Electrospray Ionization Source Geometry for Mass Spectrometry: Past, Present, and Future

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Trends in Analytical Chemistry, Vol. 25, No. 3, 2006 Trends Electrospray ionization source geometry for mass spectrometry: past, present, and future

Irina Manisali, David D.Y. Chen, Bradley B. Schneider

The geometry of an electrospray ion source plays important roles in the The technique involves a number of steps, processes of analyte desolvation, ionization, transportation, and detection in including the formation of charged drop- a mass spectrometer. We provide a brief account of the scientific principles lets, desolvation, ion generation, declus- involved in developing an electrospray ion source, and in the various geo- tering, and ion sampling. As the name metries used to improve the sensitivity of mass spectrometry. We also pre- suggests, the basis of this technique lies in sent some popular configurations currently available and outline future using a strong electric field to create an trends in this research area. excess of charge at the tip of a capillary ª 2005 Elsevier Ltd. All rights reserved. containing the analyte solution. Charged droplets exit the capillary as a spray and Keywords: Atmospheric pressure ionization; Commercial ion source design; Electro- travel at atmospheric pressure down an spray ionization; Ion source; Ion source geometry; Mass spectrometry electrical gradient to the gas conductance- 1. Brief introduction to electrospray limiting orifice or tube. Gas phase ions are Irina Manisali, then transported through different vac- David D.Y. Chen* Electrospray ionization (ESI) is a soft ioni- uum stages to the mass analyzer and Department of Chemistry, zation method, allowing the formation of ultimately the detector. ESI has success- University of British Columbia, fully been coupled with a variety of mass 2036 Main Mall, Vancouver, gas phase ions through a gentle process BC, Canada V6T 1Z1 that makes possible the sensitive analysis analyzers. Each analyzer has different of non-volatile and thermolabile com- advantages and the choice of which to use Bradley B. Schneider pounds. Consequently, the use of the ESI is based on the requirements of the appli- MDS SCIEX, 71 Four Valley Dr., cation. Comprehensive information about Concord, ON, Canada L4K 4V8 source in the field of mass spectrometry (MS) has greatly facilitated the study of the coupling of ESI with various mass large biomolecules, as well as pharma- analyzers can be found in Cole’s 1997 ceutical drugs and their metabolites. Thus, compilation of review articles on the topic the ESI source has evolved with the [2]. growth of proteomics and drug discovery research [1]. The analysis of carbohy- drates, nucleotides, and small polar mole- 2. Milestones in the evolution of cules comprises a few of the many other electrospray applications in which ESI-MS is currently used. Together with matrix assisted laser Since 1968, when Dole used ESI and a desorption ionization (MALDI), another nozzle-skimmer system to produce soft ionization technique, ESI has revolu- charged gas-phase polystyrene [3], the ESI tionized biomedical analysis. In 2002, half source has continued to undergo various the Nobel Prize for Chemistry was awar- changes in its size, material, and geome- ded to ESI developer John Fenn, who try. These transformations were made to shared it with Koichi Tanaka, who made optimize both the ionization efficiency and an outstanding contribution to the devel- the efficiency of transferring gas phase opment of MALDI. ions into the mass analyzer. * Corresponding author. The concept of the ESI source is The evolution of the electrospray-source Tel.: +1 604 822 0887; Fax: +1 604 822 2847; deceivably simple, as some aspects of its design has also reflected the coupling of E-mail: [email protected] functioning are still not well understood. the ion source to separation systems, such

