Modular, cost-effective, and portable capillary gradient liquid chromatography system for on-site analysis

https://doi.org/10.1016/j.chroma.2020.461374Get rights and content

Highlights

  • Portable and fully modular compact capillary liquid chromatography system has been developed.

  • Custom built syringe pumps with a wide range of capillary flow rates.

  • High sensitivity Z-type UV absorbance detector with custom built passively cooled housing was evaluated.

  • High repeatability of <1.5% RSD for peak area and <0.4% RSD for retention time was achieved.

Abstract

This work demonstrates the development of a compact, modular, cost-effective separation system configured to address a specific separation problem. The principles of the separation are based on gradient capillary liquid chromatography where the system consists of precision stepper motor-driven portable syringe pumps with interchangeable glass syringes (100 µL to 1000 µL). Excellent flow-rate precision of < 1% RSD was achieved with typical flow-rates ranging from 1 µL/min to 100 µL/min, which was ideal for capillary columns. A variable external loop volume and electrically actuated miniature injection valve was used for sample introduction. Detection was based upon a commercial Z-type UV absorbance flow-cell housed within a custom-built cooling enclosure (40 mm x 40 mm) which also contained a UV-LED light-source and a photodiode. System and chromatographic performance was evaluated using linear gradient elution, with day to day repeatability of <1.5% RSD (n = 6) for peak area, and < 0.4% RSD (n = 6) for retention time, for the separation of a 5 component mixture using a 50 mm X 530 µm ID C18 3 µm particle capillary column. The system can run any commercial or in-house packed columns from 50 mm to 100 mm length with IDs ranging from 200 to 700 µm. The developed portable system was operated using custom-built windows-based chromatography software, complete with data acquisition and system control.

Introduction

The development and availability of miniature and portable analytical instruments has seen irregular growth in recent years, possibly due to the timing of interest in novel applications in fields such as defense and space, remote environmental monitoring, at-site biomedical and diagnostic research and reaction monitoring in the chemical and pharmaceutical industries [1], [2], [3], [4], [5], [6]. Some of the main drivers behind miniaturization include the need to reduce the cost and time taken for analysis (e.g. at-site measurements rather than centralized analysis), improve system portability and reduce the dependency on large volumes of solvents. Achieving these goals has been possible through considerable advances in microelectronics, paving the way for smaller, smarter and more compact analytical systems [7,8].

With respect to the separation sciences, there have been a number of research efforts into the miniaturizion of high-performance liquid chromatography (HPLC) instrumentation. From a design requirements perspective, a portable HPLC should be small, light, robust, sensitive and only use small volumes of solvent. HPLC has progressed due to many factors such as the improvement in micro-electronics, the drive for green chemistry, and compatibility with syringe pumps. However, in most reported examples only some of these criteria have been addressed. In 1996 Baram reported a portable HPLC system [9] which utilized syringe pumps and demonstrated excellent chromatographic performance, but the relatively large physical size, high solvent consumption and the need to run on mains power made the system unsuitable for on-site analysis. Tulchinsky and St Angelo [10], developed a battery operated portable HPLC which had the capability of eight hours continuous operation. The group was able to integrate the system as a single unit weighing around 9.5 kg, which was controlled by a desktop personal computer. They successfully demonstrated the system with the separation of phenols from lake water samples. However, the sytem was demonstrated using conventional LC columns (4.6 mm ID) and was dependent on high solvent volumes.

The biggest challenge with portable HPLC lies in the preliminary design phase of the system, where both size and performance of each component are critical for successful outcomes. With the advent of capillary column technology, research into the miniaturization of HPLC has progressed, because smaller diameter columns use less solvent and are amenable to simpler pump arrangements. Typical of this progression is the work of Lynch et al. [11], who explored capillary HPLC separations based upon a cartridge column approach and electro-osmotic pumps, together with a 60 nL injector valve being used for sample introduction. Successful separation of a complex mixture of bovine serum albumin and myoglobin endoproteinase digests was demonstrated, although the system described relied upon a separate bench-top detector and so could not be considered a fully portable HPLC. However, Sharma et al. [12] successfully demonstrated a 24 V battery operated fully portable HPLC system, weighing ~ 4 kg, which used a nano-flow stepper motor-driven pump capable of generating pressures over 8000 psi. An on-column UV detector was used and the system was capable of performing gradient separations with excellent repeatability and reproducibility results for day to day operations. Li et al. [13] developed a portable, medium-pressure LC platform using inexpensive, off-the-shelf components (pumps, valves and detector) assembled in a plug and play arrangement. This flexible platform has recently been improved and demonstrated as a fully portable system, with optional battery operation, and applied together with a portable mass spectrometer, as a very small footprint HPLC-MS solution [14].

