Delivery of picolitre droplets to mass spectrometer

Abstract

In accordance with one or more embodiments herein, an arrangement (300) for delivering individual droplets to a chemical analysis instrument (200), e.g. a mass spectrometer, is provided. The arrangement (300) comprises: a droplet generating arrangement (310), configured for generating electrically charged droplets; and a droplet delivery arrangement (100), arranged to deliver a droplet (350) received from the droplet generating arrangement (310) to an instrument inlet (210) of the chemical analysis instrument (200). The droplet delivery arrangement (100) comprises: a quadrupole (110), arranged to electrodynamically focus droplets received from the droplet generating arrangement (310) into a collimated beam; and a sheath (120), arranged for controlling the environmental conditions within the droplet delivery arrangement (100). A method for delivering individual droplets to a chemical analysis instrument (200) is also provided. The droplets are preferably picolitre droplets.

Description

[0001] DELIVERY OF PICOLITRE DROPLETS TO MASS SPECTROMETER

[0002] TECHNICAL FIELD

[0003] The present disclosure relates generally to arrangements and methods for the controlled delivery of individual droplets to a chemical analysis instrument, preferably a mass spectrometer.

[0004] BACKGROUND

[0005] The field of chemical analysis is pushing towards the analysis of smaller and smaller sample volumes. Electrospray ionisation is commonly used for mass spectrometric analysis of aqueous solutions through the application of a high voltage across a needle, generating a plume of charged droplets, some of which result in the production of ions that are ultimately sampled into a mass spectrometer. An advance on electrospray is nanoelectrospray ionisation, where a solution is sprayed out of the tip of a very fine capillary. This approach is most appropriate when small volumes of liquid sample are available.

[0006] The article "Exploring Chemistry in Microcompartments Using Guided Droplet Collisions in a Branched Quadrupole Trap Coupled to a Single Droplet, Paper Spray Mass Spectrometer” by M.l. Jacobs et. al. (Anal. Chem. 2017, 89, 1251 1-12519) describes paper spray, in which a large electric field at the tip of a paper triangle causes a spray to form that is directed toward a mass spectrometer.

[0007] US7785897 describes generating a parent droplet, inducing a charge, and delivering the charged parent droplet to a levitation device, where the charged parent droplet is electrodynamically levitated. This causes desolvation, leading to Coulombic fissioning of the charged parent droplet into smaller progeny droplets. The movement of the levitated progeny droplets can be manipulated by an electrode assembly, and thereby be delivered to a mass spectrometer.

[0008] PROBLEMS WITH THE PRIOR ART

[0009] Mass spectrometry can only detect ions, which means that a sample to be analysed using mass spectrometry must first be ionized. The ionisation described in US7785897 requires desolvation of a parent droplet, which has the negative result of creating a disperse plume of progeny droplets that are challenging to focus efficiently into the inlet of a chemical analysis instrument. Moreover, the ionisation process could result in unwanted chemistry occurring within the droplets.

[0010] Electrospray ionisation typically requires direct infusion of relatively large sample volumes, and only a portion of the sample is ultimately chemically analysed.

[0011] Nanoelectrospray is challenging in practice due to both poor robustness of the electrospray needle and complicated operation. Similar to electrospray, only a portion of the total sample is ultimately delivered to the inlet of the mass spectrometer.

[0012] There is thus a need for more efficient approaches to enable the chemical analysis of microscopic sample volumes.

[0013] SUMMARY

[0014] The above described problem is addressed by the claimed arrangement for delivering individual droplets to a chemical analysis instrument, e.g. a mass spectrometer. The arrangement preferably comprises a droplet generating arrangement, configured for generating electrically charged droplets, and a droplet delivery arrangement, arranged to deliver a droplet received from the droplet generating arrangement to an instrument inlet of the chemical analysis instrument. The droplet delivery arrangement preferably comprises a quadrupole, arranged to electrodynamically focus droplets received from the droplet generating arrangement into a collimated beam, and a sheath, arranged for controlling the environmental conditions within the droplet delivery arrangement.

[0015] The above described problem is also addressed by the claimed droplet delivery arrangement, which is arranged to deliver an individual droplet received from a droplet generating arrangement to an instrument inlet of a chemical analysis instrument, preferably a mass spectrometer. The droplet generating arrangement preferably comprises a quadrupole, arranged to electrodynamically focus droplets received from a droplet generating arrangement, configured for generating electrically charged droplets, into a collimated beam, and a sheath, arranged for controlling the environmental conditions within the droplet delivery arrangement.

[0016] The above described problem is further addressed by the claimed method for delivering individual droplets to a chemical analysis instrument. The method preferably comprises: generating electrically charged droplets, using a droplet generating arrangement; electrodynamically focusing the droplets into a collimated beam, using a quadrupole; and delivering one of said droplets to an instrument inlet of the chemical analysis instrument, using a droplet delivery arrangement comprising a sheath arranged for controlling the environmental conditions within the droplet delivery arrangement.

[0017] This enables the delivery of an individual droplet, or a train of droplets, directly into the inlet of a chemical analysis instrument, such as a mass spectrometer, without the droplet first being converted into a spray. The entire droplet is thus delivered to the analysis instrument in droplet form. The droplets are preferably picolitre droplets.

[0018] In embodiments, the droplet delivery arrangement comprises at least one braking electrode, arranged inside the quadrupole, to which at least one braking electrode a controllable voltage of the same polarity as a droplet is arranged to be applied, in order to control the position of the droplet within the droplet delivery arrangement.

