10 mass separation - eth z · mass separation and the lorentzforce note: • all mass analyzers...

Modern Mass Spectrometry and Coupling Techniques

Rob Nieckarz Office: HCI D325

[email protected]

Special Thanks to Dr. Richard Smith at the University of Waterloo

Gas Chromatography Liquid Chromatography Capillary Electrophoresis Spectroscopy MS

MS + =

Added informaOon and greater confidence in our analysis

2

Mass Separation and the Lorentz ForceNOTE:

• All mass analyzers function on the basis of the Lorentz Force equation which describes the force exerted on a charged particle in an electromagnetic field. The particle will experience a force due to the electric field (qE), and due to the magnetic field (qv x B). Combined they give the Lorentz force equation:

F = q(E + v x B)

– F is the force (in newtons) – q is the electric charge of the particle (in coulombs) = ze– E is the electric field (in volts per meter) – B is the magnetic field (in webers per square meter, or equivalently,

teslas) – v is the instantaneous velocity of the particle (in m/s)

q = ze therefore, F = zeE+zevB

Mass Spectrometry September 2004 4

Mass resolution: Definition

Valley

Peak width at half height

By courtesy of Spektrum Akademischer Verlag

15

Mass Resolution

• The FWHM definition is easier to apply (only need one peak), but gives a resolution about twice that of the 10% valley definition

• Resolution for sector instruments is usually given as the 10% valley figure.

• High resolution has some obvious advantages:

-It allows one to resolve ions that are isobaric-The narrower a peak, the easier it is to measure its

position accurately


Why do we need high resolution?

•  To resolve single, adjacent peaks of high molecular weight compounds

•  To enhance specificity •  To determine the elemental composition of a compound:

Example: Three different molecules may have the nominal mass 28: CO = 27.9949

ΔM = 0.0112 M/ΔM = 2800 N2 = 28.0061 ΔM = 0.0252 M/ΔM = 1120 C2H4 = 28.0313


Mass resolution: peak shape

By courtesy of Spektrum Akademischer Verlag

16

Mass Resolution

• Low resolution: <2,000. Suitable only for nominal mass measurement.

• Medium resolution: 2,000-20,000. Suitable for accurate mass measurement. Resolve isotope clusters of high charge states.

• High resolution: >20,000. Better than medium resolution. You can never have too much resolution!

• In practice, there is a trade-off between resolution and sensitivity. The ions are not coming from a point source: they exit the source through a slit of finite dimensions, and cannot be perfectly focussed. Slits and lens help to compensate for this by cuttingout ions from the centre of the beam and focussing. To get veryhigh resolution, the slits have to be narrowed, which means thata lot of ions are lost.

Types of Mass Analyzers:

• Time of flight mass spectrometers (Tof) • Fourier transform ion cyclotron resonance (FTICR) • Orbitrap • Linear quadrupoles (Q -‐mass filters) • Three dimensional quadrupoles (ion traps -‐ IT) • Linear iontraps (2D) • Sector instruments • Tandem instruments

Time of Flight Mass Analyzer

Fast, simple, high mass accuracy and resoluOon

53

Time of Flight (Tof)Principle:

Ions of different mass (accelerated by the same field, V) have different velocities and thus flight times. The larger the mass the slower the ion:

K.E. = zeV = mv²/2

Ion formation:

Ions are introduced to the Tof in pulses (e.g. MALDI or orthogonal extraction from a continuous beam such as ESI)

Ion detected by analogue or time to digital converter (GHz ADC or TDC)

• Linear Tof (high mass range but low mass resolution)

• Reflectron Tof (lower mass range but high mass resolution)

54

Mass Separation: Time of Flight (Tof) MS

acceleration region(drift region)

55

Basic PrinciplesSince the initial kinetic energy of the ions is given by:

zeV = mv²/2 (i)velocity: v = (2zeV/m)1/2 (ii)time of flight: t = L/v = L[m/(2zeV)]1/2 (iii)

m/z = 2eVt2/L2 (iv)Example:

For C6H5+. and C7H7

+., (m/z 77 and 91), accelerated at 10kV, what are the velocities of these 2 ions and how long would it take them to traverse a 2m flight tube?using eqn (ii) v77 = (2x1x1.6022x10-19x10,000/m)1/2

m(kg) = 0.077/6.022x1023 = 1.279x10-25

v77 = 158,306m/s = 9498 km/h or 12.63μssimilarly for v91 v91 = 145,621m/s = 8737 km/h or 13.73μs

V is the extraction pulse potential (V)L is the length of field free drift zone (m)t is the measured time-of-flight of the ion (s)e = 1.6022x10-19C

