L.A. McDonnell et al., Eur. J. Mass Spectrom. 8, 181–189 (2002)
181
A Theoretical Investigation of Kinetic Energy of Ions
L.A. McDonnell et al., Eur. J. Mass Spectrom. 8, 181–189 (2002)
A theoretical investigation of the kinetic energy of
ions trapped in a radio-frequency hexapole ion trap
Liam A. McDonnell, Anastassios E. Giannakopulos and Peter J. Derrick*
Institute of Mass Spectrometry and Department of Chemistry, University of Warwick, Coventry, CV4 7AL, UK
Youri O. Tsybin and Per Håkansson
Ion Physics Division, Ångström Laboratory, Uppsala University, Box 534, SE-751 21, Uppsala, Sweden
The kinetic energy dependence of ions trapped in a radio-frequency (RF) hexapole ion trap has been calculated as a function of space
charge, mean free path, mass, RF potential and charge. The average kinetic energy of the ions was found to increase with increasing
space charge, mean free path and the ion charge state. For a trapped ion in a given coulombic field, the mass of the ion and the amplitude of the applied RF potential did not affect the average kinetic energy. The consequences for multipole-storage-assisted dissociation (MSAD), in which ions are accumulated for prolonged periods of time in the multipole ion trap of an electrospray ion source, are
discussed. As a result of radial stratification inside the ion trap, MSAD can lead to the preferential excitation of ions with larger m/z
values. Such discrimination would have negative consequences for the detection of labile non-covalent adducts, which are normally
detected at higher m/z values than their constituent species.
Keywords: Fourier-transform ion cyclotron resonance (FT-ICR), ion optical modelling, hexapole ion trap
Introduction
Interest in studying the ion motion in radio-frequency
(RF) hexapole ion guides rose significantly after the new insource fragmentation technique, multipole-storage- assisted
dissociation (MSAD), was developed. In this technique, ions
are accumulated inside the external hexapole ion trap of an
electrospray ion source for prolonged periods of time. The
MSAD phenomenon was first reported by Sannes-Lowery et
al.1 and has been investigated by Sannes-Lowery and
2
3
Hofstadler and by Håkansson et al. The common conclusion of both investigations was that the charge density contained in the hexapole was of primary importance.
The current mechanism for MSAD is a kind of chargeinitiated collision-activated dissociation (CAD), in which
the high charge density in the hexapole repels ions into
larger radii. The ions are forced to oscillate to higher amplitudes and, thereby, reach higher kinetic energies. Collisions
with neutral gas molecules at these higher energies serve to
instigate fragmentation. The onset of MSAD has been
shown to be a function of the kinetic energy (KE) of the
incoming ion, the ion collision frequency and the identities
of the colliding species, as well as the characteristics of the
multipole ion trap.1–3
It was found experimentally that the proportion of fragment ions increased with pressure, suggesting that increased
collision frequency outweighed any increased damping
effects.3 The observed increase in fragmentation with storage time can be interpreted as an increased number of collisions producing more fragments through imparting more
3
internal energy. The finding that the degree of fragmentation increased with RF field strength was interpreted as further proof for the proposed mechanism because, for a
specific radial position, the KE of the trapped ion is dependent on the applied RF potential. However, for a specific
space-charge field, the radial position of an ion is dependent
on the applied RF potential.4 The analytical considerations
and simulations reported here show that the kinetic energy of
a trapped ion is independent of the applied RF potential.
It has recently been suggested that, under typical
electrospray ionisation conditions, radial stratification in
which the average radial position of an ion is dependent
upon its m/z value can occur.4 The propensity of an ion for
MSAD will be affected by the number and identities of the
other ions trapped in the multipole ion trap because of the
dependence of radial position on space charge. In a similar
manner, Cooks and workers have demonstrated that the
radial distribution of an ion cloud in the 3-dimensional (3D)
© IM Publications 2002, ISSN 1356-1049
182
quadrupole ion trap increases with the number of trapped
ions and that the ion cloud dimension correlates with the
chemical mass shift (as measured in the mass spectrometer).5
These chemical shifts have been shown to be dependent on
the entire population contained in the ion trap (local space6
charge effects) and to have a compound-specific compo7
8
nent. The ion trap simulation program (ITSIM) was subsequently used to demonstrate that the compound- specific
7
shifts were due to collisions.