[0019] In embodiments, the application of a controlled voltage to the at least one braking electrode enables control of the timing of delivery of the droplet to the instrument inlet.

[0020] In embodiments, the at least one braking electrode is an annular electrode. However, the at least one braking electrode may have any shape that allows a droplet to pass.

[0021] In embodiments, the droplet generating arrangement comprises an induction electrode, configured for applying the electric charge to each droplet. The induction electrode may e.g. be annular, but it may have any shape that allows a droplet to pass. However, the electric charge may also be applied before, or during, the generation of the droplets, e.g. by charging the fluid before or during the generation of the droplets.

[0022] In embodiments, the droplet generating arrangement comprises one or more droplet dispensing devices.

[0023] In embodiments, the chemical analysis instrument is a spectrometer, preferably a mass spectrometer.

[0024] In embodiments, the arrangement comprises an imaging arrangement, arranged for monitoring droplets inside the droplet delivery arrangement.

[0025] The scope of the invention is defined by the claims, which are incorporated into this section by reference. A more complete understanding of embodiments of the invention will be afforded to those skilled in the art, as well as a realization of additional advantages thereof, by a consideration of the following detailed description of one or more embodiments. Reference will be made to the appended sheets of drawings that will first be described briefly. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figs. 1 a-b schematically illustrate arrangements for delivering individual droplets to a chemical analysis instrument, in accordance with one or more embodiments described herein.

[0027] Fig. 1 c schematically illustrates a droplet delivery arrangement, in accordance with one or more embodiments described herein.

[0028] Fig. 2 schematically illustrates a method for delivering individual droplets to a chemical analysis instrument, in accordance with one or more embodiments described herein.

[0029] Fig. 3 shows an example total ion count (TIC) chromatogram for a series of droplets.

[0030] Fig. 4 shows representative mass spectra for the droplets detected in Fig. 3.

[0031] Fig. 5 illustrates how the ion yield varies with analyte concentration and droplet volume.

[0032] Embodiments of the present disclosure and their advantages are best understood by referring to the detailed description that follows. It should be appreciated that like reference numerals are used to identify like elements illustrated in one or more of the figures.

[0033] DETAILED DESCRIPTION

[0034] The field of chemical analysis is pushing towards the analysis of smaller and smaller sample volumes. Electrospray ionisation typically requires direct infusion of relatively large sample volumes, where only a portion of the sample is ultimately chemically analysed. Nanoelectrospray is challenging in practice due to both poor robustness of the electrospray needle and complicated operation. Similar to electrospray, only a portion of the total sample is ultimately delivered to the inlet of the mass spectrometer.

[0035] The arrangement and method according to the present disclosure enables the collimation and delivery of an individual droplet, or a train of droplets, directly into the inlet of a chemical analysis instrument, without the droplet first being converted into a spray. The entire droplet is thus delivered to the analysis instrument in droplet form. The droplets are preferably picolitre droplets. A droplet delivery arrangement, comprising a quadrupole, is used to collimate droplets containing a controlled net charge into a beam and deliver them to e.g. the inlet of a mass spectrometer. The environmental conditions within the droplet delivery arrangement are controllable through a sheath arranged around it, and the droplet delivery arrangement may be arranged to have the flexibility to hold individual droplets for indefinite periods of time, e.g. to allow droplets to undergo chemical or physical change. The sheath serves to prevent ambient air currents from altering the trajectory of the droplets away from the instrument inlet - this is particularly challenging for droplets in the picolitre and smaller size range due to their aerodynamic properties. The droplet delivery arrangement operates at ambient pressure and is external to the instrument inlet of the chemical analysis instrument. Upon delivery to the instrument inlet, droplets are presumed to undergo rapid solvent loss, leading to the formation of molecular ions through an inlet ionisation mechanism.

[0036] The arrangement and method according to the present disclosure permits chemical analysis of very small amounts of sample (e.g. down to 1 pg analyte contained in 4-250 pL droplets, with a diameter of 20-80 m) and overcomes many drawbacks associated with approaches like nanoelectrospray (no glass capillary needle that can easily break or clog, no hazardous high voltage required to create a spray of charged droplets, and very high efficiency for delivery of analyte into the inlet of the mass spectrometer), where only a few picolitres of sample droplet are required. This approach provides an advantage over existing approaches in terms of the capability to sensitively analyse very small amounts of precious sample. Because the approach is droplet based, there is potential for coupling with microfluidic platforms and for timing droplet delivery to the mass spectrometer inlet to coincide with sample analysis, avoiding sample waste due to the longer duty cycles associated with certain mass spectrometry approaches. In other words, the approach can avoid sample loss, because a sample can be delivered to the chemical analysis instrument at a user- controlled time, rather than being continuously delivered even when the chemical analysis instrument is unable to accept it due to the time it takes to analyse a sample already delivered. Further, the approach permits chemical processing of the droplet before delivery to the chemical analysis instrument. This capability could be particularly useful in biological applications as some sample processing steps, e.g. enzymatic protein digestion, can be enhanced in droplets relative to macroscopic solutions, further accelerating the chemical analysis process.

[0037] The present disclosure relates generally to arrangements and methods for delivering individual droplets to a chemical analysis instrument, preferably a mass spectrometer. Embodiments of the disclosed solution are presented in more detail in connection with the figures.

[0038] Figs. 1-2 schematically illustrate embodiments of an arrangement 300 for delivering individual droplets to a chemical analysis instrument 200, preferably a mass spectrometer.

