LC/MS Crossroads

Jerry Pappas Sales Representative

265 Davidson Avenue Somerset, NJ 08873 jerry.pappas@thermo.com 732-698-2778

Comparison of Quads and Traps

Ion Traps
Mass Separation in time High sensitivity Full Scan Lower sensitivity SIM and SRM Offer multiple stages of MSn

Quadrupoles
Mass Separation in space Lower sensitivity Full Scan High sensitivity SIM and SRM Offers Only MS or MS/MS

Parent and neutral loss scans

LCQ Instrumentation

Classic

Duo
3

Deca

Duo/Deca Comparisons

LCQ DUO
400um capillary 2 octopoles One rotary pump

LCQ DECA
500um capillary 1 square quadrupole & 1 octopole 2 rotary pumps

Deca: approximately 10 x better signal than Duo

4

Current LCQ Generation

Advantage/XP Comparisons

450um Ion Transfer Tube Orthogonal Probes

550um Ion Transfer Tube Orthogonal Probes

XP: approximately 10 x better signal than Advantage

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

CI Ionization

EI

Soft
No Fragments
11

Hard

Fragments

EI and CI Mass Spectra of Ephedrine

58
100

EI
% Relative Intensity
50

0 100

20

40

60

80

100

120

140

160

180

CI

50

148 (M+H)+ 166

0 0

20

40

60

80

100

120

140

160

180

MW = 165Th
12

m/z

Ionization Techniques
200,000

ESI
15,000 1,000

APCI
Molecular Weight

TSP GC
Non Polar

FAB

PBI

Polar

13

What is API?
Atmospheric Pressure Ionization
Source Types:
1. 2.

Electrospray Ionisation (ES) Solution phase process (for the most part). APCI (Atmospheric Pressure Chemical Ionization) - Gasphase process.
Ionize the analyte (APCI) or transport ion in solution to the gas phase. Desolvate sample flow for introduction into mass spectrometer. Baffle the first vacuum region of the MS from atmospheric pressure in the source. Pump away neutrals and opposite charged ions which would otherwise interfere with the analysis of the desired polarity.

Source Purpose:
1. 2. 3. 4.

14

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

ElectrosprayBasic 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

Total control for any flow rate (200ul/min 2000ul/min)

20

LC Flow Rates

ESI:
3 L/min - 1mL/minute Optimal Flow Rate: 200 L/min Generally, higher flow rates require higher heated capillary temperatures and higher gas flow rates.

APCI:
200 L/min - 2mL/minute. Optimal Flow Rate: 500 L/min Generally, higher flow rates require more sheath and auxiliary gas, but do not require higher heated capillary temperatures.
21

Theoretical Increase in Response

Conc max = D Column 1

Column 2

Col. Diameter mm 4.6 Flow Rate ml/min 1 Theoretical Increase 1

3.0 0.5 2.3

2.0 0.2 5

1.0 0.05 21

Capillary

< 10 l / min

22

LC Additives
Acids Do not use inorganic acids (may cause source corrosion) Formic and acetic acid are recommended Bases Do not use alkali metal bases (may cause source corrosion) Ammonium hydroxide is recommended Surfactants (surface active agents) Detergents and other surface active agents may suppress ionization Trifluoroacetic Acid (TFA) May enhance chromatographic resolution, but causes ion suppression in both negative and positive ion mode Isopropyl Alcohol May Enhance Negative Ion Formation
23

Buffers (pH)
Avoid using non-volatile HPLC additives such as: Alkali Metal Phosphates Borates 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 0 100000

Tyr-Gly-Gly-Phe-Leu Leucine Enkephalin

Solvent System

200000

300000

400000

500000

Counts (protonated ion species)

27

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 Second multipole Lens

IONS IN

IONS OUT

Octapole Mount Vacuum Baffle

Analyzer Mount

31

Multipole Potential Wells

Mass Range Trans Efficiency

32

Octapole Square Quadrupole Round Quadrupole

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 wont 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

He

He He He

37

Discovery of the Effects of Helium

38

Helium as a Damping Gas

39

Ion Trap ResolutionEffect of Damping Gas

Traps injected ions by removing kinetic energy Damps ion motion to center of trap

