Polaris Q GC/MSn Ion Trap Technology

Steven T. Fannin

2

GC & GC/MS

The Column: “heart” of the Instrument

3

Maintaining GC/MS Ruggedness

“Extra Column” Effects

• Syringes

• Septa

• Liners

• Ferrules

• Gas Filters

4

Chromatography: General Overview

H = A + B/u + C u

• Resolution

• Selectivity– Spacing between two peaks

– Important role in GC confirmation analyses

• Capacity Factor (Relative Retention)– Retention relative to an unretained

compound

• Column Efficiency:

The Van Deemter equation: H = A + B/u + C u A: the multipath term (eddy diffusion)B: longitudinal diffusionC: resistance to mass transfer

Why Capillary Columns?

H = L/N

N.B: Velocity: Pressure regulated vs Flow controlled

H2 vs He vs N2

5

Common Mass Analyzers for GC/MS

• Time of Flight (TOF) - Ionized compounds/fragments from the source are directed into a flight tube. Ions are separated by virtue of their different flight times over a known distance.

• Magnetic Sector - Uses a combination of magnetic and electrical fields to sort ions. The ions are focused and resolved by passing through an electric field then a magnetic field.

• Quadrupole - consists of two sets on opposing rods. This mass analyzer uses a combination of RF and DC modulation to sort ions.

• Ion Trap - operates on a principle as the quadrupole; however ions can be stored for subsequent analysis. The ions are sorted by changing the electric field inside of the trap by manipulating the RF field and sequentially ejecting the ions from low to high mass to charge.

6

General Mass Spectrometry CharacteristicsWhat differentiates mass analyzers is how they perform mass analysis

• Mass Analysis - Common to Mass Analyzers– All determine the m/z ratio

– All measure gas-phase ions

– All operate at low pressure (<10-4 Torr) to allow appropriate mean free path of gas phase ions

• General Mass Spectrometry Instrument Characteristics– Sensitivity

– Tandem Mass Spectrometry

– Mass Range

– Resolution

– Mass Accuracy

– Scan Speed

GC/MS Ionization Methods

8

Electron Ionization: EI (“Hard Ionization”)

• Transfer of energy to a neutral molecule (in the gaseous state) to eject one of its own electrons and produce an ion (charged molecule), with a mass of m and a charge of z.

9

Example of PFTBA EI+

10

Chemical IonizationSoft Ionization Techniques

RemovableIonizationVolume

Lenses

Filament

e-To MassAnalyzer

EI Ion Volume

CI Ion VolumeCH4

11

Reagent gas reactions (methane)

m/z 16, 15, 14

m/z 17

m/z 29

m/z 28

m/z 27

m/z 41

Positive Ion Chemical Ionization

12

Proton transfer

Hydride abstraction

Adduct formation

4252

45

HCHMHCM

CHHMCHM

6252 HCHMHCM

5353

5252

HCMHCM

HCMHCM

[M+1]+

[M-1]+

[M+29]+

[M+41]+

Positive Ion Chemical Ionization

13

Common PICI Reagent Gases

• Methane [CH5+ & C2H5

+]– Protonates most organic molecules

– C2H5+ reacts with alkanes primarily by hydride abstraction

• Isobutane [C4H9+]

– Low purity (ion source gets dirty quickly)

• Anhydrous ammonia [H+(NH3)n=1-3]– Very selective protonation (nitrogen compounds)

– Forms [M+NH4]+ adduct with many compounds

– Keeps ion source clean

– Highly corrosive (short mech. pump lifetime)

• 549 & 687

• 821

• 858

Reagent GasProton

Affinity*

• 1126 & 1135

• 976

• 825

Hydride IonAffinity*

Lessfragmentation

withhigher PA

Less[M-H]+

withlower HIA

* kJ/mol

reaction must be exothermic, i.e., PA (analyte) > PA (reagent gas)

14

EI Spectrum of Heptachlor

Intensity is low for any single m/z ion.

PICI Spectrum of Heptachlor

Intensity is concentrated in [M+H]+ ion.

Spectrum is simpler.

EI vs.PICI for Pesticides

15

Adduct Formation in PICI

16

Negative Ion Chemical Ionization (EC-NICI)

• Reagent gas reactions (methane)

• Kinetic energy of electrons reduced by collisions with reagent gas

• Resonance electron capture mechanism of ionization

*44 )70( eCHeeVCH

Thermal electron

HeatABeAB * [M]-

• Reagent gas reacts with electrons to form “plasma” of thermal electrons

• Ionization is favored by molecules which have a high electron affinity – electron capture

• Useful for selective analysis in heavy matrices, e.g., pesticides in food or waste matrix.