[0039] The illustrated arrangement 300 comprises a droplet generating arrangement 310, configured for generating droplets, preferably picolitre droplets. The droplet generating arrangement 310 preferably comprises some kind of droplet dispensing device 320, such as e.g. a droplet-on-demand single-jet dispensing device which is piezo-electrically controlled to dispense a small volume of liquid (typically 5-180 pL) at a relatively high initial velocity (around 10 m / s), following the application of an appropriate voltage pulse. The droplet generating arrangement 310 may comprise a number of different droplet dispensing devices 320, for simultaneously generating different types of droplets for analysis. The droplet generating arrangement 310 may also comprise means, e.g. in the form of an induction electrode 330, for applying a small electric charge to each droplet 350. However, the electric charge may also be applied before, or during, the generation of the droplets, e.g. by charging the fluid before or during the generation of the droplets.

[0040] The illustrated arrangement 300 further comprises a droplet delivery arrangement 100, arranged to deliver a droplet 350 received from a droplet generating arrangement 310 to an instrument inlet 210 of a chemical analysis instrument 200. Fig. 1 c schematically illustrates an embodiment of a droplet delivery arrangement 100 which comprises a quadrupole 1 10, which is arranged to electrodynamically focus droplets received from the droplet generating arrangement 310 into a collimated beam. This is possible because each droplet 350 has a small electric charge, applied in the droplet generating arrangement 310. The electric charge is necessary for the use of the quadrupole 1 10, but it is an advantage of the invention that the charge is small - when using electrospray, the high voltage necessary for electrospray creation causes highly charged droplets. A low charge of the droplet 350 may provide a cleaner spectrum in the ensuing spectroscopy.

[0041] A typical quadrupole operates by applying an alternating electrical signal to four parallel metal rods, with a 180° phase shift between signals applied to adjacent rods. The resulting electric field confines a charged droplet 350 in the two lateral dimensions, allowing movement only along the central vertical axis, and countering any horizontal fluctuations. Under normal conditions, a charged droplet 350 entering the quadrupole field at the top of the central axis will fall along the full length of the axis, with a fall speed approaching the terminal settling velocity (if the quadrupole is vertical, which it does not have to be).

[0042] Inside the quadrupole 1 10, one or more braking electrodes 130 may be arranged, in order to create a local electrical field without physically obstructing the droplets. The braking electrode 130 creates an electrostatic field, which repels droplets of the same polarity. If a controllable voltage of the same polarity as a droplet 350 is applied to the braking electrode 130, the position of the droplet 350 inside the quadrupole 1 10 may be controlled. A single droplet reaches an equilibrium vertical position when the forces of gravity and gas phase drag balance the repulsion from braking electrode 130. This may be used to trap and hold one or more droplets 350 levitated inside the quadrupole 1 10, and / or to control the timing of delivery of a droplet 350 to the instrument inlet 210. The voltage applied to the braking electrode 130 controls the levitated position of the droplet 350 inside the quadrupole 110. The braking electrode could be switched on and off very rapidly, allowing droplets to be halted momentarily during transit down the quadrupole 110, for example to perform imaging analysis. Since the charge of the droplet 350 will be low, the charge applied to the braking electrode 130 can also be rather low, e.g. a few hundred volts - it only needs to be enough to keep the droplet 350 levitated by being repelled by the braking electrode 130.

[0043] The use of more than one braking electrode 130 allows the possibility to halt the droplet 350 more than once inside the quadrupole 110. Fig. 1 a schematically illustrates an embodiment comprising one braking electrode 130, and Figs. 1 b-c schematically illustrate an embodiment comprising two braking electrodes 130.

[0044] In the illustrated embodiment, the braking electrode 130 is an annular electrode, but it may have any shape that allows a droplet 350 to pass. An annular braking electrode 130 allows a droplet 350 to pass through its center, but the braking electrode 130 may be arranged to instead allow the droplet 350 to pass e.g. beside it, depending on the shape of the braking electrode 130.

[0045] The droplet delivery arrangement 100 preferably comprises a sheath 120, arranged for controlling the environmental conditions within the droplet delivery arrangement 100. The sheath ensures that ambient air currents cannot alter the trajectory of the droplets away from the instrument inlet 210, and preferably also controls the humidity inside the droplet delivery arrangement 100. The sheath 120 may create a completely closed environment, except for the droplet inlet and a small opening 140 to interface with the instrument inlet 210. There may also be one or more small gas inlets to allow environmental control. The droplet delivery arrangement 100 preferably operates at ambient pressure, while the inside of the chemical analysis instrument 200 is typically under high vacuum. There is therefore typically a constant draw of air through the instrument inlet 210 into the chemical analysis instrument 200. In the illustrated embodiment, the opening 140 in the sheath 120 is arranged to be directly in front of the instrument inlet 210, which allows the droplet 350 to be drawn into the instrument inlet 210.

[0046] In order to monitor a droplet 350 inside the droplet delivery arrangement 100, the arrangement 300 may comprise an imaging arrangement 360, e.g. comprising a zoom lens and a camera. Opposite the imaging arrangement 360, illuminating means 370, e.g. in the form of a LED with a focussing lens, may be arranged. This may allow the imaging of individual droplets.

[0047] The arrangement 300 may comprise a control system that controls its various parts. Fig. 2 schematically illustrates a method 400 for delivering individual droplets to a chemical analysis instrument 200. The method 400 may comprise:

[0048] Step 430: generating electrically charged droplets, using a droplet generating arrangement 310.

