Topic 8 Mass Spectrometry

7/29/2019 Topic 8 Mass Spectrometry

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SKA6014

ADVANCED ANALYTICAL CHEMISTRY

TOPIC 9Mass Spectrometry 1

Azlan Kamari, PhD

Department of ChemistryFaculty of Science and Mathematics

Universiti Pendidikan Sultan Idris


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Mass Spectrometry

Mass Spectrometry (a.k.a. MS or mass spec) a method

of separating and analyzingionsby their mass-to-chargeratio

MS does not involve a specific region of the

electromagnetic spectrum (because it is not directly

interested in the energies of emitted photons, electronicor vibrational transitions, nuclear spin transitions, etc)

Ion

abundance

Up to m/z = 100000!m/z

Ion

IonIon


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General Notes on Atomic and Molecular Mass

Helpful units and conversions:

1 amu = 1 Da = 1/12 the mass of a neutral 12C atom.

1 kDa = 1000 amu

Atomic weights of other elements are defined bycomparison.

Mass-to-charge ratio (m/z): the ratio of the mass of an

ion (m) to its charge (z)

Molecular ion: an ion consisting of essentially the whole

molecule


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Mass Spectrometers

A block diagram of a generic mass spectrometer:

IonizationSource

MassAnalyzer

Detector


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Ionization Sources

Electron Ionization (EI)

Chemical Ionization (CI/APCI) Photo-ionization (APPI)

Electrospray (ESI)

Matrix-assisted Laser Desorption (MALDI)

Field Desorption (FD) Plasma Desorption (PD)

Fast atom bombardment (FAB)

High-temperature Plasma (ICP)

Desorption

Gas Phase

Ionization

SourceMass

AnalyzerDetector


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EI: Electron Ionization/Electron Impact

The electron ionization

(EI) source is designedto produce gaseous

ions for analysis.

EI, which was one of theearliest sources in wide

use for MS, usually

operates on vapors

(such as those elutingfrom a GC)

Heated Incandescent

Tungsten/Rhenium Filament

Accel!

E-

Vaporized

Molecules

70 eV

IonsTo

Mass

Analyzer


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How EI works:

Electrons are emitted froma filament made of

tungsten, rhenium, etc

They are accelerated by a

potential of 70 V

The electrons andmolecules cross (usually at

a right angle) and collide

The ions are primarly

singly-charged, positive

ions, that are extracted by asmall potential (5V) through

a slit


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When electrons hit the molecules undergo

rovibrational excitation (the mass of electrons is toosmall to really move the molecules)

About one in a million molecules undergo the reaction:

M+ e- M

+ + 2e-


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Advantages:

Results in complex mass spectra with fragment ions,

useful for structural identification

Disadvantages: Can produce too much fragmentation, leading to no

molecular ions! (makes structural identification

difficult!)


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CI: Chemical Ionization

Chemical ionization (CI) is a form of gas-phase

chemistry that is softer (less energetic) than EI

Ionization via proton transfer reactions

A gas (ex. methane, isobutane, ammonia) is introduced

into the source at ~1 torr.

Example: CH4 reagent gas

CH4EI

CH4+

CH4+ + CH4

CH5+ + CH3

AH + CH5+ AH2

+ + CH4

Strong acid


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APCI: Atmospheric-Pressure Chemical

Ionization

A form of API (atmospheric pressure ionization) arange of ionization techniques that operate at higher

pressures, outside the vaccuum MS regions.

APCI a form of chemical ionization using the liquideffluent in a spray chamber as the reagent


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APCI: Atmospheric-Pressure Chemical

Ionization The APCI process:

The sample is in a flowing stream of a carrier liquid (or gas)and is nebulized at moderate temperatures.

This stream is flowed past an ionizer which ionizes the carrier

gas/liquid.

63Ni beta-emitters

Corona (electric) discharge needle at several kV

The ionized stream (which can be an LC solvent) acts as the

primary reactant ions, forming secondary ions with the

analytes.

The ions are formed at AP in this process, and are sent into the

vaccuum

In the vaccuum, a free-jet expansion occurs to form a Mach

disk and strong adiabatic cooling occurs.

Cooling promotes the stability of analyte ions (soft

ionization)


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APCI: Chemical Ionization

APCI (diagram from Agilent)

Diagram from Agilent Technologies

760 torr

10-6 torr


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APCI: Chemical Ionization

An APCI mass spectrum:



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Electrospray Ionization (ESI)

The ESI process:

Electrospray ionization (ESI) is accomplished by flowing a

solution through an electrically-conductive capillary held at high

voltage (several keV DC).

The capillary faces a grid/plate held at 0 VDC.