17

Common NICI Reagent Gases

• Methane

• Isobutane– Low purity (ion source get dirty quickly)

• Carbon dioxide– Can produce less fragmentation than methane or

isobutane

• Anhydrous ammonia– Keeps ions source clean

– Highly corrosive (short mech. pump lifetime)

Reagent Gas

• 8.6x10-10

• ~2.1x10-9

• 5.8x10-9

• 5.9x10-9

e- ThermalizationRate*

Bettersensitivitywith higher

rate* cm3/s

18

Negative Ion spectrum of the PFPA/PFPOH derivative of 11-nor-9-Carboxy-D9-THC

NICI of Carboxy THC - PFPA

19

753

Pentafluoropropionyl (PFP) Derivatives of Norepinephrine, Epinephrine and Dopamine

Analysis of Catecholamines using NICI-MS

Ion Trap vs QuadrupoleBasic Principles

21

Voltage Relationship During a Mass Scan (Quadrupole)

VRF

V+180°

RF

+Vdc

-Vdc

+1500

-1500

Complete Mass Scan

RF

Po

ten

tial

DC

Po

ten

tial

+250

0

-250

m/z

77001-1380970608

+/-(U+Vocost)

-/+(U+Vocost)Ion beam

• Ions scanned by varying the DC/Rf voltage across the quadrupoles

22

What is a Quadrupole Ion Trap?

tcosV

zo

ro

EntranceEndcap

ExitEndcap

RingElectrode

23

0 90 180 270 360R F P h ase (d e g )

-150

-100

-50

0

50

100

150

Rin

g V

olta

ge (

V)

r r

r

z z z

VVV

Potential Energy Surfaces (Ion Traps)

24

aZ

0.4

0.2

0

-0.2

-0.4

-0.6

0.5 1.0 1.5

Operating line for Mass selective stability

r stability

1.0

0.9

0.8

0.8

0.7

0.7

0.6

0.6

0.5

0.5

0.4

0.4 0.30.3

0.20.1

1.0

0.2

Z

X, Y

Operating line formass selective instability

z stability

q ~.91cut-off

qz

qz = m(r + 2

O O2 2 2

Z )

m(r + 2z ) O O

2 2 2

8eV

az = 16eU

q Z

General Principles of Stability Diagrams

• Basic Ion Trap Principles– Mathieu stability diagram and stability/reduced

parameters

• Ion trap function : “Mass Selective Instability”

• Quadrupole: Mass Selective Stability mode of scanning

• The (a,q) coordinates are simply related to m/z and the operating voltage - whereas values are related to ion motion

tcosVU

zo

ro 0a

0U

z

25

Stability Line and Mass Selective Ejection

0az/m

Vq

z

z

1.0

0.0 qz

az

0.908

Mass-selective Instability ScanningRamp RF voltage (V) to sequentiallyeject ions from low m/z to high m/z.

476 kHz

*

Mass-selectiveInstability Scanwith ResonantEjection

26

+

Trapping Injected Ions

++++ ++

22o

2

zmz4

eVD

tcosV

zo

ro

• Correct RF voltage

• Helium buffer gas

27

V

Eject (V’)

Gate Lens

Multiplier

Full Scan MS Scan Function

AGC Prescan*

Mass Analysis Scan

Ion Injection

Mass Analysis

Ion Injection

Mass Analysis

One complete scan constitutes a “microscan”

28

1

10

100

1000

10000

100000

1 10 100 1000 10000 100000 1000000 10000000

Amount of Sample (arbitrary units)

Num

ber

of Io

ns

Tra

pped

(ar

bitr

ary

units

)Fixed Ion Injection Time

10 m

s

0.1 m

s

SpaceChargeEffects

DYNAMIC RANGE: ~103

29

Ion Injection Time Optimized with AGC

250

ms*

5 µs*

*

SpaceChargeEffects

DYNAMIC RANGE: >106

Variable Time

* UserSelectable

** 5 µs PolarisQ,

10 µs LCQ,30 µs GCQ

1

10

100

1000

10000

100000

1 10 100 1000 10000 100000 1000000 10000000

Amount of Sample (arbitrary units)

Num

ber

of Io

ns

Tra

pped

(ar

bitr

ary

units

)

30

Polaris Q Tune Parameters (AGC and Injection RF)

31

Quadrupole vs. Ion Trap

Transmits one m/z ion at a time

Mass-Selective Stability scanning

Trap all m/z ions simultaneouslyMass-Selective Instability scanning

Quadrupole

Ion Trap

In full scanion traps are

more sensitive than quadrupoles.

quadrupoles use SIM to enhance

sensitivity

32

Quadupoles and Sensitivity

• Duty cycle is important for determining mass analyzer efficiency

• Efficiency of the mass analyzer:

• Ionization and mass analysis occur simultaneously: Mass resolution and scan range are important when determining duty cycle

Transmits one m/z ion at a time

Mass-Selective Stability scanning

Quadrupole

Duty Cycle for a Quadrupole

Width of transmitted ion

total width of m/z range= Duty Cycle

DutyCycleEE onTransmissierMassAnalyz

33

SIM, MIM and SRM,MRM (Target Compound Techniques)

• Single Quadrupole Technology (single-stage MS techniques)– SIM (Selected or Single Ion Monitoring)

• Set quadrupole to pass a single characteristic ion during a retention time window in the chromatogram

• Increases sensitivity 10-100X

• Lose spectral specificity

– MIM (Multiple Ion Monitoring)• Monitor 2 to 5 characteristic ions in addition to SIM quanitiation ion