[0049] Step 440: electrodynamically focusing the droplets into a collimated beam, using a quadrupole 110.

[0050] Step 450: delivering one of the droplets 350 to an instrument inlet 210 of the chemical analysis instrument 200, using a droplet delivery arrangement 100 comprising a sheath 120 arranged for controlling the environmental conditions within the droplet delivery arrangement 100.

[0051] This enables the delivery of an individual droplet, or a train of droplets, directly into the inlet of a chemical analysis instrument 200, such as a mass spectrometer, without the droplet first being converted into a spray. The entire droplet is thus delivered to the analysis instrument 200 in droplet form. The droplets are preferably picolitre droplets.

[0052] In embodiments, the electric charge is applied to each droplet 350 using an induction electrode 330 comprised in the droplet generating arrangement 310. The induction electrode 330 may e.g. be annular, but it may have any shape that allows a droplet 350 to pass. However, the electric charge may also be applied before, or during, the generation of the droplets, e.g. by charging the fluid before or during the generation of the droplets.

[0053] In embodiments, the chemical analysis instrument 200 is a spectrometer, preferably a mass spectrometer.

[0054] The method 400 may further comprise one or more of:

[0055] Step 410: arranging the chemical analysis instrument 200 to be a spectrometer, preferably a mass spectrometer.

[0056] Step 420: arranging the at least one braking electrode 130 to be an annular electrode. However, the at least one braking electrode 130 may have any shape that allows a droplet 350 to pass.

[0057] Step 460: applying a controlled voltage of the same polarity as a droplet 350 to at least one braking electrode 130 arranged inside the quadrupole 110.

[0058] Step 470: controlling the position of a droplet 350 within the droplet delivery arrangement 100, using the at least one braking electrode 130. Step 480: monitoring droplets inside the droplet delivery arrangement 100, using an imaging arrangement 360.

[0059] Step 490: timing the delivery of the droplet 350 to the instrument inlet 210, by the voltage applied to the at least one braking electrode 130.

[0060] The above steps may be effected in any order that makes technical sense, and some of the steps may be effected simultaneously with each other.

[0061] Example

[0062] Aerosol droplets are unique microcompartments containing microscopic amounts of material and exhibiting surprising chemical reactivity. Although a diverse set of tools exist to characterize the chemical composition of individual submicron particles in air, comparatively fewer approaches can chemically analyse individual, airborne picolitre droplets. This example describes a novel approach for mass spectrometric (MS) analysis of individual aqueous picolitre droplets (2-180 pL volume) containing down to 1 pg analyte mass per droplet. Individual droplets are generated using a microdroplet dispenser, imparted a small amount of net charge, and guided to the inlet of a high- resolution MS using a linear quadrupole-electrodynamic balance. Analyte molecules within the aqueous droplet are ionized using droplet assisted ionization, where droplet breakup within the MS inlet leads to generation of molecular ions. This single droplet MS approach generates clean mass spectra, permits timing of droplet delivery for chemical analysis, and, by avoiding a separate ionization stage, avoids potential artifacts arising from current electrospray-based approaches for picolitre droplet analysis. It is anticipated that this approach will permit exploration of the factors governing accelerated chemical reactions in aerosol droplets and will be suitable for sensitive analysis of particularly precious samples in different application domains.

[0063] Microcompartments like aerosol droplets are central to diverse domains including atmospheric chemistry and climate change, human health and disease transmission, and materials synthesis. Aerosols are unique because they are containerless reaction vessels with high surface area-to- volume ratios and hold only microscopic amounts of matter. A surprising observation of recent years is that some chemical reactions in aerosols can be vastly accelerated (up to 107times) compared to the same reaction in macroscopic solutions. Explanations for observations of accelerated chemistry include high reactant concentrations, interfacial confinement of reactants, and potential modification of their Gibbs energies, and interfacial electric field altering reaction pathways. However, many approaches to investigate accelerated chemical reactions in aerosol droplets have studied charged droplets generated by variations of electrospray ionization (ESI), including extractive ESI, desorption ESI, ESI / electrosonic spray, and paper spray ionization. In many applications, droplet formation and chemical analysis are tightly coupled. Moreover, these approaches can generate highly unconstrained and dynamic reaction conditions, with polydispersity in droplet size, rapid solvent evaporation altering reagent concentrations, and poor constraints on droplet charge.

[0064] A rich diversity of approaches to measure the chemical composition of single aerosol particles exists for nominally dry <10 pm diameter particles. In contrast, comparably fewer approaches exist for liquid droplets >1 pm diameter. Acoustic levitation has been coupled with MS through ionization approaches including laser desorption / ionization, field induced droplet ionization, charge and matrix-assisted laser desorption / ionization, atmospheric pressure chemical ionization, secondary ESI, and direct analysis in real time. However, acoustic levitation is generally limited to microlitre sample volumes which may not accurately represent the unique environment of aerosol droplets. Electrodynamic levitation approaches for picolitre volume aerosol droplets have been coupled with MS through ionization approaches including paper spray, corona discharge, thermal desorption glow discharge, and an open port sampling interface coupled to ESI. In these levitation approaches, droplet generation is effectively decoupled from the chemical analysis step. Moreover, relatively precise control of environmental conditions (e.g. relative humidity, which controls solute concentration) as well as droplet size and charge are possible with these single droplet approaches. Free-flowing streams of picolitre droplets have also been coupled to ESI-MS to conduct low volume, high throughput chemical analysis. An important drawback to these approaches is that they require separate ionization stages, and, for many of them, the ionization process extends over several seconds, potentially reducing the sensitivity of the approach by spreading signal out over time and introducing artifacts (e.g. undesirable reactions) that may complicate measurement interpretation.

