STANFORD UNIVERSITY
HADAMARD TRANSFORM
TIME-OF-FLIGHT MASS
SPECTROMETRY
HT-TOFMS
Magnus Wetterhall Ph.D. student
Uppsala University
Department of Chemistry
Uppsala
SWEDEN
Abstract
In this report the development of a new type of mass spectrometry - Hadamard Transform Timeof-Flight Mass Spectrometry (HT-TOFMS) - is described. The aim of this research has been to
explore the possibility of using the HT-TOFMS in the determination of biomolecules of interest.
In this sense, the HT-TOFMS has been coupled to an electrospray ionization source (ESI) due to
ESI’s compatibility with most of the separations techniques commonly used in the bioanalytical
laboratory (high-performance liquid chromatography or capillary electrophoresis). A thorough
study regarding the influence of acquisition mode on analytical performance (in particular the
scan speed) has been conducted. By varying the acquisition mode a full mass range scan speed of
6.1 kHz, mass resolution (FWHM) of 1200 amu and a sensitivity in the femtomole range has
been obtained. The results imply that the online coupling of ESI-HT-TOFMS fulfills the
expected figures of merit for bioanalysis.
2
INTRODUCTION................................................................................................................................................ 4
MASS SPECTROMETRY (MS) ............................................................................................................................... 4
HADAMARD TRANSFORM TIME-OF-FLIGHT MASS SPECTROMETRY (HT-TOFMS)................................................... 6
ELECTROSPRAY IONIZATION (ESI) ...................................................................................................................... 8
CAPILLARY ELECTROPHORESIS (CE) ................................................................................................................... 9
EXPERIMENTAL SETUP ................................................................................................................................ 10
THE ESI INTERFACE ......................................................................................................................................... 10
THE CHARGED PARTICLE MODULATOR............................................................................................................... 11
THE ELECTRONICS TO PRODUCE THE PRS .......................................................................................................... 11
THE ONLINE COUPLING OF CE-HT-TOFMS....................................................................................................... 11
RESULTS AND DISCUSSION.......................................................................................................................... 12
EVALUATION OF THE ESI INTERFACE ................................................................................................................ 12
EVALUATION OF INSTRUMENTAL CHARACTERISTICS UNDER DIFFERENT ACQUISITION MODES .............................. 17
CONCLUSIONS ................................................................................................................................................ 21
COMPARISON BETWEEN THE HT-TOFMS AND THE JAGUARTM O-TOFMS....................................................... 22
Scan speed .................................................................................................................................................. 22
Resolution................................................................................................................................................... 22
Sensitivity ................................................................................................................................................... 22
FUTURE STUDIES .............................................................................................................................................. 22
ACKNOWLEDGEMENTS ............................................................................................................................... 22
REFERENCES................................................................................................................................................... 23
3
Introduction
Mass spectrometry (MS)
Mass spectrometry (MS) is one of the most common and fastest growing detection techniques in
analytical chemistry. It is a very powerful and universal detection technique, which yields
specific structural information about each substance in a sample can be obtained. Mass
spectrometry measures the mass to charge ratio (m/z) of ions in an electrical or magnetic field in
order to identify unknown species in a sample and to determine the chemical structure of the
species. To date, any molecule or atom of interest may be satisfactory determined by MS
regardless of the matrix nature or composition. In this sense, MS is by far the most versatile and
universal analytical technique available.
All mass spectrometer instruments have the following general components: vacuum pumps, an
ion source, focusing lenses, a mass analyzer, a detector and a data handling system. Figure 1
shows a general setup of a mass spectrometer.
Ion source
Focusing lenses
Mass analyzer
Vacuum
Detector
Data system
Mass spectrum
Figure 1 General instrumental setup for MS
Even though the principle for all MS instruments is the same, there is a great diversity regarding
each general component for different instruments. To thoroughly explain the function and
diversity of each component is beyond the scope of this report, however a very short orientation
will be provided. The ions can be generated by different methods and this will be treated
separately. The produced ions have dispersion in spatial and energetic distribution. The focusing
lenses counterbalance these distributions to yield an ion beam with narrow spatial and energetic
distribution. The mass analyzer separates ions with different m/z. The detector registers the ions
and the data handling system processes the signal from the detector. It must be stressed that ions
by nature are very reactive. For this reason, MS is performed under vacuum conditions to avoid
possible collisions (that is reactions) with atoms or molecules present in the surrounding
4
atmosphere. The use of a power pumping system is required to guarantee an optimum vacuum
level (in general, below 10-6 mbar, a billion times lower than atmospheric pressure).