0165-9936/$ - see front matter ª 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2005.07.007 243 Trends Trends in Analytical Chemistry, Vol. 25, No. 3, 2006 as high performance liquid chromatography (HPLC) and electrode can be either the curtain plate of the mass capillary electrophoresis (CE). This has brought new spectrometer or a transport capillary, which will be challenges in generating ions at different flow rates, discussed later. The optimal potential difference between minimizing contamination of the interface, and opti- the sprayer and the principal counter electrode depends mizing overall efficiency. Leading instrument manufac- on experimental parameters, such as the charge state of turers are directly involved in providing innovative the analyte, the solution flow rate, the solvent compo- solutions to the industry’s demand for more sensitive, sition, and the distance between the tip and the counter reliable mass spectrometers. electrode. While different mass spectrometers may re- The purpose of this review is to present some mile- quire different applied voltages on the sprayer and the stones in the evolution of the ESI source to achieve the counter electrode, the potential difference between them designs and the capabilities currently offered by com- is similar (typically 2–5 kV) [8]. In the presence of an mercial manufacturers. We discuss some important electric field, liquid emerges from the tip of the capillary developments achieved by research groups with respect in the shape of a cone, also known as the ‘‘Taylor cone’’ to ESI design. We also present a basic theoretical back- (Fig. 1) [9]. When the electrostatic repulsion between ground on droplet formation and current theories of gas charged molecules at the surface of the Taylor cone phase ion creation in order to provide the reader with a approaches the surface tension of the solution – known better understanding of the reasoning behind the modi- as reaching the Rayleigh limit – charged droplets are fications in source design. While other atmospheric expelled from the tip. The droplets containing excess pressure ion sources use similar geometries, this review positive charge generally follow the electric field lines at will focus on only electrospray ionization. atmospheric pressure toward the counter electrode. However, trajectories will also be affected by space charge and gas flow. 3. Theory The mechanism of forming the Taylor cone is not entirely understood, but it is known that, under certain A number of publications have presented details of the conditions, the morphology of the spray emitted from the electrochemical nature of the electrospray process [4–6]. capillary tip can change [10]. The various spray modes When a large potential difference is applied between an strongly depend on the capillary voltage and are related electrode shaped as a wire and a counter electrode, a to pulsation phenomena observed in the capillary cur- strong electric field is created at the tip. In the case of ESI, rent [10]. Juraschek and Ro¨llgen showed that liquid flow a high voltage is applied to a capillary containing the rate, capillary diameter, and electrolyte concentration analyte solution. For simplicity, we consider the appli- can all impact the spray mode. Controlling the spray cation of positive potential only. Due to the electric field mode is thus crucial in achieving a stable spray and gradient at the tip, charge separation occurs in the an optimal signal. However, this becomes particularly solution as anions migrate towards the capillary walls, difficult when the mobile phase composition changes and cations travel towards the meniscus of the droplet during gradient elution conditions necessary in many formed at the tip (Fig. 1) [7]. LC-MS applications. To address this problem, Valaskovic In order to direct charged species into the mass spec- and Murphy have developed an orthogonal optoelec- trometer, a series of counter electrodes is used in order of tronic system capable of identifying many spray modes decreasing potential. Typically, the principal counter under different conditions [11]. The system automatically optimizes the ESI potential in response to changes in flow rate and solvent composition. The size of the spray droplets released from the Taylor cone, highly dependent on flow rate and capillary diameter, is critical to the efficient ionization of the HV evaporation analyte. ______Taylor + + Since a small droplet contains less solvent, desolvation cone + + ions + +++ + + + sample + + _ + + __ + _+ and and ionization can be more efficient. Because less fission ___ + + ++ _ + + solution + _ + + + neutrals + + + +_ is required to produce ions, the salt concentration in the ______+ + final offspring droplets may be lower compared to a offspring droplet that has undergone more evaporation-fission droplets cycles. As a result, the background noise in the mass spectrum may be reduced [12]. In addition, with smaller E droplets, analytes that are not surface active will have a counter electrode greater chance of being transferred to the gas phase rather than being lost in the bulk of the parent droplet Figure 1. Schematic diagram of the electrospray process. residue [12].

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The solvent is typically a combination of acidified [21,22]. In addition, counter current nitrogen gas flow water and an organic modifier. The role of the organic (Curtain Gase, further illustrated in Fig. 5) emanating solvent is to lower the surface tension of the liquid, from in front of the gas conductance limiting orifice facilitating the formation of gas phase ions. As the sol- provided additional desolvation [8]. The advantage of vent evaporates, the droplet shrinks and the electrostatic the Curtain Gas is that neutral molecules, such as sol- repulsion between charges within the droplet increases. vent, are carried away from the sampling orifice by the When the Rayleigh limit is approached, offspring drop- nitrogen flow, while charged molecules are directed lets begin to break away unevenly in a process also through the gas flow under the influence of the electric known as ‘‘coulombic fission’’ [7]. Evaporation of the field. Counter current gas flows can also aid in declus- offspring droplets leads to a new fission series and the tering as a result of collisions between ions and gas process repeats itself to produce smaller and smaller molecules. Since nitrogen is inert, it cannot form cova- droplets. The eventual creation of gas phase ions from lent bonds with the ions and clustering during transfer these droplets is thought to be a combination of two to vacuum is prevented. While its name varies depend- mechanisms known as the ‘‘ion evaporation mecha- ing on the manufacturer, counter current gas flow is an nism’’ (IEM), initially proposed by Iribarne and Thomson important feature of the ESI ion source. In addition to [13], and the ‘‘charged residue model’’ (CRM), put for- using inert gas flow, heating the ion source or gas con- ward by Dole et al. [3,14] and supported by Rollgen et al. ductance limiting orifice or capillary is also a common [15]. The fundamental difference between the two lies in way to aid declustering and desolvation [23]. the mechanism of forming gas phase ions. IEM suggests that, when the electric field on a charged droplet is high enough, single, solvated, analyte molecules carrying some of the droplet charge are ejected into 4. Historical highlights the gas phase. This happens because the potential en- ergy of the ions near the surface becomes high enough to 4.1. Development of the traditional ESI source allow evaporation to occur [16]. Although the work of Fenn and colleagues demonstrated By contrast, the CRM maintains that gas phase ions the applicability of ESI to generation of biomolecular are formed when successive fissions lead to a charged ions, it was Dole and co-workers who led the way in the droplet containing a single analyte. late 1960s by building the first electrospray based mass Although it is difficult to determine which of the two analysis system [3], as shown in Fig. 2. mechanisms is more accurate, extensive studies [17–19] A sharp hypodermic needle was used to spray poly- seem to suggest that CRM is the preferred mechanism in styrene solution into an evaporation chamber. A Teflon the case of forming charged globular proteins in the gas plate supported the needle as well as a gas inlet intro- phase. ducing nitrogen as a bath gas. A nozzle-skimmer system Kebarle also concluded that charging a single protein in and two stage differential pumping were used to reduce the evaporating droplet is due to small ions found at the pressure in the analyzer region and permit sampling of surface of the droplet [18]. The mechanism of forming the supersonic free jet without disturbing the molecular small analyte ions is still not clear. Charging analyte flow [24]. The voltage applied on the needle was main- molecules can occur through more than one process [20]. tained at approximately À10 kV (negative polarity Charge separation (in the ESI source), adduct formation, used), while the voltages on the first and second colli- gas phase molecular reactions, and electrochemical mating plates were À3 kV, and À1.4 kV, respectively. reactions may also contribute to ionization during the Dole measured mass-to-charge ratios (m/z) by deter- electrospray process. mining the retarding potential of ions through a grid Efficient transport of ions and charged droplets from preceding a Faraday cup detector. the sprayer into the mass spectrometer is challenging Intrigued by Dole’s ESI work, and having himself been and depends on parameters such as interface arrange- a pioneer of supersonic free jets as molecular beam ment and gas throughput into the instrument [8]. As gas sources, Fenn reproduced Dole’s experiments with the and ions are transported from atmospheric pressure into same observations but discontinued research in this area vacuum, strong cooling of the mixture occurs during due to technical limitations [25]. Years later, Fenn expansion. Under these conditions, polar neutral mole- reprised his experiments on molecular beams and used cules may cluster with analyte ions and it is therefore ESI coupled to a quadrupole mass analyzer for ion very important to achieve efficient desolvation within detection. Recognizing flaws in Dole’s ion source design the atmospheric pressure region. One instrument man- as well as in the interpretation of results, Fenn modified ufacturer (MDS SCIEX) introduced a method of desol- the electrospray ion source by reducing the distance vation (described in more detail in Section 5.1.2.), between the hypodermic needle and end plate contain- whereby heated auxiliary nitrogen gas is directed at an ing the nozzle [26] (Fig. 3). As a result, the applied angle from the direction of the spray (TurboIonSpraye) sprayer voltage could be significantly lower.

