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TOF and QTOF Fundamentals
March 2007Page 1
Fundamentals of TOF and QTOF
Dr. Patrick (Pat) PerkinsR&D scientist
Agilent TechnologiesLife Sciences and Chemical AnalysisSanta Clara, CA
TOF and QTOF Fundamentals
March 2007Page 2
Agenda
Time-of-Flight (TOF) Mass Spectrometry basics
• Mass measurement
• Sources of variability in the measurement
• Minimizing the variability in the measurement
Quadrupole Time-of-Flight (QTOF) MS
• Ion optics, collision cell design and performance
TOF and QTOF Fundamentals
March 2007Page 3
Definitions Used With TOF
m/z—”mass-to-charge ratio”. The mass of an ion divided by its charge, the actual result produced by all mass spectrometers. For ions with a single charge (most small molecules), this is its isotopic molecular weight plus any adduct (proton, sodium,…). Proteins and peptide ions are in general multiply-charged, and therefore additional math (deconvolution) is used to determine their molecular weights.
oa—”orthogonal acceleration”. Ions are accelerated (“pulsed”) down the flight tube in a direction perpendicular to their entry into the TOF analyzer. This minimizes the effects of ion generation and transmission on the mass measurement.
ppm—”parts-per-million”, a measure of the error in the experimental mass assignment, compared to the known (or theoretical) value.
Resolution—a dimensionless value indicating the ability of the mass analyzer to separate (resolve) ions. Higher values mean better resolving power. Several definitions exist, but the most common is M/M, the m/z value of the ion divided by its peakwidth expressed in m/z units.
Transient—one packet of ions pulsed down the flight tube and detected. Data from many pulses, typically ~10,000, are summed to create a spectrum.
TOF and QTOF Fundamentals
March 2007Page 4
Basic Ideal TOF Mass Spectrometer
KE = ½ m v2
v = d/t
t = m½ * d/(2KE)½ constant
Ideally, to measure the mass,
Measure the time and use
m = A * t2
Practically,
t = m½ * d/(2KE)½ + t0 2 constants
m = A * (t – t0)2
Detector
V
d
Vacuum
TOF and QTOF Fundamentals
March 2007Page 5
Sources of Variability in Measurement (Deviations from Ideality)
Detector
V
d
Vacuum
???
TOF and QTOF Fundamentals
March 2007Page 6
Sources of Variability in Measurement (Deviations from Ideality)
Ions don’t all start at the same plane in space relative to flight direction
Ions aren’t sitting still and have different velocities relative to flight direction
Temperature fluctuations change flight distance
Voltage pulse is not instantaneous or of invariant shape
Ions farther from voltage pulse element(s) obtain a higher velocity
Change in temperature/humidity alter pulse electronics
Poor vacuum scatters ions
Detector and/or method of detection alters arrival times
Inaccurate method(s) of locating the maximum of the mass peaks
Detector
V
d
Vacuum
???
TOF and QTOF Fundamentals
March 2007Page 7
Minimizing the Variability in Measurement (Approximating Ideality)
• Orthogonal acceleration
• Reflectron
• Beam shaping/conditioning prior to pulsing
• Higher order focusing
• Temperature control
• Humidity control
• Analog-to-Digital Conversion (ADC) vs. Time-to-Digital Conversion (TDC)
• Calibration
• Reference mass addition
• Sophisticated algorithms to locate mass peak centroids
TOF and QTOF Fundamentals
March 2007Page 8
Agilent TOF Schematic
Pulser Detector
Vacuum ~1*10-7 Torr
Internal support structure with low coefficient of thermal expansion
1 meter flight tube, 2 meter flight path
Reflectron (ion mirror) with self-compensating thermal design
Optics for beam shaping and conditioning
Flight acceleration orthogonal to ion introduction
Turbopumps
TOF and QTOF Fundamentals
March 2007Page 9
A Time-of-Flight Scan
Pulser Detector
1. Pulse ions every 100 microseconds
2. Measure at detector each nanosecond (1 GHz)
3. 100,000 data points in each transient
4. Sum ~10000 transients into one scan (1 scan/second operation)
5. Produces spectra with excellent ion statistics
20 µsec – m/z 118
46 µsec – m/z 622
90 µsec – m/z 2421
TOF and QTOF Fundamentals
March 2007Page 10
TOF Instruments: Excellent Mass Accuracy and High Resolution Simultaneously
Advantages of High Resolution and Mass Accuracy
• Excellent mass accuracy provides a high degree of confidence in compound identification
• High resolution allows distinguishing peaks separated by only small m/z values
• High resolution allows selective detection of desired (“targeted”) compounds in the presence of interferences
TOF and QTOF Fundamentals
March 2007Page 11
Greater Resolution Does Not Necessarily Yield Better Mass Accuracy
A TOF MS at 10,000 resolution cannot resolve between an ion with one C13 isotope and one with one N15 isotope. FTMS at 100,000+ resolution can.
