intro to mass spec

Introduction to Mass Spectrometry

March 2008

What is a Mass Spectrometer?

A Mass Spectrometer is a machine that weighs molecules !

0 units

12 units

12 units8 9 10 11 12 13 14 15 16

14 units8 9 10 11 12 13 14 15 16

12 units8 9 10 11 12 13 14 15 16

Outline

• Basic Chemistry• Analytical Chemistry• Mass Spectrometry

– Types of Ion Sources• EI, CI, ESI, APCI, APPI, MALDI

– Types of MS• Ion Traps, Quads, FT-ICR, TOF, Sector• MS/MS • Performance Comparisons

– Market Segments

Basic Chemistry

• Everything is made of Atoms– Atoms are made of protons, neutrons, and

electrons– Many atoms together make up molecules

Carbon

Oxygen Hydrogen

Nitrogen

6 protons (+)

6 neutrons

6 electrons(-)

Carbon Atom

Carbon

More Carbon

• 6 protons (1 mass unit each) + 6 neutrons (1 mass unit each) = 12 mass units– Electrons are negligible ( 1/3600 of mass unit)

• Some carbon (about 1%) has 7 neutrons so weigh 13 units

12.00 x 99%+13.00 x 1% = 12.01 amu

But how much does an atom weigh ?

• It was found that 12 grams of carbon contains 6.02 x 1023 atoms of carbon.( 1023 seconds have not elapsed since the beginning of time !)

• So one atom of carbon weighs 1.99 x 10-23 grams !

Caffeine

C8H10N4O2

Total Mass194 Daltons3.22x10-22 grams

Caffeine

C8H10N4O2

Total Mass194 Daltons3.22x10-22 grams

So we must devise a machine which can measure ~ 10-22 grams.

Analytical Chemistry

Instrumental Methods Chemical Methods

TitrationGravimetric AnalysisSolution Chemistry

Spectroscopy Mass Spectrometry

Optical Absorption

NMRMicrowave

Optical EmissionFT-ICRTOFQuadrupoleIon TrapLinear TrapMagnetic Sector

Gas Phase/Ionize

Detector

Separate Based on Mass/Charge

Sample

3 Elements to Mass Spectrometry(J.J. Thomson ~ 1910)

Why Ionize ?Difficult to manipulate neutral particles on molecular scale. If they are charged, then we can use electric fields to move them around.

Gas Phase/Ionize

Detector

Sample

• Electron Impact (EI)• Chemical Ionization (CI)• Electrospray (ESI)• Atmospheric Pressure Chemical

Ionization (APCI)• Photo-ionization (APPI)• Matrix Assisted Laser Desorption

and Ionization (MALDI)

Gas Phase/Ionize

Detector

Sample

• Scanning (Filter)– Linear Quadrupole– Sector

• Pulsed (Batch)– Ion Trap– FT-ICR– Time-of-Flight

Gas Phase/Ionize

Detector

Sample

• Faraday Cup• Discrete Dynode• Continuous Dynode• Multi-channel Plate

Gas Phase/Ionize

Detector

Sample

3 Elements to Mass Spectrometry(J.J. Thomson ~ 1856-1940)

So, we could come up with 6x5x4 = 120 Unique Mass Spectrometers.

In reality, not all combinations make sense, but many extra “hybrid” MS systems have value. For example Q-TOF’s and LT-FT-ICR

6 Types of Ion Sources

Ion Source Depends on Sample

Solid Sample Liquid Sample Gas Sample

EICIAPCIMALDI ESIAPPI

Make into Solution ?Make into Solid ? Turn into Gas?

Chemical Properties of analyte in gas phase ?

Chemical Properties of analyte in solution phase ?

Polarity, MW and Volatility

Caffeine

Gas Phase Ionization•EI and CI are gas phase ionization techniques•Sample is heated to cause volatilization•The molecule must have a low enough MW and polarity so that:

TBoil< TDecomposition

Electron Impact

e- e- e-

M(g) + e- M+(g) + 2e-

This reaction creates the molecular ion so is very useful. However, the excess energy from the electron can cause the molecular ion to fall apart:

IP2NeutralMolecule

Ionized Molecule

Excess Energy get redistributed throughout ion to cause fragmentation.