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N gas out Faraday Cage 2 Spray Chamber

Skimmer

N2 gas in

Liquid in

Nozzle

Pump Pump

Figure 2. Conﬁguration of Dole’s ﬁrst electrospray based mass analysis system. (Reprinted with permission from [3], ª1968, American Institute of Physics).

In contrast with Dole’s ion source, Fenn’s ESI chamber shown in Fig. 4, is currently used on Thermo Electron was built with metal walls to prevent charge build up, instruments. However, a number of other manufacturers and had significantly smaller dimensions. Fenn’s ESI have developed modified versions of this device, including source later evolved into what is known as the Fenn- heated resistive tubes [28] and heated metal capillaries Whitehouse design [27]. The additional characteristic is with different inlet/outlet dimensions [29]. The combi- a glass capillary that allows ions from atmospheric nation of a heated metal capillary and the counter current pressure to enter the first vacuum stage of the instru- gas makes possible even more efficient ion desolvation. ment. The capillary dimensions can be chosen to allow ESI-MS interfaces comprising multiple capillaries have the same flux of drying gas and ions as a regular thin- been developed for various applications, such as ion ion plate orifice [24]. Increasing the capillary diameter may reaction monitoring [30,31], separate introduction of be desirable in order to increase the acceptance and to analyte and calibrant using a multiple sprayer source decrease the chance of blockage. However, for a fixed gas [32], and improved ion transmission when used in throughput, this must be associated with a correspond- combination with an electrodynamic ion funnel [33]. ing increase in capillary length, potentially lowering Other modifications to the heated capillary inlet in- performance. clude offsetting the exit of the capillary from downstream An alternative to using the counter current gas for ion ion optics devices (such as a skimmer) [34,35]. The desolvation is the heated metal capillary, introduced by advantage of this design is that some large solvent Chowdhury et al. [23]. With this configuration, gas clusters exiting the transfer capillary are prevented from throughput depends on both capillary dimensions and entering the next vacuum stage, while the lighter ana- temperature. The heated metal capillary inlet (ion pipe), as lyte ions follow the gas flow through the ion optics.

Cylindrical Electrode

Oxygen End Plate Nozzle

Hypodermic Needle Liquid Sample Skimmer Quadrupole

Figure 3. Conﬁguration of the electrospray ion source coupled with a quadrupole mass analyzer designed by Fenn and co-workers in 1984. (Reprinted with permission from [22], ª1984, American Chemical Society).

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ESI Nozzle

Heater To ESI Skimmer Needle Ion Transfer Capillary

Ion Sweep Cone

Figure 4. Schematic of the Ion Max source from Thermo Electron using a heated metal capillary for ion desolvation and transfer.

