Fundamental LC-MS Electrospray Ionisation Instrumentation

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Mass Spectrometry

Fundamental LC-MS

Electrospray Ionisation Instrumentation

Aims and Objectives

Aims and Objectives

Aims

Explain the function of the major components of an electrospray Interface
Investigate methods of optimising signals using electrospray Ionisation

Objectives

At the end of this Section you should be able to:

List and describe the most important components of an electrospray Ionisation
interface
Demonstrate an understanding of the principles of optimising instrument response
when using electrospray ionisation

Crawford Scientific www.chromacademy.com 2
Content

Electrospray source design 3
Introduction 3
Electrospray capillary design 3
Sprayer Sampling Plate Configuration 6
Cluster Ions 9
Prevention of Cluster Ion Sampling 10
Source Cleaning 12
Ion Optics 13
Ring Electrode 13
Ion Bridges 14
Collision Induced Dissociation 14
Ion Declustering 16
References 16

Electrospray source design

Introduction

Electrospray is the dispersion of a liquid into electrically charged droplets, combining the
two processes of droplet formation and droplet charging. The process of droplet charging
is affected by three main variables:

Eluent flow rate
Liquid surface tension
Electrolyte concentration

If these parameters are not maintained at an optimised minimum level, the electrospray
process will become unstable. If any of the variables increases significantly it maybe
difficult for the electric field to produce the desired charged aerosol necessary for ion
production in the API interface. Any effects observed due to a significant increase in any
of the variables may be countered to a certain degree by increasing the capillary voltage
(and hence the effective field strength at the capillary tip), but electrical discharge may
occur, resulting in a decrease in instrument response and an unstable electrospray.

Electrospray capillary design

Standard electrospray capillaries are constructed from stainless steel or a coaxial
arrangement of fused silica and stainless steel. If the capillary is of fused silica design,
electrical contact is usually made by clamping the capillary in a metal union, however,
capillaries with silver or gold deposits upstream from the tip have also been employed for
improved electrical contact.

Conventional Electrospray Capillary (practical upper flow rate limited to 10-20L/min)

The metal composition used for stainless steel capillaries is of great importance in the
oxidative and reductive processes occurring during droplet charging and as such
continuity of composition for the metal capillaries should be ensured to give long term
robustness to analytical determinations.

Drying gas

The practical upper limit to eluent flow in pure electrospray is 10-20 L/min depending
upon the solvent composition. Capillary design may be modified to increase the tolerance
of the electrospray process to increases in eluent flow rate, liquid surface tension or
electrolyte concentration. One successful approach used in order to increase electrospray
flow rate is the introduction of a nebulising gas via a concentric tube around the capillary
(pneumatically assisted ESI).
[1]

High-flow electrospray sources (>5-10 L/min.) are normally combined with a supply of
heat within the API source housing to assist the evaporation of solvents. The evaporation
of large amounts of solvent is important to ensure that ion evaporation occurs in the
optimum position within the API source to ensure maximum transmission and reduction of
ion solvent clusters in the nozzle-skimmer region of the source.

Source heating needs to be optimized for each analytical determination to ensure the
production of the maximum amount of analyte ions in the source. Practically the use of
heated nitrogen blown into the source housing is normally employed to aid desolvation in

high flow electrospray source designs. Perkin Elmer (Sciex) (Applied Biosystems, Foster
City, CA), introduced one of the earliest instruments to incorporate pneumatically assisted
electrospray in their IonSpray design. This design used a silica capillary within a stainless
steel needle that was housed within a concentric PTFE tube. The design of the PTFE
tube is such that gas flow rates at the capillary tip are around 200 m/s.

