Miniaturized high-performance liquid chromatography instrumentation

Author’s Accepted Manuscript Miniaturized High-Performance Chromatography Instrumentation

Liquid

Kyle B. Lynch, Apeng Chen, Shaorong Liu

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S0039-9140(17)30961-X http://dx.doi.org/10.1016/j.talanta.2017.09.016 TAL17919

To appear in: Talanta Received date: 3 July 2017 Revised date: 4 September 2017 Accepted date: 6 September 2017 Cite this article as: Kyle B. Lynch, Apeng Chen and Shaorong Liu, Miniaturized High-Performance Liquid Chromatography Instrumentation, Talanta, http://dx.doi.org/10.1016/j.talanta.2017.09.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Miniaturized High-Performance Liquid Chromatography Instrumentation Kyle B. Lynch* Department of Chemistry and Biochemistry, University of Oklahoma Apeng Chen Department of Chemistry and Biochemistry, University of Oklahoma Shaorong Liu Department of Chemistry and Biochemistry, University of Oklahoma, *Corresponding author Department of Chemistry and Biochemistry, University of Oklahoma, 101 Stephenson Parkway, Norman, OK 73019, USA Tel.: +1 (405) 325 9013, Fax: +1 (405) 325 6111, Email: [email protected]

Abstract Miniaturized high performance liquid chromatography (HPLC) has attracted increasing attention for its potential in high-throughput analyses and point-of-care applications. In this review we highlight the recent advancements in HPLC system miniaturization. We focus on the major components that constitute these instruments along with their respective advantages and drawbacks as well as present a few representative miniaturized HPLC systems. We discuss briefly some of the applications and also anticipate the future development trends of these instrumental platforms.

Graphical Abstract

Highlights

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In this review, an overview of technology leading to miniaturization of an HPLC system is introduced Advancements in pumps, injectors, columns and detectors have led to a reduction in size Complete integrated and miniaturized systems, both commercial and academic, are presented Keywords: Electroosmotic pump; Nanoflow; Gradient generator; High performance liquid chromatography; Miniaturized Abbreviations EOP – electroosmotic pump HVPS – high-voltage power supply μHPLC – micro-High Performance Liquid Chromatography USB – universal serial bus

1 Introduction High Performance Liquid Chromatography (HPLC) has its roots back into the early 20th century. Martin and Synge [1] conducted initial studies in the early 1940s but advancement within the field did not take place until the 1960s when scientists realized that reducing the packing-particle diameter of the column while subsequently increasing the mobile phase velocity through an increased pressure could lead to dramatically improved separations [2-6]. It was during this time period that separation times decreased while resolutions increased showing that high performance liquid chromatography was here to stay. Leading chromatographers in the 1980s, Baram et al., constructed the first ‘portable liquid chromatograph (LC)’ weighing approximately 45 kg and utilizing a multi-wavelength photometric detector [7]. A few years later, Otagawa et al. published a paper documenting the use of another miniaturized HPLC for the onsite analysis of primary aromatic amines in coal-derived materials [8] in suspect environments. Although no specific weight was given for the system, it was considerably smaller in size than the earlier model by Baram et al. In the late 1990s, Baram et al. [9], Tulchinsky [10], and a company by the name of ICON Scientific, Inc. [11] all released new miniaturized HPLCs with continued improvements to size, power, and overall separations. The practicalities of miniaturized LC systems arise from the inherent benefits they provide. These may include but are not limited to a lower overall system solvent volume, smaller sample and mobile phase consumption, and the possible increased portability of the system. By reducing total system volume, a decrease in dwell and dead volume as well as extra-column volume is expected. The dwell volume, otherwise known as the volume difference between the systems delivery method and the front of the column, is impacted by the tubing (both internal diameter (I.D.) and length) as well as any valves/mixers that make up the total fluid path[12]. Extra-column volume, or the volume from the injector to the detector, may consist of tubing connections, detector flow cells, preheaters, injection volume, as well as the column volume. Both dwell volume and extra-column volume effect the overall separation but in very different capacities. Dwell volume may impact the separation time as well as the gradient while extra-column volume impacts the peak width, efficiency, and resolution of the separation. Along the same lines but often confused, dead-volume, or the volume of they system that is unmoving or unswept through the chromatographic system, can be a serious problem for chromatographers leading to the tailing and broadening of peaks thus compromising peak separation and quantification and in turn resulting in an overall lower peak resolution [13]. By minimizing these unwanted volumes through the overall decrease in the systems size, one hopes to improve their overall separation.

