LC/MS Crossroads
Jerry Pappas
Sales Representative
265 Davidson Avenue
Somerset, NJ 08873
[email protected]
732-698-2778
1
Comparison of Quads and Traps
Ion Traps
Quadrupoles
Mass Separation in time
Mass Separation in space
High sensitivity Full Scan
Lower sensitivity Full Scan
Lower sensitivity SIM and SRM
High sensitivity SIM and SRM
Offer multiple stages of MSn
Offers Only MS or MS/MS
Parent and neutral loss scans
2
LCQ Instrumentation
Classic
Duo
3
Deca
Duo/Deca Comparisons
LCQ DUO
LCQ DECA
• 400um capillary
• 500um capillary
• 2 octopoles
• 1 square quadrupole & 1 octopole
•One rotary pump
•2 rotary pumps
Deca: approximately 10 x better signal than Duo
4
Current LCQ Generation
5
Advantage/XP Comparisons
• 450um Ion Transfer Tube Ç
• 550um Ion Transfer Tube Ç
•Orthogonal Probes
•Orthogonal Probes
XP: approximately 10 x better signal than Advantage
6
LCQ — MSn Quadrupole Ion Trap
LC Pump
ESI
Quadrupole Ion Trap
Ion optics
Detector
Syringe Pump
Quadrupole refers to the shape of the ion confining field
inside the trap and not the shape of or number of electrodes
in the trap.
7
Mass Spectrometry Simplified
Generate
Move
S elect
D etect
8
Ion production
Ion optics
Mass filter
Electron multiplier
The Mass Spectrum
Red
Light,
all colors
Green
Prism
Blue
100
Ions,
various masses
Mass
Spectrometer
200
300
9
Trace produced by summing all observed
masses in each scan
Total Ion Chromatogram or TIC
10
Ionization vs. Fragmentation
API
Soft
No
Fragments
11
CI
Ionization
EI
Hard
Fragments
EI and CI Mass Spectra of Ephedrine
58
100
% Relative Intensity
EI
50
0
0
100
12
40
60
80
100
120
CI
20
140
160
180
148 (M+H)+
166
50
0 0
MW = 165Th
20
40
60
80
100
m/z
120
140
160
180
Ionization Techniques
200,000
ESI
15,000
1,000
Molecular
Weight
APCI
TSP
GC
Non Polar
13
FAB
PBI
Polar
What is API?
Atmospheric Pressure Ionization
Source Types:
1.
Electrospray Ionisation (ES) – Solution phase process (for
the most part).
2.
APCI (Atmospheric Pressure Chemical Ionization) - Gasphase process.
Source Purpose:
14
1.
Ionize the analyte (APCI) or transport ion in solution to the
gas phase.
2.
Desolvate sample flow for introduction into mass
spectrometer.
3.
Baffle the first vacuum region of the MS from atmospheric
pressure in the source.
4.
Pump away neutrals and opposite charged ions which would
otherwise interfere with the analysis of the desired polarity.
LCQ Classic/Duo/Deca API Probes
Electrospray Ionization (ESI)
Peek insulator
Atmospheric Pressure
Chemical Interface (APCI)
15
LCQ Advantage/XP API Probes
Electrospray Ionization (ESI)
Atmospheric Pressure
Chemical Interface (APCI)
Orthogonal ESI & APCI probes
16
Chemistry Considerations
ESI:
Ions formed by solution chemistry
Good for Thermally labile analytes
Good for Polar analytes
Good for Large Molecules (Proteins / Peptides)
APCI:
Ions formed by gas phase chemistry
Good for Volatile / Thermally Stable
Good for Non-polar analytes
Good for Small Molecules (Steroids)
17
ESI Versatility Advantage/XP
(Right: Picture represents
a low flow position,
<50uL/min)
A-F Positions for
increased ruggedness
1-5
Positions
(Left: Picture represents a
high flow position)
18
Electrospray—Basic Layout
Heated Capillary
ESI Needle
Solvent evaporation and ion release
+/- 5 kV
+
+
+
+
+
+
+
+
+
+
+
++
+
+
++
+
+
++
++
++
+
+
++
++
++
+
+
+
+ +
+
++
++ ++
++ +
+
Taylor Cone
19
+ ++
+
+
+
+
+
+
+
++
+
++
++
++
+ ++
+
+
+
+
+
+ ++
+
+
+
+
+
+++
+
+
+
+
+
+
+
+
+
+
+
+
+
APCI Position on Advantage/XP
In/Out Movable
Corona Discharge Pin
20
Total control for any
flow rate (200ul/min 2000ul/min)
LC Flow Rates
ESI:
3 µL/min - 1mL/minute
Optimal Flow Rate: 200 µL/min
Generally, higher flow rates require higher heated
capillary temperatures and higher gas flow rates.