The solution flows out of the capillary and feels the voltage

charges build up on nebulized droplets, which then begin to

evaporate

Coulombic explosions occur when the repulsion of the charges

overcomes the surface tension of the solution (holding the drop

together) known as the Rayleigh limit.

Depending on whose theory you believe

the analyte ion is eventually the only ion left

orthe analyte ion is evaporated from a small enough droplet


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A picture of two ideas for the electrospray process:

Diagram from John B. Fenn (Nobel Lecture), 2002Picture from http://www.newobjective.com/electrospray/electrospray.html

Noteions which are

surface-active will be

preferentially ionized

this can lead to ion

suppression!
http://www.newobjective.com/electrospray/electrospray.htmlhttp://www.newobjective.com/electrospray/electrospray.html


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An ESI source:



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Typical ESI Spectra

An ESI mass spectrum:



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Typical ESI Spectra

An ESI Mass Spectrum of a 14.4 kDa enzyme:

Diagram from http://www.nd.edu/~masspec/ions.html


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ESI and APCI

ESI and APCI complementary techniques:

Figure from Agilent Instruments


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ESI and APCI

ESI and APCIcomplementary techniques:

ESI APCI

Very soft ionization

can ionize thermally

labile samples

Some sample volatility

needed (nebulizer)

Ions formed in solution Ions formed in gas

phase

Singly- and multiply-charged ions [M+H]+

Singly-charged ions,[M+H]+ and [M-H]-


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Atmospheric Phase Photo-ionization

APPI can ionize things that ESI and APCI cant:


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Atmospheric Phase Photo-ionization

APPI can ionize things that ESI and APCI cant:


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Comparison of Ionization Methods

How to choose an ionization technique:

Figure from Agilent Instruments


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MALDI: Matrix-Assisted Laser

Desorption/Ionization

A method for desorbing

a sample with a laser,

while preventing thermal

degradation

A sample is mixed with a

radiation-absorbing

matrix used to help it

ionize

MALDI is mostly used for

large biomolecules and

polymers.


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MALDI: Matrix Effects The role of the matrix

Must absorb strongly at the laser wavelength

The analyte should preferably not absorb at this wavelength

Common matrices include nicotinic acid and many other

organic acids


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MALDI at Atmospheric Pressure

Advantages: fast, easy and sensitive

Disadvantages: no LC, matrix still needed

S. Moyer and R. Cotter, Atmospheric Pressure MALDI, Anal. Chem., 74, 468A-476A (2002)


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FAB: Fast Atom Bombardment

A soft ionization technique

Often used for polar, higher-mwt, thermally labile molecules

(masses up to 10 kDa) that are thermally labile.

Samples are atomized by bombardment with ~keV range Ar or

Xe atoms.

The atom beam is produced via an electron exchange process

from an ion gun.

Xee-

Xe+ + 2e-

Advantages:

Rapid sample heating reduced fragmentation

A glycerol solution matrix is often used to make it easier to

vaporize ions

Xe+accel

Xe+ (high KE)

Xe+ (high KE) + Xe Xe (high KE) + Xe+


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SIMS: Secondary Ion MS

Focused Ion Beam3He+, 16O+, 40Ar+

Beam energy 5 to 20 keV

Beam diameter 0.3 to 5 mm

Beam Hits Target

A small % of the target material is sputtered off and enters the

gas phase as ions (usually positive)

Advantages:

Imaging of ions (characteristic masses) on a surface or in

biological specimens

Surface analysis using beam penetration depth/angle

Can be used for bothatomicandmolecularanalysis

Sensitive to low levels, picogram, femtogram and lower


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Desorption Electrospray: DESI and DART

Desorption-

electrospray

ionization(DESI)

A new technique

for desorbingions using

supersonic jets

of solvents

(charged like inelectrospray)


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Inductively Coupled Plasma (ICP)

The inductively-coupled

plasma serves as anatomization and

ionization source (two-

in-one!) for elemental

studies.

Photo by Steve Kvech, http://www.cee.vt.edu/program_areas/environmental/teach/smprimer/icpms/icpms.htm#Argon%20Plasma/Sample%20Ionization

See optical electroniclecture for more details

Solution flow rates up to:

50-100 mL/min


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Mass Analyzers - Outline

Sector Mass Analyzers (Magnetic and Electrostatic)

Quadrupole Analyzers

Ion Traps

Ion Cyclotron Resonance

Time-of-Flight

and many more.

Ionization

Source

Mass

AnalyzerDetector


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Properties of Mass Analyzers

Resolution (R):

R = m/m

m = mass difference of two adjacent resolved peaks

(typically

m = mass of first peak or average

Example: R= 500 (low resolution)

resolves m/z=50 and 50.1, and m/z=500 and 501

Example: R= 150000 (high resolution)

resolves m/z=50 and 50.0003, and m/z=500 and

500.0033


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Sector Mass Analyzers

Basic Features

A sector: a geometrical construction

that has two arcs inside of one

another.

