The way to ionize a

compound

and

MS Configurations

Mass Spectrometer
Schematic

Mass Spectrometer Schematic

Atmospheric Pressure
Ionization

•

ESI (Electrospray ionization)

•

APCI (Chemical ionization)

•

APPI (Photo ionization)

Positive / Negative Modes

2.5

5.0

min

0.0e6

5.0e6

10.0e6

Int.

2:TIC(1.00)

1:TIC(1.00)

m/z280~600, +/-; Scan speed: 1000

amu/s

S/N = 124

2.5

5.0

min

0.0e6

5.0e6

10.0e6

Int.

TIC(1.00)

m/z280~600, + only/ Scan speed: 500

amu/s

S/N = 136

22.0 22.5 23.0 23.5 24.0 24.5 25.0 25.5 26.0 26.5 27.0 27.5

min

500e3

1000e3

1500e3

2000e3

2500e3

3000e3

3500e3

4000e3

4500e3

5000e3

5500e3

6000e3

6500e3

Int.

2:TIC(1.00)

1:TIC(1.00)

-

negative scan

-

positive scan

P1

Peak

P1

could not be detected in positive ion mode

MS

analyze

r

vacuum

HPLC

N

2

gas

N

2

nebulizer

3-5 kV

Electrospray Ionization
(ESI)

Electrospray (ESI)

•

The mechanism for ion generation in electrospray mass

spectrometry was first announced by the work of Fenn and

Dole, although the original publication on electrospray

dates back to 1917 from the work by Zeleny.

•

The principal outcome of the electrospray process is the

transfer of analyte species, generally ionized in the

condensed phase, into the gas phase as isolated entities.

Nanospray

Online analysis

~ 20 µm tip ID

Interface with nanoLC

Flow rate: ~300nL/min

Offline analysis (static infusion)

~ 2 µm tip ID

Flow rate: ~40nL/min

Requires pure sample free from salt

New Objective, Inc.

⊿

5

⊿

16

⊿

17

⊿

18

⊿

14

⊿

９

(M+H)

+

MH

+0

(M+NH

4

)

+

MH

+17

(M+Na)

+

MH

+22

(M+K)

+

MH

+38

(M+H+H

2

O

)

+

MH

+18

(M+H+MeOH

)

+

MH

+32

(M+H+CH

3

CN

)

+

MH

+ ９９

Molecular Ions Often Observed
in ESI-MS

NH

O

HN

R

2

H

N

R

1

H

3

C

O

N

NH

OCH

3

CH

3

CH

3

O

CH

3

CH

2

COOH

CH

3

COOH

H

3

C

O

O

Protonated molecule

(M+H)

+

Molecule with added sodium ion (M+Na)

+

COOH → COONa
1x COONa (M-H+Na+H)

+

= (M+Na)

+

2x COONa (M-H+Na-H+Na+H)

+

= (M-H+2Na)

+

•

multiply charged ions can be generated
with
ESI interface if the chemistry fits

•

hardware mass range (- 2000 m/z) is
mathematically extended up to 100.000
amu

•

acquisition has to be in profile mode

•

following deconvolution

Multiply charged ions

Electrospray Mass Spectrum
of Bovine Ubiquitin

700

800

900

1000

1100

m/z

779.44

+11

856.9681

857.47

+10

952.63

+9

714.72

+12

659.75

+13

857

858

+0.1

+0.2

+0.3

+0.4

+0.5

+0.6

+0.7

+0.9

+0.8

+1.0

M

theo

=8559.611

2
M

exp

=8559.603

Z=+10

858.5

857.5

Charge State Determination

High Resolution

– isotope peaks resolved

(1) counting isotope peaks in ONE m/z unit
(2) if the measured spacing of neighboring isotopes is (m/z),

z=1/ (m/z) or more accurately z=1.00235/(m/z)

1.00235 is the average isotope spacing

Low Resolution

- isotope peaks are not resolved

Use neighboring charge states (m/z)

1

[higher charge] and (m/z)

2

[lower charge, higher m/z]

Solve the following linear equations

for z (for (m/z)

1

) and M

(neutral mass)

(m/z)

1

Xz – z =M

(m/z)

2

X(z-1) – (z-1) =M

Deconvolution

750

1000

1250

1500

1750

m/z

0e3

100e3

200e3

300e3

400e3

500e3

600e3

Int.

1131

1060

1212

998

1305

943

1414

893

1542

849

1696

1136

1418

1064

ESI mass spectrum of myoglobin

16800

16900

17000

Mas s

0.0e6

1.0e6

2.0e6

3.0e6

4.0e6

Int.