APCI:
200 µL/min - 2mL/minute.
Optimal Flow Rate: 500 µL/min
Generally, higher flow rates require more sheath and
auxiliary gas, but do not require higher heated
capillary temperatures.
21
Theoretical Increase in Response
2
Conc max = D Column 1
Col. Diameter mm 4.6
3.0
2.0
1.0
Flow Rate ml/min 1
0.5
0.2
0.05
1
2.3
5
Theoretical
Increase
22
D
21
2
Column 2
Capillary
< 10 µl / min
LC Additives
¾Acids
Do not use inorganic acids (may cause source corrosion)
Formic and acetic acid are recommended
¾Bases
Do not use alkali metal bases (may cause source corrosion)
Ammonium hydroxide is recommended
¾Surfactants (surface active agents)
Detergents and other surface active agents may suppress
ionization
¾Trifluoroacetic Acid (TFA)
May enhance chromatographic resolution, but causes ion
suppression in both negative and positive ion mode
¾Isopropyl Alcohol
May Enhance Negative Ion Formation
23
Buffers (pH)
Avoid using non-volatile HPLC additives such as:
¾
Alkali Metal Phosphates
¾
Borates
¾
Citrates
Keep Buffer concentrations below 20 mM using volatile
salts such as ammonium acetate.
When using buffers, more frequent cleaning of the heated
capillary and API stack will be necessary
24
LC/MS Additives and Buffers Summary
Acetic Acid
Formic Acid
Ammonium Hydroxide
Ammonia Solutions
Trichloroacetic Acid (< 0.1% v/v)
Trifluoroacetic Acid (< 0.1% v/v)
Isopropyl Alcohol
(10% of organic phase)
Ammonium Acetate
Ammonium Formate
25
Proton Donors
Proton Acceptors
Chromatographic
Separation
Negative ion
formation
Buffers
Common LC/MS Solvents
Methanol
Acetonitrile
Water
Isopropanol
Dichloromethane
Chloroform
Hexane
26
Effects of Solvents and Additives on ESI
50/50 ACN/H2O 0.1% NH4OH
50/50 MeOH/H2O 0.1% NH4OH
50/50 ACN/H2O 0.02% TFA
50/50 ACN/H2O 0.05% TFA
50/50 ACN/H2O 0.1% TFA
50/50 MeOH/H2O 0.02% TFA
50/50 MeOH/H2O 0.05% TFA
50/50 MeOH/H2O 0.1% TFA
50/50 MeOH/H2O 10mM NH4OAc
50/50 MeOH/H2O 5mM NH4OAc
50/50 ACN/H2O 0.1% Formic
50/50 ACN/H2O 1% Acetic
50/50 MeOH/H2O 0.1% Formic
50/50 MeOH/H2O 1% Acetic
100 ACN
100 MeOH
100 H2O
50/50 ACN/H2O
50/50 MeOH/H2O
Solvent System
Tyr-Gly-Gly-Phe-Leu
Leucine Enkephalin
0
100000
200000
300000
400000
Counts (protonated ion species)
27
500000
API Stack
LCQClassic, LCQDUO,
LCQDECA
28
LCQ DECAXP,
LCQAdvantage
Ion Transfer Tube and Removal Tool
Ion transfer tube
Removal
tool
Heated capillary
29
Vent Prevent Mechanism
Heated Tube
in-situ
Heated Tube
removed
Tungsten Vent
Prevent
30
Ion Optics
Intermultipole
Lens
First
multipole
Lens
IONS
IN
IONS
OUT
Octapole
Mount
Vacuum
Baffle
31
Second
multipole
Lens
Analyzer
Mount
Multipole Potential Wells
Mass Range
Trans Efficiency
Octapole
Square
Quadrupole
Round
Quadrupole
32
Mass Analyzer (Ion Trap)
33
Vacuum System
Every mass analyzer must operate under vacuum in
order to minimize both ion/molecule and
molecule/ molecule collisions.