When physically connecting the t-piece or HPLC column to the API interface housing,
PEEK (polyetheretherketone) (0.1mm i.d.) tubing is preferred to fused silica tubing due to
the possible adsorption of analyte species to residual silanol species on the inner surface
of the silica capillary. Most modern instruments employ stainless steel or platinum
capillaries for electrospray to avoid similar problems with fused silica that can adversely
affect the quantitative response of the instrument. For low flow rate applications micro-
electrospray needles are available which consist of either coated or uncoated silica
capillaries with a drawn tip to allow micro-droplet formation.
[18]
Filters in the needle
assembly help to prevent blocking of the fine capillary tip (<0.5m), and the coating of the
needle helps electrical contact, negating the need for coaxial sheath liquids. Potentials of
between 0.5 and 1kV are normally sufficient to produce efficient electrospray at eluent
flow rates of less than 1 l/min. Nanoflow capillary instruments are also available which
use fused silica capillaries with 20 m i.d. that can be used in the flow rate range 100-
1000 nl/min.
[19]
The sample solution is fed to these capillaries via nitrogen-pressurised
nanovials and can provide stable electrospray for numbers of hours with sample volumes
of 1ml and less. Application areas of nanoflow electrospray systems include the study of
proteins and other solutions where sample size is extremely limited.
[2.3]

Sprayer Sampling Plate Configuration

The position of the sprayer (capillary) relative to the ion sampling orifice within the
sampling plate is of significant importance. By avoiding directly spraying at the sampling
plate orifice the maintenance interval required for API interface types may be extended
and the instance of charged droplet being sampled (as opposed to gas phase ions) is
lowered hence increasing instrument response.
[3]

The core of the electrosprayed aerosol contains larger diameter droplets than in the
extremities of the spray. It is expected that the efficiency of ion production (via ion
evaporation), will be higher at the perimeter of the aerosol. The off-axis response is also
more stable than the one observed when using on-axis.
[3,4]

In most modern instruments the sprayer position may be adjusted in one, two or three
axes by means of micrometer screws that will allow optimisation relative to the sampling
orifice.

On-axis spray
Off-axis spray
Diagonal


The distance and potential difference between the tip of the capillary and the sampling
plate determine the electric field that creates the electrospray and will influence the
performance of the spray. Optimisation of the sprayer position and capillary voltage are
interrelated and should be optimised empirically together.

Pepperpot (FISONS-Micromass)
Cross Flow (Waters)

Perhaps the most important practical advance in sprayer positioning has come with the
introduction of orthogonal source design, where the sprayer is positioned orthogonally to
the sampling orifice. This design has several advantages including, reduced down-time of
the source with decreased coating of the source elements, sampling of fewer charged
droplets relative to ions and the ability to tolerate higher flow rates.

In orthogonal spray, neutrals and non-volatile materials collide with a plate perpendicularly
located to the spray axis. Orthogonal design can be combined with a second orthogonal
extraction (LCZ or Z-Spray from Waters) or a with an off-axis extraction, such as in the
aQa (Thermo-Finnigan) disposition.
[5]

Orthogonal designs allow the use of eluent flow rates up to 1ml/min with electrospray and
gives the advantage that eluent systems containing non-volatile buffers may be used for
extended numbers of samples before source cleaning is required. Orthogonal sources
are available from several other manufacturers.

Cluster Ions

Polar molecules in the gas phase (water, solvent and eluent molecules), tend to form
clusters with ions.
[23]
Cluster ions will appear in the mass spectrum and they are often so
large that they are far outside the detectable mass range of a typical quadrupole (3000-
6000 Da):

n
O H X O H n X ) (
2 2
+ +
+

Cluster ions may collide with the source elements in the early stages of the spectrometer
and give rise to ion bursts which can result in noisy baselines in the Total or Selected Ion
Chromatogram (TIC or SIC).

One commonly applied solution to this problem allows ions to pass into the sampling
orifice but excludes water vapour and other neutral species from the entrance to the
vacuum system. This is achieved by forcing ions and neutrals in the opposite direction by
the action of an electric field and/or a flow of dried gas.

Cluster formation
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Cluster ions passing into the vacuum region of the source may be de-clustered by
collision with rapidly moving background gas molecules, imparting enough energy to
break the hydrogen bonds between the ion and the solvating cluster molecules.
Increasing the temperature and the potential difference in the first vacuum region of the
spectrometer in order to accelerate ions will both assist with ion declustering.

Prevention of Cluster Ion Sampling

A Sciex (Applied Biosystems, Foster City, CA), API source is shown. The region between
the interface plate and the sampling orifice plate is continuously flushed with dry nitrogen
that flows into the API source as well as into the vacuum region of the spectrometer.

The gas flow into the source helps to repel water, neutrals and other potential
contaminants, such as dirt and buffer salts, away from the sampling orifice, increasing the
intervals between source maintenance.