56

Example cont• From eq (iii), difference in flight time:

tA/tB = (mA/mB)1/2

• Consequently, this square root relationship causes Δt for a given Δm/z to decrease with increasing m/z

• For example:Δt/amu is calculated to be 114ns at m/z 20

to be 36ns at m/z 200to be 11ns at m/z 2000

• Tof mass analyzer depends on the ability to accurately measure these short time intervals to make it a useful MS

57

Linear Tof• Transmittance as high as 90%• Ions introduced into the flight tube have a temporal and kinetic

energy distribution which yields relatively poor mass resolution. • Kinetic energy spread can be reduced by employing Delayed Ion

Extraction

Principle of Delayed Ion Extraction:

• Ions are formed during a short pulse of a few nanoseconds• The acceleration (extraction) field is only applied after a delay of

some hundreds of nanoseconds:• At the beginning of the extraction ions with high initial velocities

have traveled further than slower ones. Therefore after the second extraction pulse they do not experience the full acceleration potential.

• Thus the initially faster ions will be accelerated less than the initially slower ions.

58

Reflectron TofSame m/z butdifferent kineticenergy

• In a reflectron Tof, the ions traverse the drift tube and penetrate into an electric field (ion mirror) where their direction is reversed.

• Faster ions (with higher kinetic energy) penetrate farther into the electric field than slower ions (with lower kinetic energy).

• Thus faster ions have a longer flight path and therefore need approximately the same flight time as the slower ions which have a shorter flight path.

59

Tof: Advantages and Disadvantages• Good mass accuracy

– reflectron ~ 5-10ppm

– limited with quadrupole MS, poor with ion traps and linear Tof

• High mass resolution

– reflectron ~5,000 to 20,000

– Quadrupole MS, ion traps and linear Tof operate closer to unit mass resolution at m/z ~ 103

• High mass range

– linear >105 Da, reflectron <104 Da

– Ion traps and quadrupoles are limited to ~6,000 Da

• Acceptable linearity for linear and reflectron Tof

– not as good as quadrupole MS, but similar to ion traps

• Very good scan-to-scan reproducibility for linear and reflectron Tof

– as good as quadrupole MS

• Very fast aquisition time (up to kHz)

Fourier Transform Ion Cyclotron Resonance Mass Analyzer

UlOmate in mass accuracy and resoluOon Expensive, difficult to operate

61

FTICR MS• Basic Construction:

– a cell where ions are trapped by intense, constant magnetic field and applied voltage

– The cell accepts ions in a “pulsed” mode from the continuous ion beam– Detection of the ions is based on the FT deconvolution of the image

current the circulating ions induce in a pair of detector plates after excitation with a resonant Rf pulse.

63

Ion Trapping and FTICR MS

• ions enter the cell (or are created internally) and they begin their cyclotron motion, orbiting around the centre of the magnetic field

• since the magnetic field is quite high (typical minimum of 4.7T, but this is increasing) the ions are trapped in the radial (x,y) direction.

• Resolving power and scan speed increase linearly with B

64

• by applying small, equal potentials to the two end or “trapping”electrodes, the ions are confined in the z or axial direction.

• ions can be confined for very long periods of time such that ion/molecule reactions or even slow unimolecular dissociation processes can be observed and monitored.

Ion Trapping and FTICR MS

m T

Cyclotron frequency Trapping oscillaKon frequency Magnetron frequency

65

FTICR MS Detection• In FT detection, all ions, regardless of their mass are detected at the

same time.

• Once ions are trapped inside the ICR cell they are excited by a fast sweep of all the Rf frequencies, exciting the ions to cyclotron motion with a larger radius.

Ions before excitation.They have their naturalcyclotron radius within the magnetic field.

67

FTICR MS - Detection

When a packet of ions (+ve) approaches an electrode, electrons are attracted from ground and accumulate in that electrode causing a temporary current.

As the ions continue to orbit, the electrons accumulate in the other electrode. The flow of electrons in the external circuit represents an image current. The amplitude of the current is proportional to the number of ions in the packet.

68

FTICR - Detection

• the frequency of the image current oscillation is the same as the frequency of the ion’s cyclotron motion which is related to mass. A small AC voltage is created across a resistor and is amplified and detected.

• using FT techniques all ion packets, each containing ions of the same mass, are detected. The decay of the image current (as the excited cyclotron orbit radius decays) is detected in time and transformed into a frequency domain signal by a Fourier transform.

Rf Excitation

Detected time domainimage current

Resulting mass domainSpectrum

Fourier Transform

Time

Time

m/z

How fast are the ions moving?