The ion source and the mass analyzer need further description. Various ion sources are
described in the literature1. The ion source used depends on the physical properties of the
sample and analyte. In the case of analyzing liquid samples electrospray ionization2 (ESI) and
matrix assisted laser desorption ionization3 (MALDI) are the most common ionization
techniques. Laser ablation4 or glow discharge ionization5 are commonly used for solid samples.
A more thorough description of ESI will be given later. The mass analyzer is the “heart“ of the
mass spectrometer and a specific technique is named after the mass analyzer used. Time-offlight mass spectrometry (TOFMS) is subsequently based on a time-of-flight mass analyzer. In
TOFMS ions are accelerated by an electric field (E) and the flight time of ions over a certain
distance is measured. Lighter ions fly faster than heavier ions and due to this a distinction
between different analytes in a sample can be made. Figure 2 shows the principle of TOFMS
Acceleration, a
Ea = Va/la
Length = l a
Field-free drift zone, d
Detector
Ed = 0
Length = l d
Figure 2 The working principle of time-of-flight mass spectrometry
Conventional time-of-flight techniques with a continuous ion source are almost exclusively
based on orthogonal extraction (OE) in which the ions are pulsed at a right angle towards
detection. The benefit of this is a very precise starting point for the flight time. However, the
sampling efficiency (duty cycle) is below 30%. This value is still higher than the one obtained in
scanning spectrometers (well below 1% if a full mass spectra is recorded). However, for
substances present at low concentration or for determination of transient signals, a higher duty
cycle is desirable. The relatively low duty cycle of the OE-TOFMS is related to the limited
repetition rate. The repetition rate is defined by the duration of a single mass spectrum (that is,
the time that the heaviest ions take to reach the detector). During the detection period, the
generated ions can not be analyzed. For a scanning spectrometer, this “dead time” is constant
regardless of the length of the mass window to be analyzed. Beyond this, the OE-TOFMS is
highly dependent on the kinetic energy of the ions. In this sense, when ions of different masses
are present in the sample, the extraction voltage will preferentially deflect some ions, introducing
errors in the measurement known as “mass bias”.
5
Hadamard transform time-of-flight mass spectrometry (HT-TOFMS)
One alternative to extracting the ions at a right angle (as in OE) is to use an on-axis
configuration. However, it is still necessary to know the starting time of the ions in order to
measure their TOF and so, there are no additional improvements of the duty cycle or repetition
rate in comparison with OE-TOFMS. In general, a pulsed voltage is applied in order to deflect
the ions, although other solutions such as the use of ion traps or continuous beam deflection
techniques have been used.
In this project, the continuous beam generated in the ionization region is constantly split into
discrete ion packets of different length following a pseudorandom sequence (PRS) of pulses
based on Hadamard type binary sequences. These individual ion packets are separated in the
flight tube and will reach the detector, providing a normal TOF mass spectrum. As the ions are
constantly reaching the detector, the overall signal will be an overlap of many time-of-flight
distributions, each one shifted following the pattern dictated by the PRS. Obviously, this rough
signal must be properly decoded to obtain a normal TOF spectrum. As the PRS has been
constructed by means of specific Hadamard matrices6, the decoding method implies the use of a
Hadamard algorithm that uses the same encoding sequence to yield a conventional TOF
spectrum. Taking into account that the method relies on the use of Hadamard matrices, the
technique has been named Hadamard Transform time-of-flight mass spectrometry (HTTOFMS).7,8
Modulation of the ion beam is accomplished using a grid (Fig 3) consisting of an interleaved
comb of alternatively positive and negative charged wires, called a charged particle modulator
(CPM). The modulation has two modes: “beam on” in which no potential is applied and the ions
reach the detector and “beam off” in which potential is applied and the ions are deflected and do
not reach the detector. Figure 4 shows the two modes.
