Advancing Miniaturized Column and Instrument Technologies for Capillary Liquid Chromatography

Increased attention on sustainability has been a key area of focus within a broad range of scientific and engineering disciplines in recent years. The “green chemistry” movement has been especially notable for its efforts to reduce the amounts and types of toxic solvents that are required for chemical synthesis at both small batch and large manufacturing scales.¹ The practice of “green analytical chemistry” has also received increased attention in recent years, with a similar focus on performing analytical measurements with lower volumes of more toxic solvents.² A number of descriptors have been used to compare methods in achieving these and other “greenness” metrics, such as the analytical method greenness score (AMGS), the Green Analytical Performance Index (GAPI), and the analytical GREEnness (AGREE)calculator scores.³ Amongst chemical measurement techniques, high performance liquid chromatography (HPLC) is one of the primary sources of chemical waste due to the use of large volumes of organic-containing liquid mobile phase mixtures.⁴ Several research groups have started to investigate the use of alternative organic modifiers for reversed phase HPLC separations, primarily focused on implementation of ethanol,⁵ dimethyl carbonate,^6,7 and carbonate esters⁸ over methanol and acetonitrile. In parallel, a strategy involving the use of capillary-scale LC separations on miniaturized instrumentation is leading to a significant reduction in mobile phase usage for routine separations.⁹ This article is focused on recent updates within this emerging approach of compact and portable capillary LC instrumentation and the ways that it is starting to impact the broader analytical community.

Innovations in Capillary LC Instrumentation and Methodology

Over the past decade, there has been a significant push to miniaturize HPLC instrumentation, with several companies focused on commercializing such technology to promote broader adoption.¹⁰ One of the primary approaches to reducing instrument size is the use of small-volume syringe pumps, which are usually much smaller than typical reciprocating piston pump designs. Because these pumps are most effective when the total mobile phase volume consumed in a separation is lower than the available syringe barrel volume, miniaturized HPLC systems are most compatible with capillary LC columns operated in the low-to-sub μL/min flow range.¹¹ This approach inherently reduces the mobile phase consumption to ~0.1-1% of a typical HPLC separation (using 4.6-mm-i.d. columns) and ~1-5% of a typical UHPLC separation (using 2.1-mm-i.d. columns).

Although the proteomics community has long implemented “nano-LC” separations with sub-μL/min flow rates on 0.075-mm-i.d. columns,¹² these newer compact LC systems are often used with 0.15 – 0.5 mm i.d. columns.¹³ Many reversed-phase columns are already available in these smaller dimensions, but moving forward, wider availability of a broader range of stationary phases typically used (HILIC, size-exclusion, etc.) will be needed to expand adoption of this instrumentation.

The other key aspect of compact and portable LC instrumentation is detector design. As absorbance detection remains one of the most widely used detection methods for routine LC separations, the use of small LED-based light sources has been a crucial step in miniaturizing these modules.¹⁴ Current LED-UV technology is readily available down to 235 nm, which is compatible with a wide range of methods typically employed for separating and detecting molecules within the pharmaceutical and biopharmaceutical fields. For other applications where lower UV wavelengths are needed, lamp sources similar to those used in standard LC instrumentation, combined with low-volume flow cells, can be used,^15–17 although this approach typically increases the overall detector size. The other detection method that is commonly coupled to compact LC systems is mass spectrometry (MS). Because of the typical sizes of mass analyzers and vacuum pumping systems, miniaturizing MS detectors has been a more challenging approach, although units that are much smaller than a typical benchtop MS system are available.¹⁸ Generally, single quadrupole and ion trap MS instruments have been most compatible with compact LCs for miniaturization of complete LC–MS systems because they are most amenable to smaller vacuum systems that also have lower power requirements.¹⁴ As LC–MS is one of the most widely used techniques in modern chemical analysis, being able to combine compact versions of both instruments can bring a truly powerful analytical methodology directly to the point-of-need.¹⁹