Electron Impact

e- e- e-

M(g) + e- M+(g) + 2e-

M+(g) A+

Fragment 1 (g) + BFragment 2 (g)•Electron energy is chosen by compromise. •Fragment Information is useful. It can help structural determination. However, many ions produce only fragments with no molecular ion remaining. Molecular ion often very unstable.•70 eV “Classical Spectra” to be used for comparisons

MW 194

H 109 m/z

55 m/z

Chemical Ionization

• EI is not appropriate for some molecules (it causes too much fragmentation)

• Instead, ionize a reagent gas (by EI) then react it with a analyte molecules

• Typically use methane or ammonia for reagent gas

CI: Form Reagent Ions First

• For Example - Methane CI1. electron ionization of CH4:

• CH4 + e- CH4+ + 2e-

– Fragmentation forms CH3+, CH2

+, CH+

2. ion-molecule reactions create stable reagent ions:• CH4

+ + CH4 CH3 + CH5+

• CH3+ + CH4 H2 + C2H5

+ – CH5

+ and C2H5+ are the dominant methane CI reagent

Methane CI Reagent Ions

– Ions at m/z 17, 29, and 41 are from methane; • H3O+ is also formed from water vapor in the

vacuum system

Reagent Ions React with Analytes• Several Types of Reactions May Occur

– Form Pseudomolecular Ions (M+1)– CH5

+ + M CH4 + MH+

– M+1 Ions Can Fragment Further to Produce a Complex CI Mass Spectrum

– Form Adduct Ions– C2H5

+ + M [M + C2H5]+ M+29 Adduct– C3H5

+ + M [M + C3H5]+ M+41 Adduct

– Molecular Ion by Charge Transfer– CH4

+ + M M+ + CH4

– Hydride Abstraction (M-1)– C3H5

+ + M C3H6 + [M-H]+

» Common for saturated hydrocarbons

EI Spectrum of Cocaine

• Extensive Fragmentation• Molecular Ion is Weak at m/z 303

Methane CI of CocainePseudo molecular Ion and Fragment Ions

Isobutane CI of Cocaine

• Soft Reagent - Less Fragmentation

Polarity, MW and Volatility

Liquid Techniques

• Depending on solvent composition and molecular properties either– APPI– ESI– APPI

• Lamp Wavelength chosen to only excite analytes not solvent/background– Low amount of

photo dissociation results

• New technique with few novel applications

• Less universal than electrospray

APCI Principles• Rapidly vaporize entire liquid flow• Ionize solvent molecules in corona

discharge• CI process ionizes sample molecules• Positive mode: proton transfer or charge

exchange• Negative mode: proton abstraction or

electron capture

APCI – Cut Away View

What applications need APCI?

• APCI works well for small molecules that are moderately polar to non-polar

• APCI works well for samples that contain heteroatoms

• Avoid samples that typically are charged in solution

• Avoid samples that are very thermally unstable or photosensitive

Why Electrospray ?

• Most Samples are delivered as liquids.– GC analysis requires heating sample to cause

evaporation– Ionization occurs through electron impact or

chemical reaction– Not all analytes are thermally stable

• Electrospray was designed to provide a gentle method of creating gas phase ions

Three Step Process1)Droplet formation 2)Droplet Shrinkage3)Gaseous Ion Formation

Electrospray process does not ionize samples !

Taylor Cone

•Solutions delivered to the tip of the electrospray capillary experience the electric field associated with the maintenance of a high potential. •Assuming a potential gradient, positive ions will accumulate at the surface. •Positively Charged Ions will “bud” off the surface when the applied electrostatic force is bigger than the surface tension.

Assisted Electrospray

NebulizingGas

LC Column Flow

High Voltage (5 kv)Low Voltage (0.5 kv)

DryingGas

Low Voltage (0.1 kv)

• Matrix Assisted Laser Desorption Ionization• Analyte co-deposited with Matrix• Laser excites matrix which transfers energy to analyte• Produces singly charged species• Typically used for large biomolecules / polymers• MALDI is a high mass/pulsed source so usually combined with TOF

5 Types of Mass Spectrometers

• Scanning (Filter)– Linear Quadrupole– Sector

• Pulsed (Batch)– Ion Trap– FT-ICR– Time-of-Flight

( Separation in Space)

( Separation in Time)

Basics of Ion Physics

maF qEF

qVmvEK 2

m – massa – accelerationB – Magnetic Fieldq – chargeE - electric fieldF – ForceK.E. – kinetic energyV – electric potentialv - velocity

Combine 1st two equations

We can control this. (volts/meter)We can

measure this.

We can control this. (volts/meter)We can

measure this.

We can deduce This !

