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Liquid beam desorption mass spectrometry for the investigation of continuous flow reactions in microfluidic chips Sandra Schulze, Maik Pahl, Ferdinand Stolz, Johannes Appun, Bernd Abel, Christoph Schneider, and Detlev Belder Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 10 May 2017 Downloaded from http://pubs.acs.org on May 11, 2017
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Analytical Chemistry
Liquid beam desorption mass spectrometry for the investigation of continuous flow reactions in microfluidic chips Sandra Schulze†, Maik Pahl†, Ferdinand Stolzǂ,§, Johannes Appunȹ, Bernd Abelǂ,§, Christoph Schneiderȹ and Detlev Belder*,† †
Institute of Analytical Chemistry, University Leipzig, Linnéstraße 3, 04103 Leipzig, Germany Wilhelm-Ostwald-Institute of Physical and Theoretical Chemistry, University Leipzig, Linnéstraße 3, 04103 Leipzig, Germany § Leibniz Institute of Surface Modification (IOM), Permoserstraße 15, 04318 Leipzig, Germany ǂ
ȹ
Institute of Organic Chemistry, University Leipzig, Johannisallee 29, 04103 Leipzig, Germany
ABSTRACT: In this work we present the combination of microfluidic chips and mass spectrometry employing laser-induced liquid beam ionisation/desorption. The developed system was evaluated with respect to stable beam generation, laser parameters as well as solvent compatibility. The device was exemplarily applied to study a vinylogous Mannich reaction performed in continuous flow on chip. Fast processes can be observed with this technique which could in future be beneficial for studying intermediates or contribute to the elucidation of reaction mechanisms.
Mass spectrometry is a highly sensitive detection technique and furthermore provides additionally structural information for analyte identification.9 Chip-MS coupling has been described in the literature.10,11,12,13,14,15,16 The analyte transfer from the solution inside the microfluidic chip to the mass spectrometer is realised via different approaches utilizing various interfaces and ionisation methods. Matrix-assisted laser desorption/ionisation (MALDI) has been used in combination with microfluidic systems using spotting techniques.17,18,19 Surface acoustic wave nebulisation (SAWN) is another interesting ambient ionisation technique for combining microfluidics with MS.20,21,22 For direct chip-MS coupling electrospray ionisation (ESI) is the predominant approach.23 The electrospray process can be initiated at the device utilising different emitter types24 like blunt chips25,26,27, capillaries or monolithic integrated emitters28,29. A prerequisite for electrospray ionisation is electric contacting of the emitter fluid to build up the electrical field necessary for the ESI-process. While ESI is an ideal ionisation and sample transfer technique for many lab-on-a-chip MS applications like chipHPLC/MS10-11, 13-15 or chip-electrophoresis/MS30 there are some limitations. Electrospray ionisation from the chip edge relies on low flow rates but is problematic at higher flow rates exceeding the nL/min range31,32 as this disrupts the formation of a stable Taylor cone. Furthermore for electrospray ionisation electrical contacting of the emitter is mandatory which can be challenging. Beside manufacturing issues, on-chip integration of respective electrodes and electrically contacting elements can also induce troublesome effects, such as electrolytic bubble generation or detrimental effects of the electrical
INTRODUCTION The concept of lab-on-a-chip technology includes the downscaling of common chemical processes of a routine laboratory into a miniature format. Such devices are regarded as an enabling technology for modern 21st century chemistry 1,2. It comprises unit operations as well as chemical reactions, separation techniques and online analysis on single microscale devices.3 In such shrunken chemical laboratories very low sample amounts are handled, which poses a challenge in realising appropriate detection techniques. For this purpose electrochemical4, optical5 and especially fluorescence detection are commonly performed in the microscale. Electrochemical detection has high potential for system integration but the application range and respective achievable sensitivities strongly depend on the utilised electrochemical technique.6 UV/Vis absorbance detection is widely used in traditional analytical equipment but often suffers from low sensitivity when performed on the microscale due to short optical pathlengths.7 While chemiluminescence detection has a rather limited scope, fluorescence detection is very popular in labon-a-chip applications as it is technically easy to implement and provides excellent sensitivity. A major drawback is however the often necessary analyte labelling. This not only requires an additional, potential troublesome8 working step but also changes the chemical and physical properties of the molecules. While fluorescence tagging is less problematic for some biological applications it can usually not be applied for monitoring common organic chemical reactions. In this context mass spectrometry (MS), as the current analytical workhorse in classical laboratories, is very appealing. 1
