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HPLC-MS with glass chips featuring monolithically integrated electrospray emitters of different geometries Carsten Lotter, Josef J. Heiland, Sebastian Thurmann, Laura Mauritz, and Detlev Belder Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04583 • Publication Date (Web): 03 Feb 2016 Downloaded from http://pubs.acs.org on February 6, 2016
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Analytical Chemistry
HPLC-MS with glass chips featuring monolithically integrated electrospray emitters of different geometries Carsten Lottera, Josef J. Heilanda, Sebastian Thurmanna, Laura Mauritza and Detlev Beldera* a
Institute of Analytical Chemistry, University of Leipzig, Linnéstraße 3, 04103 Leipzig, Germany
ABSTRACT: We present and evaluate an approach for coupling liquid chromatography in glass chips with mass spectrometry via fully integrated electrospray emitters. We developed an instrumental platform which allows a robust and reproducible operation of high performance chip chromatography coupled to mass spectrometry. A comparison of differently shaped emitters, from flat over edged to pulled geometries, revealed that all types performed equally well for typical nano-HPLC flow rates. At very low flow rates below 50 nL·min-1 very sharp, pulled nanospray emitters turned out to be mandatory for the generation of a stable electrospray.
High performance liquid chromatography (HPLC) is one of the most popular and versatile separation techniques in analytical chemistry. The miniaturisation of HPLC in the context of lab-on-a-chip technology is a very active field of research. The current status has been summarized in recent reviews.1–4 Since the first introduction of the approach in 19905 the concept and instrumentation has matured significantly, which is also reflected by the commercial introduction of HPLC-chips by companies like Agilent, Eksigent and Waters. As the winning combination of HPLC with mass spectrometry (MS) is one of the workhorses in modern analytical laboratories, mass spectrometry appears to be the natural choice as detection technique for corresponding HPLC-chip systems. The predominant ionisation and sample transfer techniques for liquid phase separations with mass spectrometry rely on the electrospray ionisation (ESI) process. Respective chip-ESI-MS approaches have been summarized by recent reviews.6–10 An important aspect in coupling microfluidic chips with ESI-MS is the sprayer configuration and geometry. First approaches were realized by spraying from the blunt end of a chip,11–14 or by attaching an external emitter.15–20 These early studies revealed the benefit of using sharp nano-electrospray21– 23 emitters in nano-flow applications such as chip electrophoresis-MS. These data build up on previous studies on ESIneedles in mass spectrometry, showing that the emitter geometry affects the spray stability and signal intensity.24 A comprehensive study of different electrospray emitters for pressure driven flow by Reschke and Timperman revealed that the ion utilization efficiency is flow rate dependent.25 From the literature it can be concluded that spraying from a small surface area, which results in a small volume of the Taylor cone, is advantageous. For chip-MS coupling the monolithic integration of such nanospray emitters proved to be beneficial compared to the external attachment of spray needles as it avoids additional extra-column band broadening. Such integrated electrospray and nanospray emitters can be fabricated for various chip materials such as different polymers26–35, silicon36–42 and glass substrates. In the context of glass chips, which benefit of excellent optical properties, chemical resistance and mechanical
rigidity, J. M. Ramsey’s group showed an interesting approach, by electrospraying from the corner of a thin chip.43 In combination with an EOF pump they developed an electrokinetically driven MS interface for various applications44–47 which was also used for coupling liquid chromatography with chip electrophoresis, enabling two dimensional separations.48 The concept of dicing a glass chip to achieve a two dimensionally confined ESI emitter was previously shown by Yue et al.49, who angled the top and bottom plate of the diced emitter. Freire et al.50 demonstrated the use of parylene-C coated glass substrates, to spray from the chip corner, while Zhu et al.51 manually grinded the tip of a glass chip into a pyramidal shape. Sainiemi et al.52 demonstrated recently the microfabrication of three-dimensionally sharp electrospray ionisation tips by isotropic etching of two glass wafers and applied it for onchip CE-MS. We introduced an approach for the monolithic integration of pulled