Recently, Varian also adopted this modification by discharge [37]. Bruins observed that the spraying positioning a heated capillary outlet off axis from a process was less dependent on the sprayer position rela- subsequent RF multipole ion guide [36]. tive to the orifice than without nebulizer assistance, and In 1987, Bruins et al. introduced the pneumatically that better sensitivity was obtained if the sprayer was assisted nebulizer for the electrospray interface, also pointed off axis instead of directly at the orifice. The known as ion spray. Fig. 5 shows the details of the ion reasoning behind the off axis geometry was that, by spray interface [37]. sampling the periphery of the spray, finer droplets entered The nebulizer gas was beneficial for coupling LC with the mass spectrometer while the larger droplets struck the ESI-MS, because it helped to stabilize electrospray for curtain plate. This led to improved performance because flow rates up to approximately 0.2 mL/min. It also tol- finer droplets are easier to desolvate. Currently, the off erated larger distances between the sprayer and the axis sprayer geometry has evolved to an orthogonal counter electrode, reducing the occurrence of corona sampling position. More detail is given in Section 5 below.

Air / N2 charged droplets sample solution N Air / N2 2

silica capillary stainless steel ions vacuum containing stainless steel tubing neutrals sample capillary holder solution

N 2 atmospheric pressure

Figure 5. Schematic of the ion spray conﬁguration developed by Bruins in 1987.

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4.2. The CE-MS interface trode and the analyte solution, thus avoiding electro- While ion spray became standard for LC-MS at higher chemical modification of analytes. An additional flows, CE-MS coupling was more complicated due to the advantage of using the sheath flow interface is minimi- large variety of solvent compositions required for sepa- zation of corona discharge as a result of reducing the ration and problems associated with electrochemistry. In ionic strength of the sprayed solvent [38]. addition, differences in flow rate requirements also Olivares et al. developed the sheathless interface in presented problems. Three common ways of interfacing 1987 by using a stainless steel capillary to create elec- CE-MS are shown in Fig. 6. trical contact with the analyte solution [39]. Accounting The most common method of CE-MS interfacing is the for about 11% of CE-MS interfaces in 2003 [40], this coaxial sheath flow interface, first described by Smith method evolved with a focus on low flow rates for im- et al. in 1988 [38]. In 2003, approximately 77% of CE- proved sensitivity. MS users employed this type of interface, due to its Other sheathless methods were subsequently devel- reproducibility and robustness [39]. By allowing a con- oped, such as sharpening the CE-capillary tip in order to ducting liquid to provide electrical contact between the produce a stable spray, while the ESI electrode was made stainless steel capillary maintained at high voltage and by installing a piece of gold wire [41] or coating the the fused silica capillary carrying the analyte solution, capillary end with a metal or alloy [42]. The advantages the interfacing of capillary zone electrophoresis (CZE) of the sheathless interface are that analyte dilution and with ESI-MS could be achieved with many buffer systems ion suppression due to the sheath flow can be eliminated. that were previously impractical. This is significant be- Sensitivity can also be improved by up to an order of cause aqueous buffers of high ionic strength are nor- magnitude over sheath-flow designs [43]. mally not tolerated by ESI-MS. Buffer ions can lead to A third method of interfacing CE-MS was the liquid- charge neutralization and corona discharge as well as junction interface developed by Henion and colleagues formation of undesirable adducts with the analyte [44,45]. A liquid junction between the CE capillary and resulting in loss of signal and high sensitivity and high the ESI emitter was built using a stainless steel tee background. By using organic solvents, such as aceto- equipped with a buffer reservoir. This interface reduced nitrile or methanol, as the sheath liquid, buffers of ionic the complication brought about by the different flow rate strength up to 0.2 M can be used. The sheath liquid requirements of CE and ESI [46], but peak broadening prevents direct contact between the high voltage elec- and mechanical difficulties limited the general applicability of this technique [42].

a nebulizer gas nebulizer gas tube 4.3. Miniaturization of electrospray sheath liquid In an attempt to reduce flow rates and thus produce smaller droplets to improve the ionization efficiency, to MS analyte/buffer solution various miniaturized ESI sources were developed [47]. Caprioli et al. [48,49] developed an on line ESI source equipped with a sprayer tip of 10–20 lm internal stainless steel separation capillary capillary/electrode for diameter and capable of operating at flow rates of 300– CZE / ESI voltage 800 nL/min. The emitter was created by burning off the application polyimide coating of a fused silica capillary tip and b tapering it by etching with hydrofluoric acid to give analyte/buffer solution to MS droplets in the micron-diameter range. Caprioli named his technique microelectrospray, a term that currently describes a flow rate range of 0.2–4 lL/min [50]. nebulizer gas separation capillary with conductive Operation in this flow-rate regime may involve various coating for ESI voltage application means for pulling silica capillaries and applying the c electrospray voltage [51]. H.V. nebulizer gas In 1995, Caprioli introduced the idea of using the LC- column tip combined with microelectrospray technology analyte/buffer solution to MS [52] to spray directly towards the mass-spectrometer inlet. This was significant because it triggered the evo- liquid junction lution of analytical LC columns towards miniaturized separation capillary versions of microbore and nanobore capillary columns necessary for low flow rate electrospray [50]. Figure 6. Schematic of the three most common ways to interface Nanoelectrospray, introduced by Wilm and Mann CE-MS: a) coaxial sheath flow; b) sheathless; and, c) liquid junc- more than a decade ago [53], was a particularly tion. successful technique due to its ability to form droplets

