Electrospray ionization source geometry for mass spectrometry: past ...

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
processes of analyte desolvation, ionization, transportation, and detection in
a mass spectrometer. We provide a brief account of the scientific principles
involved in developing an electrospray ion source, and in the various geometries
used to improve the sensitivity of mass spectrometry. We also present
some popular configurations currently available and outline future
trends in this research area.
ª 2005 Elsevier Ltd. All rights reserved.
Keywords: Atmospheric pressure ionization; Commercial ion source design; Electrospray
ionization; Ion source; Ion source geometry; Mass spectrometry
Irina Manisali,
David D.Y. Chen*
Department of Chemistry,
University of British Columbia,
2036 Main Mall, Vancouver,
BC, Canada V6T 1Z1
Bradley B. Schneider
MDS SCIEX, 71 Four Valley Dr.,
Concord, ON, Canada L4K 4V8
* Corresponding author.
Tel.: +1 604 822 0887;
Fax: +1 604 822 2847;
E-mail: chen@chem.ubc.ca
1. Brief introduction to electrospray
Electrospray ionization (ESI) is a soft ionization
method, allowing the formation of
gas phase ions through a gentle process
that makes possible the sensitive analysis
of non-volatile and thermolabile compounds.
Consequently, the use of the ESI
source in the field of mass spectrometry
(MS) has greatly facilitated the study of
large biomolecules, as well as pharmaceutical
drugs and their metabolites. Thus,
the ESI source has evolved with the
growth of proteomics and drug discovery
research [1]. The analysis of carbohydrates,
nucleotides, and small polar molecules
comprises a few of the many other
applications in which ESI-MS is currently
used. Together with matrix assisted laser
desorption ionization (MALDI), another
soft ionization technique, ESI has revolutionized
biomedical analysis. In 2002, half
the Nobel Prize for Chemistry was awarded
to ESI developer John Fenn, who
shared it with Koichi Tanaka, who made
an outstanding contribution to the development
of MALDI.
The concept of the ESI source is
deceivably simple, as some aspects of its
functioning are still not well understood.
The technique involves a number of steps,
including the formation of charged droplets,
desolvation, ion generation, declustering,
and ion sampling. As the name
suggests, the basis of this technique lies in
using a strong electric field to create an
excess of charge at the tip of a capillary
containing the analyte solution. Charged
droplets exit the capillary as a spray and
travel at atmospheric pressure down an
electrical gradient to the gas conductancelimiting
orifice or tube. Gas phase ions are
then transported through different vacuum
stages to the mass analyzer and
ultimately the detector. ESI has successfully
been coupled with a variety of mass
analyzers. Each analyzer has different
advantages and the choice of which to use
is based on the requirements of the application.
Comprehensive information about
the coupling of ESI with various mass
analyzers can be found in Cole’s 1997
compilation of review articles on the topic
[2].
2. Milestones in the evolution of
electrospray
Since 1968, when Dole used ESI and a
nozzle-skimmer system to produce
charged gas-phase polystyrene [3], the ESI
source has continued to undergo various
changes in its size, material, and geometry.
These transformations were made to
optimize both the ionization efficiency and
the efficiency of transferring gas phase
ions into the mass analyzer.
The evolution of the electrospray-source
design has also reflected the coupling of
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
capillary electrophoresis (CE). This has brought new
challenges in generating ions at different flow rates,
minimizing contamination of the interface, and optimizing
overall efficiency. Leading instrument manufacturers
are directly involved in providing innovative
solutions to the industry’s demand for more sensitive,
reliable mass spectrometers.
The purpose of this review is to present some milestones
in the evolution of the ESI source to achieve the
designs and the capabilities currently offered by commercial
manufacturers. We discuss some important
developments achieved by research groups with respect
to ESI design. We also present a basic theoretical background
on droplet formation and current theories of gas
phase ion creation in order to provide the reader with a
better understanding of the reasoning behind the modifications
in source design. While other atmospheric
pressure ion sources use similar geometries, this review
will focus on only electrospray ionization.
3. Theory
A number of publications have presented details of the
electrochemical nature of the electrospray process [4–6].
When a large potential difference is applied between an
electrode shaped as a wire and a counter electrode, a
strong electric field is created at the tip. In the case of ESI,
a high voltage is applied to a capillary containing the
analyte solution. For simplicity, we consider the application
of positive potential only. Due to the electric field
gradient at the tip, charge separation occurs in the
solution as anions migrate towards the capillary walls,
and cations travel towards the meniscus of the droplet
formed at the tip (Fig. 1) [7].
In order to direct charged species into the mass spectrometer,
a series of counter electrodes is used in order of
decreasing potential. Typically, the principal counter
sample
solution
HV
_ _ _ _ _ _ _ _ _
+
+
+ +
_ _ _ _ _ _ _ _ _ + +
+
+ +
+ + + +
_ _
_ +
_ + _+
+ +
+ _ +
+
_
Taylor
cone
+ +
E
evaporation
+ +
+ +
+ +
+
+
+
_
_
_
offspring
droplets
ions
and
neutrals
Figure 1. Schematic diagram of the electrospray process.