Frequency = 1 x 106 s-‐1

d = 0.05 m

Distance travelled = πd = 0.157 m

Speed = 0.157 m / 1x 10-‐6 s

= 157 000 m/s

= 9 424 km/hr

Time for one revoluOon = 1 x 10-‐6 s

69

FTICRMS• Very high resolution is possible. The current record is 8x108,

and routine values are 100,000 or so.• Long trapping times are possible, allowing for ion-molecule

reactions.• Good sensitivity.• Like the ion trap, the FTICR cell works well with pulsed

sources.• MSn capability• However, expensive because of the cost of superconducting

magnets and the very high vacuum requirements.• Difficult to operate

Orbitrap Mass Analyzer

The only new mass spectrometer concept to be developed in the last 30 years

The only commercial instrument that can come close to the performance of an FTICR

Orbitrap Summary

-‐ High performance mass analyzer

-‐ Resolu:on up to 200,000

-‐ Mass Range up to 50,000

-‐ High mass accuracy (1-‐2 ppm)

-‐ Non-‐destruc:ve ion analyzer

-‐ MSn possible

-‐ CID und H/D exchange possible

Quadrupole Mass Analyzer

Cheapest instrument Fast acquisiOon Omes

20

Linear Quadrupoles (2D - mass filters)

21

Linear Quadrupoles (2D - mass filters)

• Four hyperbolic rods (cheap version: circular rods) – compromise!• Opposite pairs of rods are connected electrically but are of

opposite polarity• Each pair of rods has a DC (U) + AC (V0 cosωt) Rf voltage applied:

1 pair of rods: -(U + V0 cosωt) and the opposite pair: +(U + V0 cosωt) where, ω = radial frequency = 2πf

• During a mass scan, the DC and AC voltages are ramped but the ratio of DC/AC (ie U/V0)is kept constant

• For a given DC and AC amplitude, only ions with a given m/z (or m/z range) have stable oscillations and are transmitted and can be detected

22

Quadrupole (end view)

Hyperbolic Round

Equipotential Field Lines

25

Linear Q: Equations of MotionFrom the electrical part of the Lorentz equation, we can derive the equation of motion (x and y directions) for a particle in a combination of DC and AC Rf fields the Mathieu equation:

– u represents the x or y transverse displacement. We do not consider displacement in the z direction because the electric field is 0 along the asymptotes of the hyperbolic rods.– The 2 parameters characteristc of the field (a and q) are given by:

d ud

a q uu u

2

2 2 2 0ξ

ξ+ − =( cos )

220

8ϖmr

zeUaa yx =−= 220

4ϖmrzeVqq yx

−=−=

and

26

• Where the variable ξ is the time in radians of the applied field = ωt/2

• U is the DC voltage and V is the AC Rf voltage of frequency ω• r0 is the radius of the instrument aperture

• Plotting a against q gives the Mathieu stability diagram of the linear quadrupole field - a/q = 2U/V

• Typical values are:

- U = DC voltage (~200 - 1000V)

- V = AC voltage (~1000 - 6000V, 1-2MHz),

- m = mass of ion, e = electonic charge, z = # of charges on ion

- 2r0 = distance between the rods - 1-2 cm

Linear Q: Equations of Motion

27

Stability Diagram

28

aq Space

• Note:

– Both +ve and –ve abscissa with a values ranging up to 10 and q values ranging up to 20

– In practice we only operate in the +ve area of region I

Why?

– Because in order to have a and q values >1 we would require VERY high DC and AC voltages which is not practical

29

Stability Diagram

X unstable

L1, only 1 ion has a stable trajectory allothers ions are lost therefore adjacentions are resolved from each other

L2, 3 ions have a stable trajectoryat the same time therefore these3 ions would not be resolved fromeach other

In practice, the ratio of a/q is changedby changing the DC voltage

0.1

0.80.4

0.3

0.2

a

q

Y unstable

X and Y Stable

L1

L2

L1 = L2

Operating linesa/q constant . .

.... ..

..

What would happen if noDC voltage is applied?