[0065] Inlet ionization approaches have emerged over the past decade as straightforward and versatile methods to generate molecular ions from solid and liquid samples within the MS inlet, requiring minimal sample preparation and no use of high voltages or lasers. The samples can be introduced at ambient pressure, and the ions generated are similar to those observed using ESI . A recent advance on these inlet ionization approaches is droplet assisted ionization (DAI), where liquid aerosol droplets are sampled from ambient pressure into a heated capillary inlet attached to the MS. DAI has been used to measure the chemical composition of secondary organic aerosol and explore chemical reactivity in nanoparticles. While some uncertainty remains about the ionization mechanism, a liquid droplet is thought to undergo either thermal or aerodynamic breakup within the MS inlet, leading to charge separation and the formation of charged progeny droplets, followed by successive iterations of solvent evaporation and Coulombic fission to produce molecular ions. At low inlet temperatures, the droplet may flash freeze and shatter to generate molecular ions. This ionization approach holds promise for MS analysis of finite-volume liquid aerosol droplets but has not yet been applied to aerosol particles larger than 100 nm.

[0066] This example describes a novel approach to focus, levitate, and sample individual picolitre volume droplets (2-180 pL, or 20-70 pm diameter) by coupling a linear quadrupole electrodynamic balance (LQ-EDB) with a high-resolution time-of-flight MS using DAI. Droplet properties including size, charge, and droplet age are controlled. Strong MS signals for individual droplets containing as low as 1 pg analyte are detected. The factors contributing to the sensitivity of the approach are explored, and the suitability of the technique for a wide range of analytes from small molecules to biomolecules is discussed in this example.

[0067] A Single Droplet Mass Spectrometry (SDMS) approach to characterize the molecular composition of individual picolitre aerosol droplets has been developed. Fig. 1 a illustrates the key aspects of the approach. Droplets of known composition and size are reproducibly generated using a droplet- on-demand (DoD) dispenser and guided to the MS inlet for subsequent chemical analysis using a linear quadrupole electrodynamic balance (LQ-EDB). Ionization is accomplished using DAI. An imaging assembly allows droplet size to be measured during transit through the LQ-EDB.

[0068] Chemicals

[0069] Solutions for a range of small molecules and proteins have been made for single droplet analysis. The systems examined include: angiotensin II (ApexBio), leucine enkephalin (Waters), cortisol, equine cytochrome-c, and equine myoglobin (Sigma-Aldrich). Solute concentrations and solvent compositions are described below.

[0070] Droplet Generation

[0071] Individual picolitre droplets are generated with a DoD dispenser (MicroFab, MJ-ABP-01), a piezoelectric device that controllably and reproducibly generates droplets of a desired volume upon application of a voltage pulse. The range of droplet volumes accessible spans 2 to 180 pL (20-70 pm in diameter) and is determined by the orific diameter of the dispenser and the voltage pulse applied to the dispenser. An induction electrode, positioned near the outlet orific of the dispenser, imparts a small amount of charge (order of 10s of fC, corresponding to <5% of the Raleigh limit) to the droplets upon generation. The frequency of droplet generation is user-controlled and was set to 1 Hz. The 1 Hz frequency enabled multiple droplets to be measured over a short time period while ensuring baseline resolution in the MS signal between individual droplets. Droplet Control and Guidance

[0072] The LQ-EDB is positioned external to the MS in a vertical geometry and is held at ambient pressure. It traps the dispensed droplets and guides their trajectory toward the MS inlet. This arrangement ensures that nearly 100% of the sample is delivered to the MS, minimizing sample wastage. The alternating electric field applied to the rods in the LQ-EDB confine the charged droplets in two lateral dimensions, collimating them as they transport along the central vertical axis. A charged droplet enters the quadrupole at the top of the central axis and descends along its full length with a velocity approaching the droplet's terminal settling velocity. The LQ-EDB is housed within a transparent glass sheath, which contains openings for a gas inlet and outlet, droplet injection port, and droplet outlet to the MS inlet. The sheath inhibits air currents from disrupting droplet transport and permits control of environmental conditions (e.g. relative humidity, RH). In the example, the RH was held as high as possible (>90% RH) by passing compressed air through a water bubbler before flowing the humidified gas through the enclosure. Because the ionization efficiency is sensitive to droplet water content, a high RH environment minimizes evaporation of the aqueous droplets during transit through the LQ-EDB. A braking electrode (applied with a voltage of the same polarity as the droplet charge) is positioned approximately halfway down the LQ-EDB. For the example described here, the braking electrode was activated immediately prior to performing a measurement, halting the droplet's descent while its diameter was measured by the imaging setup. The braking electrode was then switched of to allow droplet sampling into the MS for chemical analysis. Bespoke software, written in LabVI EW, controls all the electronic signals used in the LQ- EDB. The unamplified signals were generated using a multifunction DAQ (Nl USB-6343) for the quadrupole rods, induction electrode, and braking electrode, and an arbitrary waveform generator (Keysight, Trueform 33500B Series) for the dispenser pulse. These signals were amplified using a dedicated amplification unit (Biral, P9050M), which allows real-time control of the quadrupole signal (between ±1 kVAC, 0-1 kHz), induction and braking electrode signals (between ±500 VDC), and dispenser pulse (40 V pulse height, 30 ps pulse width). The voltage applied to the quadrupole was ±1 kVAC with a 200 Hz frequency.