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100–1000 times smaller in diameter than droplets and in source fragmentation, as well as improved sen- formed by conventional ESI. The technique was named sitivity, efficiency and practicality. Niessen’s 1998 and to reflect the nanometer (nm) sized droplets as well as 2003 review articles [58,59] are a good starting point the flow rate of nanoliters per minute (nL/min) [54]. for those interested in the trends that have stimulated Essentially, the solution infusion system was eliminated, ESI source evolution. Despite numerous improvements, as a few microliters of solution were transferred to the tip commercial ESI sources still retain many features of the of a gold coated glass capillary. The emitter tip was original configuration. located within a few mm of the inlet aperture, and a stereomicroscope was used to accurately position the 5.1. Source configuration and its relationship to flow sprayer in front of the inlet. By applying a low potential rate onto the conductive capillary surface, a fine spray was A parameter of critical importance in ESI-MS is the flow generated and the charged droplets were directed to- rate of the solution. Depending on the application for wards the counter electrode under the effect of the which the mass spectrometer is used, the ESI source electric field gradient. Despite an estimated efficiency of needs to be adapted to handle the in coming flow of 500 times greater than traditional ESI [54], nanoelec- solution, whether it is direct infusion, or LC or CE eluent. trospray has some limitations, including plugging of the emitter tip and lower reproducibility associated with 5.1.1. Low flow rate. While flow rates from approxi- differences in tip morphology. mately 1 lL/min up to several mL/min are typically used While the term ‘‘nanoelectrospray’’ initially referred to with the assistance of a nebulizer, traditional nanoelec- Wilm and Mann’s off line technique, evolving technology trospray relied simply on the electrostatic attraction be- has made possible the use of on line flow rates down to tens tween the mobile phase and the counter electrode to of nL/min. The term nanoelectrospray has thus expanded generate and disperse the charged droplets. The nano- to encompass both on line and off line techniques. flow regime can generally be defined as using flow rates For nanoelectrospray, the effects of tip geometry, flow of less than 1 lL/min and can extend to levels of less rate and solvent composition have been investigated by a than 1 nL/min [55]. As mentioned previously, nanoflow number of groups [55–57]. ESI can be used independently (off line) or coupled to a Motivated by the fact that, at certain solvent compo- separation system (on line), such as CE or HPLC. sitions, the sensitivity decreased dramatically, Vanhoutte Depending on experimental requirements, the commer- et al. compared the effects of various mobile phase cially available nanoelectrospray emitters vary in mate- compositions using several types of electrospray capil- rial, tip shape, diameter, and the configuration used for laries. These included combinations of uncoated or gold electrical contact. On line analyses typically use higher coated, tapered or non-tapered fused silica capillary tips, flow rates (0.1–1 lL/min), and the tips are usually fab- as well as stainless steel emitters. They found that the ricated from fused silica or metal. Off line analyses typ- gold coated, tapered tips were the most effective, because ically use flow rates in the low nL/min range, with they covered the most extensive range of solvent com- coated or uncoated glass or quartz emitters. A number of position for the tested conditions without loss of sensi- groups have also described modified nanoflow sprayers tivity [55]. with various coatings [60–62] or inserted fibers [63]. Schmidt and Karas used combinations of model com- There are many different configurations for nanoflow pounds to examine the effect of flow rate on ion signal LC-MS coupling [64], although they all include an LC [56]. They determined that, at flow rates of a few column and a narrow diameter emitter tip. The simplest nL/min, signal suppression caused by differences in combination involves coupling an upstream LC column surface activity of the analytes was insignificant com- to the emitter tip using some type of low dead volume pared to that at higher flow rates (> 50 nL/min). union [65]. The union can also serve as the electrical Li and Cole studied the often observed shift in charge contact for the electrospray process, although it is also state as a result of changing experimental parameters for possible to use conductive tips or other electrode con- nanoelectrospray [57]. Parameters such as tip diameter, figurations [64,66]. The advantage of decoupling the LC flow rate, analyte concentration and solvent composition column from the sprayer is simple, inexpensive replace- can all affect the observed ions and the charge states. ment of sprayer tips, should they become damaged or plugged. However, the coupling must be done carefully to maintain chromatographic performance. 5. Currently available commercial sources An alternative combination involves packing the LC- column material directly into the sprayer tip to eliminate Since recognition of ESI as an invaluable bioanalytical any dead volume after the column [67–70]. Tapered tool in the late 1980s, research groups have attempted sprayers containing LC column packing are available to exploit the capabilities of ESI by modifying the source commercially from companies such as New Objective geometry in order to allow a wider range of flow rates Inc. The column preparation typically involves passing a