Building on the extensive work already reported, the
results of calculations based on a potential-array model of a
hexapole ion trap that incorporates collisions and approximates the effects of space charge are presented here.
Experimental and methods
Mass spectrometry
Experimental measurements were made using 9.4 T
Bruker BioAPEX II FT-ICR mass spectrometers and external ESI sources (Analytica), which have been described in
detail elsewhere.9 The insulin and lysozyme samples were
obtained from Sigma and used without further purification.
The mono-nitrated lysozyme sample was produced by electrochemical modification of hen egg white lysozyme10 and
was reduced with dithiothreitol using the protocol developed
11
by Scigelova et al. prior to use.
Simulations
Calculations were performed using the commercial
package SIMION 3D (Idaho National Engineering Labora12
tory, version 6.0) and a user program. Apart from a skimmer being replaced with an electrode identical to the
trapping electrode, the simulated hexapole ion trap had the
same physical dimensions as those used in the Analytica ESI
sources of the Bruker FT-ICR mass spectrometers used by
the previous MSAD investigators and at the Universities of
Uppsala and Warwick.9 Specifically, the hexapole consisted
of six cylindrical rods (62 mm long, 1.1 mm diameter) with
a field radius of 1.14 mm.
A user program was written to simulate the RF field.
When programs with RF potentials were used, SIMION 6
employed a fast-refining method that involves the superposition of 3D potential arrays.12 Unless otherwise stated, the RF
field in these simulations was identical to that applied under
normal operating conditions in the Analytica ESI source of
the Bruker BioAPEX FT-ICR instruments used at Warwick
and Uppsala, namely 600 Vp–p at 5.2 MHz.
Method description and parameter definition
The approach employed was to choose typical initial
conditions and to investigate the effects of collisions and
space charge on the calculated ion trajectories. In order to
simulate space charge, a 3D potential array was used that satisfied the Laplace equation with boundary conditions V = V0
at r = 0 and V = 0 at the hexapole electrode surfaces
A Theoretical Investigation of Kinetic Energy of Ions
(1.14 mm from the hexapole axis). This approach was
adopted because Szabo and workers had demonstrated that
the shape of an ion beam in a hexapole ion guide remains
almost cylindrical even if it is operating close to the upper
limit of stability.13 Application of Gauss’s law to a cylindrical charge density leads to the following relationship for the
electric field, E:
E = ql / 2πε0r
(1)
where ql is the charge per unit length, r is the radius of the cylinder of ions and ε0 is the permittivity of free space.
The potentials applied to the space-charge array,
0.0–0.5 V, corresponded to the total charge experienced by
–13
the ion, 0–5.9 × 10 C, respectively. These values were
obtained by converting the potentials to the charge-per-unit
length experienced by the ion and multiplying by the length
of the ion cloud. As the axial motion of the ion was progressively damped, the maximum axial distribution ions that
were successfully trapped, in this case 60 mm, was used as
an estimate of the length of the ion cloud. As a rough guide, a
hexapole containing 5.9 × 10–13C of charge would have
trapped between 5.9 / t and 1.2 / t% of incoming ions for a
14
10–50 pA ion current where t is the accumulation time in
seconds.
Reflecting the generality of MSAD, the majority of simulations were run using a singly-charged positive ion of
mass 1000 Da with both trapping electrodes set at 10 V. The
initial conditions for the simulations were 0.375 mm offaxis with an initial kinetic energy of 65 eV and a velocity
parallel to the principal axis. The skimmers present in most
external ESI sources would ensure that the velocity perpendicular to the principal axis was small (relative to the parallel
velocity). The kinetic energy of 65 eV was chosen as a compromise, given the many factors affecting the entrance speed
of an ion into the hexapole.
The effects of collisions were approximated by a mean
free path and a kinetic energy loss per collision. It has been
shown that the energy distributions of ions already trapped
in a multipole can be described by a weighted sum of
Maxwell–Boltzmann distributions.15 Furthermore, in the
likely event that a characteristic temperature (or any of the
characteristic temperatures) of the ions is different from the
temperature of the bath gas, it is possible to use a weighted
average temperature to determine the relative velocity.15
Consequently, a combination of temperatures can, in principle, be assigned to a particular case study and an average
mean free path obtained. Reflecting the random nature of
collisions, the percentage kinetic energy loss per collision
was chosen randomly between 0 and a user-defined upper
limit.
