Modern Capillary Scale Liquid Chromatography Columns: An Update

Capillary-scale liquid chromatography uses columns with small inner diameters ranging from 0.075–0.50 mm operated at flow rates between 1–20 µL/min. The low flow rates required to operate these columns offer unique benefits including increased sensitivity in electrospray ionization mass spectrometry (ESI-MS), reductions in hazardous mobile phase usage, and the ability to integrate non-traditional column formats (open tubular, pillar array, and monolithic columns) into analytical workflows. In this article, we discuss the considerations required for capillary-scale LC, as well as a review of current commercial offerings of capillary-scale columns.

In the early 1970’s, advances in glass capillary manufacturing allowed for the first capillary-scale liquid chromatography columns to be produced (1). In the 50 years that have followed, the field has grown and changed considerably into what we know today. Chromatographic scale is determined by the inner diameter (i.d.) of the column, with analytical-scale columns ranging from 1.0–4.6 mm i.d. operated between 0.3–50 mL/min, and capillary-scale columns ranging from 0.075–0.5 mm i.d. operated between 1.0–20.0 µL/min (2). By reducing the flow rate while maintaining similar mobile phase linear velocities, solvent consumption can be reduced by several orders of magnitude, producing greener, cheaper, and more sustainable separations (3). At lower flow rates, the ionization efficiency of electrospray ionization (ESI) improves, increasing the sensitivity of mass spectrometric detection. These increases in sensitivity have made capillary-scale separations a staple of the ‘omics’ fields (4), which often have limited sample availability and require highly sensitive mass spectrometric detection. A limitation of the technology, until recently, was the availability of capillary-scale LC instrumentation. Over the past several years, a number of capillary-scale liquid chromatography systems have become commercially available (5–13). While these systems are just as capable at generating separation conditions (flow, pressure, gradients, etc.) as their analytical counterparts, separation performance is limited by the columns used to perform those separations. This article seeks to provide an overview of existing commercially available capillary-scale instrumentation, with a specific focus on the columns offered.

To accommodate the low flow rates of capillary-scale LC separations, the solvent delivery systems and system fluidics require additional attention when compared to the analytical scale. The two most widely used methods for low-flow solvent delivery are reciprocating piston and syringe-based pumping systems. Piston-based systems typically use two pistons offset 180˚ out of phase with each other, such that while one piston draws in solvent, the other dispenses the solvent. When using piston-based pumping systems, stroke volumes must be reduced to accurately control delivery volumes, and pulse dampening implemented to lessen fluctuations in the baseline (2). With these syringe-based pumps, a single syringe (for isocratic elution) or multiple syringes (for gradient elution) can be used. Flow is generated by drawing the solvent required for the run into the syringe and then reversing the direction of the syringe plunger and dispensing in a single push (2). By removing the switching step between pistons, near-pulseless flow is generated, removing the need for pulse dampeners. Syringe-based systems, unlike piston pumps, are unable to generate continual flow over long periods and are instead limited to the maximum volume of the syringe. This problem can be solved by increasing the syringe volume; however, larger syringe volumes at low flow rates have been shown to produce some flow pulsations resulting from the individual steps of the motor (14,15). At lower flow rates, extra-column volume must also be reduced to retain chromatographic efficiency (16). This can be achieved with the use of zero dead volume unions, small i.d. connection tubing, and face-sealing fittings (17). While discussions on the instrumentation of capillary-scale LC are outside the scope of this article, a tutorial on method translation and optimization to the capillary-scale has recently been published and readers are directed there for further information (18).

Capillary LC Columns

At the heart of every chromatographic separation is the column, which is used to facilitate the separation of compounds based on any number of chemical features. There are several notable differences between analytical- and capillary-scale columns including packing efficiency (19), heat generation and dissipation related to viscous friction (20), and the ability to use nontraditional column formats (open tubular (21) and pillar array (22)) that benefit from the reduced flow rates of capillary-scale separations.