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fields e.g. in electrochemical sensing or in some electrophoretic applications like chip-FFE/MS33. In this context laser-induced liquid beam ionisation/desorption MS34 (LILBID-MS) also named infrared matrix-assisted laser desorption/ionisation MS (IR-MALDIMS)35,36,37,38,39,40,41 is an attractive alternative. In this technique liquid micro jets are generated from nozzles42,43 with high velocity. An IR laser is directed to this liquid beam inducing desorption and ionisation of the dissolved compounds to study non-covalent interactions of inorganic as well as bio- and macromolecules in aqueous media.44,45,46,47,48 Such liquid beams can be realised by expansion of the liquid from the nozzle into vacuum. Furthermore, free liquid water droplets49 or levitated droplets50 have been investigated with LILBIDMS and the technique has been applied to study fast hightemperature reactions51. In addition, liquid jets can also be generated at ambient conditions52, which makes this technique perfectly compatible to common mass spectrometers with atmospheric pressure interfaces. Unlike in electrospray ionisation, electrical contacting of the fluid is not required in LILBID-MS. Furthermore, as the solution is emitted from a nozzle at a high velocity the common problem of sample spreading on the chip edges which complicates off-chip electrospray ionisation is less evident. It is therefore an interesting alternative to ESI for an intended coupling of microfluidic chips with mass spectrometry especially when working at very high flow rates and/or if the integration of electrodes for building up the electrospray potential poses a challenge. In this work we evaluate the potential of this technology for chip/MS with an emphasis on studying organic reactions in continuous flow. We present an approach to directly form and eject liquid jets from microfluidic reactor chips and to irradiate and evaporate the liquid beam in front of a mass spectrometer by an IR laser. For this purpose we developed microfluidic glass chips with integrated nozzles, which generate a stable liquid beam at ambient conditions. The working parameters of the system were evaluated using arginine as model analyte. The functionality of the new-established chip-LILBID-MS system is demonstrated in a proof-of-concept for the investigation of an organic model reaction.
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phy with chrome masks, wet chemical etching and bonding. Detailed description can be found in the Supporting Information (Figure S-1). Briefly, microchips were prepared using photomasks with the desired chip layout and glass slides coated with a positive photoresist which was developed after UV irradiation. After two etching steps the obtained structured bottom plate was bonded to a cover plate which contained powder blasted holes for microfluidic contacting. Integrated nozzles for liquid beam generation were generated in two different ways. One approach included cutting the chip from the sides and grinding the tip manually on a grinding disc from the top and bottom side.54 Emitter tips were also prepared by computer numerical control (CNC) milling of a cone, capillary pulling and etching with hydrofluoric acid as described previously.55 Both methods resulted in different size and shape of the nozzle opening. General liquid beam setup. Liquid beams were either generated from a fused silica capillary (40 µm ID, CS Chromatographie Service GmbH, Langerwehe, Germany) or selffabricated full glass microchips with different integrated nozzles. Solvent delivery was performed by piston pumps (SunFlow 100, SunChrom Wissenschaftliche Geräte GmbH, Friedrichsdorf, Germany and Merck Hitachi L-6200, Merck KGaA, Darmstadt, Germany) or a high pressure syringe pump (neMESYS, cetoni GmbH, Korbußen, Germany) for highly precise flow control. The microfluidic contacting was realised via polyetheretherketone (PEEK) capillaries (360 µm OD, 75 µm ID, VICI AG, Schenkon, Switzerland) which were pressed into the powder blasted holes of the chip top plate by elastomeric ferrules and PEEK screws housed in a homemade steel connection clamp.56 The liquid beam was irradiated perpendicularly by an infrared laser pulse (Opolette IR 2731, OPOTEK, Carlsbad, CA, USA; wavelength: 2925-3000 nm, frequency: 20 Hz, pulse length: 7 ns, focus point: 150 µm) which is in vibrational resonance with the OH stretch vibration. The desorbed sample was transferred to and analysed by an ion trap mass spectrometer (1100 Series, LC/MSD Trap, Agilent Technologies, Waldbronn, Germany). A schematic representation of the setup is shown in Figure 1. A photographic image of the whole setup can be found in the Supporting Information (Figure S-2).
