Quadrupole Ion-Trap Mass Analyzer EAS Workshop November 15, 2004

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]

2

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

5

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

7

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

8

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

11

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

12

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

13

Mass Selective InstabilityMass Selective Instability

q k Vm ez = ( / )

.908qz axis

“Ramp RF, Ions Leave Low m/z to High m/z”

14

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

15

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.

16

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

17

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

18

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.

19

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

20

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

21

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.

22

“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

23

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

24

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

25

Turning it up a notch!Turning it up a notch!

26

The Finnigan LTQ The Finnigan LTQ –– Linear Ion TrapLinear Ion Trap

27

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

28

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

29

LTQ Vacuum Chamber – Linear Trap and Dynodes

30

2D Ion-Trap Animation

31

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

32

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

33

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)

34

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]



10 uM

1.0 uM

0.1 uM

35

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






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.

36

MSn Peak Assignments for Buspirone/HydroxyMetabolite

N

O

O

N

NNN

122/122

265/281

222/238

123/139

152/168

37

Potential Drawbacks of Ion Trap Mass Spectrometers

that the LTQ was Designed to Overcome

Limited Storage Capacity

Inefficient Trapping

38

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

39

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

40

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

41

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

42

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....

43

Platform for Hybridization (FT/MS)Platform for Hybridization (FT/MS)

44

Questions?

Quadrupole Ion-Trap Mass Analyzer EAS Workshop November 15, 2004

Documents

Transcript of Quadrupole Ion-Trap Mass Analyzer EAS Workshop November 15, 2004