31

Conceptualizing a Q scan

0.1

0.80.4

0.3

0.2

a

q

m1 < m2 < m3

stable region of m1

stable region of m2

stable region of m3

Operating or scan line

32

Mass Range and Resolution• Depends on 5 parameters:

• Rod length (L) – 50 to 250mm• Rod diameter (r) – 6 to 15mm aligned to μm accuracy• Maximum supply voltage (Vm)• AC (Rf) fequency (f)• Ion injection energy (Vz) - ~5 volts

• From the theory of quadrupole operation the following relationship can be derived:

Mmax = 7x106Vm/f2r2

Consequently, as r and f increase, Mmax decreases and as r and f decrease, Mmax increases

33

• The resolution limit of a quadrupole is governed by the number of cycles of the Rf field to which the ions are exposed:

M/ΔM = 0.05 fL m/2eVz

Mass Range and Resolution

2

• Consequently, as both f and L increase so does resolution. If L in increased then f can be decreased and vice versa

• Scanning speeds as high as 6,000 amu/sec and mass resolution of 10,000 is attainable

34

Linear QAdvantages:

• Small and light weight ~20 cm long

• Inexpensive

• Simple to operate – complete computer control

• Low accelerating voltage – handles high source pressures better

• Full scan mass spectra and selected ion monitoring (SIM) for quantitation

Disadvantages:

• Unit mass resolution only and limited mass range

• High mass discrimination

• Rod contamination causes further imperfections in the quadrupole field – compromises resolution and sensitivity

35

Linear Q

Other applications:

• QQQ for MS/MS

• Hybrid instruments eg BEQQ and QqTof

• Ion lenses (hexapoles and octapoles)

• Collision chambers for MS/MS ie QQQ and BEQQ etc

• Prefilter – before mass resolving rods to reduce contamination

36

Quadrupole 3D Ion Trap (QIT)

Ion trap consists of three electrodes:

• ring electrode (hyperbolic shape)

• 2 hyperbolic electrodes - end caps

• Orifice for ion injection

• Orifice for ion ejection

• Pulsed introduction of ions

Cap

Cap

Ring

Cap

Cap

Ring

Cap

Cap

Ring

Cap

Cap

Ringr0

38

QIT (properties)

• Ion trap volume very small (7mm i.d.)• High sensitivity (10-18 mol) (scan mode)• High mass range : 6,000• Higher mass resolution than Q ~x2-3• High dynamic range: 106 depending on space charging• MSn capabilities• Low mass cut-off is a disadvantage• Helium is introduced intentionally into the ion trap (10–3 mbar)

– Needed as a buffer to absorb kinetic energy of incoming ions without chemical interaction so they can feel the effect of the trapping field - dampening (cooling) of oscillations

– collision partner for MS/MS and MSn

• Ions are concentrated in center of ion trap• Better resolution and better sensitivity than Q

39

QIT (ion motion)

• Between the three electrode a quadrupole field exists, which forces the ions to the center of the trap

• The farther the ion is removed from center of trap the stronger is the exerted electric force

• The ions oscillate within the trap, but with a rather complex sinusoidal motion

• The ion motion can be described by Mathieu’s differential equations

40

Quadrupole 3D Ion Trap (QIT)• For the QIT, the electric field has to be considered in 3 dimensions. The electric field can be descibed by the expression:

Φx,y,z = Φ0(r2 - 2z2)r0

2

• The Mathieu equation still applies:

• The equations of ion motion in the r and z direction are:d²z/dt² - (4e/mr0²) [(U - V cos2ωt)z = 0d²r/dt² + (2e/mr0²) [(U - V cos2ωt)r = 0

d ud

a q uu u

2

2 2 2 0ξ

ξ+ − =( cos )

41

Quadrupole 3D Ion Trap (QIT)•Solving these Mathieu type differential equations yields the parameters az and qz

az = -2a r = 16zeUm(r0² + 2z0

2)ω²

and qz = -2qr = -8zeVm(r0² + 2z0

2)ω²

Where ω = 2πf, f = fundamental Rf frequency of the trap (~1MHz)

42

QIT (Ion stability diagram)

courtesy of Spektrum Akademischer Verlag

Ring Electrode

Ring Electrode

Endcap

Endcap

q = 0.908

q < 0.908

43

QIT (stability diagram)

• Ions are only stable both in r and z direction for certain defined values of a and q

• Ions oscillate with so called “secular frequency”, f, which differs from the frequency of applied Rf field because of inertia (in addition oscillations of higher order)

• Ions of different m/z are simultaneously trapped, V determines low mass cut-off at qz = 0.908, which increases with V

44

QIT (mass selective ion stability scan)

• Mass scan is possible by increasing the amplitude of the voltage on the ring electrode (U = 0, az = 0 ie no DC voltage)

• Scan line: While scanning along this line (a=0) ions become increasingly non stable and exit the stability diagram at qz = 0.908.

• Trajectory of these ions in z- direction.

• Ions exit from trap through holes in end cap.