244 http://www.elsevier.com/locate/trac
counter
electrode
electrode can be either the curtain plate of the mass
spectrometer or a transport capillary, which will be
discussed later. The optimal potential difference between
the sprayer and the principal counter electrode depends
on experimental parameters, such as the charge state of
the analyte, the solution flow rate, the solvent composition,
and the distance between the tip and the counter
electrode. While different mass spectrometers may require
different applied voltages on the sprayer and the
counter electrode, the potential difference between them
is similar (typically 2–5 kV) [8]. In the presence of an
electric field, liquid emerges from the tip of the capillary
in the shape of a cone, also known as the ‘‘Taylor cone’’
(Fig. 1) [9]. When the electrostatic repulsion between
charged molecules at the surface of the Taylor cone
approaches the surface tension of the solution – known
as reaching the Rayleigh limit – charged droplets are
expelled from the tip. The droplets containing excess
positive charge generally follow the electric field lines at
atmospheric pressure toward the counter electrode.
However, trajectories will also be affected by space
charge and gas flow.
The mechanism of forming the Taylor cone is not
entirely understood, but it is known that, under certain
conditions, the morphology of the spray emitted from the
capillary tip can change [10]. The various spray modes
strongly depend on the capillary voltage and are related
to pulsation phenomena observed in the capillary current
[10]. Juraschek and Röllgen showed that liquid flow
rate, capillary diameter, and electrolyte concentration
can all impact the spray mode. Controlling the spray
mode is thus crucial in achieving a stable spray and
an optimal signal. However, this becomes particularly
difficult when the mobile phase composition changes
during gradient elution conditions necessary in many
LC-MS applications. To address this problem, Valaskovic
and Murphy have developed an orthogonal optoelectronic
system capable of identifying many spray modes
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
analyte.
Since a small droplet contains less solvent, desolvation
and ionization can be more efficient. Because less fission
is required to produce ions, the salt concentration in the
final offspring droplets may be lower compared to a
droplet that has undergone more evaporation-fission
cycles. As a result, the background noise in the mass
spectrum may be reduced [12]. In addition, with smaller
droplets, analytes that are not surface active will have a
greater chance of being transferred to the gas phase
rather than being lost in the bulk of the parent droplet
residue [12].

Trends in Analytical Chemistry, Vol. 25, No. 3, 2006 Trends
The solvent is typically a combination of acidified
water and an organic modifier. The role of the organic
solvent is to lower the surface tension of the liquid,
facilitating the formation of gas phase ions. As the solvent
evaporates, the droplet shrinks and the electrostatic
repulsion between charges within the droplet increases.
When the Rayleigh limit is approached, offspring droplets
begin to break away unevenly in a process also
known as ‘‘coulombic fission’’ [7]. Evaporation of the
offspring droplets leads to a new fission series and the
process repeats itself to produce smaller and smaller
droplets. The eventual creation of gas phase ions from
these droplets is thought to be a combination of two
mechanisms known as the ‘‘ion evaporation mechanism’’
(IEM), initially proposed by Iribarne and Thomson
[13], and the ‘‘charged residue model’’ (CRM), put forward
by Dole et al. [3,14] and supported by Rollgen et al.
[15]. The fundamental difference between the two lies in
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
the gas phase. This happens because the potential energy
of the ions near the surface becomes high enough to
allow evaporation to occur [16].
By contrast, the CRM maintains that gas phase ions
are formed when successive fissions lead to a charged
droplet containing a single analyte.
Although it is difficult to determine which of the two
mechanisms is more accurate, extensive studies [17–19]
seem to suggest that CRM is the preferred mechanism in
the case of forming charged globular proteins in the gas
phase.
Kebarle also concluded that charging a single protein in
the evaporating droplet is due to small ions found at the
surface of the droplet [18]. The mechanism of forming
small analyte ions is still not clear. Charging analyte
molecules can occur through more than one process [20].
Charge separation (in the ESI source), adduct formation,
gas phase molecular reactions, and electrochemical
reactions may also contribute to ionization during the
electrospray process.
Efficient transport of ions and charged droplets from
the sprayer into the mass spectrometer is challenging
and depends on parameters such as interface arrangement
and gas throughput into the instrument [8]. As gas
and ions are transported from atmospheric pressure into
vacuum, strong cooling of the mixture occurs during
expansion. Under these conditions, polar neutral molecules
may cluster with analyte ions and it is therefore
very important to achieve efficient desolvation within
the atmospheric pressure region. One instrument manufacturer
(MDS SCIEX) introduced a method of desolvation
(described in more detail in Section 5.1.2.),
whereby heated auxiliary nitrogen gas is directed at an
angle from the direction of the spray (TurboIonSpraye)
[21,22]. In addition, counter current nitrogen gas flow
(Curtain Gase, further illustrated in Fig. 5) emanating
from in front of the gas conductance limiting orifice
provided additional desolvation [8]. The advantage of
the Curtain Gas is that neutral molecules, such as solvent,
are carried away from the sampling orifice by the
nitrogen flow, while charged molecules are directed
through the gas flow under the influence of the electric
field. Counter current gas flows can also aid in declustering
as a result of collisions between ions and gas
molecules. Since nitrogen is inert, it cannot form covalent
bonds with the ions and clustering during transfer
to vacuum is prevented. While its name varies depending
on the manufacturer, counter current gas flow is an
important feature of the ESI ion source. In addition to
using inert gas flow, heating the ion source or gas conductance
limiting orifice or capillary is also a common
way to aid declustering and desolvation [23].