0V -40V

1 meter

Time of Flight MS

qVmvEK 2

Time of Flight (TOF)

+ Very high mass range + Both high resolution and high

sensitivity + Mass accuracy+ High scan speed+ Mechanically simple

m/z t2

- High vacuum critical- Demanding high voltage/ pulsed/ high

precision electronics- Expensive

Bruker, Waters-Micromass, JEOL, Analytica

Time of Flight

SECTOR MS

qvBrmvF

High resolution (60,000 at 10% valley).

MStation™ Double Focusing Magnetic Sector Mass SpectrometerFROM JEOL

Very high reproducibility Best quantitative performance of all mass

spectrometer analyzers High resolution High sensitivity High dynamic range

- Large- Expensive- Not suited for pulsed sources

FT-ICR

qvBrmvF

1347.734

1348.736

1349.741

Resolution

PROFILE Scan 35 from ...ta sept 24.04\0.01+0.036 extrste mode 1 609.xms

605.0 607.5 610.0 612.5 615.0 617.5m/z

kCounts

609.50 0.3541 1384

610.45 0.4533

611.45 0.3952

PROFILESpectrum 1A0.472 min. Scans: 3-67 Channel: 1 Ion: 2000 us RIC: 21543BP 609.50 (1384=100%) 0.01+0.036 extrste mode 1 609.xms

611612

Reserpine

Reserpine is used to treat high blood pressure. It works by decreasing your heart rate and relaxing the blood vessels so that blood can flow more easily through the body. It also is used to treat severe agitation in patients with mental disorders

Resolution ~ 1200

LC/MS/MS with data dependent acquisition using Bruker’s simple, unified Compass software package

Exact mass MS analysis to sub-ppm levels for unambiguous determination of elemental chemical

composition. Automated software to confirm composition with m/z and isotopic pattern information

Exact mass MS(n) capability for detailed structural analysis and peptide sequencing

Qh-hybrid along with CID and ECD for “top-down” proteomics (Top↓Pro™) facilitates selected gas

phase ion enrichment

Extreme resolution capability for direct analysis of complex mixtures (> 600,000 FWHM)

Wide m/z range simultaneous detection of ions (e.g. 100 - 7,000 m/z)

Sub fmol sensitivity

FT-ICR

The highest recorded mass resolution of all mass spectrometers

Powerful capabilities for ion chemistry and MS/MS experiments

Well-suited for use with pulsed ionization methods such as MALDI

Non-destructive ion detection; ion remeasurement

Stable mass calibration in superconducting magnet FTICR systems

• Limited dynamic range • High Vacuum Demands • Subject to space charge effects and ion molecule

reactions • Many parameters (excitation, trapping, detection

conditions) comprise the experiment sequence that defines the quality of the mass spectrum

• Generally low-energy CID, spectrum depends on collision energy, collision gas, and other parameters

Ion Traps, Transmission Quadrupoles and Linear Traps

• Electrodynamic quadrupole fields– Paul (University of Bonn in 1953 – Nobel Prize 1989)

• 3D and 2D traps• Created a “high resolution quad” that was 5.82 m

long !• A quadrupole field is linearly dependant on the

coordinate axis• Ions are confined or rejected based on Voltage,

Frequency, Dimension, Mass and Charge

Ion Traps and Quads

•Traps are Pulsed•Quads are Continuous•Both rely time varying electric fields (RF)

- -Splat

• Forces on ion are simple to understand• As always

Where Fz = the force in the z direction

e = charge on the particle m = mass of the particle a = acceleration Ez= electric field

maeEF zz

Ion Trap + Quadrupole Theory

dtzdmmaF zZ

)cos(222

tVUrze

dtzdmmaF

zz eEmaF

0)cos(222

ztVUmre

0)cos(22

rtVUmre

Ion Motion in an Ion Trap• After several pages of math, we can derive an equation

for ion motion as a function of time:

• These second order differentials are not trivial to solve.• Mathieu Equation ! ( solved in 1868 , sub type of Hill’s equations) • Graphical Solution – (Slightly different for Traps and Quads because of symmetry.)