A TOF MS delivers < 3 ppm mass accuracy, while FTMS is often not as good under the same sample conditions
TOF and QTOF Fundamentals
March 2007Page 12
Why is Accurate Mass Useful?
Accurate masses give possible elemental compositions
1
10
100
1000
0.1 0.05 0.01 0.005 0.001 0.0005 0.0001
Error in Mass Accuracy (amu)
176
386
882
1347
1672
5687
Po
ssib
le f
orm
ula
s MW
TOF and QTOF Fundamentals
March 2007Page 13
Accurate Mass in Small Molecules
Reserpine (C33H40N2O9) has a protonated ion at 609.28066
Single quad reports mass to +/- 0.1 = 165 ppm
Number of possible formulas using only C, H, O & N:
• 165 ppm (quad) 209
• 10 ppm 13
• 5 ppm 7
• 3 ppm 4
• 2 ppm 2
Accurate mass reduces risk of investing effort on the wrong molecule (drug discovery and development)
TOF and QTOF Fundamentals
March 2007Page 14
Data Analysis – Mass Assignment
How much is 1 ppm?m/z = 118.08625
0
10000
20000
30000
40000
50000
60000
117.6 117.7 117.8 117.9 118 118.1 118.2 118.3 118.4 118.5 118.6
m/z
Ab
un
dan
ce
Note: ADC sampling rate is 1 GHz
TOF and QTOF Fundamentals
March 2007Page 15
Data Analysis – Mass Assignment
How much is 1 ppm?m/z = 118.08625
0
10000
20000
30000
40000
50000
60000
118.05 118.07 118.09 118.11 118.13 118.15
m/z
Ab
un
dan
ce
Note: ADC sampling rate is 1 GHz
TOF and QTOF Fundamentals
March 2007Page 16
How much is 1 ppm?m/z = 118.08625
0
10000
20000
30000
40000
50000
60000
118.072 118.077 118.082 118.087 118.092 118.097
m/z
Ab
un
dan
ce
Data Analysis – Mass Assignment
Note: ADC sampling rate is 1 GHz
TOF and QTOF Fundamentals
March 2007Page 17
4 GHz ADC:What Is It?
• 4 GHz ADC for TOF and QTOF based on proprietary Agilent oscilloscope technology
– Resolution improved to • 9,000 @ 118 m/z
• 14,000 @ 322 m/z
• 16,000 @ 2722 m/z
– In-spectrum dynamic range of 4.5 decades (ten-fold increase)
– Extend mass range to 20,000 m/z
– Retrofit to existing systems
FPGAMasterAgilent
TALONADC
4GSPS8-BIT
4GSPSBUS
PULSEFrontend_A
Frontend_B
CONTROLBUS
CLOCKFPGASlave
2GSPSBUSDual Gain
Front End
SIGNAL
Real Time Signal Processing and Summing Memory
(32 staggered ADCs)
FASTDATAOUT
TOF and QTOF Fundamentals
March 2007Page 18
4 GHz ADC Resolution Example: Two Compounds Nominally 195 m/z Separated By 0.036 Da
FIA, 2 scans/sec (6713 transients/scan)2 IRM averaged over five scans
Butyl paraben[M+H]+ 195.101571 m/z
Methyl 5-acetylsalicylate[M+H]+ 195.065185 m/z
O
O
OH
O
OH
OO5x10
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2
2.1
2.2
2.3
2.4
2.5
2.6+ Scan (#7) MAS_BP_1GHz_tppOFF_dgOFF_1.d
195.088679
Counts vs. Mass-to-Charge (m/z)195 195.02 195.04 195.06 195.08 195.1 195.12 195.14 195.16 195.18 195.2
4 GHz board, 1 GHz rate
6,100 FWHM resolution