4. Historical highlights
4.1. Development of the traditional ESI source
Although the work of Fenn and colleagues demonstrated
the applicability of ESI to generation of biomolecular
ions, it was Dole and co-workers who led the way in the
late 1960s by building the first electrospray based mass
analysis system [3], as shown in Fig. 2.
A sharp hypodermic needle was used to spray polystyrene
solution into an evaporation chamber. A Teflon
plate supported the needle as well as a gas inlet introducing
nitrogen as a bath gas. A nozzle-skimmer system
and two stage differential pumping were used to reduce
pressure in the analyzer region and permit sampling of
the supersonic free jet without disturbing the molecular
flow [24]. The voltage applied on the needle was maintained
at approximately 10 kV (negative polarity
used), while the voltages on the first and second collimating
plates were 3 kV, and 1.4 kV, respectively.
Dole measured mass-to-charge ratios (m/z) by determining
the retarding potential of ions through a grid
preceding a Faraday cup detector.
Intrigued by Dole’s ESI work, and having himself been
a pioneer of supersonic free jets as molecular beam
sources, Fenn reproduced Dole’s experiments with the
same observations but discontinued research in this area
due to technical limitations [25]. Years later, Fenn
reprised his experiments on molecular beams and used
ESI coupled to a quadrupole mass analyzer for ion
detection. Recognizing flaws in Dole’s ion source design
as well as in the interpretation of results, Fenn modified
the electrospray ion source by reducing the distance
between the hypodermic needle and end plate containing
the nozzle [26] (Fig. 3). As a result, the applied
sprayer voltage could be significantly lower.
http://www.elsevier.com/locate/trac 245

Trends Trends in Analytical Chemistry, Vol. 25, No. 3, 2006
Faraday Cage
Skimmer
Nozzle
Pump Pump
N 2 gas out
In contrast with Dole’s ion source, Fenn’s ESI chamber
was built with metal walls to prevent charge build up,
and had significantly smaller dimensions. Fenn’s ESI
source later evolved into what is known as the Fenn-
Whitehouse design [27]. The additional characteristic is
a glass capillary that allows ions from atmospheric
pressure to enter the first vacuum stage of the instrument.
The capillary dimensions can be chosen to allow
the same flux of drying gas and ions as a regular thinplate
orifice [24]. Increasing the capillary diameter may
be desirable in order to increase the acceptance and to
decrease the chance of blockage. However, for a fixed gas
throughput, this must be associated with a corresponding
increase in capillary length, potentially lowering
performance.
An alternative to using the counter current gas for ion
desolvation is the heated metal capillary, introduced by
Chowdhury et al. [23]. With this configuration, gas
throughput depends on both capillary dimensions and
temperature. The heated metal capillary inlet (ion pipe), as
Spray Chamber
N 2 gas in
Liquid in
Figure 2. Configuration of Dole’s first electrospray based mass analysis system. (Reprinted with permission from [3], ª1968, American Institute
of Physics).
Oxygen
Liquid
Sample
Cylindrical Electrode
Hypodermic Needle
shown in Fig. 4, is currently used on Thermo Electron
instruments. However, a number of other manufacturers
have developed modified versions of this device, including
heated resistive tubes [28] and heated metal capillaries
with different inlet/outlet dimensions [29]. The combination
of a heated metal capillary and the counter current
gas makes possible even more efficient ion desolvation.
ESI-MS interfaces comprising multiple capillaries have
been developed for various applications, such as ion ion
reaction monitoring [30,31], separate introduction of
analyte and calibrant using a multiple sprayer source
[32], and improved ion transmission when used in
combination with an electrodynamic ion funnel [33].
Other modifications to the heated capillary inlet include
offsetting the exit of the capillary from downstream
ion optics devices (such as a skimmer) [34,35]. The
advantage of this design is that some large solvent
clusters exiting the transfer capillary are prevented from
entering the next vacuum stage, while the lighter analyte
ions follow the gas flow through the ion optics.
N 2
End Plate
Nozzle
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).
246 http://www.elsevier.com/locate/trac

Trends in Analytical Chemistry, Vol. 25, No. 3, 2006 Trends
ESI
Needle
ESI Nozzle
Ion Sweep
Cone
Recently, Varian also adopted this modification by
positioning a heated capillary outlet off axis from a
subsequent RF multipole ion guide [36].
In 1987, Bruins et al. introduced the pneumatically
assisted nebulizer for the electrospray interface, also
known as ion spray. Fig. 5 shows the details of the ion
spray interface [37].