Applications of Miniaturized Column and Instrument Technologies for Capillary LC

Because of the ability to operate the instrumentation in smaller and/or remote settings and the reduction in solvent consumption achieved by transitioning to capillary-scale separations, recent years have seen increased adoption of compact LC instrumentation across a broad range of applications. The most common application to date has been the analysis of pharmaceutical and biopharmaceutical compounds,^20,21 with some extension into dietary supplements as well.^22,23 For general analysis of the separation of pharmaceutical compounds and their impurities, including the quantitation of active pharmaceutical ingredient (API) in tablet formulations,²⁴ compact LC methods have started to achieve results that are comparable to standard benchtop systems. ^25–28

There has also been a trend in on-line reaction monitoring within the pharmaceutical industry using compact instruments, ,^16,26–28 as the smaller size aids in direct measurement of synthetic reactions across a variety of scales.²⁹ For automated sampling from reaction vessels and delivery to instruments for analysis, both standard equipment designed for use with analytical-scale separations and specialized equipment specifically focused on the smaller volumes required for capillary LC have been used. Recently in our group, efforts have been made to use these capillary LC-compatible modules for online reaction monitoring of various reactions; as shown in Figure 1, the reaction kinetics of both imine formation (Figures 1a and 1c) and Suzuki coupling (Figures 1b and 1d) reactions can be tracked by monitoring both reactant depletion and product formation over time. As part of the Enabling Technologies Consortium,³⁰ a number of major pharmaceutical companies have tested compact LC instrumentation for a variety of workflows, and it is anticipated that this trend will continue as the robustness of separation methods developed using these instruments continues to improve.

Because compact LC systems can be operated in remote settings, environmental testing applications are also being developed. Multiple demonstrations of PFAS analysis in water using compact LC coupled to smaller MS systems that are capable of operating in vehicles have been reported.^31,32 This approach enables real-time analysis across multiple geographic locations in a short time, enabling rapid remediation efforts when high PFAS concentrations are observed. Other organic pollutants in water sources, including trimethylxanthines,³³ biocides,³⁴ haloacetic acids,³⁵ and polycyclic aromatic hydrocarbons,¹⁵ have been analyzed using compact LC methodology as well. In many of these cases, because analyte concentrations are low, sample preparation strategies that allow for matrix clean-up and analyte preconcentration prior to analysis are required.³⁶ Reducing the time and complexity of sample preparation so that it can also be readily adopted in field settings will be key to further implementing these instruments in point-of-need settings.³⁷ Beyond traditional reversed-phase separations for the compound classes described above, portable ion chromatographs can be employed for real-time monitoring of ionic species, such as nitrites and nitrates, in soil samples and water sources.^38–41

The third general trend in compact LC usage is for the implementation of forensic and toxicological analysis.^42,43 A variety of compounds often observed in seized drug samples and adulterated beverages have been successfully measured using capillary LC methods with compact instrumentation.^20,44–47 One challenge in detecting many analytes in this class is that their absorbance signals increase sharply below 220 nm, which is lower than the commercially available LED-UV sources on the market. To accommodate lower wavelength UV absorbance detection, the alternative sources and miniaturized spectrophotometers mentioned above can be used. Ongoing work in our group, in collaboration with compact LC manufacturers, is focused on detecting these compounds in clinical samples, such as urine, to improve point-of-care drug monitoring and enhance the diagnostic tools available during the treatment of substance use disorders.

Outlook for Capillary LC and Compact LC Instrumentation

Although capillary LC has been around for decades and has been widely applied in proteomics analysis for nearly as long,⁴⁸ the implementation of low-flow LC separations as a replacement for standard analytical-scale LC is still in its early stages. Ongoing work to improve the repeatability and robustness of separations using columns in the 0.1 – 0.5 mm-i.d. range, as well as benchmarking equivalent (or better) performance to existing LC methods, will be needed for wider adoption. It is likely that the economic and environmental impacts of drastically reduced mobile phase solvent consumption and waste generation will be a driving factor for growth, as evidenced by companies across various industries that have already started to implement this technology within their analytical workflows. With separation performance continuing to improve and more users testing the technology for their analytical needs, the time for broader adoption of capillary LC within the world of chemical analysis may finally be here.

Acknowledgments

Support for ongoing work in this area is provided by the National Institutes of Health through award R44 DA056316. John Boughton (Rowan University) and Samuel Foster (Axcend) are acknowledged for data acquisition and visualization for Figure 1. Milton Lee (Brigham Young University and Axcend) is also acknowledged for useful feedback regarding the manuscript.

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