• Set acceptable qualifier ion “ratios” to confirm detection

• More qualifier ions boost confidence but reduce sensitivity gains

• Triple Quadrupole Technology (MS/MS Techniques)

– SRM (Single Reaction Monitoring)• Single product ion monitored

– MRM (Multiple Reaction Monitoring)• Multiple product ions monitored

34

Ion Traps and Sensitivity

• Efficiency of the mass analyzer:

• Ionization and mass analysis occur consecutively: Scan time (or rate) relative to ion accumulation is important for determining duty cycle

Duty Cycle for an Ion Trap

Ion Accumulation Time (ion gate time)

Total scan time= Duty Cycle

Trap all m/z ions simultaneouslyMass-Selective Instability scanning

External Source Ion Trap

DutyCycleEE onTransmissierMassAnalyz

Tandem MS Principles

36

Tandem Mass SpectrometryWhy use MS/MS?

• Enhanced Selectivity (Qualitative and Quantitative)– TRACE Analyses Criteria for Target Compounds

• Sensitivity and Selectivity are important– MS/MS Improves Trace Level Analyses in complex matrices and enhances

confirmatory analyses (Enhanced confirmation of identification)

• Combined with Soft Ionization techniques– Most signal in [M+H]+ ions; Added selectivity and s/n– Confirmatory assays (MW ions plus 2-3 unique ions)– Qualitative and quantitative with digital reagent gas flow

• Structural Characterization Applications– MS/MS provides unique evidence to an unknowns identity providing further

information about fragments in the MS spectrum

S/N

TRACE DSQ uses SIM to increase S

Polaris Q uses MS/MS to reduce N

37

MS/MS “Tandem-In-Space”Triple Stage Quadrupole Technology

MS/MS “Tandem-In-Time”Ion Trap Technology MS/MS and MSn Capability

MS/MS “Tandem-In-Time”Ion Trap Technology

38

MS/MS in an Ion Trap

1. Inject

2. Isolate

3. Fragment

4. Detect

39

0az/m

Vq

z

z

1.0

0.0 qz

az

0.908

476 kHz

*

Mass-selectiveInstability Scanwith ResonantEjection

How MS/MS works

qz Mp = VRF

υ(ion) = (n + β) Ω/2

How Do We Isolate Ions for MS/MS?

Ion we wish to isolate

40

m/z 1000

m/z 300

m/z 100

Fast FourierTransform

Isolation Waveforms

Time Domain Frequency Domain

41

CID using Resonant Excitation

0.908

qz

0.0

t=15 ms

0.908

qz

0.0

Product ions

How MS/MS works

qz Mp = VRF

υ(ion) = (n + β) Ω/2

42

Polaris Q Excitation Event Characteristics

P = precursor mass

Excitation “q”

0.225

0.300

0.450

qz

The choice of ‘q’ is also a function of the MS/MS lower limit of the product ion m/z range. A ‘q’ of 0.225 is 1/4Mp (where Mp is the m/z of the parent ion), and a ‘q’ of 0.3 is 1/3Mp, and a ‘q’ of 0.45 is 1/2Mp. For example, if a ‘q’ of 0.45 is used, and Mp is m/z 400, then the daughter ion lower limit that can be observed in the spectrum will be m/z 200. If the same ‘q’ is used for an Mp at m/z 800, then the daughter ion lower limit that can be observed will be m/z 400, and so on

Stability Diagram: Where parent ions reside on the q axis during the excitation event

z/m

Vqz

MS/MS

qz Mp = VRF

43

Higher qz Means Higher Energy

e16

zmq

mz4

eVD

2

o

22

z

22

o

2

z

qz = 0.225 0.30 0.45

Dz

44

Resonant Excitation qz Value

0.908

qz

0.00.225

0.908

qz

0.0

0.908

qz

0.0

0.30

0.45

Fragment ionsnot trapped

Product Ionm/z Range

FragmentationEnergy

1/4

1/3

1/2

x

2x

4x

45

V

Eject (V’)

Gate Lens

Multiplier

Tandem MS: Polaris Q MS/MS Scan Function

AGC Prescan

Mass Analysis Scan

Ion Injection

Mass Analysis

Ion Injection

Mass Analysis

Isolate

Ion Isolation

Ion IsolationResonant

Excitation

Excite

How MS/MS works for all RF-Traps

qz Mp = VRF

υ(ion) = (n + β) Ω/2

46

MS/MS Example - Chlordane

GC/MS Spectrum GC/MS/MS Product Ion Spectrum

Isolation ofPrecursor Ion

FragmentPrecursor Ion

47

Polaris Q MS/MS Parameters

MS/MS Parameters

Choice of Excitation q’s

48

MS/MS: Optimizing Conditions

Dexamethasone Product ion intensity vs Collision Voltage

0.E+00

1.E+05

2.E+05

3.E+05

4.E+05

5.E+05

6.E+05

7.E+05

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2

Collision Voltage (p-p)

Pro

du

ct Io

n In

ten

sity

‘q’ is a function of the RF voltage applied to the ring electrode during excitation

Excitation Event – higher excitation ‘q’s may provide improved conversion efficiencies (ECID = Fi / P0)