of peak tailing, and explain what to do about it.

In the final article of this series on extracolumn dispersion, we look at elution mode, post-column flow splitting, and a free calculator to use during method development.

In recent articles, we reviewed the basic concepts of extracolumn dispersion and how this phenomenon can impact the quality of an LC separation. We now specifically discuss the effects of dispersion that can occur due to tubing and detectors.

Dispersion of analyte peaks outside of chromatography columns can seriously erode the resolution provided by good columns. Here, we focus on the contribution of the sample injection step to the total level of extracolumn dispersion in an LC system.

Dispersion of analyte zones outside of the column often compromises the quality of an LC separation—particularly in smaller columns with smaller particles. We explain basic concepts in extra-column dispersion from the point of view of an entire instrument.

Liquid chromatography (LC) pumps produce mobile-phase streams with short-term variations in mobile-phase composition. We explain the impact of these waves on retention time in reversed-phase LC and what to do about it.

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.

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.

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.

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.

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.

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

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.

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.

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

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.

In this installment, tips, tricks, and suggestions are provided for best practices in reversed-phase separations, in both 1D and 2D LC, with gradient elution for minimum variations in retention 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.

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.

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

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.

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

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?

We look at problems in LC that can be traced to contaminated solvents or reagents, and how sample solvent effects can lead to poor performance in HILIC

Is an ultraviolet (UV) detector signal good for anything if the analyst is using a mass spectrometer?

An ultraviolet (UV) detector signal can be an excellent source of clues for troubleshooting problems with your LC-even if you are using a mass spectrometer. We also share another good source of clues.

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.

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.

We examine instances where contaminants are problematic in LC–MS, as well as successful methods to decrease their influence.

For reversed-phase separations of proteins, you must consider pore size,column temperature, and stationary-phase chemistry. Here are some guidelines.

Here, we see how 2D-LC can solve coelution problems that are difficult or impossible to address using peak purity tools or curve resolution methods.

A bad connection between an LC column and the rest of the system can ruin a good separation. New technologies may improve the quality of these connections and the likelihood of establishing a good connection in the first place. Understanding the issues will help you choose the best option for you.

In part II of this series, we explore how curve resolution methods advance purity assessments and illustrate one of the most popular techniques, which adds a powerful tool to the chromatographer’s arsenal.

Certain details related to mobile-phase preparation that can be assumed in reversed-phase LC separations can be much more consequential in HILIC.

Is that peak “pure”? How do I know if there might be something hiding under there? In part I of this series, we explore some of the concepts behind peak purity assessments, describe some tools that are used in commercially available software for these assessments,

Paying attention to the details of mobile-phase preparation can have a big impact on the reproducibility of HILIC separations.

What are the most useful chromatography books on your bookshelf? What are the most useful web-based resources (such as websites, downloadable documents, videos) about separation science? What are the most useful tools supporting your work (such as calculators and simulators)? In this installment, Dwight Stoll compiles input from the separation science community (both individuals and vendors) to guide you to the resources that people find most useful.

What’s not in your standard operating procedure? Documenting details can prevent headaches associated with method transfers between laboratories.

In his final column before retirement, John Dolan reflects on the changes he has seen during his 34 years as the author of “LC Troubleshooting,” and shares his short list of best practices that still hold true today.

When developing or modifying an LC separation, a common strategy is to change selectivity by choosing a new column. Here’s guidance for making that selection.

You have to be careful when adjusting gradient conditions.

We consider some of the details associated with preparing a column for storage, with an eye toward choices that will pay dividends in future use.

How much of a change can you make to a USP method without revalidating the method?

Several variables can be used to change selectivity in a liquid chromatographic (LC) separation. Here we compare the variables in an effort to prioritize which experiments will be most effective.

We’ll see how to find the “sweet spot” in terms of retention for a liquid chromatographic separation as well as how much retention change is to be expected for a selected change in mobile-phase percent organic or column temperature.

When considering column efficiency, more is not always better. We look at some ways to quickly estimate the effects of changes in column length and particle diameter rather than trying the experiments in the laboratory.

Delivering samples to the analytical column in ‘clean’ mobile phase is important for robust methods and high quality results.

