Polaris Q GC/MS

n

Ion Trap Technology

Steven T. Fannin

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GC & GC/MS

The Column: “heart” of the Instrument

3

Maintaining GC/MS Ruggedness

• • • • • “Extra Column” Effects Syringes Septa Liners Ferrules Gas Filters

Chromatography: General Overview

4

• Resolution • Selectivity –

Spacing between two peaks

–

Important role in GC confirmation analyses

• Capacity Factor (Relative Retention) –

Retention relative to an unretained compound

• Column Efficiency: H = L/N

Why Capillary Columns?

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

N.B: Velocity: Pressure regulated vs Flow controlled H 2 vs He vs N 2

5

Common Mass Analyzers for GC/MS

•

Time of Flight (TOF)

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

Magnetic Sector

The ions are focused and resolved by passing through an electric field then a magnetic field.

- Uses a combination of magnetic and electrical fields to sort ions. •

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.

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General Mass Spectrometry Characteristics

What 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 free path of gas phase ions Torr) to allow appropriate mean

General Mass Spectrometry Instrument Characteristics – – – – – –

Sensitivity Tandem Mass Spectrometry Mass Range Resolution Mass Accuracy Scan Speed

GC/MS Ionization Methods

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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+

Chemical Ionization

Soft Ionization Techniques

Filament

EI Ion Volume 10

Removable Ionization Volume e CH 4 Lenses To Mass Analyzer

CI Ion Volume

11

Positive Ion Chemical Ionization



Reagent gas reactions (methane)

m/z 16, 15, 14 m/z 17 m/z 29 m/z 28 m/z 27 m/z 41

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Positive Ion Chemical Ionization



Proton transfer

M



CH

5

  

M



M



C

2 5

  

M



H H

     

CH C

2 4

H

4



Hydride abstraction

M



C

2



5

 

M



H

  

C

2

H

6



Adduct formation

M



C

2



5

 

M



C M



C

3



5

 

M



C

2 3

H

5

H

5

    [M+1] + [M-1] + [M+29] + [M+41] +

Common PICI Reagent Gases •

Reagent Gas

Methane [CH 5 + – & C 2 H 5 + ]

Protonates most organic molecules

–

C 2 H 5 + reacts with alkanes primarily by hydride abstraction

• Isobutane [C 4 H 9 + ] –

Low purity (ion source gets dirty quickly)

• Anhydrous ammonia [H + (NH 3 ) n=1-3 ] –

Very selective protonation (nitrogen compounds)

– – –

Forms [M+NH 4 ] + adduct with many compounds Keeps ion source clean Highly corrosive (short mech. pump lifetime)

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

* kJ/mol

•

Proton Affinity*

549 & 687

Hydride Ion Affinity*

• 1126 & 1135 • 821 • 858 • 976 • 825 Less fragmentation with higher PA Less [M-H] + with lower HIA

EI vs.PICI for Pesticides

EI Spectrum of Heptachlor PICI Spectrum of Heptachlor

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Intensity is low for any single m/z ion.

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

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Adduct Formation in PICI

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Negative Ion Chemical Ionization (EC-NICI) • Reagent gas reactions (methane)

CH

4  ( 70

eV

)

e

 

CH

4  

e

 *

Thermal electron

• Kinetic energy of electrons reduced by collisions with reagent gas • Resonance electron capture mechanism of ionization

AB



e

 * 

AB

 

Heat

[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.

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Common NICI Reagent Gases •

Reagent Gas

Methane

e Thermalization Rate*

• 8.6x10

-10 • • • 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)

* cm 3 /s

• ~2.1x10

-9 • • 5.8x10

5.9x10

-9 -9

Better sensitivity with higher rate

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NICI of Carboxy THC - PFPA

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

Analysis of Catecholamines using NICI-MS

753

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Pentafluoropropionyl (PFP) Derivatives of Norepinephrine, Epinephrine and Dopamine

Ion Trap vs Quadrupole

Basic Principles

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+250 Voltage Relationship During a Mass Scan (Quadrupole) +V dc +1500 V RF • Ions scanned by varying the DC/Rf voltage across the quadrupoles 0 V RF +180 ° -250 -V dc Complete Mass Scan m/z 77001-1380 970608 -1500 Ion beam -/+(U+V o cos w t) +/-(U+V o cos w t)

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What is a Quadrupole Ion Trap?

Entrance Endcap Ring Electrode Exit Endcap r o z o V  cos  

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Potential Energy Surfaces (Ion Traps)

V 150 100 50 0 -50 -100 -150 0 90 180 RF Phase (deg) 270 360 V V r z z r r z

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General Principles of Stability Diagrams Operating line for mass selective instability 0.4

z stability a Z 0.2

0.2

0.3

b Z 0.4

0.5

0.6

1.0

0.8

0.7

0 0.1

0.2

0.3

q ~.91

cut-off -0.2

r stability -0.4

-0.6

Operating line for Mass selective stability q z = a z = m(r + 2 O 8eV 2 Z O )  2 16eU 2 2 m(r + 2z ) O O  2 0.5

q Z 1.0

0.4

0.5

0.6

0.7

b X , Y 0.8

0.9

1.0

1.5

q z • • • •

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

b

values are related to ion motion

U  V  cos   r o z o U  0  a z  0

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Stability Line and Mass Selective Ejection

a z 0.0

* 476 kHz

Mass-selective Instability Scan

with Resonant Ejection

0.908

1.0

q z q z a z  V  0 m / z

Mass-selective Instability Scanning Ramp RF voltage (V) to sequentially eject ions from low m/z to high m/z.