Unless otherwise stated, the position, velocity and
energy were sampled every microsecond, as well as at each
collision. This sampling frequency was found to provide a
good compromise for simulation time, file size, processing
time and accuracy. To enhance the statistical reliability of
L.A. McDonnell et al., Eur. J. Mass Spectrom. 8, 181–189 (2002)
183
The collisional damping model used in these simulations can be expressed as
dKE/dt = rate of collisions * KE loss per collision
= rate of collisions * KE *k
(2)
where k is the mean fraction of kinetic energy lost per collision which should be consistent throughout simulations run
under standard conditions. The absolute value of k will
depend on the random number generator used: this work used
that built into Microsoft Windows NT v4.0.
Expressing the rate of collisions as a function of the
mean free path and the kinetic energy of the ions using the
boundary condition that KE = KE0 at time t = 0 gives Equation (3)
Figure 1. Kinetic energy profiles for space-charge-free simulations using standard condisitions (see methods section). The
upper and lower curves correspond to mean free paths of 14
and 2 mm, respectively. The simulation results are shown in
black and the analytical curves are shown in light grey. The
2 mm experimental and analytical curves are in good
agreement.
(1/KEt) 1/2 – (1/KE0) 1/2 = (2/m) 1/2 × k × t / λ
(3)
where m is the mass of the ion, λ is the mean free path and KEt
is the kinetic energy at time t. As can be seen in Figure 1 (analytical curves), this expression can describe the calculated
damping. In each case the value of k was 0.145.
Kinetic energy and radial-position profile dependence on the
space charge and mean free path
the profiles obtained, the kinetic energy profiles (variation of
kinetic energy with time) detailed below were the average of
at least 12 individual trajectories in every case.
Results and discussion
Space-charge-free collisionally-damped simulations
The kinetic energy profiles for ions with mean free
paths of 2 mm and 14 mm entering a space-charge-free
hexapole array (standard conditions) are shown in Figure 1.
It is readily apparent that the shorter mean free path leads to
increased damping.
As a result of a smaller number of collisions (decreased
damping) the kinetic energy variation of the 14 mm profile
was greater than that for the 2 mm profile. The kinetic
energy profiles for mean free paths of 4–12 mm (not shown)
displayed the same pattern, in that less efficient damping
allowed the local maxima to be more pronounced for longer
mean free paths. Nevertheless, even when the trapping efficiency of the hexapole was small (the trapping efficiency for
14 mm mean free path simulations was approximately 2%),
the ions lost the majority of their initial kinetic energy (KE)
within the first millisecond.
Closer inspection of Figure 1 reveals a trough in the
kinetic energy profile for the 14 mm mean free path. The
troughs were evident whenever the ion was damped to
kinetic energies below 10 eV (trapping requirement) but the
axial component of the velocity (initial velocity) was still
dominant. The troughs were also apparent in the kinetic
energy profiles of the 10 and 12 mm mean free path trajectories. These troughs simply reflect the velocity reversals that
occurred at the first trapping electrode.
Although space-charge free, collisionless simulations
demonstrated that the kinetic energies of trapped ions
increased rapidly with radial position (data not shown). The
ions will be damped in the presence of collisions.
The kinetic energy profiles for a range of mean free
paths and charge densities were calculated. As stated earlier,
the effects of previously acquired charge density were
approximated by an extra array with a charge per unit length,
ql, dependent field.
For a specific simulation, after a few milliseconds the
average kinetic energy of the incoming ion remained fairly
constant. Figure 2 shows the kinetic energy profile of an ion
Figure 2. Kinetic energy variation with time for a space
charge = 3.54 × 10–13 C and mean free path = 10 mm simulation (other conditions as standard, see methods section) that
was sampled at twice the frequency of the applied RF field. The
close-ups reveal the fine structure contained in the kinetic
energy profile.
L.A. McDonnell et al., Eur. J. Mass Spectrom. 8, 181–189 (2002)
184
–13
(standard conditions, space charge = 3.54 × 10 C, mean
free path = 10 mm) where the trajectory was sampled at
twice the frequency of the RF field so that any resonant
motion was accurately recorded.16 As can be seen, a kinetic
energy band spanning 0–0.1 eV was obtained. The kinetic
energy band consisted of many localized maxima and minima (subharmonic motion). Further closer inspection
revealed harmonic fine structure: however, in no instance
did the harmonic fine structure lead to kinetic energy spikes
of significantly greater kinetic energy. The presence of harmonic and subharmonic components is in agreement with
17,18
previous investigations.