NEED SOLUTIONS WHICH ARE BOUND AND STABLE IN TIME

Stable Solutions to the Mathieu EquationFor a Quadrupole

2222z m4eV-

m8eU- a

Mathieu Equation for an ion trap

z stable

r stable

z stable

r stable

2222z m8eV-

m16eU- a

1.41.20.80.60.40.2 1.0

1.00.8

0.60.4

0.00.2

1.41.20.80.60.40.2 1.0 1.41.20.80.60.40.2 1.0

1.00.8

0.60.4

0.00.2

• Operated in RF only mode

• Light ions have a higher qz than heavier ions

• Ions stable in z axis when qz < 0.908

• Ions selectively ejected when RF amplitude is raised

• Light ions leave first, heavier ions later

2222z m8eV-

m16eU- a

Stability Diagram ( Area 1)

Stability Diagram for a Quad

2222z m4eV-

m8eU- a

2222z m4eV-

m8eU- a

200 100 50

V=200VU=0V

2222z m4eV-

m8eU- a

V=200VU=50V

200100

2222z m4eV-

m8eU- a

V=200VU=100V100

2222z m4eV-

m8eU- a

V=400VU=200V

Stability Diagram for a TRAP

Quad operates by selectively passing one m/z at a time.

Trap operates by collecting all ions simultaneously and then ramping them out one at a time.

2222z m8eV-

m16eU- a

1.41.20.80.60.40.2 1.0

1.00.8

0.60.4

0.00.2

1.41.20.80.60.40.2 1.0 1.41.20.80.60.40.2 1.0

1.00.8

0.60.4

0.00.2

Stability Diagram for a Trap

2222z m8eV-

m16eU- a

200 100 50

V=200VU=0V

Eject when q=0.908

2222z m8eV-

m16eU- a

200 100 50

V=300VU=0V

Eject when q=0.908

2222z m8eV-

m16eU- a

200 100 50

V=400VU=0V

Eject when q=0.908

Mass Spectrum on a Quad or Trap

Ramp RF (in trap) or ramp RF/DC in Quad

Spectrum

2222z m8eV-

m16eU- a

Potential Well Model

Need for helium buffer gas

rz mzeVDD

Secular Frequency• Ion Motion in Trap contains many frequency

components• These depend on a and q parameters

– (When q < 0.40)

0 0.2 0.4 0.6 0.8 1

m/z= 500q = 0.1816ω = 50.5 kHz

m/z= 1000q = 0.0908ω = 25.1 kHz

m/z= 1500q = 0.0605ω = 16.7 kHz

Low MassCut Off100 m/z

m/z= 106q = 0.850ω = 301.9 kHz

0 0.2 0.4 0.6 0.8 1

m/z= 500q = 0.1816ω = 50.5 kHz

m/z= 1000q = 0.0908ω = 25.1 kHz

m/z= 1500q = 0.0605ω = 16.7 kHz

m/z= 106q = 0.850ω = 301.9 kHz

Varian Eject

frequency

Amplitude

Fourier Transform

Notched Broad Band Waveform

0 0.2 0.4 0.6 0.8 1

m/z= 500q = 0.1816ω = 50.5 kHz

m/z= 1000q = 0.0908ω = 25.1 kHz

m/z= 1500q = 0.0605ω = 16.7 kHz

m/z= 106q = 0.850ω = 301.9 kHz

Frequency Notch

180kHz 240kHz

Practical Mass Spectrometer

Load Time Ion Ejection

Notched Waveform

Dipole Ejection

Mass Spectrum

Ion trap

BenefitsHigh sensitivity Multi-stage mass spectrometry (analogous to FTICR experiments) Compact mass analyzerCheap and Easy to build

Limitations•Poor quantitation •Poor inherent dynamic range •Subject to space charge effects and ion molecule reactions •Collision energy not well-defined in CID MS/MS •Many parameters (excitation, trapping, detection conditions) comprise the experiment sequence that defines the quality of the mass spectrum

Transmission Quadrupole Mass Spectrometer

BenefitsClassical mass spectra Good reproducibility Relatively small and low-cost systems Potentially good conversion efficiency for MS/MS

Limitations•Limited resolution •Peak heights variable as a function of mass (mass discrimination). Peak height vs. mass response must be 'tuned'. •Not well suited for pulsed ionization methods •Low-energy collision-induced dissociation (CID) MS/MS spectra in triple quadrupole and hybrid mass spectrometers depend strongly on energy, collision gas, pressure, and other factors.

Linear Trap

Newest Generation MS•Many of the advantages of ion traps, without normal trap limitations.•Less Space Charge Problems•MSN

•Great loading Efficiency

MS/MS • In a transmission Quadrupole, MS/MS is

done in Space– need three quads ( Triple Quad)

• In an Ion trap MS/MS is done in time.