The mobile phase pH can be a powerful tool to control retention and selectivity, but it can also get you in trouble if not controlled properly.

A reader’s problem of a method that fails the repeatability of the system suitability test serves as an example of how to approach LC method troubleshooting.

What could be causing a peak to be eluted before the column dead time? In last month’s “LC Troubleshooting” (1) we looked at problems two readers had with ghost peaks in gradient runs. This month, we’ll continue looking at submitted questions and examine one submitted by another reader of this column.

Interfering peaks or high baseline background can compromise the results of gradient LC separations.

Fluorescence detection can be a strong alternative to ultraviolet or other detectors for some compounds.

Detectors based on ultraviolet absorbance are the most common detectors in use for liquid chromatography.

A reliable autosampler is one key requirement for unattended operation of a liquid chromatograph.

Different techniques of LC mobile phase mixing can give different results… and have different problems.

Understanding how liquid chromatographic pumps operate can help streamline solving pump problems.

Retention times drop from one injection to the next. A systematic approach to troubleshooting can help to quickly identify the problem source.

How suitable is the column plate number for system suitability testing?

The silica-based packing in reversed-phase columns is not inert. Here we consider what happens when the mobile phase pH is too high or too low.

Strongly retained sample compounds can cause various changes in the appearance of a chromatogram.

Particulate matter from the sample or the system can cause havoc in the chromatogram.

How to get started in the process of identifying the problem source for column-related problems.How to get started in the process of identifying the problem source for column-related problems.

Are your solvent costs too high? Here are some ideas about how to decrease the amount of mobile phase you use.

I recently returned from a tour of teaching liquid chromatography (LC) classes to users in Minnesota, the United Kingdom, Poland, and Malta. One thing that always impresses me on such trips is that no one group has a corner on the LC problem market. The same problems pop up in most laboratories, no matter where they are located, the role of the laboratory (for example, analytical, forensic, production, research), what industry is involved, or the brands of instrumentation used.

How to distinguish between LC column overload and detector overload.

The potential causes are considered for peak distortion for the first two peaks in the chromatogram.The potential causes are considered for peak distortion for the first two peaks in the chromatogram.

Readers' questions regarding problems related to internal standard calibration of liquid chromatography methods are addressed.

Recently, I received an inquiry from a reader regarding a problem he encountered with a routine liquid chromatography (LC) method in his clinical laboratory.

When converting methods from liquid chromatography (LC) to ultrahigh-pressure liquid chromatography (UHPLC), don't get confused by flow-rate settings.

One of the most frequent times that we discover a problem with a liquid chromatography (LC) method is when we examine a data set following the analysis of a batch of samples.

Why are liquid chromatography retention times sometimes so slow to stabilize?

Have a problem with your LC system? Maybe applying a little "DDT (Don't Do That)" may help.

How can you avoid phase collapse when 5% organic solvent is too strong?

How can internal LC surfaces be made less reactive?

Stainless steel is inert – or is it?

A scouting gradient is a separation run under a standardized set of conditions and can be used to determine the complexity of a sample and estimate the difficulty of the separation. John Dolan tells why he believes using a scouting gradient is the best way to begin method development.

After exploiting the chemical factors in the separation process, chromatographers can adjust column conditions – flow rate, column size, and particle diameter – to further improve separations.

In part III of this column series, John Dolan demonstrated that systematic variation of the solvent strength can change selectivity and retention, and changing solvent type can amplify the effect. This month, he looks at three additional parameters (pH, temperature, and column type) for adjusting selectivity.

Most chromatographers can look at a chromatogram and provide a qualitative opinion about the separation, but it is equally important to be able to measure the separation quality.

LC problems fall into one of two categories: those associated with instrumentation and those associated with the separation itself. Dolan explains how to identify which kind you have and how to approach correcting the problem.

Readers submit questions about how to isolate the source of carryover in LC methods, mobile-phase temperature effects, and the care of cleaning of columns.

Inadequate mobile-phase gassing may be the single largest cause of LC problems. Columnist Dolan examines bubble-problem sources and techniques that analysts can use to eliminate excess gas in the mobile phase.

Peak shape problems don't always have a single solution.