EXPERIMENTAL SECTION Chemicals. All chemicals were used as delivered. Sulfuric acid (96%), fluorescein sodium salt, tetrahydrofuran (≥ 99.9%) and dimethylsulfoxide (≥ 99.9%) were obtained from Carl Roth (Karlsruhe, Germany). D-arginine monohydrochloride (≥ 98%), Trichloro(1H,1H,2H,2H-perfluorooctyl)silane (97%), Rhodamine B (> 95%) and Ytterbium(III) trifluoromethanesulfonate (Yb(OTf)3, 99.99%) were purchased from Sigma Aldrich (Steinheim, Germany). Acetonitrile (≥ 99.9%), methanol (100.0%) and ethanol (96%) were acquired from VWR International GmbH (Darmstadt, Germany). Ultrapure water was obtained from the ultrapure water system Smart2Pure (TKA Wasseraufbereitungssysteme GmbH, Niederelbert, Germany). (E)-4-Methoxy-N-(4-methylbenzylidene)aniline (≥ 99%) and 1,2-bissilyldienediolate (≥ 97%) were synthesised. Microchip fabrication. Microfluidic glass chips were fabricated based on common methods53 including photolithogra-
Figure 1. Schematic drawing of the microchip liquid beam desorption mass spectrometry approach. A liquid beam is generated from a microchip emitter and irradiated by a pulsed IR laser beam. Gas-phase ions are desorbed at atmospheric pressure and are transferred to the MS inlet.
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Analytical Chemistry Yb(OTf)3 catalysis in acetonitrile was studied. The starting materials were introduced into two separate 1 mL PEEK sample loops of two six-port valves before mixing and reacting in a PEEK capillary reactor or in the microchip channel. Flow rates were adjusted depending on the desired residence time in the correspondent reactor. Further solvent was added via a Tpiece or at the acceleration structure to support liquid beam generation and to dilute the reaction mixture prior to MS detection. The reaction progress was then monitored with the setup described before. Safety considerations. Hydrofluoric acid is very hazardous and should be handled with extreme care only under a ventilated hood and with appropriated protective clothing. Exposure to both liquid and vapour is very dangerous to skin and eyes. Calcium gluconate should be kept nearby for treatment in case of an emergency exposure. The employed IR laser is harmful to eyes and skin. Wearing laser protection glasses for IR light is absolutely recommended.
Mass spectrometry. The commercial ESI source of the ion trap mass spectrometer was removed and a nano-ESI spray shield was capped on the transfer capillary. The microchip was positioned in front of the MS inlet via a home-built aluminium interface frame and a xyz-translational stage (Thorlabs, Dachau, Germany). The MS acquisition parameters were set as follows: Positive ESI mode, capillary voltage: 4000 V, dry gas (N2): 4.0 L min-1, 300°C, nebulizer gas: 10.0 psi, scan rate: 20 Hz, Bruker Compass DataAnalysis 4.2. Microchip-ESI mass spectrometry. Prior to electrospray experiments the microchip emitter was cleaned with sulfuric acid and was hydrophobised with the fluorinated silane Trichloro(1H,1H,2H,2H-perfluorooctyl)silane for better electrospray performance. Similar to liquid beam experiments the mass spectrometer (micrOTOF-Q III, Bruker Daltonik GmbH, Bremen, Germany) was equipped with a nano-ESI spray shield and a xyz-translational stage for microchip positioning. A solution of arginine in methanol/water (80/20 vol%) was filled into a sample loop of a six-port valve and was delivered to the microchip via a high pressure syringe pump (neMESYS, cetoni GmbH, Korbußen, Germany). A second microchip inlet equipped with a steel capillary was flushed with methanol/water (80/20 vol%) at the same flow rate to give a final analyte concentration of 10 µmol L-1. Electrospray formation was achieved by applying a voltage of 2500 V at the spray shield while the microchip emitter was set to 0 V using the steel capillary for electrical contact. Further MS acquisition parameters were set as follows: Positive ESI mode, dry gas (N2): 3.0 L min-1, 150°C, scan rate: 2 Hz, Bruker Compass DataAnalysis 4.2. Moreover, the electrospray was illuminated by a green laser pointer (532 nm) for taking photographs with a high-speed camera (DSC-RX10M2, Sony). System evaluation. For optimisation of the parameters of the microchip liquid beam setup liquid beams were generated from a microchip as described above and arginine served as a model