Result...
Increase in resolution and sensitivity

40

Helium Effect
Helium flowing into trap
S#:1 RT:0.00 AV: SM:7G NL:2.50E7 1 T:+ p Full ms Relative Abundance 100 80 60 40 20 0 514 516 518 520 522 m/z 524 526 528 525.3 524.3 S#:23-32 RT:0.71-1.00AV: 0 SM:7G NL:5.61E7 1 T:+ p Full ms Relative Abundance 100 80 60 40 20 195.15 0 500 1000 m/z 1500 2000 524.26 1522.04 1322.06 1222.14 1122.21 1022.09 1621.97 1721.89 1821.95 1921.88

Helium shut off and not flowing into trap

S#:1 RT:0.02 AV: SM:7G NL:9.70E6 1 T:+ p Full ms Relative Abundance 100 80 60 40 20 0 514 516 518 521.8 521.2 520.7 523.9 Relative Abundance 522.6 523.0 S#:23-32 RT:0.39-0.54AV: 0 SM:7G NL:2.80E7 1 T:+ p Full ms 100 80 60 40 20 0 192.17 500 1000 m/z 1500 2000 523.01 1120.90 1320.95 1220.75 1919.96 1620.79 1520.26 1720.44

520

522 m/z

524

526

528

41

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.

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.

45

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)

~ 300 Ions
100 524.3

~ 1500 Ions
100 524.4

~ 3000 Ions
100 524.5

~ 6000 Ions
100 524.8

Relative Abundance

Relative Abundance

Relative Abundance

Relative Abundance

80 60 40 525.3 20 526.3 0 522

80 60 40 525.4 20 526.3 527.5 522

80 60 40 525.5 20 0 522 526.5 527.5

80 60 40 20 0 522 525.7

526.7

0 m/z
530

m/z

530

m/z

530

m/z

530

Good Resolution
49

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)

Target Value AGC Prescan Signal

50

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 0 100 200 Unscaled TIC (counts)

Unscaled TIC

300 Scan Number

400

500

600

Injection Time (ms)

60 50 40 30 20 10 0 0 100 200 300 Scan Number 400 500 600

Injection Time

Scaled TIC (counts)

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

Scaled TIC

100

200

300 Scan Number

400

500

600

51

Isolation of Ions

Ion we wish to isolate

qz
0.0 0.908

Ions at different qz values oscillate at different frequencies (o)

q z 2 2

52

Isolation Waveforms
~ m/z 200

q axis

.908

500 Hz

16 msec

q axis

.908

53

Why MS/MS or MSn ?

Signal to Noise Improvement

Intensity

Signal Noise S/N

Stages of Analysis
54

MS/MS Parameters

Precursor ion m/z Excitation voltage Excitation qz

Intensity

Precursor ion (Product ions)

0.0

1.0

2.0

3.0

4.0
77001-1285 970219

Excitation Voltage (V)

55

Resonant Excitation qz Value

Fragment ions not trapped Product Ion m/z Range Fragmentation Energy

qz
0.0

0.225

0.908

1/4

qz
1/3

0.0

0.30

0.908

qz
1/2

0.0
56

0.45

0.908

Ion Trap Scan Functions

1. Collect

3. Fragment

2. Isolate

4. Eject

57

Summary (Ion Trap Functions)

1) Collection 2) Isolation 3) Excitation 4) Ejection

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.

58

Summary (Ion Trap Functions)

1) Trapping 2) Isolation 3) Excitation 4) Ejection

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.

59

Summary (Ion Trap Functions)

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.

1) Trapping 2) Isolation 3) Excitation 4) Ejection

60

Summary (Ion Trap Functions)

For Scans: All

1) Trapping 2) Isolation 3) Excitation 4) Ejection

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)

61

Tune Page
convection gauge pressure < 1.5 torr?

Ion gauge Pressure <2.0x10-5 torr?

62

ESI Calibration Solution

Caffeine stock: MFRA stock: 1 mg/ml in methanol Dissolve 3.0 mg MRFA in 1 ml 50:50 methanol:water

Ultramark stock: Measure 10 l of Ultramark 1621, and dissolve it in 10 ml acetonitrile ESI calibration solution Into a clean vial pipette 100 l of caffeine stock, 5 l of MRFA stock and 2.5 ml of Ultramark stock. Add 50 l 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 90 degrees

66

HyperQuadsTM Hyperbolic Quadrupoles

TSQ 7000 1993 to 2000 r0 = 4 mm L = 250 mm

TSQ Quantum 2001 to r0 = 6 mm L = 250 mm

Forms Pure Quadrupolar Fields Reduces Fringing Effects Significantly Improves Resolution Improves Transmission Improves Peak Shapes
67