At atmospheric pressure, the mean free path of a typical
ion is only ca. 52 nm and at 1 mTorr, it is 40 m.
Without vacuum, the ions produced in the source won’t
make it to the detector.
The LCQ vacuum is maintained by a both rotary and
turbomolecular pumps
34
Ion Optics (Operating Pressures)
760 torr
1.3 torr
1.7x10-3 torr
2.0 x10-5 torr (1.0x10-5 torr He)
3.5x10-3 torr He
60 m3/hr 100 L/sec
35
220 L/sec
Steps to Ion Trap Scan Functions
Trapping- all scans
Isolation- SIM and MSn
Excitation- MSn
Ejection- all scans
36
Helium as a Damping Gas
Without Helium
+
With Helium
+
+
He
collision
+
37
+
+
He
He
He
He
+
+
Discovery of the Effects of Helium
38
Helium as a Damping Gas
39
Ion Trap Resolution–Effect of Damping Gas
ŠTraps injected ions by removing kinetic energy
ŠDamps ion motion to center of trap
– Result...
ŠIncrease in resolution and sensitivity
40
Helium Effect
Helium flowing into trap
S#:1 RT:0.00 AV:1 SM:7G NL:2.50E7
T:+ p Full ms
S#:23-32 RT:0.71-1.00AV:10 SM:7G NL:5.61E7
T:+ p Full ms
524.3
1522.04
100
Relative Abundance
Relative Abundance
100
80
60
60
40
524.26
40
525.3
20
1721.89
1222.14
1821.95
1122.21
20 195.15
0
1621.97
1322.06
80
1921.88
1022.09
0
514
516
518
520
522
m/z
524
526
528
500
1000
m/z
1500
2000
Helium shut off and not flowing into trap
Relative Abundance
100
S#:23-32 RT:0.39-0.54AV:10 SM:7G NL:2.80E7
T:+ p Full ms
522.6 523.0
100
Relative Abundance
S#:1 RT:0.02 AV:1 SM:7G NL:9.70E6
T:+ p Full ms
80
80
521.8
60
521.2
40
41
514
516
518
520
522
m/z
524
1220.75
40
20
526
528
0
1720.44
1320.95
60
523.9
520.7
20
0
1620.79
1520.26
523.01
1919.96
1120.90
192.17
500
1000
m/z
1500
2000
Ion Trap Stability Diagram
The region shaded blue indicates 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 rand z-stable regions overlap
indicates the a and q combinations under which ions will
be stable in the trap
42
Stability Diagram for Commercial Traps
V
qz = k
( m / e)
43
LCQ Scan-Out (Ejection) Rates
Normal Scan (5500 amu/sec)
Common full, SIM, or MSn (SRM and CRM)
scanning
Resolution (FWHM) = 0.50, Mass Accuracy = ±
0.05
Zoom Scan (280 amu/sec)
Increases resolution and mass accuracy across a
narrow range (allows charge state determination)
Resolution (FWHM) = 0.15, Mass Accuracy = ±
0.02
Turbo Scan (55,000 amu/sec)
Decreases total scan time of a full scan, thus
increasing number of scans across a
chromatographic peak
Resolution (FWHM) = 3.0, Mass Accuracy = ± 0.5
Used for better quantitation due to an increase
of scans across a chromatographic peak
44
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.
45
LCQ 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.