Point-of-Care (POC) analysis is becoming an ever-popular necessity in the fields of medicine [14], forensics [15], homeland security [16], and biomedical analysis [17] to name a few. However, due to traditional drawbacks of LC including size, weight, cost, and instrument complexity, HPLC has not been widely implemented in the field. With continued improvements to the various LC components including the pumping systems, injectors, columns, and detectors through the use of new machining techniques, development of smaller electronics and breakthroughs in material science, truly miniaturized HPLC systems are a reality in the 21st century leading to an ever-increasing possibility of applications. The aim of this review is to highlight some of the recent advancements in microHPLC systems (HPLC). We will focus on the major components that constitute these instruments and present a few representative miniaturized HPLC systems that have been developed to date. Schemes or approaches that miniaturize only components of an HPLC system may provide a path towards miniaturization but are not covered in detail within this paper. For example, a microfluidic chip with an LC column but a bulky fluid handling and detection system [18] is not considered to be a miniaturized HPLC. We will discuss briefly some of the applications. Representative papers are included but literatures are not exhaustively searched.

2 Miniaturized LC Components 2.1 Pumps/Gradient Generation Pumps are generally thought of as the heart of an HPLC, due to their responsibility for generating not only the high pressures required, but also the gradient to both drive and separate the sample. Commercially, piston pumps are primarily used, but depending on the application and system configuration; diaphragm [19], reciprocating [20], electroosmotic [21], and syringe [9] pumps may also be integrated. Pump Pressures ranging between 50-400 bar are typical to push the liquid through the systems entirety in a timely manner. The pumping pressure required is directly proportional to the systems backpressure caused primarily by the column [22]. Both particle diameter and column radius are key factors contributing to back pressure. Flow rates for typical liquid chromatography systems may range from tens of nL/min to several mLs/min. This orders of magnitude difference in flow rate is directly correlated to the columns pressure and detectors requirement. When constructing a new LC system, especially one that is miniaturized, careful considerations must be made into the pump’s pros and cons including its weight, power consumption, stability and reproducibility of its gradient and flow rate, a broad pressure and flow rate range, low complexity and a minimal detectable noise level. Depending on the column type and whether the system is capillary or a chipbased system, a relatively high flow (mL/min) or a nanoflow (nL/min) system

may be chosen. Most conventional HPLC pumps will be combined with a flowsplitter in order to obtain the desired lower flow rate. However, split systems may provide irreproducible results due to variable split ratios (especially when this number is high) and flow fluctuations due to the varying viscosity of the gradient solvent mixtures [23]. Due to these inherent problems with split systems, it is preferred to utilize split-less flow paths through the use of syringes or electroosmotic pumps in nano liquid chromatography systems. 2.1.1 Piston/Syringe Pump Early miniaturized systems (as covered earlier) almost explicitly adopted piston pumps, as these were the pumps that were traditionally used in commercial systems. Piston pumps may come as a single-piston reciprocating or a dual-piston pump. Early single-piston reciprocating pumps [24] had the drawback of sinusoidal pressure pulsations requiring a pulse dampener to limit these effects. High flow rates are also required when utilizing a reciprocating pump for gradient elution since the mobile phase concentration cannot change during a single disbursement of the pump. Dual-piston pumps, as utilized in an early miniaturized system by Tulchinsky et al. [10], produce a low-pulsation flow by programing the pumps 180 degrees out of phase from one another [25]. This configuration also enables a lower flow rate range for the pump but at a cost of size, weight, and complexity. Similarly, a syringe pump may be thought of as a type of piston pump. However, syringe pumps find themselves limited to a finite volume of solvent for the separation. This may not prove to be a problem as long as the separation run can be completed before the syringe volume is depleted. As with other piston pump systems, syringe pumps may be arranged in a single-syringe or dual-syringe configuration to enable gradient separations if desired. Generally speaking, the major pros for syringe pumps are attributed to their low cost and lack of complexity while still being relatively precise. Flow rates of syringe pumps are not always being stable due to the fact they are controlled through displacement rather than pressure leading to one of their underlining issues compared to that of piston pumps. With the turn of the century, piston and syringe pumps continue being the pump of choice in new miniaturized HPLC systems [26-35] primarily due to their robustness, but their major drawback of size continues being a limiting constraint for constructing miniaturized HPLC systems. Commercial companies are starting to see this trend in LC miniaturization and are beginning to offer smaller pump components to researchers who are looking to develop these miniaturized LC systems. Li et al. [31] demonstrated one such syringe pump system provided by LabSmith [36]. Other companies commercializing miniature pumps that may be applied to liquid chromatography include The Lee Company [37], Sciex [38], and Eksigent Technologies [39] which is now a subsidiary of Sciex.