Ions in the region of the sampling orifice are driven into the vacuum region by the gas flow
combined with a 600V potential difference between the interface plate and the sampling
plate. The nitrogen employed in this sense is often referred to as a Curtain Gas.
[6,7]

Gas curtain

As the nitrogen curtain gas and ions undergo expansion into the nozzle-skimmer region of
the source, significant cooling occurs. If the source and curtain gas are not heated, there
is a significant possibility of cluster ion formation occurring in this region. Therefore most
modern sources use both heated drying and curtain gas.

A method preventing cooling effects experienced through expansion and the re-formation
of cluster ions uses a heated transfer tube.
[25]
This device will preheat the mixture of ions
and neutrals prior to expansion using a heated tube of approximately 20 cm long (100
200
o
C). A further advantage of this type of device is the ability to generate ions (via ion
evaporation) from droplets that may be sampled into the nozzle-skimmer region.

An Agilent Technologies (Palo Alto, CA, USA), source is shown which employs an ion
transfer tube with metallised ends (also known as a 'dielectric capillary'), that allows the
application of an accelerating voltage that can be used to promote in-source dissociation
of cluster ions.
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Orthogonal source with dielectric capillary (heated transfer tube)

Source Cleaning

Contamination of the sampling orifice or tube can prove to be detrimental to the
performance of the instrument, in some cases leading to very frequent source cleaning
when dealing with samples in dirty matrices or when using non-volatile solvents and
buffers.

The layer of contamination inside an orifice or tube will attract a build up of charge that
can effectively stop the passage of ions, while the flow of neutrals is not affected, thus
significantly decreasing the instrument response.

Cleaning regimes will differ for instruments from different manufacturers but may include
some physical abrasion (aluminium powder is a popular choice) and / or wipe cleaning of
the sampling cone and other source components followed by sonication in a range of
solvents matched to the polarity of the contaminants (hexane, acetone and methanol are
all popular choices for source cleaning).

Dirty layer formation

The use of curtain gas, off axis spray and/or orthogonal spray can significantly increase
the interval between source cleaning, by directing the deposition of contaminant species
away from the sampling plate, orifice or ion transfer tube.

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Ion Optics

Ring Electrode

In the nozzle-skimmer region of the mass spectrometer ions are transported in the free-jet
expansion of gas, some of which will pass through the skimmer into the higher vacuum
region of the mass spectrometer.

Focusing of the ions into a narrow beam is not possible due to the effects of free jet
expansion, which tends to direct the ions and entrained neutrals and gases into a barrel
type shockwave, away from the axis of the spectrometer. However, ions may be forced to
remain closer to the axis of the beam if a tube or ring lens is placed between the nozzle
(sampling plate) and skimmer.
[8]
A voltage applied to the ring will reduce the spread of
ions (but not water vapour or neutrals), away from the axis with a subsequent increase in
instrument sensitivity (mainly due to a reduction in the noise).

Ring electrode operation

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Ion Bridges

In the high vacuum region of the spectrometer after the skimmer lenses, the pressure is
still not low enough to focus ions using traditional ion lenses. In most cases the ions are
focused using a radio frequency (RF) only hexapole or octapole ion bridge, that guides
the ion beam into the analyzing quadrupole system.

Ion bridges work on a similar principle to a quadrupole mass analyser but operate with
radio frequency potential only. The quadrupole DC component is removed, and the
application of a radio frequency potential allows ions of all m/z values to pass from one
region of the spectrometer to another.
[9]

The multipole ion bridge has the advantage of slightly focusing the ion beam, therefore
increasing transmission as well as allowing the removal of a significant amount of neutral
species which are not held within the multipole ion bridge, so increasing signal to noise
ratio.

Ion bridges operation

Collision Induced Dissociation

Classical API spectra tend to show very little fragmentation due to the 'soft' nature of the
ionisation processes. That is, during the formation of ions, the analyte molecules do not
receive enough energy to break the intra-molecular bonds.

However, fragmentation may easily be induced in one of the higher-pressure regions and
structural information can be gathered.

Acceleration of ions between the sampling orifice and the skimmer, or between the
skimmer and the RF-only multipole results in collisions of ions with the background gas.
This process is known by various different names depending upon the instrument
manufacturer and includes 'in-source collision induced dissociation (CID)', 'nozzle-
skimmer fragmentation', 'cone-voltage fragmentation', etc.