+ - + - +
Figure 3 Picture of the CPM and a schematic figure of the interleaved comb
6
" b ea m o n "
state
" beam off "
state
+ Ion
B e am
+ Ion
B e am
40
30
20
10
0
- 10
- 20
- 30
- 40
0
1
“off”
2
“on”
Figure 4 Beam on and beam off states in the multiplexing of the ion beam
The data that is actually collected is a binary sequence of transmitted and non-transmitted ion
bursts. This binary sequence is subsequently deconvoluted (‘de-multiplexed’) using a fast
Hadamard transform algorithm, that uses the same sequence applied physically at the CPM,
yielding a conventional TOF mass spectrum (Fig 5).
Raw data
Mass spectrum
2600
70 0000
2400
60 0000
Fast Hadamard
Transform
Counts
2000
1800
1600
50 0000
40 0000
Counts
2200
30 0000
20 0000
1400
10 0000
1200
0
1000
-100000
800
0
1000
2000
3000
0
4000
1000
2000
3000
4000
Bin number
Bin number
Figure 5 Hadamard transform of raw data into mass spectrum
The benefits of the HT-TOFMS are high duty cycle (50%) and ion transmission (50%), which in
turn implies fast scan speed and high sensitivity. The technique is therefore very useful when,
for instance, analyzing low concentration samples, studying kinetic reactions, characterizing
protein folding/unfolding, determining speciation and measuring fast transient signals. The low
cost and simple instrumental setup are also benefits of HT-TOFMS.
7
Electrospray ionization (ESI)
Electrospray ionization2 (ESI) is the most common ionization technique for MS analysis of
liquid samples. Modern separation techniques such as Capillary Electrophoresis, Capillary
Electrochromatography and Liquid Chromatography of samples with clinical and biomedical
origin are performed in liquids. ESI ideally suits the on-line coupling of these separation
techniques to MS. Some benefits of ESI are that it is a “soft” ionization technique in the sense
that it yields no fragmentation (cleavage) products and that the analytes often are multiply
charged. The multiple charge effect produces ions with relatively low m/z (often below 2000
amu). This is essential when analyzing large biomolecules, which often have a molecular weight
of 10 000 Da and up. It must be added that ESI is an atmospheric pressure ionization technique.
Other techniques such as fast atom bombardment (FAB) or MALDI can perform liquid
ionization, but only under vacuum conditions. These techniques therefore have low compatibility
with organic solvents that are used in chromatographic or electrophoretic separations.
There is much debate and research regarding the mechanism of ESI. The process for positive
ESI is shown in figure 6.
ESI tip
Taylor cone
Taylor jet
V ESI
Parent
+ +
+
+ +
Offspring
+
+
Counter electrode
Coulomb fission
Figure 6 The production of gas phase ions in electrospray ionization
A short description of the ESI mechanism will be given without going into too much detail. In
ESI, a high voltage is applied to the metal (metal coated) ESI capillary through which a sample
solution is emerging. The counter electrode (inlet of the MS) is often grounded. Ionic species in
the emerging sample solution undergo electrophoretic movement, moving towards the electrode
with the opposite potential in comparison to their charge. This will give rise to a Taylor cone9
where the counterbalancing force is the surface tension of the solution. If the applied electric
field is strong enough the Taylor cone will yield a Taylor jet, which in turn breaks into droplets
that contain (in the case of positive ESI) positive ions. As the droplets move towards the counter
8
electrode solvent will evaporate from the droplets, which causes an increase in surface charge
density. Eventually the ‘Raleigh stability limit’ will be reached, which is the point when the
force of electrostatic repulsion between like charges on the surface of the droplets becomes equal
to the surface tension force holding the droplets together. At this point the droplets will undergo
‘Coulomb fission’ and split into smaller ‘offspring’ droplets. The droplet fission process is
repeated and eventually the ions will be transferred to the gas phase.
The ESI process behaves as a constant current electrochemical cell2, and thus it is affected by the
concentration of the analytes and of the background electrolytes. In complex mixtures, the
presence of a high amount of an easily ionized substance might suppress the signal of another
analyte and thus the analysis will be biased. To avoid this problem, it is necessary to incorporate
a separation step prior to ESI.