Automatic Gain Control (AGC)
Prescan before the analytical scan
- Measures the # of ions in the trap for a
pre-defined time (10 ms)
-Allows software to determine optimum
ion injection time
46
No AGC Spectrum of Ultramark 1621, Caffeine,
MRFA Calibration Mixture – space charging
47
Spectrum of Ultramark 1621, Caffeine, MRFA
Calibration Mixture with AGC
48
AGC (Ion Population Control)
100
80
60
40
525.3
20
524.4
0
522
60
40
525.4
20
526.3
527.5
0
m/z
530
522
Good Resolution
49
100
80
526.3
m/z
~ 3000 Ions
530
524.5
100
80
60
40
525.5
20
0
522
526.5
527.5
m/z
~ 6000 Ions
530
Relative Abundance
524.3
Relative Abundance
Relative Abundance
100
~ 1500 Ions
Relative Abundance
~ 300 Ions
524.8
80
60
525.7
40
526.7
20
0
522
m/z
530
Poor Resolution
Calculation of Ion Time
Constant During Prescan
AGC Prescan Signal =
Number of Ions x Multiplier Gain x Prescan Time
(3 x 105 counts)
(10 ms)
Calculated Ion Time =
(how long the gate lens is “open”)
50
Target Value
AGC Prescan Signal
Unscaled TIC (counts)
Triplicate Injection of 5 nmol of MRFA (AGC ON)
2.0E+08
1.8E+08
1.6E+08
1.4E+08
1.2E+08
1.0E+08
8.0E+07
6.0E+07
4.0E+07
2.0E+07
0.0E+00
Unscaled TIC
0
100
200
300
400
500
600
400
500
600
400
500
600
Injection Time (ms)
Scan Number
60
Injection Time
50
40
30
20
10
0
Scaled TIC (counts)
0
8.0E+08
7.0E+08
6.0E+08
5.0E+08
4.0E+08
3.0E+08
2.0E+08
1.0E+08
0.0E+00
200
300
Scan Number
Scaled TIC
0
51
100
100
200
300
Scan Number
Isolation of Ions
Ion we wish to isolate
qz
0.0
0.908
• Ions at different qz values oscillate at
different frequencies (ωo)
7
52
ωo ≈
q zΩ
2 2
Isolation Waveforms
~ m/z 200
q axis
.908
500 Hz
16 msec
q axis
53
.908
Why MS/MS or MSn ?
Intensity
Signal to Noise Improvement
Signal
Noise
S/N
0
1
2
3
4
Stages of Analysis
54
5
6
MS/MS Parameters
• Precursor ion m/z
• Excitation qz
Intensity
• Excitation voltage
Precursor ion
Σ(Product ions)
0.0
1.0
2.0
3.0
Excitation Voltage (V)
55
4.0
77001-1285
970219
Resonant Excitation qz Value
Fragment ions
not trapped
qz
0.0
0.908
0.225
Product Ion
m/z Range
1/4
qz
1/3
0.0
0.30
0.908
qz
1/2
0.0
56
0.45
0.908
Fragmentation
Energy
Ion Trap Scan Functions
57
1. Collect
3. Fragment
2. Isolate
4. Eject
Summary (Ion Trap Functions)
1) Collection
2) Isolation
3) Excitation
4) Ejection
58
For Scans: All
By: Ring Electrode
Method: Alternating RF
frequency (760 kHz) at a set
amplitude along with He
dampening gas traps and
cools the ions to the center of
the trap.
Summary (Ion Trap Functions)
1) Trapping
2) Isolation
3) Excitation
4) Ejection
59
For Scans: SIM, MSn
By: Endcap Electrodes
Method: a) Tailored waveform
applied to all ions in the trap
except ion of interest
b) Thus, only ions of interest
remain in the trap.
Summary (Ion Trap Functions)
1) Trapping
2) Isolation
3) Excitation
4) Ejection
60
For Scans: MSn
By: Endcap Electrodes
Method: a) Cool ion of
interest back to set q value
(default = 0.25). b) Apply
custom RF waveform in
resonance with the set q
value, activation time
(default = 30 msec), and
optimized activation
amplitude.
Summary (Ion Trap Functions)
For Scans: All
1) Trapping
2) Isolation
3) Excitation
4) Ejection
61
By: Ring Electrode
Method: Ramp ring RF
power to increase the q values
of all ions in desired scan
range, low mass to high mass.
(i.e. Mass Selective Scanning)
Also, ramp the RF amplitude
on the endcap electrodes to
consolidate the ions to a
group (Resonance Ejection)
Tune Page
convection gauge
pressure
< 1.5 torr?
Ion gauge
Pressure
<2.0x10-5 torr?
