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Waters Unispray Ionization Source White Paper [ WHITE PAPER ] Waters UniSpray Ionization Source S. Bajic Waters Corporation, Wilmslow, UK INTRODUCTION The first commercial electrospray ionization (ESI) sources for mass spectrometers became available at the end of the 1980s. These early sources broadened the applications of mass spectrometry (MS) to biological compounds such as peptides and proteins which would typically be infused into the source at low flow rates of 1–5 µL/min. Under these low flow conditions, atomisation was achieved via a classical Taylor cone which was formed at the end of the liquid capillary following the application of a few kilovolts between the capillary and the ion inlet cone. More recently, this type of infusion-based analysis for biological applications has been superseded by nanospray ESI capillaries that operate at extremely low flow rates (10–1000 nL/min) and offer high ionization efficiency for small sample volumes. From a commercial viewpoint, the greatest leap in the utilisation of ESI sources came from the marriage of mass spectrometry with liquid chromatography (LC-MS) which enabled analytical chemists to benefit from the enhanced specificity offered by both techniques. In order to adapt ESI sources to the high flow rates (0.1–1.0 mL/min) typically UniSpray Ionization Source. used in LC, or its modern equivalent UPLC, it was necessary to aid the atomisation process with the addition of a concentric flow of high velocity nitrogen gas at the ESI tip. However, when conducting infusion experiments with a fixed analyte concentration from flow rates of 10 nL/min to 1 mL/min, it is common to observe only a 20 times increase in analyte ion signal at the higher flow rate whilst the analyte consumption has increased by a factor of 100,000. This is known to be due to the poor ionization efficiency of high flow rate ESI which is critically dependent on a number of factors such as droplet size distribution, droplet charge per unit volume, droplet evaporation rates and additional factors such as the inlet sampling efficiency. Additionally, ionization efficiency can become particularly challenging when using highly aqueous mobile phases. In this white paper, we introduce a new ionization technique called UniSpray™ which attempts to increase ionization efficiency by interacting a high velocity spray with a high voltage, cylindrical target that is positioned in an off-axis, cross-flow arrangement. A number of physical processes will be described which are believed to be important mechanisms that lead to enhanced UniSpray sensitivity when compared to ESI, viz. high Weber number droplet impacts, the Coanda effect, vortex shedding, and counter-rotating surface microvortices. 1 [ WHITE PAPER ] WHAT IS UNISPRAY? The 1.6 mm-diameter, 35 mm long, cylindrical HV target is UniSpray shares some common features with high flow constructed from a cold-drawn, 316L stainless steel wire that rate ESI sources in that the liquid flow is nebulised by a is polished to a near-mirror finish with 1 µm-grade lapping high velocity, concentric nitrogen gas flow. However, in paper. The target is connected to a 0–5 kV DC power supply comparison to ESI, the high voltage is applied to a cylindrical via a 47 MΩ current-limiting resistor. Since the target has a target electrode that is positioned in close proximity to low thermal conductance to the source housing, it rapidly the grounded nebulizer such that the near-supersonic jet reaches an equilibrium temperature that is equal to the local impinges on the target surface. A schematic of the UniSpray temperature of the heater gas (typically >250 °C for a heater source is shown in Figure 1. The source is formed by directing set-point temperature of 500 °C). In order to optimise source a high velocity nebulised jet from a grounded sprayer onto sensitivity, it is critically important to adjust the point at which a cylindrical metal target that is held at a high voltage and the collimated spray impacts on the cylindrical target. As is located between the sprayer and the ion inlet orifice of shown in Figure 1, the maximum signal intensity is typically the mass spectrometer. For singly charged analytes, it is obtained when the spray is asymmetrically positioned such conventional to use a positive potential of typically 1 kV for that it impacts on the upper right quadrant of the target. positive ion analysis and vice versa for negative ion analysis. Under these conditions, the gas flow becomes attached to a For multiply charged analytes, such as peptides and proteins, portion of the curved surface and results in asymmetric gas it is generally found that higher potentials of 3–4 kV are streamlines in the wake that are directed towards the ion required for signal optimisation. The pneumatically-assisted inlet orifice. This flow phenomenon is known as the Coanda nebulizer is formed from a 130 µm I.D. by 220 µm o.d. liquid effect. Under the influence of the Coanda flow field, ions