16953.2

+10

+12

+14

+20

Multiply charged ion

deconvolution

Advantages of ESI

•

High sensitivity to polar compounds

•

Produces multiply charged ions

•

Concentration dependent technique

•

Well suited to reverse phase solvents

Critical factors in ESI

•

Solvation plays an important role on ionization

efficiency. Surface tension effects will affect

sensitivity [phosphates].

•

Ion suppression must be carefully considered;
levels of trifluroacetic acid, sample matrix

•

Adduct ion generation must also be accounted
for in interpretation and quantitation.

MS

analyze

r

vacuum

HPLC

N

2

gas

N

2

nebulizer

Corona discharge needle 3-5 kV

200-500°C

Atmospheric pressure
chemical ionisation (APCI)

Analyte containing
aerosol

Charged
reagent gas
formed

+

+

+

+

+

+

+

+

+

++

+

+

+

+

+

+

+

+

+

+

+

+

Vapor

Heat

+

Analyte ions

Charge
transfer to
analyte

+ + +

+

APCI Process

APCI Ionisation

•

The sample needs to be thermally stable to
a certain extent

•

A protonic solvent is necessary to generate
reactant ions

•

Under normal reverse phase column
conditions, methanol/water achieves higher
ionization efficiency than acetonitrile/water.

•

Typical APCI applications are:
pesticides, drugs, azo dyes, and
steroids

MS

analyze

r

vacuum

HPLC

N

2

gas

N

2

nebulizer

200-500°C

Atmospheric pressure
photo ionisation (APPI)

Lampe

UV

hn

APPI Ionisation

•

Allows high sensitivity analysis of compounds

that have low polarity

•

Allows high sensitivity analysis of Fusarium
toxins

•

Allows not only APPI, but also a simultaneous
APCI/APPI dual ionization mode

Theory of APPI (direct
APPI)

Molecular Weight

Non-Polar

100,000

Direct APPI

UV
(hν=10 or 10.6eV)

M+

+H

Protic solvent

Analyte

Analyte molecule M is ionized to a molecular ion M

+

.(If

analyte ionization potential is below photon energy.) In
the presence of protic solvent, M

+

may extract a

hydrogen atom to form MH

+

.

M+H

+

Theory of APPI (dopant
APPI)

Molecular Weight

Non-Polar

100,000

Dopant APPI

UV
(hν=10 or 10.6eV)

M+H

+

Analyte

Solvent

Dopant

(e.g. Toluene)

M+

+

e-

+H

M+

A photoionizable dopant is delivered in large concentration to
yield many D

+

ions. D

+

ionizes analyte M by proton or electron

transfer.

M+

APCI/APPI and ESI

APCI/APPI

ESI

mass

- ca. 1000-1500

- up to 100.000

flow

50 µl - 2 mL/min

1µL - 1mL/min

analyt

unpolar

polar

solvent

unpolare solvent possible

buffer

high tolerance

N

2

< 2.5 L/min

< 1.5 L/min

Probe

ESI

APCI

APPI

Ionization

Ion

evaporation

Chemical

ionization

Photoionization

Sample

Stability

Many sample

(Soft

ionization)

Not good for heat

unstable

compounds

Not good for heat

unstable

compounds

Sample

Polarity

Polar

compounds

Middle polar

compounds

Less polar

compounds

Typical Mass Spectra of ESI-
MS

50

100

150

200

250

300

350

400

m/z

0e3

10e3

20e3

30e3

40e3

Int.

377

399

Riboflavin C

17

H

20

N

4

O

6

Exact Mass: 376.14

50

75

100

125

150

175

200

225

250

m/z

0

250

500

750

Int.

242

213

170

156

43

256

60

185

103

77

116

198

91

130

143

(a) EI spectrum of riboflavin (vitamine
B2)

N

N

NH

N

O

O

OH

OH

HO

OH

[M+H]

+

[M+Na]

+

(b) API (ESI) spectrum of
riboflavin

Dual source (ESI/APCI)

Dual ion source

heated drying gas

Compounds Analyzed by
LC/MS

Properties of compounds: a certain polarity is

essential

 With and without UV-vis absorption
 Thermally stable and labile
 Less and non-volatile
 Less polar to ionic compound
 Small to large molecules

Types of compounds:



Synthetic drugs, metabolites



Natural compounds (alkaloids, glycosides,

taxanes,
toxins, saccharides, vitamins, lipids etc)



Peptides, proteins



Non-volatile pesticides, herbicides etc



Surfactants, dyes and various organic

additives

O

HO

O

O

OH

N

O

O

O

O

O

OH

MALDI

Matrix-Assisted Laser

Desorption/Ionization

N

O

H

O

OH

4-hydroxy-picolinic acid

m = 139.05 Da

NH

2

OH

O

anthracinic acid

m = 137.05 Da

negative mode

OH

O

OH

N

-cyano-4-hydroxy-

cinnamic acid (CHCA)

m = 189.07 Da

O

H

OH

OH

O

2,5-dihydroxy-

benzoic acid (DHB)

m = 154.03 Da

positive mode

OH

O

O

H

O

O

sinapic acid (SIA)

m = 224.07 Da

small, aromatic,

organic acids:

UV-absorbing,

proton donor

Common matrices for MALDI-TOF
MS

MALDI sample
preparation

addition of 0.3 µl Matrix solution

MALDI

•Analyte is dissolved in solution with excess matrix (>10

4

).