2.1.2 Electroosmotic Pump The growing trend in micro analytical systems, including miniaturized HPLC systems, has allowed the prospect for electroosmotic pumps (EOPs) to be popularized in recent decades [40-42]. EOPs utilize electroosmosis through charged porous media (pumping elements) to generate pressure and flow. Electroosmotic pumps offer a cost-effective and simplistic method for producing the necessary flow rates and pressures required for liquid chromatographic systems while having several other inherent benefits. For example, this style of pump naturally creates pulse-free flows, unlike most traditional pump systems. The liquid profile of EOPs more resemble a plug rather than parabolic in shape with pressure driven systems. The rate and direction of flow is easily manipulated through a change in the voltage magnitude and polarity [43]. These pumps are readily miniaturized and integrated in both lab-on-chip and traditional systems, all the while reducing complexity due to no moving parts. Liu’s group has developed different electroosmotic pump configurations for use in portable LC systems. With the advent of monolithic packed columns for electroosmotic pumping systems, step [44, 45], linear [46, 47] and programmable [48] gradients were all obtained. Pumps capable of generating 1200 bar [49] have been reported, however lower pressures (~120 bar) are more routinely used to drive separations at a flow rate of ~200 nL/min. Although electroosmotic pumping systems are small in size and have low dwell volume compared to conventional pumping systems, limitations to EOP do exist. These may include pump-solution incompatibility with high organic contents, flow rate fluctuations due to condition variations at the pumping element surfaces, unstable voltage sources, and/or chemical breakdown within the pumping element itself. Each of these limitations lend to the fact that EOPs have never been commercialized in the 20 years of their existence. Although EOP pressures and flow rates are approaching the workable range for LC separations and its size lends itself to a miniaturized HPLC system, further research is needed to provide stable flow rates and a reproducible and programmable gradient before they will ever reach the market. 2.1.3 Constant-Pressure Pump Two types of constant-pressure pumps are available today for incorporation into a liquid chromatography system. These include a pressure-regulated air chamber and a pneumatic pressure amplifier. A pressure-regulated air chamber has

been used as a constant-pressure source for open tubular liquid chromatography [50-52]. On the other hand, pneumatic pressure amplifiers have found their place in the new field of Ultrahigh-Pressure Reversed-Phase Liquid Chromatography (UPLC). UPLC, developed by Macnair et al. [53] in the late 1990s, operates at pressures greater than 1300 bar (much higher than the 400 bar for standard HPLC systems) and takes advantage of the high efficiency offered by small column particles. The major concern of using a constant-pressure rather than constant flow rate source is the fluctuation in flow rate with changing flow resistance. This may arise with changes in eluent viscosity, column flow

resistance, temperature, etc. However, this issue may be overcome through the addition of a flow sensor and the use a feedback loop. Because of its simplicity and low-cost, this kind of pump may prove to be an option in the development of inexpensive microHPLC systems, however, at the current time, constant-pressure pumps are not utilized compared to constant-flow within LC systems. 2.2