Also included in Figure 2 are the collisions the ion experienced. The collision frequency was much less than that of
the fine (harmonic) structure and the subharmonic structure.
Furthermore, the average time between collisions (≈ 177 s
for this simulation) was much greater than the standard sampling time interval of 1 s. As the smallest interval between
collisions recorded was greater than 20 s, the 1 MHz sampling rate was more than sufficiently accurate to record any
trends pertinent to collisional activation and was sufficient
to describe fully the subharmonic oscillations.
The kinetic energy band shown above was observed in
all of the simulations. The kinetic energy profiles for a 2 mm
mean free path as a function of charge are shown in Figure 3.
The kinetic energy band of the ion increased from 0–0.01 eV
to 0–0.1 eV as the space charge increased from 0 to
5.9 × 10–13 C. For each mean free path investigated, from 2 to
14 mm, the kinetic energy band of the ion increased as space
charge increased. As expected, the ions were repelled into
larger radii where they were forced to oscillate at higher
amplitudes and thereby gained more kinetic energy.
In addition to the finding that the width of the kinetic
energy band increased with increasing space charge, it was
also found that the width of the band increased slowly with
an increasing mean free path. For example, the average
kinetic energy for a 12 mm mean free path was about 50 %
larger than that for a 2 mm mean free path. Decreased damp-
Figure 3. Kinetic energy profiles for a 2 mm mean free path as a
function of space charge, all other conditions as standard (see
methods section).
Figure 4. Mean kinetic energy surface of trapped ions as a
function of mean free path and space charge obtained using
standard conditions (see methods section). To omit the contribution from the ion’s initial energy the average kinetic energy
was determined between 2500 and 15000 µs.
ing allowed the ions to gain more kinetic energy. The average kinetic energy of the ion (determined between 2 500 s
and 15 000 s so as to omit the contribution from the initial
kinetic energy of the ion) as a function of mean free path and
space charge is shown in Figure 4.
With regard to collisional activation, the trend to higher
kinetic energies for longer mean free paths should be offset
by the fact that ions with a smaller mean free path experience
more collisions. The number of collisions increased with
decreasing mean free path and with increasing space charge
(Figure 5). This increase was much more rapid for a smaller
mean free path in combination with higher space charge.
Figure 5. Dependence of the number of collisions on mean free
path and space charge.
L.A. McDonnell et al., Eur. J. Mass Spectrom. 8, 181–189 (2002)
Although ions with a shorter mean free path acquired
less kinetic energy under higher space-charge conditions,
the rapid increase in collision frequency with decreasing
mean free path was more pronounced. As a result, once
space charge is significant, slow-heating collisional
activation19 can increase with a decreasing mean free path
because the increase of ion kinetic energy, due to the higher
number of collisions, can outweigh the decrease in the average kinetic energy of the ion. This finding is in agreement
with previous experimental results in which the degree of
fragmentation was found to increase with an increased background gas pressure.2,3
Kinetic energy and radial-position profile dependence on
mass, charge and RF-potential amplitude
It was recently reported that the radial position of an ion
trapped in a hexapole guide is dependent upon the mass-to4
charge ratio of the ion. This inference came as a direct result
of the need for balance at the ion’s equilibrium position
between the field due to space charge, Esc, and the effective
*
RF field, E (derived from the effective potential of the adia15,20
batic approximation ). In the absence of collisions, the
4
radial position of an ion is given by
1/4
2
2
rq = a(qL/q3 Qmax) ; q3 = 12qVRF/mω a ; Qmax = 3πε0VRF /2 (4)
where qL is the charge per unit length experienced by the ion
and will depend on the m/z of the ion as well as the number
and the identities (m/z) of the other ions present in the
hexapole4 and VRF. Further, q3 is the hexapole stability param15
eter for that ion, q is the charge of the ion, VRF is the RF
potential, m is the mass of the ion, ω is the angular frequency
of the RF field and a is the field radius of the hexapole which
is defined as the minimum distance from the hexapole axis to
185
an electrode surface (1.14 mm for the simulations reported
here).