62
ESI Calibration Solution
Caffeine stock:
1 mg/ml in methanol
MFRA stock:
Dissolve 3.0 mg MRFA in 1 ml 50:50 methanol:water
Ultramark stock: Measure 10 υl of Ultramark 1621, and dissolve it in
10 ml acetonitrile
ESI calibration solution
Into a clean vial pipette 100 µl of caffeine stock, 5 µl of MRFA stock and
2.5 ml of Ultramark stock. Add 50 µl of glacial acetic acid and 2.34 ml
50:50 methanol:water
63
Ion Trap Animation
64
The Eighth Generation Triple Quad
TSQ 15,
TSQ 45,
TSQ 46,
TSQ 70,
TSQ 700,
TSQ 7000,
TSQ,
TSQ Quantum
65
Smaller because…
8degrees
25 cm quad
25 cm quad
TSQ 7000
90 Degree
Square
quad
collision
cell
25 cm quad
Quantum
25 cm quad
66
90 degrees
HyperQuadsTM Hyperbolic Quadrupoles
TSQ 7000
1993 to 2000
r0 = 4 mm
L = 250 mm
• Forms Pure Quadrupolar Fields
• Reduces Fringing Effects
• Significantly Improves Resolution
• Improves Transmission
• Improves Peak Shapes
67
TSQ Quantum
2001 to …
r0 = 6 mm
L = 250 mm
Quadrupole Mass Analyzer
+
+
+
+
The
ion is transmitted along the quadrupole in a
stable trajectory Rf field. The
ion does not have a
stable trajectory and is ejected from the quadrupole.
68
How does the Quadrupole work ?
The quadrupole consists of four parallel rods. The opposing rods
have the same polarity while adjacent rods have opposite polarity.
Each rod is applied with a DC and
an RF voltage. Ions are scanned by
varying the DC/Rf quadrupole
voltages.
Only ions with the selected mass
to charge ratio will have the
correct oscillatory pathway in the
Rf field.
-ve
+ve
69
Effect of Peak Width On Transmission
HYPERQUAD
T
r
a
n
s
m
%
i
s
s
i
o
n
ROUND RODS
100
80
60
40
20
0
2
1.5
0.7
0.5
Peak Width FWHM
70
0.2
0.1
Effect of Peak Width on Resolution
12000
Quantum operating at
0.1 FWHM at m/z 1000
R = 10,000
Quad 0.7 FWHM
Quad 0.1 FWHM
10000
Sector
Resolution
8000
6000
4000
Quantum, API 4000, Ultima
at 0.7 FWHM at m/z 1000
R = 1428
R is relatively flat across m/z
2000
0
0
200
400
600
m/z
71
800
1000
1200
The Power of Resolution
z Separation of ions with same
nominal m/z value
z Unequivocal determination of
charge state (ESI)
z High resolution precursor ion
selection for MS/MS
z High resolution product ion for
charge state determination
72
Effect of changing resolution on peak shape
1.0 FWHM
0.3 FWHM
73
0.7 FWHM
0.2 FWHM
0.5 FWHM
0.1 FWHM
Resolution vs. Intensity
1.0 FWHM
2.5e6
74
0.7 FWHM
2.1e6
0.5 FWHM
1.9e6
Resolution vs. Intensity
0.3 FWHM
1.5e6
75
0.2 FWHM
0.1 FWHM
1.4e6
0.8 e6
Quadrupole Mass Analyzer
Š If one MS scan between m/z 100 and 500
is completed in one second, then each
m/z will be allowed to pass for 2.5 ms.
1000 ms
400 amu
= 2.5
ms/amu
+
+
+
+
+
+
76
To
detector
Quadrupole Mass Analyzer
Š And, for the same peak, for example, the
quadrupole performs 5 complete scans
from 100 – 500 Da each taking 1 sec.
100 – 500 Da
100 – 500 Da
100 – 500 Da
100 – 500 Da
100 – 500 Da
77
5 sec
Ion Trap Pre-Scan
Š The length of time that the trap stays
open to collect ions is determined by a
pre-scan which measures total ion current
(prevents space charging, so no ghost
peaks…)
Pre-Scan
78
5 sec
Ion Trap Pre-Scan Cont’d
Š Across a peak 5 sec. wide the trap might
fill and empty 5 times. So, a group of ions
are collected ca. every 1 sec – each group
is then ejected to the detector, smaller
ions first.
1 sec
1 sec
1 sec
1 sec
1 sec
79
5 sec
Comparison of Quads and Traps
For the quadrupole,
each m/z is scanned
(sequentially)
to the detector for 2.5
ms.
+ +
+
+
80
But for a trap, each m/z (and
all m/z at the same time) is/are
collected for ca. 750 ms (taking
pre-scan and interscan times
into account) and then scanned
to the detector.