Injectors

2.2.1 Valve-Based Injector Injectors, due to their inherently small size, have not undergone the same transformation over the years as other components of the HPLC. However, when choosing a detector, a researcher must choose an injector from one of two categories: stop-flow or continuous-flow injectors. No matter what injector one chooses, low dead-volume, high switching precision, and minimal flow disturbance are always key characteristics sought to reduce band broadening and increase resolution. While the majority of previous prototypes and commercial systems use continuous-flow injection systems [8, 10, 11, 54], they typically see an increased dead-volume for this integration. Stop-flow, on the other hand, allows for much lower dead-volume compared to its counterpart and most prototypes developed within the last few years are using this injection method with either manual control or an actuated switch [33, 34, 46]. 2.2.2 Chip-Based Injector With the development of microfluidic chromatography, the integration of an injection scheme onto the chip has allowed for an even further decrease in deadvolume compared to that of stop-flow injection [55]. The two most frequently used injection techniques on a chip-based platform, gated and pinched injection, are electrokinetically driven. These injection modes are performed with high reproducibility with almost no dead-volume making them an almost perfect method. Harrison et al. [56] tried to adapt a lab-on-chip injection method for HPLC in the early 1990s, but it was not until recently where systems began to be constructed with promising separations [18, 21, 57-59].

2.3 Columns When determining which column is appropriate for a new system, consideration of backpressure, flow stability, detectors and dwell volume should be taken into account. While small columns utilize little mobile phase and thus generate minimal waste making them ideal for miniaturization, they may pose challenges in generating a stable gradient at low flow rates, finding suitable detectors, and finding an applicable pump for the high backpressure. 2.3.1 Capillary

Since the early HPLC systems, it has been known that in order to increase resolution and decrease separation times, packing particle sizes and column dimensions need to be minimized. The advent of microcolumns (0.5-1.5 mm i.d.) lead to the development of capillary-based columns (0.1-0.05 mm i.d.) and then to microcapillary columns (50-200 μm i.d.) [60] in the late 1970s. Tsuda et al. [61] as well as Jorgenson’s groups [62, 63] continued to pioneer columns with the further reduction of column diameter to 5-500 μm i.d. . During this development of microcapillary columns, much research went into the slurry packing of fused silica particles in order to increase the columns homogeneity and thus improve its efficiency and reproducibility [62, 64-70]. Jorgenson, in one of these developments, decreased the particle diameter to 1.5 and 1 μm i.d. showing that extremely high efficiency columns (<200,000 plates/m) could be developed [71]. Through this decreased column size and particle diameter a new type of liquid chromatography, ultrahigh-pressure liquid chromatography (UHPLC) where pressures may reach levels higher than 1100 bar [53], has been developed. Researchers soon realized that although decreasing the packing particles size can lead to more than one million plates/m [72], very high pressure pumps were required to drive these separations. This requirement thus limited the application of these columns to miniaturized liquid chromatography systems. Monolithic capillary columns, conceived in the early 1990s, addressed this backpressure issue by replacing a packed slurry with polymerized monomers within the column [7375]. To date, separation efficiencies with monolithic columns between 100,000 and 250,000 plate/m have been obtained [76-78] and with added functionalization including organic polymers [79, 80] and zwitterionic functionalization as well as incorporation of metallic nanoparticles, metal organic frameworks, and carbonbased nanomaterials, monolithic capillary-based columns continue being on the forefront of column research. Filled columns, whether they are packed or contain a monolith, represent one subclass of capillary-based columns. Open tubular capillary columns, in turn, represent the other. Open tubular capillary columns have only recently (last two decades) begun to make an impact on chromatographic separations compared to that of filled columns. Open tubular columns can exist as porous layer open tubular (PLOT) [81-85] or wall coated open tubular (WCOT) columns [86-88]. Improvements in coatings of open tubular capillary columns have allowed for an increase in column efficiency while taking advantage of a smaller inner diameter that minimizes the effect of dispersion compared to that of packed columns. However, generally speaking, the overall length of the open tubular column must be increased in order to create an equivalent separation to that of a filled column. 2.3.2 Chip-based columns Like injectors, columns’ transition to chip-based systems have made large strides in the past couple decades. Leading to fully functional HPLC systems, packed silica, polymerized monolithic columns, and open tubular capillary-based columns have all been developed and integrated onto chips [18, 21, 57, 89-91].