Therefore under conditions of identical charge per unit
length
rq ∝ m1/4
(5)
In the adiabatic approximation, the average kinetic
energy of the fast oscillatory motion is given by20
2
KE = q (q/m) × (3VRF / 2aω) × (r/a)
4
(6)
By substituting Equation 5 for r in Equation 6, it is found that,
under identical conditions (constant charge per unit length),
the ion kinetic energy is predicted to be independent of mass.
The same approach can be used to study the effects of varying
the RF potential. Under otherwise identical conditions
rq ∝ (1/VRF ) 1/2
(7)
Therefore, under conditions of identical charge per unit
length, the average kinetic energy of the trapped ion is predicted to be independent of the RF potential. (A summary of
several methods used to calculate average ion kinetic energies in radio-frequency fields is included in Todd and
March’s recent review on the earlier days of ion traps.21)
Radial stratification and kinetic energy independence
with respect to ion mass were investigated by comparing the
radial position and kinetic energy profiles of singly-charged
ions of masses 500, 1000 and 1500 Da (mean free
path = 10 mm and space charge = 3.54 × 10–13 C). In agree4
ment with the predictions of Tolmachev et al. and those
detailed above, it was found that the average radial positions
of the heavier ions were greater than those of the lighter ions
(data not shown) and that the kinetic energy profiles for the
Figure 6. Kinetic energy and radial position dependence on the ion’s charge state. In agreement with the analytical expressions,
the kinetic energy is directly proportional to the charge on the ion even when the mass-to-charge ratios of both ions are the same
whereas the radial position is dependent on m/z only. The simulations were run using a 10 mm mean free path and 3.54 × 10–13 C
of space charge.
186
A Theoretical Investigation of Kinetic Energy of Ions
Figure 7. Microspray spectra of mono-nitrated lysozyme (20 µM in 1 : 1 water + acetonitrile containing 1% formic acid). Accumulation time: (a) 20 s and (b) 4 s.
trapped ions were independent of the ion mass and RF potential.
It was also predicted that the kinetic energy of the ion
would be directly proportional to the number of charges on
the ion. The results of simulations using ions of 10 times the
molecular mass but the same m/z entering the hexapole at the
same speed (m = 10,000 Da, z = 10, KE0 = 65 eV, mean free
–13
path = 10 mm and space charge = 3.54 × 10 C) are shown
in Figure 6. After the ions have been damped (2500 s), the
kinetic energy band of the heavier ion is about 10 times
larger than that of the singly-charged ion [Figure 6(a)]. Also
in agreement with predictions, the average radial position of
the ion is independent of the charge state of the ion: it is
dependent only on the mass-to-charge ratio [Figure 6(b)].
For the conditions used, the average kinetic energy of
the ion is given by
KEaverage / eV = z × [– 10–4 + (6.7 ± 0.3) × 1010 × γ +
–3
(1.4 ± 1) × 10 × λ]
(8)
where γ is the total space charge contained in the hexapole (in
C) and λ is the mean free path (in mm). As expected from
Equation 2, the kinetic energy increased linearly with space
charge (average R2 value = 0.99). The mean free path
dependence also appeared to be linear (including the error
2
bars from each individual plot of KE vs space charge, the R
value for the mean free path variation was 0.98).
The average kinetic energy of an ion was approximately
0.025 × z eV. Thus, the similar fragments that have been
obtained by the slow-heating techniques MSAD, SORI3 and
22
IRMPD can be explained by the similar energetics
involved, especially considering that SORI is routinely performed on multiply-charged species.
Experimental view of the radial stratification phenomenon
A comparison of the FT-ICR mass spectra of reduced
10
mono-nitrated hen egg white lysozyme, obtained under
MSAD and non-MSAD conditions, is shown in Figure 7. For
clarity, the data from the MSAD experiment [Figure 7(a)]
has been enlarged. Presumably, the lower intensity of the
MSAD spectrum reflects the larger number of species present in the hexapole.