5x10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
2.6
2.8
3
3.2
3.4+ Scan (#7) MAS_BP_4GHz_tppOFF_dgOFF_1.d
195.100392
195.066356
Counts vs. Mass-to-Charge (m/z)195 195.02 195.04 195.06 195.08 195.1 195.12 195.14 195.16 195.18 195.2
4 GHz 8,900 FWHM resolution
5x10
0
0.10.2
0.3
0.4
0.50.6
0.7
0.8
0.91
1.1
1.2
1.31.4
1.5
1.6
1.71.8
1.9
22.1
2.2
2.3
2.42.5
2.6
2.7
2.8
+ Scan (#7) MAS_BP_4GHz_tppON_dgOFF_2.d
195.101392
195.065349
Counts vs. Mass-to-Charge (m/z)195 195.02 195.04 195.06 195.08 195.1 195.12 195.14 195.16 195.18 195.2
Error = + 0.8 ppm
Error = - 0.9 ppm
4 GHz, transient peak picking
14,000 FWHM resolution
0.036 Da
5x10
0
0.25
0.5
0.75
1
1.25
1.5
1.75
2
2.25
2.5
+ Scan (#7) MAS_BP_1GHz_tppOFF_dgOFF_1.d
195.088679
Counts vs. Mass-to-Charge (m/z)195 195.01 195.02 195.03 195.04 195.05 195.06 195.07 195.08 195.09 195.1 195.11 195.12 195.13 195.14 195.15 195.16 195.17 195.18 195.19 195.2
4 GHz board, 1 GHz rate
6,100 FWHM resolution
5x10
0
0.25
0.5
0.75
1
1.25
1.5
1.75
2
2.25
2.5
2.75
+ Scan (#7) MAS_BP_4GHz_tppON_dgOFF_2.d
195.101392
195.065349
Counts vs. Mass-to-Charge (m/z)195 195.01 195.02 195.03 195.04 195.05 195.06 195.07 195.08 195.09 195.1 195.11 195.12 195.13 195.14 195.15 195.16 195.17 195.18 195.19 195.2
Error = + 0.8 ppmError = - 0.9 ppm
4 GHz, high resolution mode 0.036 Da
14,000 FWHM resolution
5x10
0
0.5
1
1.5
2
2.5
3
+ Scan (#7) MAS_BP_4GHz_tppOFF_dgOFF_1.d
195.100392
195.066356
Counts vs. Mass-to-Charge (m/z)195 195.01 195.02 195.03 195.04 195.05 195.06 195.07 195.08 195.09 195.1 195.11 195.12 195.13 195.14 195.15 195.16 195.17 195.18 195.19 195.2
4 GHz 8,900 FWHM resolution
TOF and QTOF Fundamentals
March 2007Page 19
Automatic Tuning and Calibration
Automated tuning done infrequently
Automated calibration done daily
TOF and QTOF Fundamentals
March 2007Page 20
View of Calibration Results
TOF and QTOF Fundamentals
March 2007Page 21
Inlets – Electrospray Ion SourceDual sprayer design for sample and mass reference compound
Reference Sprayer
Analytical Sprayer
TOF and QTOF Fundamentals
March 2007Page 22
Automated Internal Reference Mass Correction
100 200 300 400 500 600 700 800 900 1000m/z, amu
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Re
l. Int. (%
)
279.091691
579.155974
121.050925 301.073403922.010400
Reference Masses
TOF and QTOF Fundamentals
March 2007Page 23
Empirical Formula Confirmation Report
Formula input by submitter and system calculates monoisotopic mass
Extracted ion chromatogram covering specified adducts
Mass spectrum for major peak
Zoomed spectrum covering adduct range
Calculated mass error results
TOF and QTOF Fundamentals
March 2007Page 24