(Technically, a pie slice!)

Types:

Magnetic

Electrostatic Combination (e.g. double-focusing)


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Magnetic Sector Mass Analyzers

Ion kinetic energy:

V

erB

z

m

2

22

2

2

1

mvzeVT

BzeVFm

rmvFc

2

mc FF

Forces:

Only ions with equal

forces will pass:

Therefore:

Where:

Tis kinetic energyzis charge on ion

e is electron charge (1.60 x 10-19 C)

B is magnetic field (T)

v is velocity (m/s)

Vis the accelerating voltage

m is the mass


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Electrostatic Sector Mass Analyzers

2

v

reV

z

m

Therefore:

Ion kinetic energy:

221 mvzeVT

eVFm

rmvFc

2

Mc FF

Forces:

Only ions with equal

forces will pass:V

can be varied to bring ions ofdifferent KE (and different m/z

ratio to the exit)


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Double-Focusing Sector Mass Analyzers

If a batch of ions of equal

m/z but with differentkinetic energies enters a

magnetic sector

instrument, this will result

in a spread-out beam

Soution: minimize

directional and energy

differences between ions

of the same m/z.

Example of a double-

focusing MS: the Nier-

Johnson geometry


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Double-Focusing Sector Mass Analyzers

Another design, the Mattauch-Herzog geometry

This geometry is analogous to CCD-based opticalelectronic spectroscopy systems, while Nier-Johnson

instruments are similar in nature to traditional scanning

monochromator spectrometers.


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Time-Of-Flight (TOF) Mass Analyzers

The principle of Time-of-flight mass analysis:

A batch of ions is introduced into a chamber by anpulse of accelerating current.

This chamber has no fields, and is a drift tube

Since the ions have the same kinetic energy, their

velocities vary inversely with their mass during theirdrift.

Notes:

Typical flight times are 1-30 us Lighter ions arrive at the detector first

2

21 mvT


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Delayed extraction anything you can do to

tighten the KE spread will help a TOFinstrument

m/zis mass-to-charge ratio of the ion

Eis the extraction pulse potential (V)

s is the length of flight tube over which E is applied

dis the length of field free drift zone

tis the measured time-of-flight of the ion

zeEsmvT 221

2

2

v

eEs

z

m

2

2

d

t

eEsz

m

Ti Of Fli ht (TOF) M A l


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Time-Of-Flight (TOF) Mass Analyzers The reflectrona method of compensating for different ion KEs

Figure from http://www.abrf.org/ABRFNews/1997/June1997/jun97lennon.html


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The reflectrona method of compensating for different ion KEs

Figure from http://www.abrf.org/ABRFNews/1997/June1997/jun97lennon.html


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Quadrupole Mass Analyzers

The quadrupole (named for its electrical structure) is one of the

simplest and most effective mass spectrometers.



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How a quadrupole works:

Most important points:

It is easier for an applied AC field to deflecta light ion than a heavier ion

Conversely, it is easier for an AC field to

stabilize a light ion

Using this knowledge a combined AC/DC

potential is applied to the rods. Via the DC,the ion is attracted to one set of rods and

repelled by the other

The DC serves to stabilize heavy ions in one

direction (high pass filter). The AC serves to

stabilize light ions in the other direction (lowpass filter).

The ion must pass through the quadrupole to

make it to the detector


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Another view and the concept

of the mass scan

Images from http://www.jic.bbsrc.ac.uk/SERVICES/metabolomics/lcms/single1.htm

Light ion:

(ex. m/z = 100)

Dragged by AC

Heavy ion:

(ex. m/z = 500)

Dragged by DC

Just right:

Dragged by both,

But equally balanced

I T M A l


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Ion Trap Mass Analyzers

Ion trap: a device for

trapping ions and confiningthem for extended periods

using EM fields

Used as mass analyzers because

they can trap ions and eject themto a detector based on their mass.

Theory is based on Mattieus work

on 2nd order linear differential

equations (in the 1860s), and onWolfgang Pauls Nobel Prize

winning implementations


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The stability region of an

ion trap based ondifferential equations

22

0

8

mr

eU

az

22

0

4

mr

eVqz

)cos(0 tVU

Most ITMS systems dont

use DC (U), i.e. only qzis

controlled


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Layout of an ion trap mass analyzer:

Diagram courtesy of M. Olsen, GlaxoSmithKline

+

Main RF

Ring

Endcap

Lenses

Octopole

Optimized Asymptote Angle

End Cap

Shutter

Focus

Electron Multiplier

Conversion Dinode

Low Amplitude Dipole Field

(1/3 frequency of main RF)

++

++

++


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The BrukerEsquire ESI

ITMS - a typical

ion-trap LC-MS

system:

I C l t R


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FT-ICR:a FT-based mass spectral method that offers

higher S/N, better sensitivity andhigh resolution

Also contains a form of ion trap, but one in which ion

cyclotron resonance occurs.

