quadrupole ion-trap mass analyzer eas workshop november 15, 2004
TRANSCRIPT
Quadrupole Ion-Trap Mass AnalyzerEAS Workshop
November 15, 2004
Jerry PappasThermo Electron
Finnigan Life Sciences Mass Spectrometry265 Davidson AvenueSomerset, NJ 08873
E-mail: [email protected]
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Schematic of an Ion Trap Schematic of an Ion Trap
1st RF OnlyQuad
2nd RF Only Quad
Ring Electrode
Endcaps
3
LCQ 3LCQ 3--D Ion TrapsD Ion Traps
4
Basic Ion Trap ComponentsBasic Ion Trap Components
rozo
E n tranceE ndcap
E x itE ndcap
R ingE lec trode
)cos( tV resres Ω
)cos( tV rfrf Ω
-
IonIn jec tion
IonE jection
+
Schematically, the layoutof an ion-trap resemblesa slice through a quadrupole
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Helium as a Damping (Buffer) GasHelium as a Damping (Buffer) Gas
+
++
+Without HeliumWithout Helium
He
collision He
He
He
He
+
++
+
With HeliumWith Helium
6
Helium as a Damping GasHelium as a Damping Gas
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Purposes of Buffer GasPurposes of Buffer Gas
• Trap injected ions by removing KE• Damps ions to center of ion trap• Collision gas for MS/MS
Results...
Increase sensitivityIncrease resolution
i.e. resolution is improved by giving narrower and larger peaks
Increase fragmentation efficiency
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Ion Stability in the TrapIon Stability in the Trap
eUm
q eV
mr zz
= -
= -+
16
82
2
0
2
0
2 2
r z+20
2
0
2( )Ω
( )Ω
zaa = variable solution
q =solution
e= charge of trapped ion
U=DC Voltage
V=RF amplitude
m = mass of ion
Ω = constant summarizing mathematics
z0 = distance between centre of trap to either endcap
R0 = internal radius of ring electrode
Controlled by a culmination of differential equations Controlled by a culmination of differential equations termedtermed Reduced Mathieu Equations:Reduced Mathieu Equations:
DC on the Ring
RF on the Ring
Mass Ring Parameters
9
Excel Trapping Field Animation
10
Ion Trap Stability DiagramIon Trap Stability DiagramThe region shaded blue indi-cates a (DC) and q (RF) values which provide stable trajectories in the r-direction
The region shaded yellow indicates the z-stable a and q combinations
The green area where the r-and z-stable regions overlap indicates the a and q combi-nations under which ions will be stable in the trap
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Ion Stability DiagramIon Stability Diagram
0 .0 0 .2 0 .4 0 .6 0 .8 1 .0 1 .2 1 .4q z
-0 .8
-0 .6
-0 .4
-0 .2
0 .0
0 .2
a z
β r
β z
0 .1 0 .2 0 .30 .4
0 .5 0 .60 .7
0 .81 .0 0.
0 .10 .2
0 .3
0 .4
0 .5
0 .6
0 .7
0 .80 .9
1 .0a eU
m r z
q eVm r z
z
z
= −+
= −+
1 62
82
02
02 2
02
02 2
( )
( )
Ω
Ω
0
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Mass Selective InstabilityMass Selective Instability
“There are two landmarks in [the history of Quadrupole Ion Traps].
The first is the invention of the ion trap [in 1953] by Wolfgang Paul and Hans Steinwedel, which was recognized by the award of the 1989 Nobel Prize in Physics . The second is the discovery, announced in 1983, of the mass selective instability scan by George C. Stafford, Jr. On these two landmarks rests the entire field of ion trap mass spectrometry.”
Raymond March, John F. J. Todd in the preface of
Practical Aspects of Ion Trap Mass Spectrometry
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Mass Selective InstabilityMass Selective Instability
q k Vm ez = ( / )
.908qz axis
“Ramp RF, Ions Leave Low m/z to High m/z”
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Automatic Gain Control (AGC)Automatic Gain Control (AGC)
Prescan before the analytical scan
•Measures the # of ions in the trap for a pre-defined time (10 – 30 ms)
•Allows software to determine optimumion injection time
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What is AGC and Why Is it Important?What is AGC and Why Is it Important?
Camera AE
• Too much light degrades the image stored on film, causing a loss of color and image resolution.
• Too little light results in dark picture with no fine details visible.
• Cameras with high quality light meters and AE controls produce high quality pictures over a wide dynamic range of lighting conditions.
LCQ/LTQ Series AGC
• Controls amount of ions (light) entering the ion trap (film)
• Too many ions degrade the spectral quality in the trap, causing loss in mass resolution and mass assignment. Too few ions result in poor sensitivity to low level or minor components.