analyte. Laser power and wavelength were varied and the influence on the signal intensity of arginine (10 µmol L-1 in water) was determined. For solvent screening experiments arginine was dissolved to 10 µmol L-1 in mixtures of different solvents with 1-80% water. Investigations of the acceleration structure. Solutions of 1 mmol L-1 fluorescein sodium salt and 1 mmol L-1 rhodamine B in acetonitrile/water (80/20 vol%) were introduced into sample loops of two six-port valves and delivered via high and low pressure syringe pumps (neMESYS, cetoni GmbH, Korbußen, Germany). Both fluorescent dyes were mixed at the Yjunction of the chip and were guided through the meandering reaction structure. At the acceleration structure acetonitrile was added as further solvent and the influence on dilution was studied. Therefore, the microchip was placed on an inverse epi-fluorescence microscope (IX50, Olympus, Hamburg, Germany) and a mercury lamp (HBO 130 W/2, Osram GmbH, Augsburg, Germany) served as excitation source. Excitation and emission light were separated by a standard U-MWIB filter set (Olympus). The experiments were observed using a 10-fold objective (CPlan, Olympus) and a digital camera (D90, Nikon). Continuous flow reaction. As a model reaction the vinylogous Mannich reaction of the imine (E)-4-methoxy-N-(4methyl-benzylidene)aniline and 1,2-bissilyldienediolate under
RESULTS AND DISCUSSION A prerequisite for the generation of a stable liquid beam emerging from a microfluidic channel of a glass chip is the design and manufacturing of a respective nozzle. For this purpose we investigated two different nozzle types which were obtained from glass chips either by pulling and etching or by grinding. The tip manufacturing process builds up on our previous work for the generation of differently shaped electrospray emitters.54,55 The tip openings and geometries of chips generated in the present contribution were characterised by light microscope and scanning electron microscope (SEM) images which are provided in the Supporting Information (Figure S-3). The channel cross section of ground emitters shows the same size and shape as the channel of the remaining chip. The usage of a 30 µm photomask in this chip area and the described etching protocol results in tip openings of about 79 µm x 20 µm, which act as straight jet nozzles. Tapered nozzles are prepared in analogy to our previously described nanospray emitters by a protocol using CNC milling with subsequent pulling of a heated glass pin and etching.55 Such pulled and etched emitters are characterised by an elliptical shape of the channel cross section. The opening dimension can be adjusted by the applied etching time. Such prototype glass microchips where manufactured in-house within one or two working days depending on the emitter type as documented in the Supporting Information (Section 1). Both chip types were connected to pumps via capillaries and metal-clamps to investigate liquid beam generation from the different tips using water as medium. Delivering a flow rate to a microchip leads to the formation of a drop at the tip opening which grows and finally tears down. The usage of a sufficient flow rate depending on the tip opening and pressure results in the formation of a liquid beam which is stable for a certain length of several centimetres (Figure 2a). The beam decays into droplets after a critical length as described in the literature for liquid beams from common nozzles.42
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irradiated by the IR laser this resulted in significant appearance of analyte ions and respective MS data. A typical mass spectrum obtained with a laser power of 30 mW at a wavelength of 2925 nm is shown in Figure 2c. These laser parameters were optimised in preceding experiments (see Supporting Information, Figure S-4). As evident the mass spectrum is dominated by two major signals – one for the monomer (m/z 175.1) and the other for the dimer (m/z 349.2) of the analyte. After this successful first proof of concept we further evaluated and compared the microchip-LILBID-MS approach with more common microchip-ESI-MS experiments. In these studies a solution of the analyte was pumped through the chip and the desorption/ionisation was either performed by ESI applying an electrical field or by LILBID in two sets of experiments