Comparison of Quads and Traps
A trap will fill similar to
filling a glass with water. All
ions entering the trap will be
collected until the trap fills
with a pre-defined amount of
ions (AGC target value).
At any one particular
instant, a quad will only
scan/look for only one
m/z. All other m/z will
be ignored. In this
example, each m/z is
scanned for 2.5 ms.
+ +
+
+
81
Comparison of Quads and Traps
Š So, for full scan MS, a trap will give
better sensitivity – because there are
more ions representing each m/z
arriving at the detector for each scan.
+ +
+
+
82
How Much More???
Š Accounting for pre-scan/interscan
timing, the trap produces ca. 300 times
(750 ms/2.5 ms) for the “collection” of
each m/z compared to a quadrupole (i.e
2 orders of magnitude).
+ +
+
+
83
But…
What if I wanted to pass (filter) only one
m/z ion to the detector (i.e. SIM or SRM) –
then I could spend more time on that ion…
+ +
+
+
84
Comparison of Quads and Traps
Yes, on a quadrupole,
interscan times are
relatively short and so
the quadrupole remains
fixed on that one ion – a
duty cycle of close to
100%
+ +
+
85
But a trap will still only
collect ions in “batches”and prescan/interscan
times afford a duty cycle of
about 75%
+
What is a Duty Cycle?
Definition:Time taken to acquire 1 scan and be ready to acquire
the next one
Duty Cycle
Scan 1
ISD
Scan 2
Interscan delay (ISD) is the time taken to return all
system voltages to the start values and reach a stable
state
This is ‘dead’ time and should be minimized
86
In Addition…
Š While the beam instrument is continuously
detecting one particular m/z – a trap builds a
curve from an “average” over each collection
time – and the points are least frequent at
the most important region for quantitation
(the “take off”).
87
For Example…
Š SRM of 5 pg Alprazolam with LCQ Deca
gives a %RSD of 6.11 while…
Š SRM of 750 fg Alprazolam with TSQ 7000
gives a %RSD of 1.87.
Š Signal to noise ratio (S/N) are similar
(20:1 and 28:1 respectively).
88
RT: 4.26
SN: 20
100
90
Relative Abundance
80
70
SRM 5pg Alprazolam
with LCQ Deca
%RSD 6.11
60
50
40
30
20
10
0
SRM 750fg Alprazolam with TSQ
RT: 4.41
SN: 28
100
90
80
70
60
%RSD 1.87
50
40
30
20
10
0
0.0
89
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Time (min)
4.5
5.0
5.5
6.0
6.5
7.0
7.5
The Effect of Ion Trap Scan Speeds on
Quantitative Performance
• In general, faster scanning produces more points
across a chromatographic peak, hence better
precision and lower LOQ’s.
• In fact, for an ion-trap, scan speed refers only to
the time taken to scan ions from the trap during
mass analysis.
• Scan speed does not refer to the total analytical
cycle. In an ion trap device, an MSn analytical
scan comprises at least four events:
90
The Effect of Ion Trap Scan Speeds on
Quantitative Performance
1- AGC Pre-scan
2- Ion Injection (usually the rate-determining step)
3- Isolation and activation of the parent ion within
the trap
4- Scanning the ions out of the trap (mass analysis)
91
MS Scan Function
Mass
Analysis
Ion
isolation
Ion
Injection
AGC Prescan
92
Analytical Scan
Ion
Activation
Scan Terminology
Prescan
Mass Analysis
1st Microscan
Prescan
Mass Analysis
2nd Microscan
Complete Scan Cycle
93
Prescan
Mass Analysis
3rd Microscan
Save Data
The Effect of Ion Trap Scan Speeds on
Quantitative Performance
• The injection time constitutes the majority of the
total scan time.
• For good quantitative reproducibility, it is
necessary to take enough points to precisely
determine the chromatographic peak particularly
at the take-off point.
• Increasing the scan speed in the trap does not
significantly increase the number of data points
taken across the peak.