The nebulizer gas was beneficial for coupling LC with
ESI-MS, because it helped to stabilize electrospray for
flow rates up to approximately 0.2 mL/min. It also tolerated
larger distances between the sprayer and the
counter electrode, reducing the occurrence of corona
Heater
Ion
Transfer
Capillary
To
Skimmer
Figure 4. Schematic of the Ion Max source from Thermo Electron using a heated metal capillary for ion desolvation and transfer.
silica capillary
containing
sample
solution
stainless steel
capillary holder
sample solution
stainless steel
tubing
discharge [37]. Bruins observed that the spraying
process was less dependent on the sprayer position relative
to the orifice than without nebulizer assistance, and
that better sensitivity was obtained if the sprayer was
pointed off axis instead of directly at the orifice. The
reasoning behind the off axis geometry was that, by
sampling the periphery of the spray, finer droplets entered
the mass spectrometer while the larger droplets struck the
curtain plate. This led to improved performance because
finer droplets are easier to desolvate. Currently, the off
axis sprayer geometry has evolved to an orthogonal
sampling position. More detail is given in Section 5 below.
Air / N2
Air / N2
E
neutrals
charged
droplets
ions
atmospheric pressure
Figure 5. Schematic of the ion spray configuration developed by Bruins in 1987.
N 2
N
2
vacuum
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Trends Trends in Analytical Chemistry, Vol. 25, No. 3, 2006
4.2. The CE-MS interface
While ion spray became standard for LC-MS at higher
flows, CE-MS coupling was more complicated due to the
large variety of solvent compositions required for separation
and problems associated with electrochemistry. In
addition, differences in flow rate requirements also
presented problems. Three common ways of interfacing
CE-MS are shown in Fig. 6.
The most common method of CE-MS interfacing is the
coaxial sheath flow interface, first described by Smith
et al. in 1988 [38]. In 2003, approximately 77% of CE-
MS users employed this type of interface, due to its
reproducibility and robustness [39]. By allowing a conducting
liquid to provide electrical contact between the
stainless steel capillary maintained at high voltage and
the fused silica capillary carrying the analyte solution,
the interfacing of capillary zone electrophoresis (CZE)
with ESI-MS could be achieved with many buffer systems
that were previously impractical. This is significant because
aqueous buffers of high ionic strength are normally
not tolerated by ESI-MS. Buffer ions can lead to
charge neutralization and corona discharge as well as
formation of undesirable adducts with the analyte
resulting in loss of signal and high sensitivity and high
background. By using organic solvents, such as acetonitrile
or methanol, as the sheath liquid, buffers of ionic
strength up to 0.2 M can be used. The sheath liquid
prevents direct contact between the high voltage elec-
a nebulizer gas
sheath liquid
stainless steel
capillary/electrode for
CZE / ESI voltage
application
b
nebulizer gas
c
H.V.
nebulizer gas tube
analyte/buffer solution
analyte/buffer solution
separation capillary
separation capillary with conductive
coating for ESI voltage application
liquid junction
nebulizer gas
to MS
analyte/buffer solution
separation capillary
to MS
to MS
Figure 6. Schematic of the three most common ways to interface
CE-MS: a) coaxial sheath flow; b) sheathless; and, c) liquid junction.
248 http://www.elsevier.com/locate/trac
trode and the analyte solution, thus avoiding electrochemical
modification of analytes. An additional
advantage of using the sheath flow interface is minimization
of corona discharge as a result of reducing the
ionic strength of the sprayed solvent [38].
Olivares et al. developed the sheathless interface in
1987 by using a stainless steel capillary to create electrical
contact with the analyte solution [39]. Accounting
for about 11% of CE-MS interfaces in 2003 [40], this
method evolved with a focus on low flow rates for improved
sensitivity.
Other sheathless methods were subsequently developed,
such as sharpening the CE-capillary tip in order to
produce a stable spray, while the ESI electrode was made
by installing a piece of gold wire [41] or coating the
capillary end with a metal or alloy [42]. The advantages
of the sheathless interface are that analyte dilution and
ion suppression due to the sheath flow can be eliminated.
Sensitivity can also be improved by up to an order of
magnitude over sheath-flow designs [43].
A third method of interfacing CE-MS was the liquidjunction
interface developed by Henion and colleagues
[44,45]. A liquid junction between the CE capillary and
the ESI emitter was built using a stainless steel tee
equipped with a buffer reservoir. This interface reduced
the complication brought about by the different flow rate
requirements of CE and ESI [46], but peak broadening
and mechanical difficulties limited the general applicability
of this technique [42].
4.3. Miniaturization of electrospray
In an attempt to reduce flow rates and thus produce
smaller droplets to improve the ionization efficiency,
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
diameter and capable of operating at flow rates of 300–
800 nL/min. The emitter was created by burning off the
polyimide coating of a fused silica capillary tip and
tapering it by etching with hydrofluoric acid to give
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].
Operation in this flow-rate regime may involve various
means for pulling silica capillaries and applying the
electrospray voltage [51].
