DEPARTMENTS OF CHEMISTRY AND BIOCHEMISTRY

Shabaz Mohammed October 20, 2015

GRADUATE COURSE IN MASS SPECTROMETRY: LECTURE 2Mass Analysers

MassFilterInlet

Ion Source Detector

DataSystem

High Vacuum SystemTurbo pumpsDiffusion pumpsRough pumpsRotary pumps

Sample PlateTargetHPLCGCSolids probe

MALDIESIIonSprayFABLSIMSEI/CI

TOFQuadrupoleIon TrapFTMS

Microch plateElectron Mult.Hybrid Detec.

PC’sUNIXMac

Mass Analyzers

Quadrupole Analyzer (Q)

Ion Trap Mass Analyzer (QIT)

Time-of-Flight Analyzer (TOF)

Orbitraps(Orbi)

Electromagnetism

Force or F = q1

4πεr2 or V1-V2d

Lens at Voltage V1

Lens at Voltage V2

q2

rd

charge

q2 x q2 x

q1

q2

+

0V +200V

Same situation as+1000V +1200V

-200V 0V

It’s the difference that matters

In mass spectrometry we work with charges and lenses

Electromagnetism

+

0V -200V

Same situation as+1000V +800V-200V -400V

It’s the difference that matters

In mass spectrometry we work with charges and lenses

Electromagnetism

Work or energy W= V1-V2d

Lens at Voltage V1

Lens at Voltage V2

d

q2 x

q2

x d

q is often expressed in multiples of e (charge on an electron)i.e energy is expressed in eV (electron volts)

Electromagnetism

+

0V -200VWork or energy W= V1-V2

dq2 x x d

Potential energy

300 eV

0 eV

If q is 1 charge then:Loses 200 eV of potential energyGains 200 eV of kinetic energy

Electromagnetism

Time-of-flight Mass Analyzer

Analyser performs the equivalent of the 100m sprint for ions

Start line Finish line

•Ions are given the same amount of energy through a pulse (Energy is proportional to charge and the applied potential. E=zeV, z is number of charges, e is the amount of charge on an electron, V is volts)

2

2mvzeV

which can be turned into 2

2

Lt

eVzm

•So….

•Distance to finish line is established and stopwatch (t) is accurate to 1 nanosecond or better…

•Ions then move at the speed determined by their mass (E=0.5mv2, v for velocity (L/t))i.e. velocity goes down as mass goes up

Known Distance (L)

ToF: Problems

Starting position is not always the same!

A) Temporal: Ions of the same mass can form at different times but have the same KE.

D) Direction: There is also the problem that when an ion forms, it may do so with an initial velocity in the opposite direction.

C) Kinetic Energy: Ions of the same mass can form with different KEs.

B) Spatial: Ions of the same mass form having the same KE but in different parts of the source;

Step 1: No applied electric field. Ions spread out.

++ +

Ions of same mass, different velocities

Step 2: Field applied. Slow ions accelerated more than fast ones.

0 V.

0 V.

++ +

Step 3: Slow ions catch up with faster ones.

20 kV.

20 kV.

0 V.

0 V.+++

Ion formation problem solution: Time Lag Focussing/Delayed extraction

Improving resolution: The reflectron

Reflectron is an electrostatic hill for the ions to try and overcome

In reality they fail and are turned around

Ions that have a higher velocity will have more kinetic energy and will climb the hill further before being turned around.

21.30 kV

Improvement of resolution.

1560 1566 1572 1578 1584 1590Mass (m/z)

1616.7

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity

4700 Linear Spec #1 MC=>SM3=>MC=>MC[BP = 1570.7, 1617]

Linear mode+ Delayed extraction

÷ Reflector

Resolution = 3,500

1560 1566 1572 1578 1584 1590Mass (m/z)

2191.4

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity

4700 Linear Spec #1 MC=>SM3=>MC=>MC[BP = 1571.8, 2191]

Continuous extraction

÷ Delayed extraction

÷ Reflector

Resolution = 500

1560 1566 1572 1578 1584 1590Mass (m/z)

2642.4

010

2030

40

5060

7080

90100

% In

tens

ity

4700 Reflector Spec #1 MC=>MC[BP = 1570.7, 2642]

Resolution = 10,000

Reflector mode

÷ Delayed extraction

+ Reflector

Reflector mode+ Delayed extraction

+ Reflector1560 1566 1572 1578 1584 1590

Mass (m/z)

1751.0

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity

4700 Reflector Spec #1 MC=>MC=>MC[BP = 1570.7, 1751]m/z = 1570.68

Resolution = 20,000

Quadrupole Mass Analyser: Description

-(U+ Vcost

+ (U+ Vcost

x

y

z x

y

zx

y

x

y

2r

•Consists of Four Metal Rods

•Opposite Rods have same voltage and adjacent rods have opposite voltage

•The voltage is a combination of a constant potential (U) and an alternating potential (V)

-[U+Vcos(t)]

+[U+Vcos(t)]

t1 t3t2 t4

Taken from Roman Korner: quadrupole mass analyzers. BM58/BMP58 Course 1998.