Pass only 195

RF ONLY-Pass Everything-Collisions with Ar cause fragmentation

Scan from 100-195 Look at daughter ions

Q1 Q2 Q3

Triple Quad

Ion Trap

Why MS/MS

• Unknown Identification• Potentially two compounds of interest

have the same mass ( and same retention time)

• Quantitation improvements ( background signal reduced)

Problem: Thiabendazole in Grapefruit Extract

• Antifungal agent, thiabendazole (TBZ) must be below 10 ppb in the processed grapefruit

• Major matrix interferent: Similar retention time Similar spectrum Concentration much greater than TBZ

Interferent

MS, MS/MS, and MS/MS/MS of TBZMatrix peak

Matrix peak

No matrix peak

MS (500 pg)

MS/MS (10 pg)

MS/MS/MS (10 pg)

20180-220

20117465-220

SelectScanningProduct Ion Scan

SelectPrecursor ion Scan

Scanning Neutral Loss Scan

Scanning

Quadrupole 1MS 1

Quadrupole 2Collision Cell

Quadrupole 3MS 1

Real Life System

PROFILE Scan 35 from ...ta sept 24.04\0.01+0.036 extrste mode 1 609.xms

605.0 607.5 610.0 612.5 615.0 617.5m/z

kCounts

609.50 0.3541 1384

610.45 0.4533

611.45 0.3952

PROFILESpectrum 1A0.472 min. Scans: 3-67 Channel: 1 Ion: 2000 us RIC: 21543BP 609.50 (1384=100%) 0.01+0.036 extrste mode 1 609.xms

611612

Reserpine

Reserpine is used to treat high blood pressure. It works by decreasing your heart rate and relaxing the blood vessels so that blood can flow more easily through the body. It also is used to treat severe agitation in patients with mental disorders

PROFILE Scan 85 from ...ultip charge ions\4500-cytochrome c-6-17-04.xms

500 750 1000 1250 1500 1750 2000m/z

kCounts

721.00 1.3347

765.65 0.6099 1186

816.43 0.3790 2316

874.45 0.4061 1507

941.55 0.4655

1020.13 0.4678

413 1112.72 0.4881

247 1223.87 0.6065

1359.76 0.5506

1529.57 0.5139

1747.75 0.7672

PROFILESpectrum 1A4.898 min. Scans: 10-160 Channel: 1 Ion: 500 us RIC: 159835BP 816.43 (2316=100%) 4500-cytochrome c-6-17-04.xms

Cytochrome C – MW 12220

+12+11

m/z = mass/charge

nHnHMW

Market Segments and Where Varian Sits

Single QuadrupoleSingle Quadrupole$204 M$204 M

Triple QuadrupoleTriple Quadrupole$ 14 M$ 14 M

Ion TrapIon Trap$50 M$50 M

SectorSector$5 M$5 M

TOFTOF$7 M $7 M

GC/MS mass analyzer type

GC/MS Initial Sales $280M

33%25%

13%Single QuadrupoleSingle Quadrupole

$114 M$114 M

Triple QuadrupoleTriple Quadrupole$221 M$221 MIon TrapIon Trap

$140 M$140 M

Sector/FTICRSector/FTICR$30 M$30 M

Q-TOFQ-TOF$128 M$128 M

API TOFAPI TOF$65 M$65 M

LC/MS mass analyzer type

2004 LC/MS Initial Sales $698M

Agilent Bruker JEOL MicroMass

Sciex Thermo Varian

Single Quad 1 2 1 1 1Triple Quad 2 4 4 1Sector 3 1 1FT-ICR 2 13D Trap 3 3 2 1Linear Trap 1 1TOF 1 1 1 2

TOF/TOF 2

Q-TOF 2 4 1TOTAL (LC/MS) 5 10 4 11 6 10 3

The High-end LC/MS VendorsHigh-end TQ (55%) Waters, Thermo, ABI

High-end Ion Traps (23%) Bruker/Agilent, Thermo

LTQ -Thermo

LC-TOF, TOF-TOF, Q-TOF (13%)

Q-Trap (5%)Magnetic sector (4%)

High-end LC/MS Vendor Market Share

Markets served by the high-end LC/MS

Academic, Food/AG, Indep.Test 40M (5%) 14M (15%) 16M (6%)

Total Market $330M

Varian participates in less than 25% of the market, with a 1% overall market share

A Mass Spectrometer is a machine that weighs molecules ! (by measuring the mass to charge ratio of ions)

SourceEICIESIAPCIAPPIMALDI

DispersionTOFFT-ICRSectorQuadTrap

DetectorFaraday CupChanneltronMCP

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