• AGC ensures excellent quality MS, SIM and MS/MS spectra, as well as excellent sensitivity over a wide dynamic range.
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AGC (Ion Population Control)AGC (Ion Population Control)
530m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
524.3
525.3
526.3
530m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
524.4
525.4
526.3527.5
530m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
524.5
525.5
526.5527.5
530m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
524.8
525.7
526.7
522 522 522 522
~ 300 Ions ~ 1500 Ions ~ 3000 Ions ~ 6000 Ions
Poor ResolutionGood Resolution
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Major Strengths of Ion Trap Mass Spectrometers that make them Valuable Tools for Various
ExperimentsFull-Scan MSn Sensitivity
Resonant Step-Wise Excitation
“Universal” Excitation Conditions
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Full-Scan MSn Sensitivity
M1
M2
M3RF
3D Ion Trap: RF voltage across the ring electrode produces a quadruple field that maintains the ions over the mass range of interest in stable trajectories within the trap. As one ion mass is being scanned out of the trap to the detector, the remaining ions remain stably trapped. Excellent full-scan MSn sensitivity.
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Full-Scan MSn Sensitivity (continued)
Quadrupole: A combination of RF and DC voltages across the rods produces a stable trajectory for only one ion at a time. As one ion mass is being scanned out to the detector, the remaining ion masses are lost. Decreased full-scan MS and MS2 sensitivity.
M1
M2
M3
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Resonant Step-Wise ExcitationTriple Quadrupole Collision Cell
P F1
F1
F1
F2
F2
Acceleration Voltage
F3
Depending on the collision energy and collision pressure, one can get a combination of MS2, MS3…MSn
occurring simultaneously. This can make it difficult to trace a fragment back to its parent which complicates spectral interpretations.
argon
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Unique Features of Ion Traps…Unique Features of Ion Traps…
Mass separation in time – Isolation of the ions of interest and subsequent dissociation (MS/MS) are done in the same “chamber”.
Also…Ion Trap (resonant excitation) : Excitation energy is in resonance with only one mass at a time. Fragments, once formed, can not be further excited unless they are purposely selected for next stage of MS. Allows one to take apart a molecule in a controlled, step-wise fashion.
But…
Triple Quad (nonresonant excitation) : Acceleration voltage applied equally to all masses. Get a mix of ms2, ms3…msn
products.
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“Universal” Excitation ConditionsTriple Quadrupole Mass
SpectrometerIon Trap Mass Spectrometer
Inte
nsity
of F
ragm
ent X
Inte
nsity
of F
ragm
ent X
Less variability between compounds in an ion trap mass spectrometer
Cmpd B
NCE ~ 30% Cmpd A
Cmpd A
Cmpd B
Collision EnergyCollision Energy
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Other Interesting Aspects of Ion Traps…Other Interesting Aspects of Ion Traps…
Data Dependent Scanning
A (1, 2)B (1, 2)
C (1)Threshold
1. Full Scan MS∗
2. Full Scan MS2
*
* ion selected
Intensity
Time
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Dynamic Exclusion Dynamic Exclusion -- MS and MS/MS ofMS and MS/MS of CoelutersCoeluters
407
407
452
365
206
Threshold
MS/MS
MS
Time
407
407
452
206
MS
MS/MSMS/MS
MS MS/MS377
MSMS
452 MSMS231
m/z
MS255 377
m/z
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Turning it up a notch!Turning it up a notch!
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The Finnigan LTQ The Finnigan LTQ –– Linear Ion TrapLinear Ion Trap
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LTQ LTQ –– SchematicSchematic
400 L/sec300 L/sec
Detector1
Source
Tube Lens
Skimmer
SquareQuadrupole Octopole
BackLens
BackSection
FrontSection
Ion TransferTube 3x10-3
Torr He
Inter-multipole Lens 1
CenterSection
Detector2
FrontLens
Quadrupole
8.3 L/sec 25 L/sec
Ion TransferTube
Square
Tube Lens
Electrospray
Split Gate Lens
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Basic Linear Trap StructureBasic Linear Trap Structure
X
ZFront
Section
CenterSection
BackSection
12 mm
37 mm
12 mm
Slot: 30 mm x .25 mm
R0 = 4 mmHyperbolic Rods
X0 > Y0
Y
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LTQ Vacuum Chamber – Linear Trap and Dynodes
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2D Ion-Trap Animation
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How Does the LTQ Save Time?