The reduced size of the capillary-scale column produces several unique benefits for the column. As solvent flows through the column, heat is produced through friction between the mobile phase and the stationary phase. Heat is then dissipated through the column walls, producing a temperature gradient between the center and edges of the column. With a reduced inner diameter comes reduced heat generation and increased heat dissipation, reducing the negative impacts of this radial temperature gradient on chromatographic performance (23).

Analytical-scale and capillary-scale columns packed with stationary phase particle supports also differ in their bed morphology (19). Previous studies have used confocal laser scanning microscopy (CLSM) for capillary columns (24) and focused-ion-beam scanning electron microscopy (FIB-SEM) for larger i.d. columns to study the effects of bed morphology on band broadening. For analytical-scale columns, it was determined that column beds consist of three distinct regions: an ordered layer at the column wall (up to ~1.5 particle diameters from the wall surface), a more dense but randomly packed layer around the column wall (~70 particle diameters thick), and a less dense bulk packing region that fills the remaining column volume (25). In situations where the denser wall region consists of the majority of the column bed, such as on the capillary-scale, higher efficiencies can be obtained. While this is not the case across all commercial columns and geometries, capillary columns offer the potential for uniquely efficient separations compared to larger i.d. columns.

Capillary-scale columns come in a variety of lengths, i.d.’s, particle porosities, and particle sizes. A compilation of the existing commercially available options for capillary columns is listed in Table I. Across the commercial landscape, columns and their particles are offered in various geometries; however, several combinations are commonly used. Column i.d.’s tend to be 0.075 mm, 0.15 mm or 0.3 mm, with 0.1 mm, 0.2 mm, and 0.5 mm options also available. As the i.d. of the column decreases, so too does the operating flow rate range, with 0.15 mm i.d. columns requiring ~4x less flow than 0.3 mm i.d. columns to maintain a similar mobile phase linear velocity. While this does reduce the solvent consumption even further, efficiency losses from extra-column band broadening are exacerbated as the column volume decreases. Columns tend to be either 50 mm, 100 mm, or 150 mm in length; however, lengths as low as 5 mm, or as high as 2000 mm are also available. Particle diameters range from <2 µm to 5 µm and can be found in both fully porous and superficially porous formats. Particles as large as 10 µm in diameter are available, but are uncommon at these smaller column i.d.’s due to challenges in packing. Column pressure limits are similar to those found with analytical-scale columns, with most ranging from 400 bar to 1000 bar. These high-pressure limits allow for faster analysis times, enabling high-throughput separations. The commercial offerings cover a broad range of physical dimensions, enabling the translation of most separations to the capillary scale.

When translating a separation to the capillary scale, the best option is to select a column of identical chemistry. Although capillary columns are offered in reversed-phase liquid chromatography (RPLC), normal phase liquid chromatography (NPLC), ion exchange chromatography (IEX), hydrophilic interaction liquid chromatography (HILIC), (hydrophobic interaction chromatography) HIC, and size exclusion chromatography (SEC) functionalities, an identical column chemistry cannot always be commercially sourced. In these cases, a column with similar selectivity should be chosen. For reversed-phase applications, the column selectivity database (26) can be used to identify the closest match by using the hydrophobicity subtraction model (HSM)(27). The HSM is used to model a column's selectivity based on a number of factors, including hydrophobicity, hydrogen bond acidity and basicity, ion affinity, and solute permeability. Columns with similar HSM scores are expected to produce similar retention times for a given analyte. The HSM can also be used for initial column selection, by choosing a column with a high affinity towards a given analyte property to increase the expected retention. In other separation modes, columns featuring similar chemistry, particle size, and particle porosity can be substituted to produce similar separations.