http://www.elsevier.com/locate/trac 249 Trends Trends in Analytical Chemistry, Vol. 25, No. 3, 2006 slurry containing column packing material through a HPLC eluent tapered sprayer [71,72]. Sometimes, a small frit is included inside the tip of the sprayer to help confine the packing material. The elimination of post column dead volume helps to ensure optimal chromatographic per- 1.5 torr orthogonal formance. In addition, it has been suggested that the nebulizer ion transfer capillary presence of packing material in the tapered sprayer acts asymmetrical similar to a filter, extending tip lifetimes by preventing lens plugging. The drawback with this configuration is that nebulized to MS tip replacement due to blockage or damage requires the analyte solution entire column to be replaced. Electrical contact can be established at the sprayer tip or distal to the tip end. Amirkhani and co-workers recently compared the atmospheric pressure efficiency of four different sheathless electrospray emitter configurations for a nanoflow LC system [73]. Two of the configurations used on-column emitters with the applied voltage either at the outlet or the inlet end of the column, and the other two had emitters coupled to columns, with the electrical connection at either the Figure 7. Schematic of an ESI source configuration developed by Agilent Technologies. sprayer tip or the low dead volume union. It was demonstrated that all the configurations worked equally well, provided the connections were made with minimal electrospray process [75]. The purpose of the electrode is dead volume [73]. to help initiate and sustain electrospray. Conversely, Currently, most major MS manufacturers (e.g., Thermo other groups have attempted to use various types of Electron, MDS SCIEX, Agilent Technologies, and Waters/ auxiliary electrodes [76–78] to focus ions at atmospheric Micromass) make the nanoelectrospray source available. pressure. The number of applications using electrospray at low flow Also exploiting the advantages of orthogonal sampling rates is very large, and outside the scope of this paper. is the Waters/Micromass Zspraye [79,80] interface, Wood et al.’s 2003 review article is a good starting point shown schematically in Fig. 8. The spray is first sampled and a source of references [47]. orthogonally through the sampling cone into a low pressure chamber. An extraction cone (skimmer) is ori- 5.1.2. High flow rate. If the flow rate is in the range ented at a right angle relative to the axis of the spray to 0.05–3 mL/min, sensitivity can be an issue, due to the sample for a second time into the next differentially decrease in ionization efficiency resulting from large pumped vacuum stage. The double orthogonal sampling droplet size. ESI-MS is widely interfaced with LC, so high system prevents solvent and neutral molecules from flow rates are often necessary. One solution to this entering the analyzer, resulting in reduced chemical problem is to employ a splitter that allows only a limited background [81]. Larger cone apertures are used to volumetric flow rate to reach the MS interface. Under these conditions, part of the eluent is wasted and connection dead volume is a common problem. A more practical approach adopted by manufacturers is to sample the spray from a region, peripheral to the main droplet trajectory, where the mist is much finer. This is done by simply re-orienting the sprayer relative to the interface so that the fine droplets from the exterior of the spray plume can enter the sampling inlet, while the majority of large droplets are directed away from the entrance [74]. To minimize contamination, many major MS manufacturers now sample orthogonally from the spray plume for many applications (e.g., Agilent Tech- nologies, Waters/Micromass, and MDS SCIEX). An example of this is shown in Fig. 7 for an Agilent Tech- nologies source incorporating an additional asymmetrical lens. The asymmetrical lens is essentially a half-full, partial Figure 8. Schematic of the ZsprayTM source configuration used by cylinder electrode operated at high voltage during the Waters/Micromass.