By increasing the potential difference between the skimmer and the quadrupole V(S-Q)
or between the nozzle and skimmer V(N-S)), the energy imparted to the analyte
molecule through increasing frequency and energy of collisions can be enough to cause
intra-molecular bonds to be broken and for fragmentation to occur.
[10,11]
In the case of
larger molecules, such as peptides and proteins, the excess energy can often be
absorbed in several vibrational modes and high potential differences are required to
fragment these kind of molecules.
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CID working principle

The major advantage of this technique for the production of spectra containing a greater
amount of structural information is its simplicity. Only one voltage needs to be adjusted
and there is no need for switching or adjustment of collision gases and no retuning of ion
optics.

It is possible to test the degree of CID using probes.
[11]

In negative ion mode the degree of CID can be estimated using the drug naproxen that
normally shows the [M-H]
-
ion as the only at m/z 229. With small nozzle-skimmer potential
differences the naproxen molecule readily loses CO
2
giving rise to a base peak at m/z
185.

In the positive ion mode the [M+H]
+
ion of dibutyl phthalate at m/z 279 can be used as a
test ion. Fragmentation to m/z 149 takes place readily even under mild CID conditions,
making declustering of ions impossible with these labile sample ions.

Mass spectrum of naproxen (used to estimate the degree of CID in negative ion mode)

Fragmentation
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Ion Declustering

Collision Induced Dissociation can also be used to improve the baseline noise and
increase the signal-to-noise ratio in LC-MS experiments.
[9,12]
When ions pass through the
sampling orifice into the vacuum region the background density of neutral gas ions falls
rapidly. If ions moving in a low-density gas are accelerated by the nozzle-skimmer region
mild-CID may be affected, which will be enough to cleave the hydrogen bonds inside the
ion-water or ion-neutral gas clusters. Further, heating of the ion clusters, which occurs
during collisions, will also aid desolvation of the cluster ions.

Moderate acceleration of clusters is effective and widely used to decluster ions, leading to
a reduction in baseline noise and the numbers to cluster ions detected. A disadvantage of
this type of approach is the moderate scattering of the ion beam that is associated with
ion-neutral gas collisions that may lead to a small reduction in the numbers of ions
passing through the skimmer element and into the mass analyser (i.e. reduced
spectrometer transmission).

Ion declustering process by using CID

References

1. A. P. Bruins, T. R. Covey and J. D. Henion, Anal. Chem. 59, (1987), 2642.
2. M. R. Emmett and R. M. Caprioli. J. Am. Soc. Mass Spectrom. 5, (1994), 605.
3. M. S. Wilm and M. Mann. Anal. Chem. 68, (1996), 1.
4. A. P. Bruins, T. R. Covey and J. D. Henions. Anal. Chem. 59, (1987), 2642.
5. K. Hiraoka. Rapid Commun. Mass Spectrom. 6, (1992), 463.
6. J. Abian. The Coupling of Gas and Liquid Chromatography
with Mass Spectrometry. J. Mass Spectrom. 34, (1999), 157 168.
7. C. K. Meng and J. B. Fenn. Org. Mass. Spectrom. 26, (1991), 542.
23. C. M. Whitehouse, R. N. Dreyer, M. Yashamita, J. B. Fenn. Anal. Chem. 57, (1985),
675.
8. I. C. Mylchreest, M. E. Hail and J. R. Herron. United States Patent 5, 157, 260,
October 20, 1992.
9. M. E. Hail and I. C. Mylchreest. Presented at the 41
st
ASTM Conference on Mass
Spectrometry and Allied Topics. May 31 Jun 1, 1993, San Francisco, CA, 745.
10. R. D. Smith, J. A. Loo, C. J. Barinaga, C. G. Edmonds and H. R. Hudspeth. J. Am.
Soc. Mass Spectrom. 1, (1990), 53.
11. R. D. Smith and C. J. Barinaga. Rapid Commun. Mass Spectrom. 4, (1990), 54.
12. A. P. Bruins in Electrospray Ionisation Mass Spectrometry R. B. Cole [ed.] John
Wiley and Sons Int. 1997, 132-133.
Declustering
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Fundamental LC-MS Electrospray Ionisation Instrumentation

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