As can be seen, under non-MSAD conditions [Figure
7(b)], parent ions in the 9+ to 16+ charge states were formed,
the 13+ charge state being the most intense. However, after
MSAD [Figure 7(a)], the only remaining parent ions were
the 14+ ions, despite the 13+ charge state being more
intense. Radial stratification inside the hexapole can explain
the counterintuitive result that, under a prolonged residence
time inside the hexapole, the lower charge states of mononitrated lysozyme were preferentially fragmented, despite
intramolecular space charge causing the higher charge states
23
to have a lower fragmentation threshold. Thus, as the lower
charge states would have been located at larger radial positions, the space charge they experienced would have been
greater (for the cylindrical distribution of ions in a
13
hexapole, the net space charge experienced by an ion is
24
determined only by those ions of smaller radius ). The simulations reported above show that, as a result of this increased
space charge, the lower charge states would have had a
higher average kinetic energy and would have collided more
L.A. McDonnell et al., Eur. J. Mass Spectrom. 8, 181–189 (2002)
often. Consequently, the collisional activation of the lower
charge states was greater.
Under identical space-charge conditions, the higher
charge states would have been expected to have more energy
and to collide more often. As was shown in Figure 6, the
average kinetic energy of an ion varies linearly with its
charge state and Clemmer et al. have shown that the higher
charge states of lysozyme are physically larger.25 Consequently, under identical space charge conditions, the higher
charge states would be expected to fragment first. The finding that the lower charge states fragmented first indicates
that the extra space charge experienced by the lower charge
states, as a result of radial stratification, outweighs the
charge-state dependency of the ion kinetic energy and mean
free path.
In the instrument used to obtain the above results, a
9.4 T Bruker BioAPEX II FT-ICR with an Analytica
9
electrospray ion source, the hexapole ion trap formed part of
the ion source and was located 3.75 mm downstream from
the capillary exit (the skimmer used to collimate the ion
beam also acted as one of the trapping plates for the
hexapoles). The ions were accumulated in the ion trap for a
user-defined period of time before being injected into the
FT-ICR cell. For these experiments, the total signal intensity
(per unit time) was quite low and an accumulation time of
approximately 20 s was required to achieve appreciable
fragmentation [see Figure 7(a)]. A corollary of the above
explanation is that 20 s was sufficient for the ion cloud to
equilibrate and that the relatively low flux of incoming ions
from the capillary (the flux of ions was not halted) was insufficient to significantly alter this distribution.
Experimental view of the kinetic energy dependence
In agreement with the adiabatic approximation predictions, the average kinetic energy of the trapped ion for a
given space-charge field (charge per unit length) was found
187
to be insensitive to the applied RF potential over the range
200–600 Vp–p (data not shown).
As the potential experienced by an ion at a specific
radius is dependent on the applied RF potential and the space
charge was thought to be constant (the spectrum was insensitive to RF potential for shorter accumulation times), the
observation that the degree of fragmentation decreased with
decreasing RF potential was interpreted as indicating that
the ions were driven to a lower KE as a result of the lowered
3
RF field. However, the results reported here indicate that for
a given space-charge field, the radius of the ion increases
with decreasing RF potential, which in turn leads to the ion
kinetic energy being insensitive to the RF potential.
As the kinetic energy of the ions would have been insensitive to the applied RF field if space charge was constant, it
is now thought that the decrease in the degree of fragmentation with decreasing RF potential is due to a decrease in the
space charge present in the hexapole. Although the signal
intensity was found to be insensitive to the applied RF potential for a short accumulation time, the ion-intensity profile of
the ESI ion source indicates that the signal intensity obtained
after a longer accumulation time, as used for the MSAD
experiments, could have a significant RF potential
dependence.
The total ion intensity obtained from 20 M lysozyme
(the unreduced form is very difficult to fragment by MSAD)
as a function of accumulation time is shown in Figure 8(a).
As can be seen, for the conditions used there was a ≈ 1 s
delay before appreciable signal intensity was observed (no
ion signals were observed for an accumulation time under
0.6 s). The sigmoidal line shape is indicative of a low initial
trapping efficiency that increases with the number of trapped
ions before tailing off as the ions leak out. The upper limit
corresponds to the working charge capacity of the ion trap,
which has been predicted to be dependent on the applied RF
potential.26 The ion intensity profiles obtained for insulin
Figure 8. Total-ion intensity dependence on hexapole accumulation time. (a) Electrospray ionization of 20 µM lysozyme in 1 : 1
(v / v) water + acetonitrile containing 1% (v / v) formic acid. Unreduced lysozyme was used as it is difficult to fragment. (b) Nanoelectrospray ionization of 25 µM insulin in 1 : 1 (v / v) water + methanol containing 1% (v / v) acetic acid. The upper curve corresponds to VRF = 300 V (600 Vp–p), and the lower curve to VRF = 200 V. The initial concentration of analyte was so high that insulin
signals were obtained even at almost zero accumulation time.