and etched three dimensionally sharp nanospray emitters in glass chips53 and applied them for the MS coupling of chip electrophoresis54–57, chip free-flowelectrophoresis58 and for on-chip reaction monitoring59. While numerous reports on coupling low pressure microfluidic separation devices with MS can be found in the literature there is only few methodical work on coupling high pressure HPLC-chips with mass spectrometry.60–62 An exception is the application oriented field utilizing the commercial chipHPLC-MS systems from Agilent and Waters. Waters Corp. recently launched a ceramic LC-chip (ionKey/MS™) with an attachable ESI emitter. Agilent Technologies developed a fully integrated polyimide chip27 (Chip Cube™) including an enrichment column and a laser sharpened ESI emitter, where the microfluidic contacting is elegantly solved by integrating the laminated polyimide chip into a 6-port valve. The observation that chip-electrophoresis-MS is very popular in academia while instrument companies launch solely chip-HPLC-MS systems reflects both the commercial importance of HPLC compared to capillary electrophoresis and the technical challenges in operating microfluidic devices at high pressures and flow rates. While we developed approaches for coupling chip electrophoresis with mass spectrometry and strategies to realize
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HPLC on glass chips with fluorescence detection63–66 in previous work, we now combine these technologies to realize HPLC-MS with glass chips. For this purpose we engineered a semi-automated HPLC-MS chip system. It combines high precision on-chip injection of picoliter sample volumes, high performance chromatographic separation and efficient coupling to mass spectrometry. A special focus of the study is the evaluation of suitable emitter geometries.
EXPERIMENTAL SECTION Chemicals and materials. The chemicals thiourea (99%), capsaicin (≥99%), dihydrocapsaicin (90%), 7-amino-4methylcoumarin (99%), 2-aminoanthracene (96%), 3aminofluoranthene (90%), benzanthrone (90%), LC-MS grade formic acid (98%), 3-(trimethoxysilyl)propyl methacrylate (98%), 1,3-butanediol diacrylate (98%), 2,2-dimethoxy-2phenylacetophenone (99%) and trichloro(1H,1H,2H,2Hperfluorooctyl)silane (97%) were purchased from SigmaAldrich GmbH (Taufkirchen, Germany). Gradient grade methanol, n-heptane (p.a.) and chloroform (p.a.) were acquired from VWR International LLC (Radnor, USA). High purity water was obtained from a Smart2Pure purifying system (18.2 MΩcm, TKA Wasseraufarbeitungssysteme GmbH, Niederelbert, Germany). Microchip design and fabrication. The microfluidic glass chips were designed in cooperation with and produced by iXfactory (Dortmund, Germany) by common photolithography, wet-etching and bonding methods. Briefly, a borosilicate glass slide (BOROFLOAT® 33, 1.1 mm thick) was micro-structured in a two-stage wet etching process with hydrofluoric acid. A cover glass slide with sputtered platinum electrodes and powder blasted access holes was then fusion bonded to the structured chip. The bottom slide also contains powder blasted holes to grant access to the electrodes. Due to the compact design, the diced chips measure only 10 mm · 45 mm · 2.2 mm and allow therefore a high yield per produced wafer. The layout of the microfluidic chip is shown schematically in Figure 1. It features an injection cross, a column chamber, a sheath flow channel and two platinum electrodes. The column chamber is 35 mm long and restricted by two particle retaining elements. It can be filled with particulate stationary phase material by a slurry packing process via a packing channel, located in the middle of the column. All channels up to the end of the column are 90 µm wide and 40 µm deep with typically rounded corners from the anisotropic wet etching process. Particle retaining elements are realized in a two-step etching process, allowing a reduction in channel width and height to 40 µm and 10 µm, respectively. After the column the channel remains 10 µm deep with a width of 60 µm. It has fluidic contact to the first and second platinum electrode with a sheath channel joining the main channel in between the two electrodes. The sheath channel contains a filtering element to prevent clogging of the electrospray emitter. After a U-turn close to the end of the chip, the main channel ends in an outlet port. Column and Emitter Manufacturing. The column was integrated via a slurry packing method, based on our previous work.64 Briefly, the packing setup consisted of a 6-port valve, connected to a conventional HPLC pump and the chip via capillary tubing. A homemade clamp was attached to the packing channel port of the chip to realize the fluidic connection between chip and tubing. The particulate material (ProntoSIL