Capillary electrophoresis (CE)
Capillary electrophoresis10 (CE) is a widely used analytical technique. Some benefits of CE are
its high separation efficiency and low sample consumption. The CE separation is performed in
narrow fused silica capillaries. The inner diameter of the capillary is usually between 5 and 100
µm and the outer diameter is between 200-400 µm. Figure 7 show the general setup for capillary
electrophoresis.
EOF
µe+
µe-
- -
N
+
+
Fused silica capillary
Detector
+
Inlet buffer
vial
High voltage
power 5-60 kV
Outlet buffer
vial
Fig 7 Instrumental setup for Capillary electrophoresis
The working principle for CE is that a buffer filled capillary is immersed into two buffer vials
and a high voltage (5-60 kV) is applied over the capillary. This will cause a migration of the
ionic species in the capillary. This migration is called the electrophoretic mobility (µe). The
cationic (positive charged) substances will move towards the cathode (- electrode) and the
9
anionic (negative charged) substances will move towards anode (+ electrode). Neutral
substances will be unaffected by the imposed electrical field. The rate of the electrophoretic
mobility is dependent on the analytes charge to size ratio. Small highly charged analytes will
migrate faster than large analytes with a low charge. There is a second movement in the
capillary called the electroosmotic flow (EOF) that is dependent on the buffer and the inner
surface of the capillary. The inner surface of the capillary will attract buffer ions with opposite
charge to the surface yielding an inner static layer (Stern layer) and an outer diffuse layer (Outer
Helmholtz Plane). The buffer ions will move towards their counter electrode pulling the buffer
solution in that direction. In summary, the electrophoretic mobility give rise to separation of
ionic substances and the EOF will cause a net movement of all substances in one direction, the
detection end of the capillary.
Experimental setup
The HT-TOFMS instrument is shown in figure 8. Three components of the instrument were
identified as “weak” points that were crucial to improve. These components were the ESI
interface, the CPM and the electronics used to produce the PRS.
Heaters
Modulator
Octopole
TOF chamber
Focusing
lenses
ESI tip
Steering plates
Vacuum pumps
Amplifier
MCP Detector
Phase I
Pseudorandom
Sequence generator
Phase II
Reflectron
Signal
Vacuum pumps
Pre amplifier
Trigger
Clock
Multichannel
scaler
Computer
Figure 8 Schematic figure of the HT-TOFMS instrument
The ESI interface
The ion source is one of the most important components to optimize and have control over in MS
since it is where the ions to be measured are produced from the sample. One of the goals of this
10
project is to couple separations to the MS. A sheathless ESI interface called the fairy dust 11
technique was implemented and evaluated on the instrument. In the fairy dust method the
emitter end of a fused silica capillary is tapered and a coating of polyimide and fine gold powder
is applied. This will yield a durable and stable high voltage contact for the ESI.
Continuous infusion experiments of tetra alkyl ammonium salts, polymers, peptides and proteins
where conducted in order to evaluate the ESI and overall instrumental performance. A thorough
study regarding how the length of the PRS influences characteristics of the MS such as scan
speed, mass resolution and sensitivity has been performed using continuous infusion ESI-HTTOFMS of a reserpine sample. Three parameters of the PRS were investigated: the length of the
PRS (the positive integer n was changed between 11 and 13), the acquisition bin width
(measuring time interval) and the modulation bin width (multiplexing time interval).
The charged particle modulator
The unique feature of HT-TOFMS is the Hadamard multiplexing of the on-axis ion beam. The
previous construction of the CPM was a tedious and quite expensive procedure. A skilled user
could manufacture one CPM in about 3 days. With the newly developed setup a skilled person
can fabricate a CPM within 1-2 hours. Different polymer materials have also been explored
when constructing the CPM. A thorough evaluation of the performance for the newly developed
CPM’s is yet to be conducted.
The electronics to produce the PRS
The electronics used for production of the PRS have until recently been in-house designed and
in-house assembled. The benefit of this is that a freedom regarding modifications of the
electronics is obtained. The drawbacks are high noise levels due to improper shielding, difficulty
when switching to different PRS lengths, and the low robustness of the setup. The second
generation of the electronics has been developed on printed circuit boards, thus avoiding the
drawbacks of the previous generation.