[0073] Droplet Imaging

[0074] Droplets in the central axis of the LQ-EDB were illuminated with a low-power alignment laser (Thorlabs, 5 mW, 532 nm), allowing visual detection of the droplets. In addition, a brightfield imaging assembly permitted the droplets to be observed in more detail. This imaging assembly consists of an objective (Motic, Plan apo 20x), zoom and focusing module (Navitar, Resolv4K), and camera (JAI, GQ-2400M-USB). Illumination was provided using an LED (Thorlabs, M455L3) with focusing lens. The image dimensions for each zoom setting were calibrated using a micrometre graticule. Collectively, the imaging system enables high resolution, high magnification visualization of individual droplets. The droplet images permit relatively precise retrieval of droplet diameter (±2 pm) and, consequently, droplet volume.

[0075] Single Droplet Mass Spectrometry

[0076] The LQ-EDB assembly is coupled with a high-resolution quadrupole time-of-flight MS (Waters, Synapt XS) by positioning the bottom opening of the LQ-EDB enclosure against the sample cone inlet of the MS. After transiting the central axis of the LQ-EDB, droplets exit the electric field of the LQ-EDB and continue to fall vertically (10 mm) until they approach the MS inlet. The droplet is then aspirated into the MS inlet by the continuous 2 L / min MS inlet flow where the droplet rapidly desolvates to generate molecular ions through an inlet ionization mechanism presumably similar to that proposed for DAI or solvent assisted ionization. In the example, the MS was operated in sensitivity mode to detect either positive or negative ions, with mass spectra typically collected over a 50-1200 m / z range (unless otherwise specified with a 0.1s scan time). Mass spectra were generated in centroid mode. The source conditions were optimized and held constant at the following values: source temperature 60-80 °C, sampling cone voltage 0 V, source offset voltage 30 V, trap and transfer collision energy off capillary voltage 1-1.5 kV.

[0077] Results

[0078] An example TIC chromatogram recorded by the MS for a uniform series of 39 pL (42 pm diameter) aqueous droplets containing 320 pM angiotensin II (corresponding to 12.9 pg or 12 fmol of angiotensin II analysed per droplet) dispensed at 1 Hz is shown in Fig. 3. The series of peaks at 1s intervals in the chromatogram corresponds to analysis of individual picolitre droplets. Droplets generating very low TICs were observed to miss or clip the MS inlet due to small fluctuation in trajectory during the aspiration process. By defining a successful SDMS measurement as one that generates at least 20% of the largest measured signal across all droplets (thick line in the upper diagram of Fig. 3), the droplet hit rate approaches 95%, with a 33% standard deviation in maximum TIC intensity per droplet. The signal-to-noise ratios for the TICs of the detected droplets range between 4 and 16. Before delivery to the inlet, droplets have uniform size (<0.2%), charge (<3%), and composition. Therefore, observed variations in the TIC primarily arise from small variations in the efficiency of droplet sampling into the MS inlet or synchronization between droplet sampling and MS acquisition. The lower diagram of Fig. 3 magnifies a portion of this chromatogram, showing that virtually all ions arising from an individual droplet are contained within a single 0.1 s scan (the highest time resolution available on the MS using standard user settings), with baseline resolution achieved within 1 -2 scans. This fast time scale for single droplet analysis is similar to that reported for a rapid droplet sampling interface where larger picolitre and nanolitre droplets are ejected onto an open-face capillary with a continuous flow of electrosprayed solvent. By contrast, single droplet analysis by other approaches (e.g. paper spray ionization or open port sampling interface), which rely on ejected droplets dissolving into larger volumes of solvent before electrospray, results in signal from a single droplet being spread out across at least an order of magnitude longer time scale (many seconds). The absence of analyte dilution within another solvent, along with the very short time scale over which analyte within an individual droplet is detected, suggests relatively high sensitivity for this approach compared to other single droplet analysis approaches.

[0079] Representative mass spectra for the droplets detected in Fig. 3 are shown in Fig. 4. The upper diagram in Fig. 4 shows a single droplet mass spectrum for angiotensin I I, with [M + H]+and [M+2H]2+molecular ions at 1047 m / z and 524 m / z, respectively, clearly resolvable from the noise. The magnitude of the noise envelope (i.e. the magnitude of the largest nonmolecular ion peak) is <8% of the [M+2H]2+signal. Owing to droplet-to-droplet variation in signal intensity (see Fig. 3), some improvement in the cleanliness of the mass spectrum arises from averaging signal from multiple droplets. The lower diagram in Fig. 4 shows that by integrating the signal across five droplets (64.5 pg of analyte consumed) the mass spectral noise is reduced to <5% of the [M +2H]2+ion. Integrating over additional droplets leads to diminishing reductions in spectral noise: integrating across all 60 droplets in Fig. 3, the noise level is still 4.4% of the [M+2H]2+ion signal.

[0080] Analysis of all droplets detected in Fig. 3 shows that the relative abundance of the +1 and +2 charge states of the molecular ion exhibit little variation between droplets, with the +2 charge state accounting for 65±9% of the all molecular ions detected. Perhaps unsurprisingly, the largest droplet-to-droplet variations in the relative intensities occurs for droplet mass spectra where the total ion count is low. Additionally, isotopic distributions for single droplets can be clearly resolved. Although there is a slight deviation from expected isotopic abundances for a single droplet, after integrating across only five droplets the isotopic abundance is consistent with expected values.