Companies such as Agilent [92], ParaFluidics, Waters, and the late Nanostream all have commercialized and released chip-based cartridges for the incorporation into HPLC systems.

2.4 Detectors As the size of chromatographic systems continue to decrease, the stress is placed for the detection systems to complement the separations. Extensive amounts of research and review articles have gone into various detection methods including Raman Spectroscopy [15, 16, 93, 94], UV, Visible and NIR Spectroscopy [95100], Laser and LED Induced Fluorescence [101-106], electrochemical detectors [107-112], X-ray fluorescence [113-115]. The majorities of these miniaturized detectors are designed around the detection of a specific class of compounds but may be adapted to supply the needs of others. The simplest class of detectors to miniaturize and thus integrate into an HPLC system is absorbance detectors. Although absorbance can give a great detail of information, the researcher is limited to a specific subset of substances based on what wavelength of light he/she chooses. Mass spectrometry on the other hand can provide the researcher with a great deal of information both qualitatively and quantitatively. Miniature mass spectrometer development and commercialization are progressing rapidly [116] and many applications are anticipated as HPLC and miniaturized mass spectrometer are integrated with one another. 2.4.1 Absorbance Detectors Absorbance detectors have existed since the beginning of liquid chromatography. The first commercialized fixed wavelength detector was announced in 1978 for use with HPLC [117]. Since then, advancements in electronics, light sources, and optics have lead to the decrease in detector sizes. One of the greatest breakthroughs in technology enabling the advancement in microsized detectors is the use of light emitting diodes (LEDs) as the light source. Within the last decade, numerous absorbance detectors have been developed, most integrating LEDs both in the visible and UV region, as their light sources [118-124]. Laser Induced Fluorescent (LIF) detection is a highly sensitive technique for analyzing native fluorescent and fluorescently tagged molecules. LIF detectors are highly suitable for miniaturized LC systems due to their low sample consumption, short testing time, high sensitivity, a relative low complexity of their components and are easily integrated into microfluidic chip-based systems . Compact LIF detectors continue to be developed throughout the academic community. Fang et al. [101] developed a handheld LIF detector with a 450 nm laser diode and tested the prototype with CE, flow cytometry, and droplet analysis. Novak et al. [125] developed a low-cost miniaturized LIF detector for lab-on-chip applications with sensitivities in the nanomolar region and incorporated lock-in amplification for measurements under ambient light. The last notable LIF detector to be recently developed utilized a solid-state 488 nm capable of detecting labeled protein

separations to the attomolar region and was developed by Weaver et al. [102]. Although LIF detectors lend themselves to miniaturized LC systems with their high sensitivities and small size, the researcher is limited to applications based not only on the laser wavelength chosen but also the inherent need for a fluoraphore, whether that be native or labeled, to exist in the first place. 2.4.2 Electrochemical Detectors and Conductivity Detectors Although currently not as popular as absorbance detectors, electrochemical detectors (ECDs) possess the highest potential to be integrated with microfluidic conduits. For detection of electrochemically active compounds (phenols, aromatic amines, and antioxidants to name a few), ECDs often offer a high degree of selectivity and sensitivity [126]. Contributing factors that make ECD a potential choice for miniaturized HPLC systems include electrode composition and its respective potential, nM detection limits, a linear dynamic range over four magnitudes, and noise levels in the nano to picoamperes. One such detector system, capacitively coupled contactless conductivity detection (C4D) [127] is an attractive zero-dead-volume electrochemical detection scheme for HPLC when measurable admittance changes of an analyte are detectable. Although commercial capillary detectors exist, they tend to be expensive and bulky with a large portion devoted to the electronics. By creating an ECD to specific to the detection needs of the HPLC system and integrating the systems electronics, a reduction in the bulkiness of the detector is possible. In recent years, microfluidic coupled ECDs have been developed with applications including detection of antibiotics [107], antigens in immunoassays [109], creation of biosensors [110, 128], and industrial sensors [111, 112]. 2.4.3 Miniaturization in Mass Spectrometer Mass spectrometry (MS) is generally seen as universal and superior compared to most other HPLC detection methods. Mass spectrometers, however, are usually bulky, delicate, and expensive, which limits its utilization in miniaturized HPLC systems. Within the last decade, the advancement and miniaturization of mass spectrometers has taken great strides and a variety of both articles and reviews [116, 129, 130] have been published. Research into miniature mass spectrometry can be grouped into the following categories: miniature ion trap MS [131-134]; ambient pressure miniature MS [135-137]; and fully integrated systems [138-144]. Although the thought of a fully integrated and miniaturized LC/MS system could prove to have a wide reaching benefits in a variety of fields and applications, vacuum requirements of LC/MS continue to pose a challenge inhibiting a truly miniature mass spectrometer due to the inherent size of vacuum pumps. Several companies are beginning to commercialize miniature mass spectrometers to capitalize on the need for highly sensitive and informative field analyses [145148]. In recent years, miniaturized MS has allowed advancement in numerous fields such as medicine, forensics, and agriculture through eliminating barriers