• Linear scan function

47

Space Charging

530m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

524.3

525.3

526.3

5300

20

40

60

80

100524.4

525.4

526.3527.5

5300

20

40

60

80

100524.5

525.5

526.5

527.5

5300

20

40

60

80

100 524.8

525.7

526.7

522 522 522 522

~ 300 Ions ~ 1500 Ions ~ 3000 Ions ~ 6000 Ions

Good resolutionand mass accuracy

Poor resolutionand mass accuracy

Courtesy of Agilent

48

QIT (space charge)

• With increasing number of ions trapped the space charge increases

• Space charge distorts the electric field

• Deterioration of resolution, sensitivity and mass accuracy

Solution:

Pre-scan or measure in real time to control the number of ions (or more correctly, the number of charges)

in the trap (a maximum of ~103 - 104)

49

Linear (2D) traps

• Similar idea to 3D traps with a “new” 2D geometry

• Rf only quads with DC voltage end electrodes

• Larger size than 3D IT – higher ion capacity (~x50) therefore fewer space charge problems

• More than one design for this type of trapping instrument

• Hybrids such as QQQ where Q3 can also be used as a linear trap and LT-FTICR

50

AxialTrapping

Exit Lens

Radial Trapping RF Voltage

Radial Trapping RF Voltage

AxialTrapping

DCVoltage

Resonance Excitation

Trapping Forces in a Linear Ion Trap

Courtesy of Sciex

52

Linear Ion Trap vs 3D Trap

No low mass cut-off

Trapping Efficiency: >10

Detection Efficiency: doubled

Overall Efficiency: >10

Ion Capacity (Spectral): >20

Scan Rate (amu/sec): 4x

Highly Efficient MSn: 5x over 3D IT

Electric and MagneOc Sector Mass Analyzer

The ‘original’ commercial MS systems

High resoluOon, but comes at the cost of sensiOvity

detector!

5

m/z = eB2r2/2V

• Therefore specific values of V and B allow ions unique in m/z to pass to the detector. Variations in V or B will cause ions to collide with the walls of the flight tube therefore at any unique value of V or B only one specific ion will be passed to the detector. In practice only B scans are preferrred when generating full scan data over a large (>50Da) mass range

• One exception to this is when high resolution, accurate mass measurements are made where Vacc scanning is preferred as voltages can be controlled and measured much more accuartely than can B

Mass Separation: Magnetic Fields

7

Deflection of ions of different masses in a constant magnetic field

•This is how Aston’s original mass spectrograph operated!• In modern instruments, the magnetic field is scanned to bring ionsof different m/z ratios successively to the detector

8

Directional (angular) focusing of a magnetic field

Divergent ions of the same m/z will be brought into focus by a magnetic field

9

Mass Separation: Magnetic Fields

• One significant drawback with employing B scans is that the initially accelerated ions have a kinetic energy spread which exhibits itself as increased peak width ie low resolution.• To overcome this problem an electric sector (ESA) is combined with the magnetic sector to produce what is called a double focusing instrument.

19

“New” Developments in Magnetic Sector Instruments

• Large, high field magnets

– Mass range up to 10,000 Da at full accelerating potential (10 kV) for analysis of large biopolymers

– Example: bovine insulin (MW 5734)

• Laminated magnets– To reduce magnetic hysteresis– Total cycle time < 1 sec, fast scanning

8

C15

C9 C21

Analysis of diesel fuel sample 1:1000 in DCM, 60m DB5 column

Petroleum Hydrocarbons

Analysis of diesel fuel: •  typically by GC or 2D GC-‐(TOF)MS

•  what other compounds can be detected with ultra high resoluKon mass spectrometry?

9

Zoom

Diesel 1:1000 in MeOH + 0.2% formic acid

10

Zoom

Analysis of diesel fuel sample

11

Zoom


12

C17H34ONa+1 (0.03 ppm)

C16H30O2Na+1 (0.00 ppm)

C15H26O3Na+1 (0.28 ppm)

C14H22O4Na+1 (0.11 ppm)

C13H18O5Na+1 (0.18 ppm)

C17H18O2Na+1 (0.00 ppm)

C13H18O4K+1 (0.11 ppm)

C15H26O2K+1 (0.14 ppm)

C18H22ONa+1 (0.40 ppm)

C18H22K+1 (0.32 ppm)


13

110,000 FWHM

2048K data points 29,000 FWHM



32K data points

C17H18O2Na+1 (0.00 ppm)


14

110,000 FWHM




32K data points


15

110,000 FWHM




32K data points


16

110,000 FWHM




32K data points


10 mass separation - eth z · mass separation and the lorentzforce note: • all mass analyzers...

Documents