[0081] Size, Concentration, and Charge Dependencies of Ion Yield

[0082] Fig. 5 shows the measured TIC and ion yield as a function of angiotensin I I concentration (holding droplet volume constant at 58 pL, see the upper diagram of Fig. 5) and as a function of droplet volume (holding analyte concentration constant at 320 pM, see the lower diagram of Fig. 5). This shows how the SDMS ion yield varies with analyte concentration and droplet volume, and provides key information about the ionization mechanism. Each data point represents the average value calculated across all detected droplets in a 60s measurement window. Error bars represent the standard deviation. Across nearly three orders of magnitude in analyte concentration, increasing analyte concentration within a droplet increases the TIC. At the same time, the ion yield per droplet decreases with increasing analyte concentration. This observation highlights the importance of the protic solvent water to the ionization process. For instance, when analysing plumes of submicron aerosol, drying the aerosol to low relative humidity or using a nonpolar solvent resulted in orders- of-magnitude decreases in ion signal. The dependence of ion yield on analyte concentration reflect the ability of water to donate charge to the analyte as well as its apparent ability to induce charge separation during droplet breakup. Consequently, although a higher absolute analyte mass in a droplet leads to a greater signal intensity, the ionization process becomes less efficient suggesting the SDMS approach is best suited to analysis of dilute droplets. In the example, aqueous droplets containing down to 2.4 pg analyte in a 58 pL volume (48 pm diameter) droplet were successfully analysed with good analyte intensity. Droplets containing lower masses of analyte were not routinely observed, mainly due to challenges in optimizing droplet delivery, as such droplets individually generate relatively little signal (owing to the small amount of analyte they contain). Extrapolating of the log-log relationship shown in the upper diagram of Fig. 5 and assuming the absolute level of background noise remains constant across all droplet sizes (at 100 counts), the current measured limit-of-detection could hypothetically be improved to 100 fg analyte. It is noted that if this approach would be utilized to explore chemical kinetics in aerosol droplets, reactions would generally occur at a fixed RH, mitigating any RH-dependence on the sensitivity of the approach.

[0083] Similarly, droplet volume also plays a key role in the ionization process. As droplet volume increases from 2.5 pL to 195 pL, the TIC increases by approximately 1 order of magnitude. This increase is because the total analyte mass delivered to the MS increases from 0.9 pg to 65.1 pg across the investigated droplet volume range. By contrast, ion yields are about an order of magnitude larger for the 2.5 pL volume droplets compared to the 195 pL droplets. In other words, ionization is more efficient for smaller droplets. This observation is consistent with size-dependent analyses previously performed on submicron particles analysed using DAI, demonstrating that analytes in smaller droplets are more efficient ionized, as well as with trends in ionization efficiency using (nano-)ESI. In the example, the minimum droplet size is limited by what can be generated using the DoD dispenser. However, extrapolating the observed trends suggests that single droplet analysis is in principle feasible at least until the droplet volume falls below 100 fL (i.e. 5 pm diameter) for angiotensin II droplets at the same concentration, an extrapolation roughly consistent with analysis of ionization trends for plumes of submicron aerosols. Note that when single droplet analysis becomes challenging, signal can still be detected by integrating across multiple droplets. The log-log plots of the TIC dependence on analyte concentration and droplet volume in Fig. 5 are both linear with a gradient 0.5. The ion yields presented in Fig. 5 fall between 106and 108across the studied ranges in droplet volume and angiotensin I I concentration. Given the observed increase in ion yield with both decreasing volume and decreasing analyte concentration, ion yields better than 105could potentially be achieved in the future by leveraging these phenomena in concert. These ion yields compare very favorably to those calculated for submicron plumes under similar conditions, 10’9, and even approach the ion yields achieved using ESI under similar conditions, 10-5, despite the far lower absolute quantities of analyte required in SDMS and absence of highly charged droplets. Indeed, since individual picolitre droplets can be detected using this approach, only a tiny fraction of the bulk volume used to load the dispenser is typically consumed, and any unused sample can be extracted for other purposes.

[0084] The relative importance of droplet charge on ionization using the SDMS approach has also been explored. As described earlier, a small amount of charge is imparted onto the droplets to collimate them using the LQ-EDB. The total charge is limited by the maximum induction electrode voltage, which imparts 10s of fC per droplet (i.e. <5% of the Rayleigh limit for 50 pL droplets). Droplet charge was systematically varied between ±100 fC and quantified by depositing droplets into a Faraday cup that was positioned below the LQ-EDB and connected to an electrometer (Keithly 6514 / E). For angiotensin I I, both positive and negative molecular ions were detectable, irrespective of the polarity of the charge initially applied to the droplet, although in general more positive ions were detected than negative ions. Total ion count does not vary significantly with net initial droplet charge, consistent with the hypothesis that the protic solvent and droplet breakup process (rather than initial droplet charge) are key to ionization. It is possible that droplet charge may play a minor role in the ionization process but its effect is obscured by droplet-to-droplet variations in ion signal over the investigated range of droplet net charge.