associated with the size and weight restraints of conventional MS. Also, the complexity of these miniature devices is generally far less, enabling the adaptation of these detectors by the nonscientific community. Currently, miniature MS allows for clinical diagnostics [136] and surgical procedures [142], bomb threats [129, 149], hazardous material detection [132, 141], or food contaminants [150] to be tested and analyzed in real time.

3 Functional Systems Table 1 Pump Type Electroosmotic Lynch 2017 [46] Ishida 2015 [21] Syringe Piston Sharma 2015 [34] Sharma 2014 [33] Boringa 1998 [151] Baram 1996 [9] Dual-Piston Commercial [11] Commercial [54] Tulchinsky [10]

Weight (kg)

Dimensions (cm)

3

20 x 20 x 17.5

2

26 x 18 x 21

4.5

31 x 18x 14

1.75

-

10

28 x 43 x 15

14

53 x 20 x 30

Injector Stop-flow (60 nL) Continuousflow (20 nL) Stop-flow (60 nL) Stop-flow (60 nL) Stop-flow (100 nL) Auto-stopflow (1-100 μL)

Column

Detector

Silica RP

fixed-λ UV*

Micro-chip RP

Micro EDC

Monolith

fixed-λ UV

Monolith

fixed-λ UV

AEX

EDC

Silica RP

multi-λ UV

Continuousfixed-λ UV flow (20 nL) Continuousfixed-λ UV flow (20 nL) Continuous9.5 41 x 25 x 23 Silica RP fixed-λ UV flow (20 nL) ContinuousOtagawa [8] Silica RP EDC flow (20 nL) Table 1. Fully Developed miniaturized HPLC systems. The various miniaturized HPLC systems over the years classified based on their weight, size, flow type, column, and detector. 3.5

12 x 19

Miniaturized liquid chromatography systems lend themselves towards increased portability. In order to successfully construct a portable device, one must take into consideration the overall weight of the system, ability to provide both isocratic and gradient elution, a stable flow rate, and ease of operation. Advancements in pumping systems, columns, and detectors have driven the development of micro Total Analytical Systems (microTAS). Several groups have coupled these microTAS with liquid chromatography to create portable devices as summarized in Table 1 and several key developments are summarized below. 3.1

HPLC Cartridge [46] Lynch et al. published an HPLC cartridge [46] as a proof of concept prototype resulting from a culmination of recent work regarding gradient generation systems [44, 45, 47-49] as discussed previously. This cartridge, as seen in Figure 1, demonstrated the feasibility of utilizing electroosmotic pumps and a series of valves (60 nL injector, 10 port selection valve, and 12-port two way valve) to separate complex mixtures (bovine serum albumin and myoglobin digests) even though the system did not include a detector and thus is not considered a fully integrated LC system. By developing a miniature high voltage power supply and using the inherent small nature of electroosmotic pumps, Lynch et al. were able to create a gradient on a very small-scale system platform that was powered entirely by a computer’s USB port.

