Strategies to Combat Extra-Column Volume and Dispersion in LC Analysis

by | Feb 28, 2024 | Liquid Chromatography

Balancing extra-column volume with sensitivity and signal-to-noise ratios is crucial for reducing extra-column dispersion.

Producing narrower peaks in liquid chromatography is essential for enhancing analytical precision and efficiency. Over recent decades, significant progress has been made thanks to advancements in particle sizes and column technology. Yet, despite these leaps in miniaturization, the conventional layout of chromatographic systems—characterized by long connection tubing between components—has remained unchanged. This discrepancy could amplify the impact of ‘extra-column’ effects—the processes outside the column that contribute to the broadening of chromatographic peaks.

Extra-Column Volume vs. Dispersion in Liquid Chromatography

Extra-column volume (ECV) refers to the physical volume from the injector to the end of the detector, including tubing, fittings, autosampler, and detector flow cell. On the other hand, extra-column dispersion (ECD) is a process that leads to the broadening of a peak between the injector and the detector, excluding the column itself.  “Think of extra-column volume as the cause and dispersion as the effect,” explains Tom Jupille, founder and moderator of Chromatography Forum. 

Not all extra-column volume is equivalent, cautions Jupille. For instance, the influence of the volume before the column can be lessened if the solvent used for diluting the sample is much weaker than the mobile phase. Additionally, if a fitting is not assembled correctly, it can significantly increase dispersion due to a ‘mixing chamber’ effect caused by changes in the tubing's diameter.

Desmet and Broeckhoven examine the gap between advancements in chromatographic column technology and instrument design, pointing out that columns now exceed the performance of the instruments. They review methods for measuring extra-column dispersion, emphasizing the difficulty in accurate quantification due to each method's specific strengths and weaknesses. This highlights the necessity for a balance between simplicity and precision in these measurements.

Strategies to Reduce Extra-Column Dispersion

Studies of ultra-high pressure liquid chromatography (UHPLC) columns packed with sub-2-μm particles revealed extra-column volume significantly affects separation efficiency, especially as column diameter decreases. Both apparent retention factors and pressures deviated from expected values due to extra-column volume, with the largest discrepancies observed in the smallest diameter columns. The study suggests optimizing the ratio of extra-column volume to column void volume to about 1:10 to achieve 80% or higher of the theoretical efficiency, emphasizing the importance of adjusting inlet tubing diameter for enhanced column performance.

Reducing extra-column dispersion requires trade-offs. Decreasing injection and detector volumes impacts the signal-to-noise ratio and sensitivity, while smaller tubing diameters can lead to increased pressure drops. Smaller detector cells reduce band broadening but can raise noise and lower signal intensity. One emerging approach is to fine-tune injection through fixed loop systems instead of conventional flow-through mode, as discussed in a recent Separation Science food satefy webinar.

Reducing Dispersion in 2D-LC

Two-dimensional liquid chromatography (2D-LC) employs two distinct separation phases with varying chemistries to achieve superior resolution of complex samples compared to one-dimensional LC. However, extra-column dispersion is heightened in this mode for several reasons. The initial separation stage in 2D-LC produces small, focused peaks; dispersion before the second stage can dilute these, reducing resolution. Further dispersion in the second stage broadens peaks, compromising effectiveness.

Two recent studies have explored solutions for minimizing band broadening in spatial 2D-LC systems. One approach leveraged rapid prototyping through 3D-printed microfluidic devices, finding that omitting spacers in the 2D separation area and incorporating stationary-phase material in the 1D channel significantly reduced band broadening. Additionally, the study discusses the pressure limits and printability of the fabricated devices, contributing to the optimization of spatial 2D-LC systems.

Another effort used numerical simulations and tightly coiled loops to analyze the impact of various factors, including flow rates, loop volume, and the interface loop's physical configuration, on analyte dispersion. Results show that loops yield significantly narrower peaks with less tailing than straight capillaries, reducing peak variance by 20–40%.


Effective management of extra-column volume and dispersion is essential, requiring a nuanced approach to system design and optimization. This is particularly vital in 2D-LC, where the precision of separation significantly influences the analytical outcome. As the field progresses, innovative technology and system optimizations will be key to overcoming these obstacles. For more insights, strategies, and discussions on navigating these challenges, join the conversation at Chromatography Forum.

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