188
were found to agree qualitatively with these predictions [see
Figure 8(b)].
The difference between the sigmoidals itself resembles
the sigmoidal profile: it is initially very small and then
increases with accumulation time before reaching a plateau,
which corresponds to the difference between the upper limits of the two sigmoidals. With regard to ion accumulation in
a hexapole ion trap, this plateau corresponds to the difference between the charge capacity of the ion trap for the two
RF potentials.
After a short accumulation period, the small decrease in
total-ion intensity accompanying a decrease in the charge
capacity of the hexapole could be within the boundaries of
experimental uncertainty (see experimental scatter in Figure
8). However, after a longer accumulation time, the reduced
charge capacity became more pronounced. As a result, the
fact that the measured ion intensity was experimentally
insensitive to RF potential for shorter accumulation times
does not mean that this was also the case for the MSAD
experiments that used a longer accumulation time.
The accumulation time required for an experimentally
measurable decrease in ion intensity becomes smaller as the
charge capacity of the hexapole is further reduced. Thus, the
observation that the ion signal, after a 1 s accumulation
period, was found to decrease for Vp–p < 400 V but not for
Vp–p = 400–600 V can be explained as the former reduction
was experimentally observable whereas the latter was not.
Therefore, the observation that the degree of fragmentation
decreased with RF potential (accumulation time = 3 s) could
have been due to a reduction in the charge capacity of the
hexapole and not to a reduction in the average kinetic energy
of the trapped ion, in agreement with the results presented
here and adiabatic approximation predictions.26
Analytical considerations
The observation of preferential fragmentation as a result
of radial stratification shows that the space charge from lowmass ions (these low-mass ions may not be trapped or
detected in the FT-ICR cell but are nevertheless contained in
the hexapole ion trap) could result in the preferential fragmentation of the high-mass ions of interest. This would be
especially true for the detection of non-covalently bound
adducts27 which are normally detected at higher m/z ratios
than their constituent species. Methods to eliminate the contribution from lower-mass ions include operating the
multipole as a mass filter, changing the RF characteristics of
the multipole or using an additional multipole to filter the
ions prior to accumulation.15 The recently reported work of
Belov et al., concerning the control of fragmentation and
discrimination in a quadrupole linear ion trap, indicates
potential alternative solutions to discrimination and fragmentation in higher-order multipoles.29,30
A Theoretical Investigation of Kinetic Energy of Ions
Conclusions and outlook
The general explanation for MSAD is still collisional
activation of the ions in higher energy trajectories as a result
of space-charge repulsion. The kinetic energy of the trapped
ions correlates strongly with the charge density and more
weakly with the collision frequency; however, longer mean
free paths allowed more pronounced kinetic energy fluctuations to occur, which could contribute significantly to
collisional activation. For a given space-charge field, the
average kinetic energy of the trapped ion was found to be
independent of its mass and the amplitude of the applied RF
potential but was directly proportional to its charge state.
Radial stratification in the hexapole results in ions of
higher m/z experiencing a larger space-charge field. The
resulting m/z dependence of the ion’s kinetic energy is
thought to be the cause of the observation that the lower
charge states of reduced mono-nitrated lysozyme were preferentially fragmented despite intramolecular space charge
favouring the opposite effect.
The calculated kinetic energies were of the order
0.025 × z eV, where z is the elementary charge of the ion.
This value is similar to the energy of a single infrared photon
and to the ion kinetic energy during sustained off-resonance
irradiation (SORI) excitation.22 The similar energies
involved explain the similar fragments that have been
obtained using MSAD, SORI, and infrared multiphoton
dissociation.3,22
Acknowledgements
We would like to acknowledge Dr John Heptinstall for
the production and purification of mono-nitrated lysozyme
and both Peter N. Joanes and Dr Xidong Feng for their technical assistance. The assistance and collaboration of Bruker
Daltonics is gratefully acknowledged. This work was supported by the EPSRC, BBSRC and Avecia.
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Received: 3 December 2001
Revised: 18 January 2002
Accepted: 22 January 2002
Web Publication: 8 April 2002