• Improved Cycle Time– More mass spectra can be acquired across narrow chromatographic peaks
• Increased information content– Simultaneous acquisition of MS2 spectra from co-eluting metabolites– Acquisition of MS2 spectra from low- level metabolites in complicated
matrices• Increased confidence in structural assignments based on spectral features
– Multiple spectra can be compared for reproducibility
• Improved Sensitivity– Lower levels of metabolite detected
• Less sample preparation required• Less re-running of samples required
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Improved Cycle Time of the LTQ
3 seconds
TSQ MS MS2 MS2
LCQ MS MS2 MS2 MS2
LTQ MS MS2 MS2 MS2 MS2 MS2 MS2 MS2 MS2 MS2 MS2
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Test System• Buspirone incubated with microsomes from male Spraque-Dawley
rats– Buspirone was incubated at concentrations of 0.1 uM, 1.0 uM, and 10 uM– Time points at 0 min and 60 min were acquired for each concentration– Samples were diluted in half with acetonitrile at the appropriate time to
quench the reactions– Samples were centrifuged to precipitate the proteins– 10 µL of supernatant from each sample was injected without any
preconcentration
N
O
O
N NN
N
Buspirone (MH+: 386)
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Improved Sensitivity of the LTQ
100 150 200 250 300 350 400m/z
0
20
40
60
80
1000
20
40
60
80
100
Rel
ativ
e A
bund
ance
0
20
40
60
80
100122
281150238
359123 148 307282239210151 219
122
281150 238
359123 282 307148 239151 219210
122
281150 238
298 344 385210 359200139 308243122
NL: 9.13E4buspirone_4_12_2004_35#1709 RT: 10.60 AV: 1 SB: 37 10.94-11.94 , 9.00-10.28 F: ITMS + c ESI sid=15.00 d Full ms2 [email protected] [ 100.00-415.00]
NL: 7.00E3buspirone_4_12_2004_33#1673 RT: 10.56 AV: 1 SB: 27 10.92-11.89 , 9.62-10.23 F: ITMS + c ESI sid=15.00 d Full ms2 [email protected] [ 100.00-415.00]
NL: 6.52E2buspirone_4_12_2004_31#1661 RT: 10.58 AV: 1 SB: 32 11.00-12.00 , 9.59-10.28 F: ITMS + c ESI sid=15.00 d Full ms2 [email protected] [ 100.00-415.00]
10 uM
1.0 uM
0.1 uM
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Using Higher Order MSn Spectra to Postulate Metabolite Structures
MS
MS3
MS2
MS5
MS4
Instrumentcontinuously cycles
between these 9 scan events
MS3
MS2
MS5
MS4
1st most intense ion
2nd most intense ion
1st most intense fragment
1st most intense fragment
1st most intense fragment
1st most intense fragment
1st most intense fragment
1st most intense fragment
This scanning sequence is designed to obtain MS5 data on a smaller number of components. It is useful for when an initial MS2 screen has been performed that limits the
number of possible metabolites that need to be monitored to those on a parent mass list.
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MSn Peak Assignments for Buspirone/HydroxyMetabolite
N
O
O
N
NNN
122/122
265/281
222/238
123/139
152/168
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Potential Drawbacks of Ion Trap Mass Spectrometers
that the LTQ was Designed to Overcome
Limited Storage Capacity
Inefficient Trapping
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Storage Capacity
• There is a maximum number of ions that can be stored within an ion trap at any one time– When filled beyond this maximum, “space charging” will occur
• “Space charging” can degrade spectral resolution, mass accuracy, and sensitivity
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3D-Trap: Ions need to be confined to a small volume at center of trap for optimum performance
Ring Electrode
Storage Capacity (continued)
2D-Trap: Ions can spread out in the axial dimension without degrading performance
The ion storage capacity of the 2D-trap is more than 20x greater than that of the 3D-trap
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Trapping Efficiency in a 3D-Trap
++
-
-A positively charged ion is repelled from the trap by a
positive electrical field
A 3D RF field exists which is unfavorable for ion trapping except for during a narrow
phase window
Ring Electrode
Ion Exit
Ion Entrance
+
-A positively charged ion is
pulled through the trap by a negative electrical field
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Trapping Efficiency in the LTQ 2D-Trap
2D Trap
In the LTQ 2D trap, the RF field is perpendicular to the ion entrance axis which reduces the problems associated
with ion injection in the 3D trap. This results in a 10-15x improvement in trapping efficiencies
3D Trap
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Summary Summary –– Analytical ImprovementsAnalytical Improvements• Increased Trapping Efficiency (55-75%)• Increased Trapping Capacity (~20,000 ions)• Increased Sensitivity (Dual detectors-2x better detection efficiency).
Increased Dynamic Range (4-5 orders of linear Dynamic range)Increased S/N for full Scan MSPractical MSn experimentsFaster Scan times (16,700 amu/sec – Normal Mode)Resolution (FWHM) 30,000 (m/z 1522) at 27 amu/sec Method for DD Neutral loss scanning capability
Which means....
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Platform for Hybridization (FT/MS)Platform for Hybridization (FT/MS)
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Questions?