In 1995, Caprioli introduced the idea of using the LCcolumn
tip combined with microelectrospray technology
[52] to spray directly towards the mass-spectrometer
inlet. This was significant because it triggered the evolution
of analytical LC columns towards miniaturized
versions of microbore and nanobore capillary columns
necessary for low flow rate electrospray [50].
Nanoelectrospray, introduced by Wilm and Mann
more than a decade ago [53], was a particularly
successful technique due to its ability to form droplets

Trends in Analytical Chemistry, Vol. 25, No. 3, 2006 Trends
100–1000 times smaller in diameter than droplets
formed by conventional ESI. The technique was named
to reflect the nanometer (nm) sized droplets as well as
the flow rate of nanoliters per minute (nL/min) [54].
Essentially, the solution infusion system was eliminated,
as a few microliters of solution were transferred to the tip
of a gold coated glass capillary. The emitter tip was
located within a few mm of the inlet aperture, and a
stereomicroscope was used to accurately position the
sprayer in front of the inlet. By applying a low potential
onto the conductive capillary surface, a fine spray was
generated and the charged droplets were directed towards
the counter electrode under the effect of the
electric field gradient. Despite an estimated efficiency of
500 times greater than traditional ESI [54], nanoelectrospray
has some limitations, including plugging of the
emitter tip and lower reproducibility associated with
differences in tip morphology.
While the term ‘‘nanoelectrospray’’ initially referred to
Wilm and Mann’s off line technique, evolving technology
has made possible the use of on line flow rates down to tens
of nL/min. The term nanoelectrospray has thus expanded
to encompass both on line and off line techniques.
For nanoelectrospray, the effects of tip geometry, flow
rate and solvent composition have been investigated by a
number of groups [55–57].
Motivated by the fact that, at certain solvent compositions,
the sensitivity decreased dramatically, Vanhoutte
et al. compared the effects of various mobile phase
compositions using several types of electrospray capillaries.
These included combinations of uncoated or gold
coated, tapered or non-tapered fused silica capillary tips,
as well as stainless steel emitters. They found that the
gold coated, tapered tips were the most effective, because
they covered the most extensive range of solvent composition
for the tested conditions without loss of sensitivity
[55].
Schmidt and Karas used combinations of model compounds
to examine the effect of flow rate on ion signal
[56]. They determined that, at flow rates of a few
nL/min, signal suppression caused by differences in
surface activity of the analytes was insignificant compared
to that at higher flow rates (> 50 nL/min).
Li and Cole studied the often observed shift in charge
state as a result of changing experimental parameters for
nanoelectrospray [57]. Parameters such as tip diameter,
flow rate, analyte concentration and solvent composition
can all affect the observed ions and the charge states.
5. Currently available commercial sources
Since recognition of ESI as an invaluable bioanalytical
tool in the late 1980s, research groups have attempted
to exploit the capabilities of ESI by modifying the source
geometry in order to allow a wider range of flow rates
and in source fragmentation, as well as improved sensitivity,
efficiency and practicality. Niessen’s 1998 and
2003 review articles [58,59] are a good starting point
for those interested in the trends that have stimulated
ESI source evolution. Despite numerous improvements,
commercial ESI sources still retain many features of the
original configuration.
5.1. Source configuration and its relationship to flow
rate
A parameter of critical importance in ESI-MS is the flow
rate of the solution. Depending on the application for
which the mass spectrometer is used, the ESI source
needs to be adapted to handle the in coming flow of
solution, whether it is direct infusion, or LC or CE eluent.
5.1.1. Low flow rate. While flow rates from approximately
1 lL/min up to several mL/min are typically used
with the assistance of a nebulizer, traditional nanoelectrospray
relied simply on the electrostatic attraction between
the mobile phase and the counter electrode to
generate and disperse the charged droplets. The nanoflow
regime can generally be defined as using flow rates
of less than 1 lL/min and can extend to levels of less
than 1 nL/min [55]. As mentioned previously, nanoflow
ESI can be used independently (off line) or coupled to a
separation system (on line), such as CE or HPLC.
Depending on experimental requirements, the commercially
available nanoelectrospray emitters vary in material,
tip shape, diameter, and the configuration used for
electrical contact. On line analyses typically use higher
flow rates (0.1–1 lL/min), and the tips are usually fabricated
from fused silica or metal. Off line analyses typically
use flow rates in the low nL/min range, with
coated or uncoated glass or quartz emitters. A number of
groups have also described modified nanoflow sprayers
with various coatings [60–62] or inserted fibers [63].
There are many different configurations for nanoflow
LC-MS coupling [64], although they all include an LC
column and a narrow diameter emitter tip. The simplest
combination involves coupling an upstream LC column
to the emitter tip using some type of low dead volume
union [65]. The union can also serve as the electrical
contact for the electrospray process, although it is also
possible to use conductive tips or other electrode configurations
[64,66]. The advantage of decoupling the LC
column from the sprayer is simple, inexpensive replacement
of sprayer tips, should they become damaged or
plugged. However, the coupling must be done carefully
to maintain chromatographic performance.