Expanded View of Results
TOF and QTOF Fundamentals
March 2007Page 25
TDC versus ADC
Time to Digital Conversion only measures time of arrival of first ion at a given m/z value
• More sample means more ions means earlier arrival
• Requires higher acquisition rate than ADC and peak intensity matching to accurately assign mass
• Reduced dynamic range
Analog to Digital Conversion (ADC) records time and number of ions arriving
• Sample concentration does not impact (maximum) arrival time
• Provides wider dynamic range
How much is 1 ppm?m/z = 118.08625
0
10000
20000
30000
40000
50000
60000
118.05 118.07 118.09 118.11 118.13 118.15
m/z
Abun
danc
e
TOF and QTOF Fundamentals
March 2007Page 26
Dynamic Range Example for TOF Technology: 400x Difference in Abundance
TOF and QTOF Fundamentals
March 2007Page 28
Precision of the Mass Measurement in TOF Depends on Number of Ions Sampled
TOF and QTOF Fundamentals
March 2007Page 29
High Throughput Chemical Library AnalysisAnalysis of 140 real screening compounds under ultra-fast RRLC/TOF conditions (90s cycle time 1000 samples/day):
2 outliers not shown, 16 compounds could not be ionized by ESI+
-5.00
-4.00
-3.00
-2.00
-1.00
0.00
1.00
2.00
3.00
4.00
0 20 40 60 80 100 120 140
Err
or [p
pm]
Results from automated formula confirmation report – no manual interaction!
LC: Water/ACN(0.1%TFA), 5-100%B in 0.7min, 60°C, 1.5ml/minUV-Detection: 210 – 500 nm, 80Hz MS-Detection: 120-1200Da, 8Hz, Split 1:7.5 Injection: 1µl, online sample dilution by injector program (determined the cycle time!)
TOF and QTOF Fundamentals
March 2007Page 30
High Throughput Chemical Library AnalysisMass error histogram of the analysis of 140 real screening compounds under ultra-fast LC conditions (90s cycle time 1000 samples/day):
2 outliers not shown, 16 compounds could not be ionized by ESI+
!
0
5
10
15
20
25
30-5
.0
-4.5
-4.0
-3.5
-3.0
-2.5
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
91% within ±2.0 ppm
ppm
n p
er
0.2
5p
pm
71% within ±1.0 ppm
TOF and QTOF Fundamentals
March 2007Page 31
Mass Accuracy – EnvironmentalExtremes
Injecting every 15 minutes, from 11º - 36º C and 10 - 95 %Relative Humidity
Mass Accuracy
-5-4-3-2-1012345
0 50 100 150 200 250 300
Reserpine Injection
Mas
s E
rro
r, p
pm
TOF and QTOF Fundamentals
March 2007Page 32
XIC of +TOF MS: 334.4 to 335.4 amu from Sample 18 (Blank Poultry+0.5ppb) of Nitrodfuransequence1.wiff Max. 2.0e5 cps.
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5Time, min
0.0
5000.0
1.0e4
1.5e4
2.0e4
2.5e4
3.0e4
3.5e4
4.0e4
4.5e4
5.0e4
5.5e4
6.0e4
6.5e4
6.8e4
Inte
ns
ity, c
ps
123.06
123.05
NPAMOZ at Unit Mass Resolution 335.1352 -0.7+0.3 (similar to quadrupole)
???