When an ion travels through a strong magnetic field, itstarts circulating in a plane perpendicular to the field

with an angular frequency c:

m

zeB

r

vc



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How ICR works:

The ions are circulated in a field

An RF field is applied to match the cyclotron frequency of the ionsthis field brings them into phase coherence(forming ion packets)!

The image current is produced as these little packets of ions get

near the plates. The frequency of the image current is characteristic

of the ion packets m/z ratio.

http://www-methods.ch.cam.ac.uk/meth/ms/theory/fticr.html

Ion Cyclotron Resonance and Magnetic Field


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Ion Cyclotron Resonance and Magnetic Field

Parallels between NMR/EPR and ICR:

B

B= q B

m

=

B

Picture courtesy Prof. Alan Marshall, FSU/NHMFL


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The OrbitrapTM:A Hybrid Trap Between IT

and ICR

The Orbitrap is a recently developed electrostatic iontrap with FT/MS read-out of image current, coupled

with MS/MS

Advantages

Ease of use

Resolving power (superior to TOF)

Precision and accuracy

Versatility, dynamic range

A lower-resolution, more economical ICR

LTQ O bit h ti


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LTQ Orbitrap schematic

API Ion source Linear Ion Trap C-Trap

Orbitrap

Finnigan LTQ Linear Ion Trap

Differential pumping

Differential pumping

Image/animation from Thermo Electron Inc. See A. Makarov et al.,Anal. Chem.2006,78, 2113-2120.

LTQ Orbitrap Operation Principle


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LTQ Orbitrap Operation Principle

1. Ions are stored in the Linear Trap

2. . are axially ejected

3. . and trapped in the C-trap4. . they are squeezed into a small cloud and injected into the Orbitrap

5. . 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



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The axial oscillation frequency follows the formula

Where = oscillation frequencyk = instrumental constant

m/z = mass-to-charge ratio

zm

k

/

Frequencies and Masses

Many ions in the Orbitrap generate a complex

signal whose frequencies are determined using a

Fourier Transformation



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Multiple-Stage MS: MS-MS, and MSn

Also known as Tandem MS or MSn

Mass

Analyzer

Mass

Analyzer

Multiple quadrupoles are very common (e.g. triple-quad or

QQQ systems, EB for double-focusing, Q-TOF for quad time-

of-flight)

Why tandem MS? Because of the possibility of doing CIDcollisionally induced dissociation. Ions are allowed to collide

with a background gas (He) for several millliseconds, prior to

analysis. Allows for MSn experiments in an ion trap.

Comparison of Mass Analyzers


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Comparison of Mass Analyzers

A brief overview of the properties of common mass

analyzers

Analyzer Cost Scan speed Resolution

Double-

focusing

High Slow High

Quadrupole Low Medium Low-medium

Trap Low Medium Medium

TOF Medium Medium Medium-high

ICR High Fast High


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Detectors for Mass Spectrometry

Electron multipliers: like a

photomultiplier tube. Ions strike asurface, cause electron emission. Each

successive impact releases more

electrons.

Faraday Cups: Ions striking a cup cause

charge to flow across a load. The

potential across the load is monitored.

Ionization

Source

Mass

AnalyzerDetector

Figure from D. W. Koppenaal, et al.;Anal. Chem., 77; 2005, 418A-427A.

Detectors: Electron Multipliers


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Detectors: Electron Multipliers

Electron multiplier (EM): most common design in current

use

High gain (107), low noise, good dynamic range (104-106) Several designs:


D t t Oth


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Detectors: Others

Super-conducting tunner junction high mass

range, used with MALDI Can detect fmol of 150 kDa proteins

Can measure both energy and arrival time (2D MS

plots of m/z vs. kinetic energy)

Focal-plane array detectors/CCD

Like in electronic spectroscopy, much more

challenging to design for ion detection

Would combine well with mini-traps or other smallMS systems



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MS-Chromatography Interfaces

GC-MS: gas eluent from a column is piped directly to the

MS source

LC-MS: the ionization methods themselves serve as

interfaces techniques like ESI, APCI and APPI work on

liquid phase samples. The methods are generally

tolerant to RP LC solvents and some NP solvents.

Some buffers can quench ionization of analytes though:

Bad: Phosphate leaves a solid upon evaporation.

Also ionizes preferentially. Bad: any other non-volatile additives are also bad

Good: TFA, ammonium acetate, formic acid

Good: lower concentrations,

Topic 8 Mass Spectrometry

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