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C18 SH, 3µm, BISCHOFF Analysentechnik u. -geraete GmbH, Leonberg, Germany) was suspended in methanol (MeOH) (5 mg·mL-1) and filled into the sample loop (~ 50 µL) of the 6-port valve. By switching the valve, the slurry was transferred to the chip. The column was packed and compressed at 200 bar for 30 min in an ultrasonic bath. The packing channel is located in the middle of the column chamber, ensuring a uniform and proper compression. After relaxation of the system, the packing channel was sealed via laser assisted photo polymerization. A detailed description of the setup is provided in a previous publication.66 In brief, a solution made up of 4 µL 3-(trimethoxysilyl)propyl methacrylate, 746 µL 1,3-butanediol diacrylate, 100 µL methanol and 5 mg 2,2dimethoxy-2-phenylacetophenone was purged with nitrogen and filled into the sample loop of the packing apparatus. The chip was flushed with the polymerization solution for 5 min at 100 bar. After relaxation, the chip was placed on a microscope equipped with a piezo-electric scanner and the packing channel was sealed off at the column junction by space resolved irradiation using a pulsed 355 nm laser with about 2 µW power output. Remaining monomer was removed by flushing with methanol. The electrospray emitter tips for chip-LC-MS hyphenation were manufactured in three alternative ways. Three dimensionally sharp nanospray emitters were realized by computer numerical control (CNC) milling, pulling of the heated pin and etching with hydrofluoric acid. The detailed manufacturing process and a performance evaluation of these monolithic emitters were shown in previous work.53,55 Another approach was to manually grind off the chip on a grinding wheel, sharpening the end of the chip from all four sides to a pyramidal shape. Alternatively, a wire saw (Logitech Limited, Glasgow, UK) was utilized to cut a corner at the end of the elution channel, or to generate a flat blunt end. Before first use all emitters were cleaned with ethanol, surface activated with 1 M NaOH for 30 min and hydrophobized with a solution of 15 vol% trichloro(1H,1H,2H,2H-perfluorooctyl)silane in 4:1 n-heptane:chloroform for 10 min. Contact angle measurements using an aqueous solution yielded values of about 90°, while the mainly utilized elution composition of MeOH/H2O (80/20 vol%) with 0.1% formic acid resulted in an contact angle of about 70°. Instrumentation. The fluidic circuitry was realized via a 10-port and a 6-port valve (100 µm bore, CHEMINERT C72MPKH-4670D and C72MPKH-4676D, VICI AG, Schenkon, Switzerland) and three piston pumps (1260 Iso Pump, 1200 Nano Pump, 1200 Cap Pump, each used in normal mode with electronic flow sensor bypassed, Agilent Technologies Inc., Santa Clara, USA). The valves and the microchip were interconnected via PEEK capillaries (360 µm OD, 50 or 75 µm ID, VICI AG, Schenkon, Switzerland), the microfluidic chip contacting was realized via homemade steel connection clamps. For precise positioning a XYZ linear translation stage (OWIS GmbH, Staufen i.Br., Germany) and two cameras (KJL Sicherheitssysteme GmbH & Co.KG, Hamburg, Germany) were utilized. The on-chip emitter was held at ground potential via the integrated electrodes, while the orifice of the mass spectrometer (6520 Q-TOF, Agilent Technologies Inc., Santa Clara, USA) was set to 2000 to 3000 V, depending on the emitter geometry. The chip was positioned at a distance of approximately 2 mm in front of a micro bore inline spray shield. The MS was used in TOF-only mode, with 5 L·min-1 drying gas, 300°C gas temperature, 175 V fragmentor voltage, 100 – 1000 m/z mass range and an acquisition rate of 4 spectra
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per second. The whole setup was controlled by Clarity (DataApex, Prague, Czech Republic). Microchip Operation. The chip operation and injection method is based on previously published work65. Briefly, the
6-port valve is equipped with a sample loop, allowing the injection of sample into the flow coming from the sample pump. The
Figure 1. Schematic illustration of the utilized HPLC-MS-chip. a) Microfluidic glass chip with six powder blasted access holes and two platinum electrodes. The green and yellow line mark the positions where the chip can be cut open to implement an ESI emitter for sheathless (green line) or sheath-flow (yellow line) operation mode. b) Magnification of the on-chip injection cross with a particle retaining element at the column head. c) Microscopic image of the post column region with electrode and sheath channel junctions. The insert shows an enlarged view of the filter element in the sheath channel.