250 http://www.elsevier.com/locate/trac Trends in Analytical Chemistry, Vol. 25, No. 3, 2006 Trends compensate for transmission losses. The ability to dis- This probe configuration is referred to as Heated Elec- assemble and to clean the sampling cone without trospray Ionization (H-ESI) and it tolerates the use of breaking vacuum is an additional advantage contribut- higher flow rates than previously possible on Thermo ing to the ruggedness of the Zspray source. Electron instruments. Although some MS manufacturers (e.g., MDS SCIEX, Waters/Micromass, and Agilent Technologies) have op- 5.2. Interface ted for orthogonal sampling, Thermo Electron uses a Various ion sampling interface configurations have been sampling angle at 60 from the ion optics axis. In its Ion developed [8]. The sampling orifice can be built in a disc, Max source, the function of the angled position of the in the top or the bottom of a cone, or in glass or metal sprayer is similar to the orthogonal one. tubes. Some MS manufacturers now equip their instru- Sprayer orientation is not the only important param- ments with conical interfaces. Intuitively, the conical eter when it comes to optimizing sensitivity while using shape is meant to improve the electric field density at the high flow rates. For conventional high flow rate ESI sampling orifice in an attempt to improve ion transmis- sources, the nebulizer gas is an essential component. Air sion. To avoid potential contamination, the sampling or nitrogen is typically used to disperse the emerging cone can be heated to evaporate residuals from its solution into small droplets and to direct the droplets on surface. Since high temperatures are required for this the trajectory chosen for optimal sampling. purpose, ceramic materials have often been used for the To vaporize the large amounts of solvent emerging interface. Ceramic produces no outgassing, and is from the sprayer as efficiently as possible, manufacturers thought to have low sample adsorptivity, thus reducing have introduced additional features to assist the nebu- memory effects on the interface surface [84]. lizer gas. MDS SCIEX developed the TurboIonSpray source [21,22], in which heated nitrogen gas that is released from a unit external to the sprayer is used to 6. Multiple sprayers assist evaporation of the spray droplets at atmospheric pressure. More recently, MDS SCIEX took this design a Commercial multiple sprayer systems for ESI have step further, creating the TurboVe source, shown in evolved mainly as a result of industry’s increasing Fig. 9. In this case, two heated auxiliary nitrogen sources requirements for high throughput sample analysis, par- are oriented to achieve very efficient desolvation. The ticularly for pharmaceutical samples. With a multiple improved desolvation permits stable, sensitive operation sprayer source and suitable equipment, LC-MS analyses for flow rates of greater than 1 mL/min [82]. can be done quickly and efficiently. However, the first Thermo Electron also integrated auxiliary nitrogen gas attempts at building multiple ESI sprayers were moti- for desolvation purposes, except that it flows directly vated by other reasons. Smith et al. [30,85] were the first from the nozzle of the Ion Max source [83]. Similar to the to describe the coupling of two ESI sprayers in order to TurboV, the Ion Max uses a high temperature source generate ions of opposite polarity and study ion-ion heater incorporated into the housing of the ion source. chemistry. McLuckey et al. also interfaced up to three ion sources (two ESI sources and one atmospheric sampling glow discharge ionization (ASGDI) source) onto a quadrupole ion trap mass spectrometer in order to control the charge states of reactant and product ions [86,87]. Other multiple source combinations have also ESI been described, incorporating ESI with sources such as probe heater atmospheric pressure chemical ionization (APCI) [88,89]. Using a linear array of capillary electrodes, Rulison et al. demonstrated the feasibility of increasing sample heated nitrogen throughput by using parallel capillaries [90]. They also observed that the Taylor cones at the ends of the array were being deflected due to end effects caused by the electric field. In 1994, Kostiainen and Bruins proposed that multiple sampling orifice spray sprayers could be used to improve ion current stability under high flow rate conditions (i.e., above 200 lL/min) exhaust [91]. Four years later, Kassel and Zeng developed a dual ESI source for purification and simultaneous analysis of combinatorial libraries using two separate LC-MS eluent TM Figure 9. Schematic of the TurboV source used by MDS SCIEX. streams [92]. Although this development paved the way

http://www.elsevier.com/locate/trac 251 Trends Trends in Analytical Chemistry, Vol. 25, No. 3, 2006 for further efforts in high throughput parallel analysis, effects, only a small percentage of the spray plume is Kassel’s system required knowledge of the particular actually sampled. There is still significant room for analyte that each sprayer was generating. Dual ESI improvement in ion sampling efficiency. sources have also been used to introduce an internal calibration solution separately from the analyte solution 7.1. High velocity nebulizers [93–96]. This configuration provides benefits compared One approach developed by Zhou et al. [106] involved with a single solution containing both analyte and installing an industrial air amplifier between the elec- calibrant because it avoids preferential ionization. trospray probe and the sampling orifice. The concentric, In 1999, Kassel et al. [97] and De Biassi et al. [98] high velocity gas flow was meant to control the expan- described indexed multiple sprayer systems that allow sion of the spray plume as well as assist in the desolva- sequential sampling of each spray by a mass spectrom- tion of ions. In addition, focusing of ions could be eter, thus making it possible to identify ions from their achieved by applying an optimized voltage of up to 3 kV respective eluent stream. onto the amplifier. Thus, when both the air amplifier and Currently, publications in which fast LC-MS analyses the high voltage were used, Zhou’s ion source configu- of samples are discussed indicate that the Waters/ ration seemed to provide some improvement in ion- Micromass multiplexed ESI source (MUX) is the most sampling efficiency. Suppression of background chemical commonly used for the analysis of multiple LC eluent noise was also observed. streams [99–101]. Tiller’s 2003 review paper provides useful references regarding biological applications using 7.2. Improved nanoflow configurations the MUX system as well as other types of parallel anal- Schneider et al. developed a novel interface for nanoflow ysis [102]. The MUX system attaches to the Zspray ESI (Fig. 10) that provides two separate atmospheric source of the mass spectrometer and is fitted with either pressure regions for droplet desolvation as well as two four or eight channels. As a rotating aperture allows regions for removing unwanted particles [107]. only one spray at a time to reach the sampling cone, The first desolvation stage makes use of the traditional spectra of compounds eluting from individual separation counter-current gas to eliminate neutral particles and columns can be quickly acquired. solvent. Next, charged particles, ions and gas travel However, a significant problem with MUX-type through the heated laminar flow chamber, where addi- systems is a decrease in sensitivity of approximately tional desolvation takes place at optimal temperature. three-fold at high flows [103]. Yang et al. attributed the Once they reach the particle discriminator area, ions are drop in sensitivity to several reasons, such as difficulty in carried by the gas streamlines through the discriminator optimizing sprayer position and lower desolvation space and into the sampling orifice, while large charged efficiency due to large amounts of solvent introduced particles with high momentum escape from the gas into the source in combination with a desolvation gas streamline trajectory under the influence of the strong that was counter-current relative to the spray instead of electric field. This source design provided improved sig- the traditional coaxial flow. The sensitivity decrease is nal and ion current stability for flow rates from a few nL/ expected to be larger for nanoflow operation, where min up to 1 lL/min as well as improved performance for sprayer position can be more critical. gradient elution nanoLC-MS in both positive-ion and In 2002, Schneider et al. [104] developed an alternative negative ion mode [108]. multiple sprayer system that appeared to solve the sensitivity problem. By installing an atmospheric pressure ion 7.3. Desorption electrospray ionization lens [76,105] on each sprayer of a dual sprayer and a four Cooks and co-workers recently described a new method sprayer ESI source, they were able to maintain high sen- of ionization in which analytes are desorbed from sitivity by optimizing the applied lens potential as well as surfaces by nebulizing the charged droplets of an sprayer position. An additional advantage of the system electrospray plume at a surface [109]. This technique is was that sprayers could be enabled or disabled by referred to as desorption electrospray ionization (DESI). changing the lens potential, potentially a faster method The exact mechanism of ion formation in DESI is not than using mechanical devices. well understood, although the technique has been successfully used for analyzing a number of samples, including pills and TLC plates [110]. Efficient sampling of 7. Recent developments in ESI sources desorbed ions requires careful optimization of the angles and the spacings between the surface, the electrospray The quest for a superior source design capable of effec- emitter, and the mass spectrometer inlet. tively focusing more of the electrospray generated ion cloud through the sampling aperture of the instrument is 7.4. Coupling ESI to micro-analytical systems still on going. Due to gas phase collisions occurring In the rapidly expanding area of microchip chemical under atmospheric pressure conditions and space charge analysis, currently known as ‘‘Micro-Total Analysis