3.2 Hand-Portable HPLC [32, 34] Sharma et al. characterized and reported two functioning portable LC systems in 2014 and 2015. The first, an isocratic nanoflow pumping system was integrated with a fixed wavelength (254 nm) UV detector. The system was demonstrated through the isocratic separation of 6 different benzene compounds over the course of 19 minutes [33]. It showed promising results; however, the limitation of isocratic gradient does not allow for complex sample analysis. Shortly after the publication of the first system, a modification to the pumping system from a single syringe pump to a dual syringe pump system allowed for programmable gradient elution shown in Figure 2. The system weighed in at a total of ~4.5 kg and was capable of generating up to 550 bar pumping pressure, a 60 nL injection volume, and a maximum gradient loop capacity of 74 μL with a typical flow rate at 350 nL/min. Sharma et al. characterized the system using a three-component pesticide mixture as well as a five phenol mixture [34].

3.3 Ishida [21] The last notable portable liquid chromatograph system within recent years was one developed by Ishida et al. [21] in 2015 and is shown in Figure 3. It consisted of a battery-operated system integrated with an electroosmotic pump and a microfluidic device containing an integrated column and electrochemical detector. The flow

rate ranged from 0 to 10 μL/min with a high degree of precision due to the use of flow sensors. The overall weight of the system was 2 kg, proving to be one of the more lightweight systems ever developed and had an operating time of 24 h with dry batteries. The chip itself was designed to minimize the dead volume between the column end and the detector; limiting it to only 10 nL. The column was a packed ODS particle reverse-phase column and the system performance was tested using standards of alkylphenols, catecholamine, catechin, and amino acids[21]. This is the first complete system integrating a microfluidic chip and shows a promising future for microfluidic HPLC systems. Although no other complete chip based HPLC systems have been created, multiple groups continue to provide research into the field of chip-based HPLC [91, 152154] Two groups in recent years worth noting are Geisser et al. [155]and Yin et al. [18]. Geisser et al. has focused their attention on the monitoring of microchip HPLC separations through the use of coherent anti-stokes Raman scattering (CARS) as well as epi-fluorescence through the use of lasers and LEDs as their light sources respectively. Yin et al. has focused on the integration of microchip HPLC with mass spectrometry. They were able to combine an enrichment column, separation column, a nanospray tip for MS, and the fittings needed to connect these components together.

4

Applications

4.1 Pharmaceutical In recent years liquid chromatography has become a gold standard technique used in the pharmaceutical industry and accounts for the leading analytical technique within the field[156]. From prep liquid chromatography for drug discovery to the last steps of validation and quality assurance before a compound is released to the public; liquid chromatography continues being at the forefront of drug research. Although miniaturized HPLC systems may never find a home within large corporation laboratories for drug development; early drug discovery could definitely benefit from on site parallel-HPLC for high-throughput analysis or a system for direct sample collection and analysis to be further studied elsewhere [157-160]. Welch et al. [161] studied two commercial systems at the time, Nanostream’s Veloce 24 channel microfluidic HPLC [162] and Eksigent’s 800 8 channel HPLC [39] and compared their performance for high throughput LC separations for pharmaceutical process research. The Eksigent system was found to provide very fast gradients and separations leading to the conclusion that on-chip parallel chromatographic techniques have great analyzing potential for pharmaceutical research. Sample preparation is one of the most essential part, however, remains one of the most difficult at the micro-scale. Detection methods for a microTAS system also probe to be a challenging task but with sensitive detection methods such as LIF and mass spectrometry taking strides towards

miniaturization, a microTAS/LC system will likely become a reality in the near future.

4.2 Biomedical The separation and analysis of biological samples for detection of diseases and infections continues to be a source of quality research. Lazar et al. [163] has developed a microfluidic LC system capable of positively identifying 77 proteins, 39 of which are known as specific cancer biomarkers. He later incorporated his system with an on-chip MALDI-MS detection for detection of proteins with biomarkers for breast epithelial cell extracts. Through the use of biomarkers such as antigen/antibody complexes [164], or compounds indicative of trauma or an infectious disease or tumor [165, 166], scientists have been able to develop POC analytical techniques through liquid chromatography. Meagher et al. [167] showed the capability of an microfluidic electrophoretic immunoassay platform to detect biological toxins. As research into POC devices increases, miniaturized LC systems may become the forefront of disease detection and prevention through the use of biomarkers and immunoassays.