10-port valve allows the distinction in two different fluid flow situations. In infusion mode, the sample flow is directed to one branch of the on-chip injection cross, while an auxiliary pinch pump keeps the second branch free of sample. In this way, sample is flushed over the injection cross along the column head, creating a sample plug directly on the column. In elution mode the 10-port valve disconnects both pumps necessary for the injection from the chip. Now only the elution pump delivers eluent to the chip, precisely to the connection port previously held free of sample by the pinching flow. Remaining sample in the injection cross is immediately flushed out and the elution begins. In both modes the branches of the on-chip cross which are not connected with pumps are connected to restriction or waste capillaries via the 10-port valve. A detailed scheme of the fluidic connections can be seen in Figure 2. For all measurements a 10 s long injection was utilized. Typical flow rates are 20 µL·min-1 for sample and pinch pump and 101000 µL·min-1 for the elution pump, resulting in a column flow of 15-1400 nL·min-1 at a split ratio of about 700:1. For column flow measurements see Figure S-1 in the supporting information. An essential aspect of this setup is the generation of an on-chip split in both modes. This allows the generation of narrow injection plugs directly on the column head, eliminating pre-column band broadening and the generation of a nano-flow over the column without the need for special nanopump equipment.
of the current contribution is the hyphenation of this technology to mass spectrometry. This builds up on our expertise in coupling chip electrophoresis with mass spectrometry, utilizing glass chips with monolithically integrated nanospray tips.55,53 In order to realize an efficient and versatile hyphenation to mass
RESULTS AND DISCUSSION In previous works we developed approaches for high quality HPLC in microfluidic glass chips,65,66 where the separations were monitored by fluorescence detection. The scientific focus
Figure 2. Scheme of the fluidic connections in a) injection and b) elution mode. Red and green lines symbolize connection capillar-
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ies, where green lines deliver solvent to the chip and red lines end in the waste via restriction capillaries. The inserts I) and II) describe the fluid flow situation in the on-chip injection cross in injection and elution mode, respectively. Length and inner diameter of used PEEK capillaries (outer diameter: 360 µm): (1) 20 cm, 100 µm; (2) 20 cm, 100 µm; (3) 14 cm, 75 µm; (4) 30 cm, 75 µm; (5) 20 cm, 75 µm; (6) 14 cm, 75 µm; (7) 20 cm, 75 µm; (8) 20 cm, 75 µm & 35 cm, 50 µm; (9) 25 cm, 75 µm; (10) 14 cm, 75 µm; (11) 50 cm, 50 µm; (12) 100 cm, 150 µm; (13) 15 cm, 75 µm.
Figure 3. Schematic illustration of the developed instrumental platform for chip-HPLC-MS hyphenation. The platform replaces the sprayer unit of the mass spectrometer. Fluidic connections within the platform are realized via capillary tubing (not shown for simplicity). Crucial parts: 1 6-port valve, 2 10-port valve, 3 XYZ linear translation stage, 4 cameras, 5 chromatographic chip with homemade fluidic connection clamps in front of the MS orifice, 6 connection to piston pumps via 1/16” tubing, 7 sample injection port with syringe.