and delivery capillary (stainless steel) that is surrounded by a charged droplets are directed towards the ion inlet which is 330 µm I.D. nebulizer tube (stainless steel) with a restriction surrounded by a cone gas nozzle that supports a drying gas length of 10 mm. Nitrogen gas is delivered to the nebulizer flow of nitrogen at 150 L/hr. tube at a gauge pressure of 7 bar. Under these conditions, UniSpray sources are generally found to give enhanced the nitrogen jet will be near supersonic at a distance of a few ionization efficiency when compared to high flow rate ESI millimetres from the nebulizer tip. The distance over which the which, in turn, can lead to enhanced analytical sensitivity. inner capillary protrudes from the nebulizer tube is adjustable Some general characteristics of the UniSpray source have and is typically found to optimise at very small protrusions been studied by Lubin et al1. These authors compared the (<0.2 mm) where the spray appears highly collimated. In a signal intensity obtained by ESI and UniSpray for small conventional manner, the nebulizer is also surrounded by an pharmaceutical compounds (16 analytes in positive ion annular heater that delivers hot nitrogen gas at a flow rate of mode and 7 in negative ion mode) with acidic, basic and typically 1200 L/hr. neutral mobile phase types. Figure 2 shows the signal gains (UniSpray ion intensity divided by ESI ion intensity) obtained at various mobile phase compositions and for three different flow rates. Here, each point is an average of the pooled data for each compound and each mobile phase type. From these plots it can be concluded that the signal intensity observed with the UniSpray source is, on average, higher than the ESI signal for all mobile phase compositions with relatively higher gains observed under higher aqueous conditions. The full data set exhibited a strong compound dependence where, whilst UniSpray gains in excess of x20 were observed under certain conditions, other analytes could occasionally give greater responses with an ESI source. In view of the performance enhancements outlined above, the following sections will consider some underlying physical mechanisms that are unique to the UniSpray source and may contribute to the observed increase in ionization efficiency or ion sampling efficiency. Figure 1. A schematic of the UniSpray API source. Waters UniSpray Ionization Source 2 [ WHITE PAPER ] Figure 2. A comparison of the average signal intensity ratios for UniSpray and ESI versus mobile Figure 3. Typical droplet size distribution for a pneumatically phase composition for 16 positive ion analytes and 7 negative ion analytes (from reference1). assisted ESI probe at a flow rate of 0.5 mL/min of water (Malvern Instruments Spraytec system). DROPLET SIZE DISTRIBUTION: THE LIMITATION OF HIGH experimental techniques where, in particular, FLOW RATE ESI the ping-pong drift cell method4 more closely In the traditional ESI model, charged droplets are formed by positive and resembles the dynamics of LC/MS sources. negative charge separation in the high electric field region at the tip of the The diameter of a droplet, dp, after an evaporation liquid capillary. At very low flow rates (<1 µL/min), this process proceeds time, t, an evaporation rate, s, and an initial with extremely high efficiency to yield highly-charged, sub-micron droplets droplet diameter, d0, is given by that almost instantaneously give rise to gas phase ions due to Rayleigh 2 2 dp = d0 − st (i) disintegration processes2. However, at the high flow rates typically used in LC and UPLC-MS (100–800 µL/min), it is necessary to use a high velocity where s has been experimentally determined as 2 -1 nebulizer gas to atomize the liquid flow, a process which is known to 1250 and 6500 µm s for water and acetonitrile, 4 produce larger droplets with a lower charge per unit volume and hence respectively . According to equation (i), Figure lower ionization efficiency3. 4 shows a plot of initial droplet diameter versus droplet diameter after an evaporation period Whilst the use of a high velocity gas is advantageous from an atomisation of 100µs for water and acetonitrile. From these viewpoint, it has the disadvantage of reducing the residence time of data, it becomes apparent that (i) droplets above droplets between the ESI capillary and the ion inlet orifice of the mass 2 µm in diameter do not undergo significant spectrometer, which, in turn, reduces the time available for droplet evaporation in this time frame and (ii) rapid evaporation which is critical to the Rayleigh disintegration process. evaporation only occurs for diameters below In-house measurements, using Phase Doppler Anemometry (PDA) and the “knee” of the curve, which corresponds Laser Diffraction Particle Sizing (LDPS) reveal that the ESI and UniSpray to approximately 0.4 and 1.0 µm for water nebulizers typically produce initial droplet size distributions that peak at and acetonitrile, respectively.
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