TOF and QTOF Fundamentals
March 2007Page 33
NPAMOZ 335.1352 +/- 0.1 (300 ppm)
XIC of +TOF MS: 335.0 to 335.2 amu from Sample 18 (Blank Poultry+0.5ppb) of Nitrodfuransequence1.wiff Max. 1.7e5 cps.
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5Time, min
0.0
2000.0
4000.0
6000.0
8000.0
1.0e4
1.2e4
1.4e4
1.6e4
1.8e4
2.0e4
2.2e4
2.4e4
2.6e4
2.8e4
3.0e4
3.2e4
3.4e4
Inte
ns
ity, c
ps
123.06
S/N = 70:1
TOF and QTOF Fundamentals
March 2007Page 34
XIC of +TOF MS: 335.1 to 335.2 amu from Sample 18 (Blank Poultry+0.5ppb) of Nitrodfuransequence1.wiff Max. 9.3e4 cps.
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5Time, min
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
6500
7000
7500
8000
8500
9000
Inte
ns
ity, c
ps
NPAMOZ 335.1352 +/- 100ppm (335.09939 - 335.16641)
S/N = 103:1
TOF and QTOF Fundamentals
March 2007Page 35
NPAMOZ 335.1352 +/- 20ppm (335.1262 - 335.1396)
XIC of +TOF MS: 335.1 to 335.1 amu from Sample 18 (Blank Poultry+0.5ppb) of Nitrodfuransequence1.wiff Max. 1.0e4 cps.
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0Time, min
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
2000
2100
2200
2300
2400
2500
2600
2700
2800
2900
3000
3100
3200
Inte
ns
ity, c
ps
123.05
S/N = 214:1
TOF and QTOF Fundamentals
March 2007Page 36
Why QTOF?
All the benefits of TOF
plus
accurate mass information on the substructure fragments produced by MS/MS
and
selectivity of accurate mass product ions
yields
even greater confidence in the confirmation of the structure
and
even better selectivity for quantitation
TOF and QTOF Fundamentals
March 2007Page 37
The analysis of impurities in Amoxicillin by Q-TOF
NH
O
HNH2 N
O
SH
CH3
CH3
H COOH
H
OH OH
NH
NH
O
O
NH
S
CH3
CH3
H COOH
1 3
1 Amoxicillin, 3 Impurity: Amoxicillin penilloic acid
3x105.5
00.250.5
0.751
1.251.5
1.752
2.252.5
2.753
3.253.5
3.754
4.254.5
4.755
5.25
Abundance vs. Mass-to-Charge (m/z)100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400
+ Product Ion (10.969 min) (366.1127[z=1] -> **) Cal10ng-10V.d
160.04243.9 ppm for MS/MS
114.0367207.0783
366.1327-0.90 ppm for MS
247.1216
Degradation of Amoxicillin
160.0432
366.1325
Confirmation of the identity of the degradation product by accurate mass measurement in MS and MS/MS mode with subsequent empirical formula calculation
Optimization of collision energy for targeted Q-TOF MRM and calibration for quantitative analysis of degradation products
TOF and QTOF Fundamentals
March 2007Page 38
Agilent QTOF Design Incorporates Cumulative Hardware Innovations
Ion opticsCommon with Q & QQQ
Flight tubeCommon with TOF
Octopole 1
DC Quad
Collision cellCommon with QQQ
Rough Pump
Turbo Turbo Turbo
Quad Mass Filter (Q1)
Collision CellLens 1 and 2
Octopole 2
Ion Pulser
Ion Mirror
Detector
Turbo
TOF and QTOF Fundamentals
March 2007Page 39
Innovations in Front-End Ion Optics Deliver Better Sensitivity Across a Broad Mass Range
10X sensitivity advantage Key components contributing to sensitivity • Dielectric capillary • Small diameter octopole ion