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Trapping Injected Ions

V  cos   D z  eV 2 4 mz 2 o  2 + r o z o + ++ + • •

Correct RF voltage Helium buffer gas

Full Scan MS Scan Function

Mass Analysis Ion Injection Ion Injection Mass Analysis V Gate Lens Eject (V’) Multiplier 27 AGC Prescan*

One complete scan constitutes a “microscan”

Mass Analysis Scan

Fixed Ion Injection Time

100000 10000 1000 100 10

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1 1 10 100 1000 10000 100000 Amount of Sample (arbitrary units)

DYNAMIC RANGE: ~10 3

1000000 10000000 Space Charge Effects

Ion Injection Time Optimized with AGC

100000 10000 1000 100 10

Space Charge Effects * User Selectable ** 5 µs Polaris

Q

, 10 µs LCQ, 30 µs GCQ 29

1 1 10

DYNAMIC RANGE: >10 6

100 1000 10000 100000 Amount of Sample (arbitrary units) 1000000 10000000

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Polaris Q Tune Parameters (AGC and Injection RF)

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Quadrupole vs. Ion Trap Quadrupole

quadrupoles use SIM to enhance sensitivity

Transmits one m/z ion at a time Mass-Selective Stability scanning

Ion Trap

In full scan ion traps are more sensitive than quadrupoles.

Trap all m/z ions simultaneously Mass-Selective Instability scanning

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Quadupoles and Sensitivity Quadrupole • Duty cycle is important for determining mass analyzer efficiency • Efficiency of the mass analyzer:

E MassAnalyz er



E Transmissi on



DutyCycle

Transmits one m/z ion at a time Mass-Selective Stability scanning

Duty Cycle for a Quadrupole

Width of transmitted ion total width of m/z range = Duty Cycle

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

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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

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Ion Traps and Sensitivity External Source Ion Trap • Efficiency of the mass analyzer:

E MassAnalyz er



E Transmissi on



DutyCycle

Trap all m/z ions simultaneously Mass-Selective Instability scanning

Duty Cycle for an Ion Trap

Ion Accumulation Time (ion gate time) Total scan time = Duty Cycle

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

Tandem MS Principles

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Tandem Mass Spectrometry

Why 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

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MS/MS “Tandem-In-Time”Ion Trap Technology MS/MS and MS n Capability MS/MS “Tandem-In-Time” Ion Trap Technology MS/MS “Tandem-In-Space” Triple Stage Quadrupole Technology

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1. Inject

MS/MS in an Ion Trap

3. Fragment 4. Detect 2. Isolate

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How Do We Isolate Ions for MS/MS?

a z 0.0

Ion we wish to isolate

* 476 kHz

Mass-selective Instability Scan

with Resonant Ejection

0.908

1.0

q z q z a z  V  0 m / z

How MS/MS works q z M p = V RF υ(ion) = (n + β) Ω/2

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Isolation Waveforms

m/z 1000 Fast Fourier Transform m/z 300 m/z 100 Time Domain Frequency Domain

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CID using Resonant Excitation

0.0

How MS/MS works q z M p = V RF υ(ion) = (n + β) Ω/2

0.0

0.908

q z t=15 ms Product ions 0.908

q z

42 Polaris Q Excitation Event Characteristics

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

q z  V m / z

MS/MS q z M p = V RF

q z

Excitation “q”

0.225

0.300

0.450

P = precursor mass 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

Higher q z Means Higher Energy

D z D z  eV 2 4 mz o 2  2  mq z 2  2 z 2 o 16 e

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q z = 0.225

0.30

0.45

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Resonant Excitation q

z

Value

q z 0.0

0.225

q z 0.0

0.30

q z 0.0

0.45

Fragment ions not trapped Product Ion m/z Range Fragmentation Energy 1/4 x

0.908

0.908

0.908

1/3 1/2 2x 4x

Tandem MS: Polaris Q

MS/MS Scan Function

Mass Analysis Ion Injection Ion Isolation Ion Isolation Mass Analysis Ion Injection Resonant Excitation 45

How MS/MS works for all RF-Traps q z M p = V RF υ(ion) = (n + β) Ω/2

V Gate Lens Isolate Excite Eject (V’) Multiplier AGC Prescan Mass Analysis Scan

MS/MS Example - Chlordane GC/MS Spectrum GC/MS/MS Product Ion Spectrum

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Isolation of Precursor Ion Fragment Precursor Ion

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Polaris Q MS/MS Parameters

MS/MS Parameters

Choice of Excitation q’s

MS/MS: Optimizing Conditions

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

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Excitation Event – higher excitation ‘q’s may provide improved conversion efficiencies (E CID =



F i / P 0 )

Dexamethasone Product ion intensity vs Collision Voltage

7.E+05 6.E+05 5.E+05 4.E+05 3.E+05 2.E+05 1.E+05 0.E+00 0 0.2

0.4

0.6

0.8

1 1.2

Collision Voltage (p-p)

1.4

1.6

1.8

2