The online coupling of CE-HT-TOFMS
One major consideration when coupling CE online with ESI-MS using bare fused silica
capillaries is the irreproducible EOF. This will bias the separation and compromise the
performance of the ESI. One solution to this problem is to coat the inner surface of the capillary.
The capillaries used when coupling CE online with ESI-MS had a MAPTAC12 coated inner
surface. In the MAPTAC procedure the silanol groups of the capillary inner surface are
derivitized with quaternary amino groups. This will produce a positively charged inner surface
that yields reproducible EOF and minimizes analyte-inner surface interactions for positive
charged analytes. Experiments on CE-ESI-HT-TOFMS are underway as this report is being
written.
11
Results and Discussion
Evaluation of the ESI interface
Continuous infusion ESI-HT-TOFMS spectra of various analytes of interest are shown in figures
9-14. Figure 9 and 10 show mass spectra for continuous infusion of tetra butyl ammonium
(TBA, Mw 242.28 Da) ions and tetra ethyl ammonium (TEA, Mw 129.16 Da) ions respectively.
Both samples contained 100 µM analyte in 50/50 acetonitrile/deionized water, the flow rate was
set at 500 nL/min, ESI potential at 2 kV and the orifice was heated to 120°C.
242.45
Intensity
120000
60000
144.37
0
0
100
200
300
400
500
600
700
800
900 1000
m/z
Figure 9 Mass spectrum for continuous infusion ESI-HT-TOFMS of TBA
12
129.57
Intensity
200000
100000
242.45
57.93
104.3
0
0
1 00
2 00
3 00
4 00
5 00
6 00
7 00
8 00
9 00 10 00
m/z
Figure 10 Mass spectrum for continuous infusion ESI-HT-TOFMS of TEA
In figure 9 a dominant peak at m/z 242.45 can be seen, which is the TBA. A small peak at m/z
144.37 can also be observed. It is not known if this peak is an artifact originating from the
multiplexing or if it is an impurity in the sample. In figure 10 the dominant peak at m/z 129.57 is
the TEA. The small peak at 242.45 is TBA and the appearance of TBA in this spectrum is due to
a memory effect in the capillary from previous TBA experiments. The small peaks at m/z 57.93
and 104.3 are probably artifacts or impurities in the sample.
Figure 11 show a mass spectrum collected for a binary mixture of TBA and TEA, 100 µM each
and in the same conditions mentioned above.
13
242.45
TBA
1600 00
TEA
N+
Intensity
Ion suppression
800 00
N+
TBA
129.57
TEA
0
0
1 00
2 00
3 00
m/z
Figure 11 Mass spectrum for continuous infusion ESI-HT-TOFMS of TBA and TEA
This mass spectrum shows the interesting phenomenon of ion suppression. The ion suppression
can in this case be understood by comparing the structure of TBA and TEA (shown in figure 11).
TBA has 4 butyl groups coupled to the nitrogen atom and TEA has 4 ethyl groups coupled to the
nitrogen atom. The longer alkyl groups in TBA imply that TBA is more hydrophobic than TEA.
The higher hydrophobicity of TBA will yield a higher surface activity for TBA than for TEA in
the ESI produced droplets. In other words, TBA will be more abundant on the surface of the
droplets than TEA and subsequently more easily transferred into the gas phase than TEA. The
presence of TBA in the sample will thus suppress the signal of the TEA.
Both TBA and TEA have a permanent positive charge and are therefore easily transferred into
the gas phase in ESI. In figure 12 a mass spectrum of polypropylene glycol (PPG) with a narrow
molecular weight (Mw) distribution around 450 g/mole is shown. PPG has no permanent charge
and the ions therefore have to be produced in the ESI. The PPG sample contained 100 µM
analyte in 50/50 methanol/deionized water, the flow rate was set at 250 nL/min, ESI potential at
2 kV and the orifice was heated to 110°C.