guide • High frequency RF octopole• Lens 2 RF (transmission of
higher masses)• Hyperbolic post-filter and
quadrupole
TOF and QTOF Fundamentals
March 2007Page 40
Innovations in Front-End Ion Optics Deliver Better Sensitivity Across a Broad Mass Range, cont.10X sensitivity advantage Lens 2 RF (transmission of higher masses) Hyperbolic post-filter and quadrupole
TOF and QTOF Fundamentals
March 2007Page 41
1st Quadrupole
Beam shaper
HEXAPOLE RODS
Entrance lens Exit lens
Beam shaper
3rd Quadrupole
Agilent 6410 Collision Cell Position
TOF and QTOF Fundamentals
March 2007Page 42
Collision Cell Design and Electrical Drive
Acceleration Potential
to 10 V
RF Voltage = 10-550 v
•Hexapole construction
•2R0 = 4.5 mm
•Length = 150 mm
•Experimental press. range = 0.1 – 20mTorr
•In coming ion energy range = 0 – 250eV
•Accelerating potential range = 0 – 10V
•RF Drive voltage range 10 – 550v
TOF and QTOF Fundamentals
March 2007Page 43
Transmission Velocity (Latency) Results
Simulation Studies
• 3-D ion optics modeling (100-600sec)
Experimental Results
• Fast precursor ion selection (SRM)
• Linked scan latency
• Gated ion beam measurements
TOF and QTOF Fundamentals
March 2007Page 44
3x10
00.2
0.40.60.8
11.21.41.6
1.82
2.2
2.42.62.8
Abundance vs. Acquisition Time (Min)0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9
+EIC (281.0 m/z) MRM (309.0 m/z); from alprazolam 100 A Dwell with 200 pg OC -101505-28V_2uL.d
7.9291.396 6.3004.6653.0301 1
Dwell time evaluation 200 pg Alprazolam 100, 20, 5 ms dwell times
3x10
00.2
0.40.60.8
11.2
1.41.61.8
22.22.4
2.62.8
Abundance vs. Acquisition Time (Min)0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9
+EIC (281.0 m/z) MRM (309.0 m/z); from alprazolam 20 A Dwell with 200 pg OC -101605-28V_2uL.d
7.9163.025 4.6581.396 6.2981 1
3x10
0
0.25
0.5
0.75
1
1.25
1.5
1.75
2
2.25
2.5
2.75
3
Abundance vs. Acquisition Time (Min)0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9
+EIC (281.0 m/z) MRM (309.0 m/z); from alprazolam 5 A Dwell with 200 pg OC -101605-28V_2uL.d
7.9306.2894.664
3.0411.419
1 1
3x10
0
0.20.4
0.60.8
11.2
1.41.6
1.8
22.2
2.42.6
2.8
Abundance vs. Acquisition Time (Min)1.18 1.2 1.22 1.24 1.26 1.28 1.3 1.32 1.34 1.36 1.38 1.4 1.42 1.44 1.46 1.48 1.5 1.52 1.54 1.56 1.58 1.6 1.62 1.64 1.66 1.68
+EIC (281.0 m/z) MRM (309.0 m/z); from alprazolam 5 A Dwell with 200 pg OC -101605-28V_2uL.d
1.4191 1
Dwell 100 ms 20 ms 5 ms
Area 14860 13605 13202% RSD 0.61 0.74 2.25
5 ms dwell
TOF and QTOF Fundamentals
March 2007Page 45
Beam Turn-off Characteristics
10
100
1000
10000
100000
-500 0 500 1000 1500 2000
microseconds
Arb
. Un
its .
mz922 mz118
Collision Cell Clearing Profile
0 V collision energy 5 V Applied Axial Potential
600 sec
350 sec
TOF and QTOF Fundamentals
March 2007Page 46
Broad Mass Range Transmission and Transmission Efficiency – Why Hexapole?
Mass Range Transmission
0
0.2
0.4
0.6
0.8
1
1.2
0 500 1000 1500Mass (m/z)
Tran
smis
sion
.