94
Scan speeds in ion traps
Pre-scan
Pre-scan
Quantitation m/z 500 Isol
scanning
135-510
/Activ
Isol
/Activ//Download
Download
Pre-scan
Pre-scan
60
375
amu/sec
60msec
msec
375amu
amu@
@13,000
13,000
amu/sec
Isol
Injection
Isol/Activ
/Activ//Download
Download
80
Injectiontime
time80
Pre-scan
60
msec
Pre-scan
60
msec
375
amu
@
13,000
amu/sec
30
Total
scan
375 amu @ 13,000
amu/sec
30
Total
scantime
time
Isol/Activ/
80
Isol/Activ/Download
Download
Injection
time
wide
Injection time80Scans
500
Scans//10
10sec
sec500
widepeak
peak
375
amu/sec
70
375amu
amu@
@5500
5500
amu/sec
70
Total
scan
time
670
Total scan time
670
Injection
500
Injectiontime
time Scans
/
10
sec
wide
15
Scans / 10 sec500
widepeak
peak
15
Total
Totalscan
scantime
time
710
710
Scans
Scans//10
10sec
secwide
widepeak
peak
14
14
95
00msec
msec
80
80
30
30
500
500
610
610
16
16
The Effect of Ion Trap Scan Speeds on
Quantitative Performance
• The only significant way to increase the sampling
rate across the peak is to reduce the injection
time which can be achieved in two ways:
1- Set a lower max injection time which reduces the
number of ions in the trap, hence sensitivity
2- Increase the efficiency of the source and lenses
to improve the transmission of ions; I.e filling the
trap to the same level in a shorter period of time.
96
Data Dependant Acquisition of MSn Spectra
Data Dependant Acquisition: “Intelligent” decision-making software that
selects precursor ion for MSn experiments based on user-define criteria.
Critical for metabolite screening experiments.
Scan event 1
Full-Scan MS
Scan event 2
Full-Scan MS2
Software selects most intense
ion from scan event 1 as
precursor ion for ms2
experiment in scan event 2,
provide that its intensity is
above a user selected threshold
m/z
97
m/z
“Dynamic Exclusion”-- MS and MS/MS of Co-Eluters
MS
MS/MS
MS/MS
MS
MS/MS
377
407
MS
MS
MS
MS
MS
452
Threshold
365
231
Time
MS
407
m/z
206
255
452
m/z
98
m/z
MS/MS
377
m/z
Comparison of Quads and Traps
Major Strengths of Triple Quads
• SRM Sensitivity
• Neutral Loss Scan Mode
• Parent (Precursor) Scan Mode
Major Strengths of the LCQ Deca XP Plus
• MSn Scan Mode
• Full Scan MS/MS Sensitivity
• Consecutive Reaction Monitoring (CRM)
99
What are neutral loss scans ?
• Both Q1 and Q3 are scanned together
• Q3 is offset by the neutral loss under
investigation
• The precursor ions collide with Argon gas in
Q2 to create fragment ions
• Only those compounds which give a fragment
having that specific loss are detected
• Since both Q1 and Q3 are scanning, neutral
loss scan mode is slower than any other mode
100
Neutral loss scans
Neutral loss scans are used for screening experiments where a
group of compounds all give the same loss
H2 N
- m/z 84
H2 N
N
N
H2 N
H2 N
N
NH2
- m/z 84
HO
N
101
N
N
N
N
NH2
H2 N
- m/z 84
H2 N
N
N
- m/z 84
HO
N
N
What are precursor ion scans ?
• Precursor ion scans also known as parent ion scans
• Q1 is scanned
• Q3 is set to allow only a fragment ion of one m/z to
pass; (Q3 fixed)
• ions collide with Argon gas in Q2 to create fragment or
product ions
• Only those compounds which give that specific
fragment ion are detected
102
Precursor ion scans
Precursor ion scans are used for screening experiments where a
group of compounds all give the same fragment ion
m/z 192
NH2
m/z 162
N
N
H 2N
N
HO
N
N
N
N
m/z 192
N
m/z
84
H 2N
N
H 2N
N
m/z 238
103
N
m/z 268
Comparison of MSn in a Triple Quad verses
an Ion Trap Instrument.
Triple Quad(nonresonant excitation): Acceleration voltage applied
equally to all masses. Get a mix of ms2, ms3…msn products.
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.
104
Comparison of Quads and Traps
Ion Traps
Quadrupoles
Mass Separation in time
Mass Separation in space
High sensitivity Full Scan
Lower sensitivity Full Scan
Lower sensitivity SIM and SRM
High sensitivity SIM and SRM
Offer multiple stages of MSn
Offers Only MS or MS/MS
Parent and neutral loss scans
105