4.3 Forensics Conventional liquid chromatography and gas chromatography systems have been and continue being the instruments of choice for forensic analysis [15]. In current available literature, the vast majority of applications of LC within forensics involves detection of illicit drugs [168], metabolites [169], and poisons through MS. LC has also been utilized, although not extensively, to analyze, fiber and lipstick dyes [170], gunshot residues [171], amino acid residues from fingerprints, and both chemical and biological warfare agents. Hsieh et al. [160] was able to quantify eight common penicillin dosages in a variety of liquid and tissue samples using nano-LC. Both the limit of quantification (LOQ) and limit of detection using UV detection were shown to be better than that of typical ion trap mass spectrometry. Typical LC instruments, however, are impractical for field analysis and sample degradation may occur from the time of sample collection until analysis can take place. Also, sample size may be an issue when conducting forensics. All of these factors point to the need of a more portable, miniaturized system with comparable sensitivity to conventional detection methods.

4.4 Environment and Agriculture As with the applications mentioned above, conventional methods utilized in agricultural and environmental analysis lend themselves only to a qualified and dedicated research laboratory. Wastewater analysis from factories or mining and drilling locations soil analysis after a contamination source occurs are both examples of when field-testing for toxic compounds would be useful [172-175]. An immediate analysis and mapping of a man-made disaster could lead to a more

timely and better response from agencies such as the EPA within the United States [176]. The agricultural sector in recent years has invested time and money into research pertaining to herbicides and pesticides and their residual impact on both the environment and human consumption of the crops [177].

5 Prospective One of the current trends of advanced analytical chemistry is the miniaturization of analytical instruments. A HPLC system often leads to increased separation speed, reduced sample/reagent consumption and a lower instrument cost compared to that of a traditional system. Another particularly important application of HPLC is the capability of parallel analysis with multiple instruments (microTAS) in the same space as the footprint of a conventional system. However, miniaturizing an HPLC system also brings inherent challenges. A HPLC may not prove to be as robust as a conventional HPLC system because some delicate components are sensitive to environmental condition (such as mechanical vibration, temperature or electrostatic/magnetic field) changes. In addition, a reduced amount of sample is injected into a HPLC for analysis, which may also lead to decreased reproducibility. With the continued advancement of precision machining and operation automation, we expect that these challenges can be overcome soon. Currently, the cost of commercialization of miniaturized HPLC systems by the instrument industry has not been warranted by the demand for such instruments globally. However, miniaturized equipment offers unique benefits that cannot be matched by traditional, large-scale equipment, especially in the areas of POC applications. With the development and perfection of miniaturized mass spectrometers [116] and personal devices (such as cellular phones, smart watches, etc.), we anticipate HPLC will be routinely used in remote field, biotech companies, doctor’s offices, etc. As these applications increase and demand for these types of instruments grow, commercialization of these instruments may once again gain in popularity.

Competing Interest Statement I declare that I have no significant competing financial, professional or personal interests that might have influenced the performance or presentation of the work described in this manuscript.

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Figure 1. Cartridge system developed by Lynch et al. The proof of concept system included an innovative electroosmotic pump and miniaturized HVPS. (A) A schematic as to how the system works along with the different components. (B) Three-dimensional rendering of the µHPLC system made within the laboratory. (C) Comprised of an EOP system, high voltage power supply, a 12-port 2-position valve, a 10-port stream selector valve, a 60 nL injector, capillary column, and a UV detector.

Figure 2. Hand-portable HPLC system developed by Sharma et al. It utilizes custom pumps developed by Vici to deliver a consistent flow rate and reproducible gradient. They utilize a miniaturized LED detector built in-house as their detector.

Figure 3. Fully integrated microchip HPLC system. Through the use of an electroosmotic pump and on-chip column and detector, Ishida et al. developed this functional miniaturized HPLC system capable of flow rates into the µL/min range while utilizing an electrochemical detector to identify their analytes of interest.