Quadrupole Hexapole Octopole
Variables include:
•Number of poles (i.e. quad, hex, octo)
•Inscribed diameter (R0)
•Drive Frequency
Evaluation included:
•Theoretical modeling
– Calculation, Simulations
•Experimental results
TOF and QTOF Fundamentals
March 2007Page 47
Full Mass Range With Single Parameter Set on QTOF (m/z 100 – 3000), No Switching
5x10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
2.6
2.8
3
3.2
3.4
3.6
3.8
4
Abundance vs. Mass-to-Charge (m/z)0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000
+MS (0.317-0.384 min) TTI_after calib_IRM_MS2_1Hz.d (BP=436212) #2
391.28380
1221.99081
622.02836922.00964
223.20489
1521.97242
1821.95480
2121.93491
2421.91573
2721.89678
TOF and QTOF Fundamentals
March 2007Page 48
Mass Accuracy in MS/MS ModeMS Calibration is Maintained over Broad Collision Energy
3x10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
Abundance vs. Mass-to-Charge (m/z)0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400
+MS (0.075-0.249 min) MS/MS(2121.93311 m/z) MS2_on_IRM_2HZ.d (BP=1598) #6
229.92086
2121.93167
1477.927061789.92031
1165.92259853.92438541.92207
4.8
pp
m
1.3
pp
m
0.9
p
pm
1.6
pp
m 1.2
pp
m
3.2
pp
m
0.7
pp
mCE=150.5 eV @ 10 mTorr
3.4
pp
m
5.1
pp
m
3.8
pp
m
1.6
pp
m
3x10
0
1
2
3
4
5
6
7
Abundance vs. Mass-to-Charge (m/z)50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050
+MS (0.101-0.275 min) MS/MS(922.00983 m/z) MS2_on_IRM_2HZ.d (BP=7578) #6
922.00753789.98942
677.97608
229.92169 321.92867565.96291
453.94855
175.98726
1.2
pp
m
2.1
pp
m
1.4
pp
m
0.2
pp
m
0.4 ppm
0.7 ppm 2.5 ppmCE=66.5 eV @ 10 mTorr
2.6
pp
m 2.3
pp
m
2.6
pp
m
2.0
pp
m
TOF and QTOF Fundamentals
March 2007Page 49
Internal Reference Mass Correction
Fundamentals: The header of each acquired spectrum has reference mass corrected “a” and “t0” coefficients. They are calculated based on the presence of user selectable ions in each MS spectrum. Running averaging is supported.
Extensions to MS/MS operation:
MS/MS (DDE): Survey scan coefficients are copied into the headers of the subsequent MS/MS spectra.
MS/MS (Manual/Targeted): MS scans are auto inserted into the specified MS/MS spectra acquisition cycle. The updated coefficients of these “background” generate MS spectra are copied into the headers of the subsequent MS/MS spectra.
TOF and QTOF Fundamentals
March 2007Page 50
0 2 4 6 8 10 12 14 160
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
550016.27
13.83
9.20 11.43
6.994.95
2.84
STD ESI: 0.25 mL/min flow Q1=256 (2.5 wide), CE=10v, 167 (0.1 wide) EIC
60 80 100 120 140 160 180 200 220 240 2600
50
100
150
200
250
300
350
400
450
499 167.0805
257.1350
168.0836179.0656
71.0826 117.0875 152.0602
MS/MS spectrum at 1 pg. Background subtracted
200 fg500 fg
1000 fg 2000 fg
Dilution Series of Diphenhydramine on Q-TOF
TOF and QTOF Fundamentals
March 2007Page 51
Benchmarking - Small Molecule Sensitivity200 pg MDMA (3,4-methylenedioxymethamphetamine)
0.2 0.6 1.0 1.4 1.8Time, min
0
5.0e4
1.0e5
1.5e5
2.0e5
2.5e5
3.0e5
3.5e5
4.0e5
4.5e5
5.0e5
0.68
S/N = 230
! !
Agilent QTOF Breadboard
0.5 1.0 1.5 2.0 2.5 3.0Time, min
0
50
100
150
200
250
300
350
400
1.61
S/N = 10
Other QTOF