An alternative combination involves packing the LCcolumn
material directly into the sprayer tip to eliminate
any dead volume after the column [67–70]. Tapered
sprayers containing LC column packing are available
commercially from companies such as New Objective
Inc. The column preparation typically involves passing a
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Trends Trends in Analytical Chemistry, Vol. 25, No. 3, 2006
slurry containing column packing material through a
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 performance.
In addition, it has been suggested that the
presence of packing material in the tapered sprayer acts
similar to a filter, extending tip lifetimes by preventing
plugging. The drawback with this configuration is that
tip replacement due to blockage or damage requires the
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
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
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
dead volume [73].
Currently, most major MS manufacturers (e.g., Thermo
Electron, MDS SCIEX, Agilent Technologies, and Waters/
Micromass) make the nanoelectrospray source available.
The number of applications using electrospray at low flow
rates is very large, and outside the scope of this paper.
Wood et al.’s 2003 review article is a good starting point
and a source of references [47].
5.1.2. High flow rate. If the flow rate is in the range
0.05–3 mL/min, sensitivity can be an issue, due to the
decrease in ionization efficiency resulting from large
droplet size. ESI-MS is widely interfaced with LC, so high
flow rates are often necessary. One solution to this
problem is to employ a splitter that allows only a limited
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 Technologies,
Waters/Micromass, and MDS SCIEX). An
example of this is shown in Fig. 7 for an Agilent Technologies
source incorporating an additional asymmetrical
lens.
The asymmetrical lens is essentially a half-full, partial
cylinder electrode operated at high voltage during the
250 http://www.elsevier.com/locate/trac
asymmetrical
lens
orthogonal
nebulizer
nebulized
analyte solution
HPLC eluent
atmospheric pressure
1.5 torr
ion transfer capillary
to MS
Figure 7. Schematic of an ESI source configuration developed by
Agilent Technologies.
electrospray process [75]. The purpose of the electrode is
to help initiate and sustain electrospray. Conversely,
other groups have attempted to use various types of
auxiliary electrodes [76–78] to focus ions at atmospheric
pressure.
Also exploiting the advantages of orthogonal sampling
is the Waters/Micromass Zspraye [79,80] interface,
shown schematically in Fig. 8. The spray is first sampled
orthogonally through the sampling cone into a low
pressure chamber. An extraction cone (skimmer) is oriented
at a right angle relative to the axis of the spray to
sample for a second time into the next differentially
pumped vacuum stage. The double orthogonal sampling
system prevents solvent and neutral molecules from
entering the analyzer, resulting in reduced chemical
background [81]. Larger cone apertures are used to
Figure 8. Schematic of the Zspray TM source configuration used by
Waters/Micromass.

Trends in Analytical Chemistry, Vol. 25, No. 3, 2006 Trends
compensate for transmission losses. The ability to disassemble
and to clean the sampling cone without
breaking vacuum is an additional advantage contributing
to the ruggedness of the Zspray source.
Although some MS manufacturers (e.g., MDS SCIEX,
Waters/Micromass, and Agilent Technologies) have opted
for orthogonal sampling, Thermo Electron uses a
sampling angle at 60° from the ion optics axis. In its Ion
Max source, the function of the angled position of the
sprayer is similar to the orthogonal one.
Sprayer orientation is not the only important parameter
when it comes to optimizing sensitivity while using
high flow rates. For conventional high flow rate ESI
sources, the nebulizer gas is an essential component. Air
or nitrogen is typically used to disperse the emerging
solution into small droplets and to direct the droplets on
the trajectory chosen for optimal sampling.
To vaporize the large amounts of solvent emerging
from the sprayer as efficiently as possible, manufacturers
have introduced additional features to assist the nebulizer
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
assist evaporation of the spray droplets at atmospheric
pressure. More recently, MDS SCIEX took this design a
step further, creating the TurboVe source, shown in
Fig. 9. In this case, two heated auxiliary nitrogen sources
are oriented to achieve very efficient desolvation. The
improved desolvation permits stable, sensitive operation
for flow rates of greater than 1 mL/min [82].
Thermo Electron also integrated auxiliary nitrogen gas
for desolvation purposes, except that it flows directly
from the nozzle of the Ion Max source [83]. Similar to the
TurboV, the Ion Max uses a high temperature source
heater incorporated into the housing of the ion source.
sampling orifice
exhaust
ESI
probe
heater
spray
heated nitrogen
Figure 9. Schematic of the TurboV TM source used by MDS SCIEX.
This probe configuration is referred to as Heated Electrospray
Ionization (H-ESI) and it tolerates the use of
higher flow rates than previously possible on Thermo
Electron instruments.
5.2. Interface
Various ion sampling interface configurations have been
developed [8]. The sampling orifice can be built in a disc,
in the top or the bottom of a cone, or in glass or metal
tubes. Some MS manufacturers now equip their instruments
with conical interfaces. Intuitively, the conical
shape is meant to improve the electric field density at the
sampling orifice in an attempt to improve ion transmission.