14
PPG Narrow Mw distribution <450>
HO
H
O
1 2 0 00 0
n= 7
n
505
n=5 to 10
n= 8
In tensity
389.22
n= 6
6000 0
563.07
330.68
n= 9
184.53
n= 5
621.44
272.45
n=10
0
20 0
40 0
60 0
m/z
Figure 12 Mass spectrum for continuous infusion ESI-HT-TOFMS of PPG
In figure 12 several peaks with a ∆m of 58-59 can be observed. These peaks represent the Mw
distribution of PPG. The monomer unit structure of PPG is also given in figure 12 and the
number of monomer units is given for each peak in the mass spectrum. The Mw distribution is
very useful for mass calibration of the instrument in this mass range.
A mass spectrum of the peptide Bradykinin (Mw 1059.57 Da) is shown in figure 13. The sample
contained 100 µM analyte in 100 % methanol, the flow rate was set at 500 nL/min, ESI potential
at 2 kV and the orifice was heated to 110°C. Figure 14 shows the mass spectrum for the protein
Cytochrome C (Mw 12384 Da).
The sample contained 50 µM analyte in 50/50
methanol/deionized water 2.5% acetic acid, the flow rate was set at 500 nL/min, ESI potential at
2 kV and the orifice was heated to 110°C.
15
532.76
600000
Bradykinin 2H
Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg
+
H2N
C
HN
NH
H2C
CH2
H2C
NH2
CH
C
H2N
O
N
O
C
OH
O
H2C
H2C
C
N
C
NH
N
CH
O
CH
400000
C
HN
C
O
H
C
HN
H2C
NH
NH
CH2
C
NH
O
HN
CH
CH C
C
OH
O
H2C
CH
O
O
Intensity
H2C
563.07
200000
+
Bradykinin 2H + CH 3 OH
0
40 0
50 0
60 0
70 0
m /z
Figure 13 Mass spectrum for continuous infusion ESI-HT-TOFMS of Bradykinin
8 2 6 .82
8 8 5 .84
7 7 5 .12
14H
12000
I n tensit y
16H
+
+
9 5 3 .92
13H
7 3 0 .22
6000
17H
+
1033 .07
+
12H
+
0
600
800
1000
m/z
Figure 14 Mass spectrum for continuous infusion ESI-HT-TOFMS of Cytochrome C
16
Figures 13 and 14 show that analysis of peptides and fully intact proteins can easily be
conducted. In figure 13 two peaks are observed, a dominant peak at m/z 532.76 which is the
doubly protonated bradykinin and a smaller peak at m/z 563.07. The peak at m/z 563.07 is the
doubly protonated bradykinin in a cluster formation with the solvent methanol. The reason for
cluster formation in this case is that the bradykinin sample was prepared in pure methanol.
Cluster formation is often observed under such circumstances. Figure 14 shows the phenomenon
of multiple charging (multiple protonation) in ESI. It is due to multiple charging that intact
proteins with a high mass can be analyzed in ESI-MS.
Evaluation of instrumental characteristics under different acquisition modes
The unique feature of HT-TOFMS is, as previously mentioned, the multiplexing of the
continuous ion beam. The influence of the PRS length is shown in figures 15-18. Throughout
these experiments a 100 µM reserpine (Mw 608.68 Da) in 50/50 methanol/deionized water 0.1%
acetic acid solution was analyzed. The ESI voltage was set at 2 kV and the orifice temperature
was set at 110°C.
3.5e+5
3.0e+5
Intensity
2.5e+5
2.0e+5
11 bits PRS
1.5e+5
1.0e+5
5.0e+4
0.0
- 5 .0e+4
0
200
400
600
800
1000
800
1000
7e+5
6e+5
Intensity
5e+5
4e+5
12 bits PRS
3e+5
2e+5
1e+5
0
-1e+5
0
200
400
600
1.6e+6
1.4e+6
Intensity
1.2e+6
1.0e+6
13 bits PRS
8.0e+5
6.0e+5
4.0e+5
2.0e+5
0.0
- 2 .0e+5
0
200
400
600
800
1000
Time-of-flight (µs)
Figure 15 Comparison of 11-13 bits PRS multiplexing of a reserpine sample
17
Figure 15 shows the effect of varying PRS length, a shorter PRS yields a faster scan speed but
also a smaller full mass range. The Felgett equation13 implies that the signal to noise ratio (SNR)
will increase by 2 as the PRS length is doubled. Figure 16 the compares SNR for 11,12 and
13 bits PRS. The SNR increase clearly follows the Felgett equation.