Pillar array columns, often referred to as microfabricated or µ-pillar array columns, harness lithography-based microfabrication to create perfectly ordered arrays of micro-pillars etched into a silicon wafer.PharmaFluidics, now owned by Thermo Fisher Scientific, introduced µPAC columns around 2021. The design enables capillary flow rates (1–15 µL/min), which are ideal for proteomics and metabolomics areas of study. The uniform geometry of the devices yields extremely low back pressures, exceptional column-to-column reproducibility (e.g., <1 % CV in peak area), and high plate counts, especially valuable when handling minute sample volumes such as in high-sensitivity bioanalytical separations. Pillar array columns have been noted as part of a broader “nontraditional column formats” trend, which emerged in reviews of new LC products (28). These formats deliver comparable or superior separation efficiency relative to conventional packed or monolithic columns, while offering design flexibility for flow dynamics and minimal dispersion. Pillar array columns may still be poised to play a leading role in future high-performance separations, especially in workflows requiring ultra-high reproducibility and low-volume sample handling.

Open‑tubular liquid chromatography (OTLC) typically employs a narrow capillary coated on its inner walls with a thin stationary-phase layer instead of packed particles or monoliths. The origins of the technique date back to Tsuda in the 1970’s (29), however, there has been a renewed interest driven by recent advances in instrumentation, capillary fabrication techniques, and nanoparticle-coated supports (30). OTLC’s intrinsic benefits include high separation efficiency (due to reduced mass-transfer resistance), very low back pressure, small sample volume requirements, and reduced solvent consumption, rendering it attractive for green chromatography initiatives. However, OTLC's also have their limitations: poor sample loading capacity, challenges in reproducible column manufacture, and sensitivity constraints. Novel engineering approaches (e.g., porous-layer open‑tubular supports, nanoparticle coatings) have recently mitigated many of these barriers, reigniting research interest, resulting a resurgence in open‑tubular formats.

Despite the seemingly broad coverage of commercially available capillary-scale columns, limitations arise when seeking less common phases, or when attempting to directly match the exact phase of an existing workflow. For example, when sourcing a size exclusion column, a capillary equivalent of identical length and particle size may be commercially available, but not at the desired pore size, limiting functionality. Additionally, for columns using mixed mode functionalities such as RPLC/IEX (commonly used for oligonucleotide separations), commercial availability becomes limited. Even among common functionalities such as a standard C18, limitations are found in the availability of high/low pH compatible phases, or the packing of those particles into biocompatible hardware. Although these gaps can be filled through the use of bulk packing services, such as PremierLCMS, CoAnn, and Dr. Maisch, these limitations in readily available commercial offerings leave many hesitant to consider implementing capillary LC into their workflows.

As the field of capillary LC continues to grow, so too will its commercial offerings. The field of chromatography shifted from 4.6 mm i.d. columns to 3.0-mm-i.d. columns for their solvent-saving benefits. The field again, shifted from 3.0-mm-i.d. to 2.1-mm-i.d. columns for further reductions in solvent consumption. It is clear that there is trend in shifting to smaller and smaller chromatographic scales. This trend is expected to continue, with analytical scale columns of 1.0 mm and 1.5 mm i.d. becoming more common (31) and the growing adoption of capillary-scale LC systems. This trend should result in wider availability of low-flow LC products, not just in the number of available columns and phases, but across every aspect of the system including tubing, fittings, detectors, and pumping systems as well.

Capillary-scale liquid chromatography has a number of benefits to offer. The reduced inner diameters allow for separations at microliter per minute flow rates, reducing solvent consumption by several orders of magnitude when compared to analytical scale. This reduction in solvent also extends to the harmful modifiers often used, including PFAS compounds such as trifluoroacetic acid (TFA) and hexafluoroisopropanol (HFIP). This makes capillary separations a greener and more cost-effective technique compared to larger scales. Lower flow rates also provide greater ionization efficiencies, and by extension greater sensitivities when coupled to ESI-MS. This offers considerable benefits to the ‘omics’ fields, which often require high sensitivity MS detection, with limited sample availability. Compact and portable systems have also leveraged capillary-scale separations to provide point of need analysis capabilities. The field of capillary scale liquid chromatography has a long way to go before it rivals the commercial offerings of other scales; however, it is clear that the interest and adoption of capillary separations is on the rise.

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