To avoid potential contamination, the sampling
cone can be heated to evaporate residuals from its
surface. Since high temperatures are required for this
purpose, ceramic materials have often been used for the
interface. Ceramic produces no outgassing, and is
thought to have low sample adsorptivity, thus reducing
memory effects on the interface surface [84].
6. Multiple sprayers
Commercial multiple sprayer systems for ESI have
evolved mainly as a result of industry’s increasing
requirements for high throughput sample analysis, particularly
for pharmaceutical samples. With a multiple
sprayer source and suitable equipment, LC-MS analyses
can be done quickly and efficiently. However, the first
attempts at building multiple ESI sprayers were motivated
by other reasons. Smith et al. [30,85] were the first
to describe the coupling of two ESI sprayers in order to
generate ions of opposite polarity and study ion-ion
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
been described, incorporating ESI with sources such as
atmospheric pressure chemical ionization (APCI)
[88,89].
Using a linear array of capillary electrodes, Rulison
et al. demonstrated the feasibility of increasing sample
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
sprayers could be used to improve ion current stability
under high flow rate conditions (i.e., above 200 lL/min)
[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
streams [92]. Although this development paved the way
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Trends Trends in Analytical Chemistry, Vol. 25, No. 3, 2006
for further efforts in high throughput parallel analysis,
Kassel’s system required knowledge of the particular
analyte that each sprayer was generating. Dual ESI
sources have also been used to introduce an internal
calibration solution separately from the analyte solution
[93–96]. This configuration provides benefits compared
with a single solution containing both analyte and
calibrant because it avoids preferential ionization.
In 1999, Kassel et al. [97] and De Biassi et al. [98]
described indexed multiple sprayer systems that allow
sequential sampling of each spray by a mass spectrometer,
thus making it possible to identify ions from their
respective eluent stream.
Currently, publications in which fast LC-MS analyses
of samples are discussed indicate that the Waters/
Micromass multiplexed ESI source (MUX) is the most
commonly used for the analysis of multiple LC eluent
streams [99–101]. Tiller’s 2003 review paper provides
useful references regarding biological applications using
the MUX system as well as other types of parallel analysis
[102]. The MUX system attaches to the Zspray
source of the mass spectrometer and is fitted with either
four or eight channels. As a rotating aperture allows
only one spray at a time to reach the sampling cone,
spectra of compounds eluting from individual separation
columns can be quickly acquired.
However, a significant problem with MUX-type
systems is a decrease in sensitivity of approximately
three-fold at high flows [103]. Yang et al. attributed the
drop in sensitivity to several reasons, such as difficulty in
optimizing sprayer position and lower desolvation
efficiency due to large amounts of solvent introduced
into the source in combination with a desolvation gas
that was counter-current relative to the spray instead of
the traditional coaxial flow. The sensitivity decrease is
expected to be larger for nanoflow operation, where
sprayer position can be more critical.
In 2002, Schneider et al. [104] developed an alternative
multiple sprayer system that appeared to solve the sensitivity
problem. By installing an atmospheric pressure ion
lens [76,105] on each sprayer of a dual sprayer and a four
sprayer ESI source, they were able to maintain high sensitivity
by optimizing the applied lens potential as well as
sprayer position. An additional advantage of the system
was that sprayers could be enabled or disabled by
changing the lens potential, potentially a faster method
than using mechanical devices.
7. Recent developments in ESI sources
The quest for a superior source design capable of effectively
focusing more of the electrospray generated ion
cloud through the sampling aperture of the instrument is
still on going. Due to gas phase collisions occurring
under atmospheric pressure conditions and space charge
252 http://www.elsevier.com/locate/trac
effects, only a small percentage of the spray plume is
actually sampled. There is still significant room for
improvement in ion sampling efficiency.
7.1. High velocity nebulizers
One approach developed by Zhou et al. [106] involved
installing an industrial air amplifier between the electrospray
probe and the sampling orifice. The concentric,
high velocity gas flow was meant to control the expansion
of the spray plume as well as assist in the desolvation
of ions. In addition, focusing of ions could be
achieved by applying an optimized voltage of up to 3 kV
onto the amplifier. Thus, when both the air amplifier and
the high voltage were used, Zhou’s ion source configuration
seemed to provide some improvement in ionsampling
efficiency. Suppression of background chemical
noise was also observed.
7.2. Improved nanoflow configurations
Schneider et al. developed a novel interface for nanoflow
ESI (Fig. 10) that provides two separate atmospheric
pressure regions for droplet desolvation as well as two
regions for removing unwanted particles [107].
The first desolvation stage makes use of the traditional
counter-current gas to eliminate neutral particles and
solvent. Next, charged particles, ions and gas travel
through the heated laminar flow chamber, where additional
desolvation takes place at optimal temperature.
Once they reach the particle discriminator area, ions are
carried by the gas streamlines through the discriminator
space and into the sampling orifice, while large charged
particles with high momentum escape from the gas
streamline trajectory under the influence of the strong
electric field. This source design provided improved signal
and ion current stability for flow rates from a few nL/
min up to 1 lL/min as well as improved performance for
gradient elution nanoLC-MS in both positive-ion and
negative ion mode [108].