1.6e+6
450
1.4e+6
400
350
S/N
1.2e+6
300
250
Intensity
1.0e+6
200
150
8.0e+5
0
1e+7
2e+7
3e+7
4e+7
5e+7
6e+7
7e+7
8e+7
Squared MLPRS Length
6.0e+5
11 bit PRS
12 bit PRS
13 bit PRS
4.0e+5
2.0e+5
0.0
-2.0e+5
128
129
130
131
132
133
Time-of-flight (µs)
Figure 16 Comparison of SNR for 11, 12 and 13 bits PRS for a reserpine sample
The choice of PRS length has a clear effect on the characteristics of the instrument. For instance,
with a fixed acquisition bin width of 100 ns a 11 bits PRS yields a maximum scan speed (full
mass range 1.5 kDa) of 4.9 kHz. For the same acquisition bin width a 13 bits PRS yields a
maximum scan speed (full mass range 24 kDa) of 1.2 kHz. Another effect is, as mentioned
before, that the SNR will increase by 2 as the PRS length is doubled.
The ESI is the most crucial step in ESI-MS, as an unstable ESI will decrease the reproducibility
of the results. In figure 17 an estimation of the sensitivity of the instrument and the stability of
the ESI is shown.
18
11 bit PRS
12 bit PRS
13 bit PRS
Regression
1e+5
800
1e+4
17 femtomoles
600
400
Counts
Signal Intensity (counts)
1e+6
200
0
1e+3
-200
-400
90
100
110
120
130
140
150
160
170
Time-of-flight (µs)
1e+1
1e+2
1e+3
1e+4
1e+5
Femtomoles electrosprayed
Figure 17 Signal intensity versus femtomoles reserpine electrosprayed
In figure 17 it can be seen that analytes in the low femtomole (10-15 mole) range can readily be
analyzed and that the ESI is very stable over time. The measurements for the different PRS
lengths where conducted on different days and the results imply that the reproducibility of the
method is very good.
By varying the multiplexing modulation bin width and the acquisition bin width for a 12 bit PRS
the investigation of the influence on the resolution was performed. The effect on the resolution
is shown in figure 18.
19
12 bit PRS
100 ns modulation bin width
100 ns acquisition bin width
12 bit PRS
40 ns modulation bin width
40 ns acquisition bin width
1e+5
7e+5
6e+5
8e+4
5e+5
6e+4
3e+5
Intensity
Intensity
4e+5
50000 summed sp ectra
10000 summed sp ectra
4e+4
50000 summed sp ectra
10000 summed sp ectra
2e+5
2e+4
1e+5
0
0
- 1e+5
129.0
129.5
130.0
130.5
131.0
- 2e+4
129.0
131.5
129.5
TOF (µs )
130.0
130.5
131.0
131.5
TOF (µS)
12 bit PRS
100 ns modulation bin width
50 ns acquisition bin width
12 bit PRS
40 ns modulation bin width
20 ns acquisition bin width
5e+5
40000
4e+5
30000
3e+5
Intensity
Intensity
20000
2e+5
50000 summed sp ectra
10000 summed sp ectra
50000 summed sp ectra
10000 summed sp ectra
10000
1e+5
0
0
- 1e+5
129.0
129.5
130.0
130.5
131.0
-10000
129.0
131.5
TOF (µs )
129.5
130.0
130.5
131.0
131.5
TOF (µs )
Figure 18 Modulation and acquisition bin width effect on resolution of a reserpine sample
Decreasing modulation bin width and acquisition bin width (faster multiplexing) yields a higher
resolution but it also induces more noise in the measurements. The highest resolution (m/∆m
FWHM at m/z 608.68) that could be achieved in this study was around 1200 amu.
A summary of how the parameters of the PRS are influencing the characteristics of the
instruments is given in table 1.