0VU

V

cos(t)

t1

t2

t3

t4

Voltage

Quadrupole Mass Analyser: Description

Ions are given a small amount of kinetic energy (1 - 2 eV) to allow them to traverse the quadrupole to the detector (assuming they have stable trajectories)

Quadrupole Mass Analyser: Description

Quadrupole Mass Analyser: Scanning

time

volta

ge

Complete mass scan

products of fib a m+2h collsion gas =6.8e-4, ce=40

200 400 600 800 1000 1200 1400m/z0

100

%

SM100602 1 (3.073) Daughters of 769ES+ 4.72e7767.92

767.73

766.62644.28

643.85

643.35642.92

444.37273.38185.80

388.01 459.89

766.49756.87

645.59

768.04

1076.57

1074.90904.59

903.60902.73777.73

1074.46905.58906.33

1077.44

1078.621262.09 1349.29

Quadrupole Mass Analyser: Scanning

Quadrupole Mass Analyser

•Used to be one of the cheapest mass spectrometers on the market

•Very good for precursor ion scanning and selective reaction monitoring

-(U+ Vcost

+ (U+ Vcost

x

y

z x

y

zx

y

x

y

2r

-[U+Vcos(t)]

+[U+Vcos(t)]

Quadrupole Ion Traps: Description

Taken from: Web Site of Chemistry Department Purdue University,Indiana , USA

RF voltage is applied to ring electrode to produce a three-dimensional quadrupole electric field for trapping ions.

Quadrupole Ion Traps: Description

Taken from: Web Site of Chemistry Department. Purdue University, Indiana , USA

Quadrupole Ion Traps

2D Ion Trap Mass Analyzer – ’LTQ’

A Little Bit About Orthogonal ToFs

Important Factors in on-axis MALDI-ToF:

•Resolution and mass accuracy depend on knowing exact distance, time and energies given to the ions

•Ions form in the same direction as ToF analyser and so the source conditions are important factors in mass accuracy and resolution e.g.

•Starting position of ions •Starting kinetic energy and direction of ions

SOURCE

DETECTOR

TOF

Collisional Cooling of ions (usually in a quadrupole) before they enter the ToF region helps further improves ToF sensitivity and Resolution.

Cooling allows better positioning of ion packet into pusher region of the ToF

A Little Bit About Orthogonal ToFs

SOURCE

DETECTOR

TOFReducing the ions velocity here

Allows better focusing of ions here

Collisional cooling of ions increases mass accuracy and resolution

A Little Bit About Orthogonal ToFs

The Orbitrap

+-

Very high negative potential

+

Positive ion moving very fast

Positive ion attracted and has its path bent

Attraction of rod is high,Velocity of ion is high..Ion makes an orbit around rod Similar to a satellite around the earth

LTQ Orbitrap Hybrid Mass Spectrometer

API Ion source Linear Ion Trap C-Trap

Orbitrap

Finnigan LTQ™ Linear Ion Trap

Differential pumping

Differential pumping

LTQ Orbitrap Operation Principle

1. Ions are stored in the Linear Trap2. …. are axially ejected3. …. and trapped in the C-trap4. …. they are squeezed into a small cloud and injected into the Orbitrap5. …. where they are electrostatically trapped, while rotating around the central electrode

and performing axial oscillation

The oscillating ions induce an image current into the two outer halves of the orbitrap, which can be detected using a differential amplifier

Ions of only one mass generate a sine wave signal

Detection:Fourier Transform

Samples are typically not just 1 ion but several and on top there will be ions with several different m/z values.

Typical image current for a ‘simplish’ mixture looks like this:

Time

How do we deduce the individual frequencies?

Detection:Fourier Transform

Luckily waves do not affect each other and so within the ‘messy’ image current, the waves are present intact.

Fourier transform is a mathmatical technique which can deduce the frquencies present.

Resolution

The longer you measure, the better the spectrum….more datapoints for final deduction

The axial oscillation frequency follows the formula Where w = oscillation frequency

k = instrumental constantm/z = …. well, we have seen this before

zmk/

Frequencies and Masses

Many ions in the Orbitrap generate a complex signal whose frequencies are determined using a Fourier Transformation

Collision induced dissociation

MS 1 Collision cell MS 2

Mixture

Potential energy

-10 eV

-50 eV

DeltaKineticenergy

40 eV

0 eV

Accelerate ionsinto collision cell

50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600m/z0

100

%

sm117501 1 (0.043) Cm (1:23) TOF MSMS 556.30ES+ 708

500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150m/z0

100

%

sm207803 17 (0.950) Cm (11:20) TOF MS ES+ 7.02e3

50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600m/z0

100

%

sm117502 18 (0.781) Cm (6:19) TOF MSMS 556.30ES+ 1.64e3

MS 1 Collision cell MS 2

Mixture

A mass spectrum of a mixture Isolation of an ionIn our case a protonated peptide

A mass spectrum of the fragmentsProduced by the ion

Collision induced dissociation

Q Exactive

D30

Remove neutrals

mass selection

high resolution MS and MS/MS

Beam CIDfragmentation

Q- ToFs

Orbitrap Fusion CID

mass selection

Beam CIDfragmentation

Redesigned instrument capable of: 15 MSMS HCD (orbitrap) per second20 MSMS CID (ion trap) per second

Ion trap CIDfragmentation

Mass Analyzers

Quadrupole Analyzer (Q) Low (1 amu) resolution, fast, (relatively) cheap

Ion Trap Mass Analyzer (QIT) Fair resolution, all-in-one mass analyzer

Time-of-Flight Analyzer (TOF) Good resolution, exact mass, fast, no upper m/z limit, costly

Orbitraps(Orbi) High resolution, exact mass, costly