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 0.2 0.1

Peak Width FWHM

70

Effect of Peak Width on Resolution

12000 Quad 0.7 FWHM 10000 8000 Quad 0.1 FWHM Sector
Quantum operating at 0.1 FWHM at m/z 1000 R = 10,000

Resolution

6000

4000 2000

Quantum, API 4000, Ultima at 0.7 FWHM at m/z 1000 R = 1428 R is relatively flat across m/z

0 0 200 400 600 800 1000 1200

m/z
71

The Power of Resolution

Separation of ions with same nominal m/z value Unequivocal determination of charge state (ESI) High resolution precursor ion selection for MS/MS High resolution product ion for charge state determination

72

Effect of changing resolution on peak shape

1.0 FWHM 0.7 FWHM 0.5 FWHM

0.3 FWHM

0.2 FWHM

0.1 FWHM

73

Resolution vs. Intensity

1.0 FWHM 0.7 FWHM 0.5 FWHM

2.5e6

2.1e6

1.9e6

74

Resolution vs. Intensity

0.3 FWHM 1.5e6 0.2 FWHM 1.4e6 0.1 FWHM 0.8 e6

75

Quadrupole Mass Analyzer

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
+

+ + +

To detector

76

Quadrupole Mass Analyzer

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

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 Contd

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. 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.

+ + +
80

Comparison of Quads and Traps

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. 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).

+ + +
81

Comparison of Quads and Traps

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???

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% But a trap will still only collect ions in batchesand prescan/interscan times afford a duty cycle of about 75%

+ + +
85

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
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
SRM of 5 pg Alprazolam with LCQ Deca gives a %RSD of 6.11 while SRM of 750 fg Alprazolam with TSQ 7000 gives a %RSD of 1.87. Signal to noise ratio (S/N) are similar (20:1 and 28:1 respectively).

88

100 90 80 Relative Abundance 70 60 50 40 30 20 10 0

RT: 4.26 SN: 20

%RSD 6.11

SRM 5pg Alprazolam with LCQ Deca

SRM 750fg Alprazolam with TSQ

100 90 80 70 60 50 40 30 20 10 0 0.0 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

RT: 4.41 SN: 28

%RSD 1.87

89

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 LOQs. 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
Ion isolation Ion Injection Ion Activation Mass Analysis

AGC Prescan
92

Analytical Scan

Scan Terminology

Prescan

Mass Analysis

Prescan

Mass Analysis

Prescan

Mass Analysis

1st Microscan

2nd Microscan Complete Scan Cycle

3rd Microscan

Save Data

93

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 0 msec 0 msec 80 80 30 30 500 500 610 610 16 16

Quantitation m/z 500 Isol /Activ //Download scanning 135-510 Isol /Activ Download
60 msec 375 amu @ 13,000 amu/sec 60 msec 375 amu @ 13,000 amu/sec Isol /Activ //Download time80 Injection Isol /Activ Download time80 Injection Pre-scan 60 msec Pre-scan 60 msec 375 amu @ 13,000 amu/sectime Total scan time 375 amu @ 13,000 amu/sec 30 Total scan 30 Isol/Activ/ Download Isol/Activ/ Download time80Scans / 10 sec500 peak Injection time80Scans / 10 sec500 peak wide Injection wide 375 amu @ 5500 amu/sec time 375 amu @ 5500 amu/sec time Total scan 70 670 Total scan 70 670 Injection time Scans / 10 sec500 peak Injection time Scans / 10 sec500 peak wide 15 wide 15 Total scan time Total scan time Scans //10 sec wide peak Scans 10 sec wide peak
95

Pre-scan Pre-scan

710 710 14 14

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

m/z

97

Dynamic Exclusion-- MS and MS/MS of Co-Eluters

MS MS/MS MS/MS MS MS MS 452 Time MS MS

MS
407

MS/MS
377

Threshold
231 365

MS

407 452

m/z

206 255

MS/MS
377

m/z

m/z 98

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 N H2 N N

- m/z 84

H2 N N H2 N N

- m/z 84
N

NH2 N H2 N N

NH2

- m/z 84
H2 N N

HO N

- m/z 84

HO N

101

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 162
N N H 2N N NH2 N

m/z 192

HO N

m/z 192
N N

m/z 84

H 2N N H 2N N

m/z 268

m/z 238

103

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, ms3msn 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
Mass Separation in time High sensitivity Full Scan Lower sensitivity SIM and SRM Offer multiple stages of MSn

Quadrupoles
Mass Separation in space Lower sensitivity Full Scan High sensitivity SIM and SRM Offers Only MS or MS/MS

Parent and neutral loss scans

105