20
Table 1 Summary of multiplexing influence on instrumental characteristics
PRS Length
Time interval of
Acquisition bin
Mass
modulation (ns)
width (ns)
Resolution
Scan speed (Hz)
Maximum
at m/z=608.68
1000 scans
Maximum mass
range KDa
(S/N)
(50000 scans)
11
12
100
294
4885
5 (20)
1.5
50
514
4885
5 (28)
1.5
20
601
4885
5 (20)
1.5
100
307
2442
2 (38)
6.0
50
435
2442
2 (28)
6.0
40
777
6105
6 (10)
1.0
20
1105
6105
6 (8)
1.0
100
100
415
1221
1 (57)
24.0
50
485
1221
1 (31)
24.0
60
60
433
2035
2 (23)
8.7
30
845
2035
2 (17)
8.7
40
1232
3052
3 (9)
3.8
20
1203
3052
3 (9)
3.8
100
100
40
13
40
Conclusions
The three “weak” points of the HT-TOFMS instrument have been improved. The focus in this
report has been on the improvements made for the ESI source. Stable and reproducible ESI
performance is crucial for the analysis of liquid samples. The results shown in this report clearly
indicate that the HT-TOFMS instrument is well suited for online coupling of separation
techniques.
The possibility of tailoring the analytical HT-TOFMS performance by a correct selection of the
PRS has also been demonstrated. For n=13, high S/N, large mass range (24 kDa), and a fullmass scan speed of 1.2 kHz have been obtained. Decreasing n to 11 yielded an increased fullmass scan speed of 6 kHz, making the system highly compatible with fast transient signals, such
as those that occur in CE, µHPLC, GC, etc. The maximum full mass range resolution obtained
in this study was 1200 (m/∆m) for a mass peak at 608 amu.
One important question is how well the HT-TOFMS instrument performs in comparison to
commercially available instruments. A comparison between the HT-TOFMS instrument and a
commercially available state of the art instrument; the JAGUARTM O-TOFMS (Leco, St. Joseph,
MI USA), is given below.
21
Comparison between the HT-TOFMS and the JAGUARTM O-TOFMS
Scan speed
Depending on the PRS the HT-TOFMS has a full mass range scan speed of 1.2-6.1 kHz. The
mass range is also dependent on the PRS. For a scan speed of 1.2 kHz the full mass range is 124 000 Da and for a scan speed of 6.1 kHz the full mass range is 1-1000 Da. A number of
spectra must be summed in order to distinguish the analyte peaks. Typically 100 spectra must be
summed for the HT-TOFMS which yields a final scan speed of 12-60 Hz for the instrument.
The JAGUAR TM O-TOFMS has a full mass range scan speed of 4-5 kHz for a fixed mass range
of 1-6000 Da. The collected mass spectra are summed in a number of 50 and upward yielding a
final maximum scan speed of 100 Hz.
Resolution
The resolution for the HT-TOFMS is dependent on the modulation and acquisition bin width and
of course the instrumental setup for this specific instrument. In this study a maximum resolution
(FWHM) of 1200 was achieved for m/z 608.68. The JAGUARTM O-TOFMS has a maximum
resolution of 2000 for the same m/z.
Sensitivity
The sensitivity is solely dependent on the specific instrumental setup. For the HT-TOFMS
instrument a sensitivity of low femtomole (10-15) range was achieved. The JAGUARTM OTOFMS has sensitivity in the low attomole (10-18) range. The sensitivity is a key feature as it is
influencing the scan speed. A better sensitivity implies that a fewer number of scans must be
summed and thus an increased scan speed.
Future studies
We are currently working on the online coupling of various separation techniques to the HTTOFMS instrument. Capillary electrophoresis, Capillary electrochromatography and chip based
separation techniques are of great interest.
Another project in the pipeline is the construction of the second-generation HT-TOFMS
instruments.
Acknowledgements
I would like to thank Professor Richard N. Zare at Stanford University for giving me the
opportunity to spend six very exciting months in the Zarelab at Stanford University. I would
also like to acknowledge my colleagues in the HT-TOFMS project Ph.D. Facundo M Fenandez,
Ph.D. Jose M Vadillo and Joel R. Kimmel. It has been a true pleasure working with you.
I would like to acknowledge Professor Karin Markides and Associate Professor Jonas Bergquist
at Uppsala University for constant support and enthusiasm regarding my visit at Stanford
University.
22
A special acknowledgement to Professor Stig Hagström at Stanford University and the
Wallenberg Foundation/ The Wallenberg Research Link. Their support has been invaluable for
me during my visit at Stanford University.
I thank you all.
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