[0085] Conclusion

[0086] A novel approach for MS of individual picolitre volume droplets is disclosed in this example. Individual droplets are generated by a droplet dispenser, imparted a small amount of net charge, and guided to the MS inlet using a LQ-EDB. Analyte molecules are ionized using DAI, where the polar solvent in a liquid droplet imparts charge to the analyte molecules contained within during droplet breakup within the MS inlet. There are several benefits of this approach to analysing picolitre droplets. Sample preparation is minimal, and the droplet generation and manipulation processes are segregated from the ionization and analysis steps. Indeed, the chemical analysis step is complete in milliseconds within the MS inlet, compared to chemical analysis requiring several seconds to tens of seconds for comparable single droplet MS approaches, reducing the potential for artifacts arising from the ionization process (e.g. further reaction) affecting measurement interpretation. The approach can characterize droplets across a wide volume range (2-180 pL) currently limited only by the droplet generation technique. This SDMS approach permits efficient chemical analysis of individual droplets containing low pg quantities of analyte. The SDMS approach generates clean mass spectra and permits timing of droplet delivery to the MS, potentially yielding benefit over continuous flow approaches for applications using MS with a lower duty cycle. This SDMS approach will facilitate explorations of the factors governing chemical reactions in aerosol droplets, improving the mechanistic understanding of atmospheric aerosol and cloud droplet chemistry. Additionally, this SDMS technique may leverage the reaction accelerations inherent in aerosol droplets for reaction discovery and optimization processes. The approach may also have broader utility for efficient analysis of picoscopic samples in different application domains. The foregoing disclosure is not intended to limit the present invention to the precise forms or particular fields of use disclosed. It is contemplated that various alternate embodiments and / or modifications to the present invention, whether explicitly described or implied herein, are possible in light of the disclosure. Accordingly, the scope of the invention is defined only by the claims.

Claims

CLAIMS1. Arrangement (300) for delivering individual droplets to a chemical analysis instrument (200), preferably a mass spectrometer, the arrangement (300) comprising: a droplet generating arrangement (310), configured for generating electrically charged droplets; and a droplet delivery arrangement (100), arranged to deliver a droplet (350) received from the droplet generating arrangement (310) to an instrument inlet (210) of the chemical analysis instrument (200), wherein the droplet delivery arrangement (100) comprises: a quadrupole (110), arranged to electrodynamically focus droplets received from the droplet generating arrangement (310) into a collimated beam; and a sheath (120), arranged for controlling the environmental conditions within the droplet delivery arrangement (100).

2. Arrangement (300) according to claim 1 , wherein the droplet delivery arrangement (100) further comprises at least one braking electrode (130), arranged inside the quadrupole (110), to which at least one braking electrode (130) a controllable voltage of the same polarity as a droplet (350) is arranged to be applied, in order to control the position of the droplet (350) within the droplet delivery arrangement (100).

3. Arrangement (300) according to claim 2, wherein the application of a controlled voltage to the at least one braking electrode (130) enables control of the timing of delivery of the droplet (350) to the instrument inlet (210).

4. Arrangement (300) according to claim 2 or 3, wherein the at least one braking electrode (130) is an annular electrode.

5. Arrangement (300) according to any one of claims 1-4, wherein the droplet generating arrangement (310) comprises an induction electrode (330), configured for applying the electric charge to each droplet (350).

6. Arrangement (300) according to any one of claims 1-5, further comprising an imaging arrangement (360), arranged for monitoring droplets inside the droplet delivery arrangement (100).

7. Droplet delivery arrangement (100), arranged to deliver an individual droplet received from a droplet generating arrangement (310) to an instrument inlet (210) of a chemical analysis instrument (200), preferably a mass spectrometer, the droplet generating arrangement (100) comprising: a quadrupole (110), arranged to electrodynamically focus droplets received from a droplet generating arrangement (310), configured for generating electrically charged droplets, into a collimated beam; and a sheath (120), arranged for controlling the environmental conditions within the droplet delivery arrangement (100).

8. Droplet delivery arrangement (100) according to claim 7, further comprising at least one braking electrode (130), arranged inside the quadrupole (110), to which at least one braking electrode (130) a controllable voltage of the same polarity as a droplet (350) is arranged to be applied, in order to control the position of the droplet (350) within the droplet delivery arrangement (100).

9. Droplet delivery arrangement (100) according to claim 8, wherein the application of a controlled voltage to the at least one braking electrode (130) enables control of the timing of delivery of the droplet (350) to the instrument inlet (210).

10. Arrangement (300) according to claim 8 or 9, wherein the at least one braking electrode (130) is an annular electrode.

11. Method (400) for delivering individual droplets to a chemical analysis instrument (200), preferably a mass spectrometer, the method (400) comprising: generating (430) electrically charged droplets, using a droplet generating arrangement (310); electrodynamically focusing (440) the droplets into a collimated beam, using a quadrupole (110); anddelivering (450) one of said droplets (350) to an instrument inlet (210) of the chemical analysis instrument (200), using a droplet delivery arrangement (100) comprising a sheath (120) arranged for controlling the environmental conditions within the droplet delivery arrangement (100).

12. Method (400) according to claim 11 , further comprising applying (460) a controlled voltage of the same polarity as a droplet (350) to at least one braking electrode (130) arranged inside the quadrupole (110), and thereby controlling (470) the position of the droplet (350) within the droplet delivery arrangement (100).

13. Method (400) according to claim 12, wherein the applying (460) of the controlled voltage to the at least one braking electrode (130) enables timing (490) of the delivery of the droplet (350) to the instrument inlet (210).

14. Method (400) according to claim 11 or 12, further comprising arranging (420) the at least one braking electrode (130) to be an annular electrode.

15. Method (400) according to one of claims 11-14, wherein the electric charge is applied to each droplet (350) using an induction electrode (330) comprised in the droplet generating arrangement (310).

16. Method (400) according to one of claims 11-15, further comprising monitoring (480) droplets inside the droplet delivery arrangement (100), using an imaging arrangement (360).

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