7.3. Desorption electrospray ionization
Cooks and co-workers recently described a new method
of ionization in which analytes are desorbed from
surfaces by nebulizing the charged droplets of an
electrospray plume at a surface [109]. This technique is
referred to as desorption electrospray ionization (DESI).
The exact mechanism of ion formation in DESI is not
well understood, although the technique has been
successfully used for analyzing a number of samples,
including pills and TLC plates [110]. Efficient sampling of
desorbed ions requires careful optimization of the angles
and the spacings between the surface, the electrospray
emitter, and the mass spectrometer inlet.
7.4. Coupling ESI to micro-analytical systems
In the rapidly expanding area of microchip chemical
analysis, currently known as ‘‘Micro-Total Analysis

Trends in Analytical Chemistry, Vol. 25, No. 3, 2006 Trends
Curtain Plate
Tee and Nanospray
Tip with Nebulizer
Curtain Gas
Systems’’ (l-TASs) and the related ‘‘Lab-on-a-chip’’, ESI
has become the preferred interface method for MS
detection [111–113]. More than a decade after being
introduced [114], l-TASs have been used in a wide
range of biological applications, drug discovery, and
clinical diagnostics. References regarding applications
can be found in review papers [115–120].
The interest in miniaturized analytical systems rose
due to the potential for integrating time consuming
steps, such as sample preparation, chemical reaction,
separation, and detection, into a fast, highly automated
technique, while at the same time using minute
sample quantities. Constantly evolving microfabrication
techniques adapted from the semiconductor
industry are used to produce the microfluidic devices
[117,121,122].
Although on-chip optical detection is often used in
microfluidic applications, MS detection is desirable due to
its suitability for analyzing biological molecules. In
addition, the low flow rates (nL–lL/min) used with
microfluidic devices are optimal for the ESI source.
However, integrating ESI-MS with a microchip is not
easy. Initial work involved using traditional ESI emitters,
such as a section of fused silica capillary or a nanoelectrospray
needle attached to a specific microchannel exit,
but these types of sources were prone to contamination
problems from bonding agents, poor reproducibility, and
dead volume problems [121,123,124]. Work has
therefore focused on building the ESI emitter as part of
the microdevice and a range of materials, from silicon
based to polymers, has been tested for fabrication of the
ESI emitter [117,121].
Agilent Technologies has introduced a polymer based
HPLC-MS microfluidic chip [125]. The chip contains
(TM)
Particle Discriminator
Space
Heated Laminar Flow
Chamber
Teflon Spacer
Orifice
Figure 10. Schematic of the particle discriminator interface developed by MDS SCIEX.
enrichment and analytical columns packed with reversephase
material, a face-seal rotary valve for switching
between loading and separation positions, and a conical
ESI emitter created directly on the chip structure by laser
ablation. Gradient reverse phase separation of peptides
has been achieved at flow rates of 100–400 nL/min. The
system was reported to be stable with various solvents,
sensitive, and robust. Since the various connections
necessary for traditional nanoelectrospray were eliminated,
dead volume problems as well as analyte dispersion
were minimized [125].
While the combination of microfluidic devices with MS
may have great potential, many barriers still need to be
overcome. Currently, CE methods appear to be most
common for sample separation on microfluidic chips,
because they are less complex to miniaturize than HPLC
[126]. Difficulties, such as integrating pumping systems
for controlling flow and creating gradients, still limit
widespread commercialization of microfluidic-LC systems
[127].
For high throughput analyses, Advion BioSciences
successfully introduced a fully automated nanoelectrospray
system, called the NanoMatee. This microchip
device contains an array of nozzles to which the sample
is automatically delivered for MS analysis. Short analysis
times are possible due to a simplified spray optimization
procedure [128,129]. Although the NanoMate was initially
intended for use in combination with microfluidic
separation devices [128], it has been integrated with
sources from other manufacturers (e.g., Waters/Micromass,
MDS SCIEX, and Thermo Electron). The key
advantages of the NanoMate include elimination of
sample carry over and simplification of the tuning procedure
[130].
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Trends Trends in Analytical Chemistry, Vol. 25, No. 3, 2006
8. Conclusion and future outlook
Because ESI-MS is used in many areas of chemistry,
there is an immense number of publications covering
modifications to the ion source for various applications.
This article has attempted to sketch a basic picture of
how ESI works and how it has evolved, and continues to
do so, towards new designs. Undoubtedly, commercial
ESI sources will continue to evolve to meet growing
demands for improved sensitivity and robustness. Further
improvements are likely to include increasing the
gas throughput to sample a larger number of ions, even
though these types of approaches are typically associated
with higher costs due to the increased pumping
requirements. Research is also likely to continue to increase
the ion sampling efficiency by electrostatic and
aerodynamic focusing, without the need to increase
system pumping. With MS more popular than ever, it is
fascinating to see that the capabilities of ESI-MS are still
expanding.
Acknowledgement
The authors thank Dr. Damon Robb for valuable discussions
on many topics included in this article.
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