Commercially available portable HPLCs have been relatively few in number over the years, with instruments such the Milichrom system typical of the earlier systems [15], weighing in at 17 kg, and equipped with a 120 mm capillary column and variable wavelength UV absorption detection over the range 190- 360 nm. More recently three new systems have been made available to the market [16], the Alltesta HPLC Analyser from SIELC Technologies is designed to use 4.6 mm and 3.2 mm column formats and flow rates (0.6 – 1.0 mL min-1). This system weighs ~ 10 kg and the instrument is aimed at delivering an HPLC solution at one tenth the cost and size of benchtop systems targeting environmental field testing and educational purposes. The Smart HPLC instrument from both PolyLC and Supercritical Fluid Technologies (http://www.supercriticalfluids.com/) is a fully portable HPLC weighing 11.3 kg with an embedded fixed wavelength detector at 415 nm targeting the screening of HbA1c and inherited blood disorders. The system is a fully functional gradient standard format HPLC, with the footprint of a mid-sized brief case and despite the fixed wavelength detector the company suggests other LED wavelengths are available and easily exchanged (280 nm and 255 nm). However, with a flow cell volume of 15 µL the system is ideally suited to high flow rates. More recently, a compact 6.8 kg system, the Axcend Focus nano LC, was released and is capable of performing gradient separations with 150 µm ID capillary columns containing sub-2 µm particulate stationary phases, delivering pressures up to 10,000 psi. With this instrument the developers are focusing its application on field analysis and drug testing.

The pharmaceutical industry has also strongly influenced the pursuit of portable HPLC instruments. HPLC is one of the key quality assurance technologies used in the pharmaceutical industry. Throughout this industry, a key application driver for miniature HPLC is the need for portable systems to be applied to direct analysis at the point of product manufacture, in order to preserve sample integrity [3,[17], [18], [19]. In November 2019, the Enabling Technologies Consortium of the pharmaceutical industry (https://www.etconsortium.org/compact-hplc) identified a need for a compact HPLC in reaction monitoring as there was no suitable instrument availble to meet their requirements.

Working towards these emerging needs, the present study reports the development of a separation instrument to fit the niche where the separation demands are low (about 10–15 sample components or less), run times should be short (<10 min), the system has to be compact, the solvent and power demand is low and the system is overall cost-effective given the limitations on the requirements of the number of components to be resolved. The separation requirement for reaction monitoring has been the driving force of the development. Here the demand is to have a compact instrument close to the reactor (e.g. inside the fume hood) and quantitate a small number of sample components (starting material, product, and a small number of impurities) at a frequency of 5–10 min. Other areas of interest is the in-field deployment in the environmental analysis sector, where the instrument can be taken to the site of analysis and be powered either by its own battery or by a car battery and the educational sector where the instrument can be used in the teaching environment to demonstrate the principles of chromatography by, for example, measuring the caffeine content of beverages. From the earliest concept we decided to build an instrument to serve a certain application (fit-for-purpose) rather than compete with standard benchtop HPLC. To this end modularity was important so we are able to customize the instrument to serve a certain task. Where possible, the system has incorporated off-the-shelf components making it easier to fabricate without losing system performance. Some of the drawbacks noted in earlier portable HPLC platforms have been taken into consideration and improved, with an overall design focused on delivering performance with simplicity and versatility – the modular architecture enabling “interchangeable” elements such as the syringe pumps, valves and detectors.

Section snippets

Materials

Pump components:

NEMA 11 stepper motors were purchased from US Automation (Anaheim, CA, USA) while the aluminum motor housing was fabricated in-house. All syringes are gas tight range – 100 µL (005,250), 250 µL (006,250), 500 µL (007,250) and 1 mL (008,105) are from Trajan Scientific and Medical (Ringwood, VIC, Australia). The plunger button was modified to connect to the stepper lead screw and the syringe front screw was fitted with an adaptor for tubing connection.

Valves:

All valves were

System architecture

The aim of this work was to design and develop an inexpensive modular, automated separation system based on the principles of gradient capillary LC with a small portable footprint. For demonstration purposes, the whole system was designed to fit into a briefcase (300 mm x 450 mm x 120 mm) and in total weighed 7.2 kg (Fig. 1a). Fig. 1b shows a schematic representation of the portable instrument and the various components therein. The system hardware was based on an I2C communication protocol and

Conclusion

This research focused on building a cost-effective system without compromising the performance of the specific measurement. A syringe pump based, fully modular, separation system has been developed and demonstrated to give performance comparable to commercial LC systems, with overall system performance for peak area <1.5% RSD and retention time <0.4% RSD. The ability of the system pumps to generate pressures close to 4000 psi allows the use of capillary columns with fully porous particles down

CRediT authorship contribution statement

Lewellwyn J. Coates: Methodology, Writing - original draft, Investigation, Validation, Software. Shing C. Lam: Methodology, Resources, Validation, Formal analysis. Andrew A. Gooley: Methodology, Formal analysis, Project administration, Writing - review & editing, Funding acquisition, Supervision. Paul R. Haddad: Formal analysis, Project administration, Writing - review & editing, Funding acquisition, Supervision. Brett Paull: Formal analysis, Project administration, Writing - review & editing,

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This research was conducted by the Australian Research Council (ARC) Training center for Portable Analytical Separation Technologies (IC140100022). LJC is a recipient of Australian Government Research Training Program Scholarship. University of Tasmania and Trajan Scientific and Medical are gratefully acknowledged for their support and financial contribution. We would like to thank Frank Riley and Angel Diaz from Pfizer, Groton, CT for providing the 5-component test mixture and technical

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