using the same chip. In these studies we investigated a wide flow rate regime from 5 µL min-1 to 200 µL min-1. It is evident from Figure 2d where the obtained signal intensity of arginine at different flow rates is displayed that chip-ESI-MS is best suited for low flow rates while LILBID is best at high flow rates. These experiments reveal the perfect complementary nature of the two chip-MS techniques. While chip-ESI does not work above 50 µL min-1 as this deteriorates the formation of the Taylor cone, this was about the lowest flow rate to generate a liquid beam from the same emitter. These findings correspond well with microscopic images recorded during the flow dependent beam or spray as shown in Figure S-5 in the Supporting Information. After successful optimisation of the technique with regard to flow rates and laser settings we investigated the usability of alternative co-solvents. So far LILBID-MS has mainly been applied in the studies of biomolecules using aqueous media, alcohols and respective buffers.58 As the technique is especially attractive to monitor fast processes in a flow, such as the generation of intermediates in organic flow chemistry, we evaluated the compatibility with alternative solvents employed in organic synthesis. For these studies a microchip with a pulled emitter (tip opening: 17 µm x 8 µm, flow rate: 125 µL min-1) was utilised. Five water-miscible solvents, namely methanol, ethanol, tetrahydrofuran (THF), acetonitrile (MeCN) and dimethylsulfoxide (DMSO) were investigated in detail (Table 1). The analyte was diluted to 10 µmol L1 in a water/solvent-mixture and introduced into the microchip.
Figure 2. a) Microchip in front of the mass spectrometer inlet with liquid beam and laser position coloured by a red diode. b) Enlarged view of the marked area of a). c) Mass spectrum of 10 µmol L-1 D-arginine in water after microchip LILBID-MS. d) Signal intensities of 10 µmol L-1 D-arginine in methanol/water (80/20 vol%) after microchip-ESI-MS (orange) and microchipLILBID-MS (blue) at different flow rates. Relative intensity stands for the intensity of the arginine monomer signal (m/z 175.1) at each flow rate divided by the maximum intensity.
The flow rates which were necessary for generating a stable liquid beam were determined for both types of chips using water as a solvent. Ground emitters with the above mentioned size of the tip opening required flow rates of 250 µL min-1 for liquid beam generation. For pulled emitters with a tip opening of 34 µm x 17 µm a reduced flow rate of 125 µL min-1 was sufficient due to the smaller tip opening. The apparent pump pressures, affected by channel length and dimensions as well as the tip opening were recorded as well. In these studies the pressure for ground emitters were in the range of 35 bar to 44 bar whereas pulled emitters just required 24 bar to 27 bar. While we used glass chips in the present study this pressure range is also compatible with polymeric microfluidic devices such as polydimethylsiloxane (PDMS) or Thermoset Polyester (TPE)57 which are more commonly prototyped in academic laboratories. After method optimisation, we were able to generate liquid beams from both emitter types. While with pulled emitters a stable beam generation is possible at lower flow rates in the range of 100 µL min-1, ground emitters need a higher flow rate of 250 µL min-1. After we were able to reliably generate water jets from the edge of microfluidic glass chips we moved on to the initial detection of an analyte from the liquid beam by combining the system with an IR laser in front of a mass spectrometer. For this purpose an aqueous solution of D-arginine was pumped through a tapered-tip microchip (tip opening: 17 µm x 8 µm, flow rate: 100 µL min-1) and the water jet was irradiated with an IR laser just in front the orifice of the mass spectrometer as shown in Figure 2a. The process was monitored by simultaneously recording mass spectra. As soon as the liquid beam was
Table 1. Solvent screening experiments with 10 µmol L-1 D-arginine and microchip-LILBID-MS detection in different solvents.
solvent [vol%]
20
40
60
80
water [vol%]
80
60
40
20
90
99
10
1
3
absolute intensity [10 counts] methanol
23.9
30.8
36.8
33.5
n.d.
32.3
ethanol
n.d.
n.d.
n.d.
n.d.
n.d.
26.1
THF
30.9
37.0
37.1
29.7
n.d.
32.3
acetonitrile
36.4
43.2
42.6
38.2
12.0