spectrometry we designed a new chip layout. As schematically illustrated in Figure 1 the developed glass chip includes an onchip cross for injection and flow splitting and a column channel which is defined by two particle retaining elements. Two platinum electrodes were included to define the electrospray potential when connected to a high voltage source. An optional sheath flow channel joins the elution channel in between the electrodes. For MS coupling the chip has to be diced to open the outlet channel for the electrospray. The layout offers two alternative options as indicated by the cutting lines in Figure 1. The first cutting line (green line in Figure 1) just behind the first embedded platinum electrode is intended to enable a sheath-less electrospray ionisation process. If the chip is cut after the sheath flow channel along the second cutting line (yellow line in Figure 1), the chip can be used in a sheath-flow operation mode to add for example modifiers to facilitate the electrospray process. In order to reliably connect the HPLC-chip to high pressure pumps and to couple the device with a mass spectrometer a platform was developed. The setup brings all necessary components in close spatial proximity and allows straightforward and reliable handling and positioning of the microchip in front of the MS orifice. Figure 3 shows a schematic drawing of the setup. It replaces the sprayer-unit of the ESI-source at the mass spectrometer inlet. The key components of this platform are a 10-port and a 6-port valve, which allows direct switching of the channel flows for injection and elution. The eluent flow is generated by three piston pumps, which are connected to the
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platform via integrated adapters, allowing the transition from 1/16” tubing to 360 µm capillaries. The microchip is fluidly connected by homemade high pressure tight connection clamps. The exact positioning of the spray tip is realized via a XYZ linear translation stage and two cameras. A key step for the efficient coupling of particulate chromatography with mass spectrometry is the choice of the electrospray emitter. As finely pulled emitter tips proofed to behave best in our previous studies on coupling chipelectrophoresis with mass spectrometry, this was also the starting point for
Figure 4. Base peak chromatogram (BPC) via sheathless chipHPLC-MS with pulled emitter of a sample containing (1) 20 µg·mL-1 7-amino-4-methylcoumarin, (2) 15 µg·mL-1 2aminoanthracene, (3) 15 µg·mL-1 3-aminofluoranthene and (4) 50 µg·mL-1 benzanthrone. Column: ProntoSIL C18 SH, 3 µm, 35 mm. Mobile phase: MeOH/H2O (85/15 vol%) with 0.1% formic acid. Linear velocity: 3.4 mm·s-1.
chip-HPLC-MS coupling. We were able to implement sharp emitters at the end of the HPLC-chip in an analogous way as previously shown for electrophoresis chips. This is a two stage mechanical process with an initial CNC-milling step followed by pulling the heated pin. With this approach we realized the first successful chip-HPLC-MS experiments with the shown setup. A representative chromatogram of the isocratic separation of four compounds within 30 s is shown in Figure 4. However, due to a coincidental break down of the CNCmilling machine we prototyped another emitter as a practical expedient by simple grinding. To our surprise the simple grinded pyramidal shaped but blunt emitter gave comparable results in chip-HPLC-MS as the sharp pulled emitter. This finding was further evaluated in a set of experiments to thoroughly characterize both types of chip emitter. For a first and simple spray performance test two chips were prepared with a pulled and a grinded emitter. By infusion of 80% methanol and 20% H2O with 0.1% formic acid as additive via the sheath channel (about 300 nL·min-1), the stability of the formed electrospray was tested. The relative standard deviation of the total ion count (TIC) for the pulled and the grinded emitter tip over the period of two hours were 4.2% and 3.7%, respectively. Both emitter types produce a stable electrospray. For further evaluation of their performance, packed chips with both emitter geometries were prepared and operated in sheathless mode. Chromatograms of capsaicin and dihydrocapsaicin as sample analytes were recorded at a broad range of eluent flow rates for both emitter geometries. It was anticipated that the potentially larger fluid volume of the Taylor cone at the grinded emitter could affect band broadening and signal intensity. In order to explore these behaviours we studied and compared
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the achievable separation efficiencies and peak areas. In this series of experiments we found that the two emitter types performed similar at high flow rates. At flow rates below about 50 nL·min-1 a stable spray and corresponding signal could only be obtained with the pulled emitter. The results of this set of experiments are summarized in Figure 5 together with magnified images of the spray tips in action. Representa-
tive chromatograms are shown in Figure 6. The van Deemter plot in Figure 5b displays that the potentially bigger emitter area of the grinded emitter does not negatively influence the observed plate heights. Figure 5c shows an increase in peak area with decreasing flow rate with a steep increase at very low flow regimes which can only be accessed by
Figure 5. Experimental evaluation of emitter tip designs. Magnified images of the LC chip in front of the MS orifice with a) grinded and b) pulled emitter. ESI potential: 2000 V (pulled emitter), 3000 V (grinded emitter); column flow: about 450 nL·min-1 MeOH/H2O (80/20 vol%); illuminated with a 523±10 nm