252 http://www.elsevier.com/locate/trac Trends in Analytical Chemistry, Vol. 25, No. 3, 2006 Trends

Curtain Plate Heated Laminar Flow Chamber

Curtain Gas (TM)

Orifice

Tee and Nanospray Tip with Nebulizer Particle Discriminator Space

Teflon Spacer

Figure 10. Schematic of the particle discriminator interface developed by MDS SCIEX.

Systems’’ (l-TASs) and the related ‘‘Lab-on-a-chip’’, ESI enrichment and analytical columns packed with reverse- has become the preferred interface method for MS phase material, a face-seal rotary valve for switching detection [111–113]. More than a decade after being between loading and separation positions, and a conical introduced [114], l-TASs have been used in a wide ESI emitter created directly on the chip structure by laser range of biological applications, drug discovery, and ablation. Gradient reverse phase separation of peptides clinical diagnostics. References regarding applications has been achieved at flow rates of 100–400 nL/min. The can be found in review papers [115–120]. system was reported to be stable with various solvents, The interest in miniaturized analytical systems rose sensitive, and robust. Since the various connections due to the potential for integrating time consuming necessary for traditional nanoelectrospray were elimi- steps, such as sample preparation, chemical reaction, nated, dead volume problems as well as analyte disper- separation, and detection, into a fast, highly auto- sion were minimized [125]. mated technique, while at the same time using minute While the combination of microfluidic devices with MS sample quantities. Constantly evolving microfabrica- may have great potential, many barriers still need to be tion techniques adapted from the semiconductor overcome. Currently, CE methods appear to be most industry are used to produce the microfluidic devices common for sample separation on microfluidic chips, [117,121,122]. because they are less complex to miniaturize than HPLC Although on-chip optical detection is often used in [126]. Difficulties, such as integrating pumping systems microfluidic applications, MS detection is desirable due to for controlling flow and creating gradients, still limit its suitability for analyzing biological molecules. In widespread commercialization of microfluidic-LC systems addition, the low flow rates (nL–lL/min) used with [127]. microfluidic devices are optimal for the ESI source. For high throughput analyses, Advion BioSciences However, integrating ESI-MS with a microchip is not successfully introduced a fully automated nanoelectro- easy. Initial work involved using traditional ESI emitters, spray system, called the NanoMatee. This microchip such as a section of fused silica capillary or a nanoelec- device contains an array of nozzles to which the sample trospray needle attached to a specific microchannel exit, is automatically delivered for MS analysis. Short analysis but these types of sources were prone to contamination times are possible due to a simplified spray optimization problems from bonding agents, poor reproducibility, and procedure [128,129]. Although the NanoMate was ini- dead volume problems [121,123,124]. Work has tially intended for use in combination with microfluidic therefore focused on building the ESI emitter as part of separation devices [128], it has been integrated with the microdevice and a range of materials, from silicon sources from other manufacturers (e.g., Waters/Micro- based to polymers, has been tested for fabrication of the mass, MDS SCIEX, and Thermo Electron). The key ESI emitter [117,121]. advantages of the NanoMate include elimination of Agilent Technologies has introduced a polymer based sample carry over and simplification of the tuning pro- HPLC-MS microfluidic chip [125]. The chip contains cedure [130].

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