•Sample/matrix mixture is dried on a target and placed in the MS
vacuum.

Requirements for a satisfactory matrix:

•It must co-crystallize with typical analyte molecules
•It must absorb radiation at the wavelength of the laser
(usually 337 nm)

•To transfer protons to the analyte it should be acidic

Typical successful matrices for UV MALDI are aromatic carboxylic

acids.

MALDI-TOF MS: basic principles

io n d e t e c to r

analyte molecules
incorporated in
matrix crystals

in vacuum
< 10

-7

Torr

io n d e t e c to r

+

+

laser pulse:
desorption of matrix and
analyte molecules,
ionization by charge
transfer

MALDI-TOF MS: basic principles

io n d e t e c to r

+

+

+

+

build-up of an
electromagnetic
field

acceleration of ions

MALDI-TOF MS: basic principles

io n d e t e c to r

+

+

+

+

separation of ions in a
field free drift range of
a fixed length by
velocity
(Time Of Flight)
L 1 m

no further
acceleration!

MALDI-TOF MS: basic principles

io n d e te c to r

+

+

+

+

mass-independent detection
of ions at a detector

time resolved output to
oscilloscope and computer

MALDI-TOF MS: basic principles

38

MALDI-TOF MS:

Matrix Assisted Laser Desorption/Ionization

Time-of-Flight Mass Spectrometry

ion detector

+

+

+

+

+

+

template

matrix/analyte

crystals

acceleration

zone

field-free

drift range

grid electrode

desorption

ionization

acceleration

separation

detection

m

z =

2eU

L²

t²

m: mass

z: charge

U: accelaration voltage

L: path length

t: time

e: elemetary charge

Ionization Methods for
Biomolecule Analysis

MALDI

•Very long sample lifetime;

repeated measurements
possible

•Good for mixtures
•Matrix peaks can interfere at

MW <600

•Salt tolerant
•Low maintenance
•Generate ions with few

charges

Electrospray

•Online LC/MS

possible

•Poor for mixtures

without LC

•Quantitation

possible

•Good for MW

<600

•Generate highly

charged ions

polarity

Non-Polar

Very polar

Molecul
ar
Weight

10,000

1,000

100

ESI

APCI

APPI

100,000

Mass spectrometry

MALDI

GCMS

LCMS

MALDI TOF MS

EI/ CI

MS configurations

Different types of MS

Aebersold and Mann (2003) Nature 422, 198-207

Single quadrupole MS

analyzer

Sourc

e

Detector

Nonresonant
Ion

Resonant
Ion

DC and RF
voltages

Quadrupole Mass Analyzer

Just RF applied Ion Transmission
Device

DC and RF applied Mass Analyzer

Ion Optics

A device for manipulating ion beams.
A mass spectrometer consists of many ion optical
components

Uses a combination of RF and DC voltages to operate as a mass

filter.

•

Mass analyzer.

•

Mass selection device

•

Ion transport device (RF-only collision cell).

Quadrupole Mass
Analyzer/Filter

+ U + V cos t

-U - V cos t

Mass scan and stability diagram

Working mass range: 2 to 2000 m/z

Resolution: ~ 2*m (a mass of 200 can be

analyzed with a resolution of 400 FWHM)

Linearity for quantitation: dynamic range

~ 10

5

Mass filter

• Figure A) A light ion will be dragged a large distance by the alternating field, and will find

itself in stronger and stronger regions of field. It will quickly collide with an electrode and

disappear

• Figure B) A very heavy ion will not be affected much by the alternating field, but will

gradually drift in the constant part of the field (the DC part). The alternating field is not strong

enough to drag it back as it wanders, so it also collides with an electrode, and is lost.

• Figure C) An ion that is the right weight drifts slightly in the constant part of the field, but is

always dragged back by the alternating part. The alternating part, however, is not quite strong

enough to make it spiral out of control into an electrode. Thus an ion just the right size is

stable in this quadrupole field and reaches the end, where it can be measured.

Quadrupoles theory and operation

•

By gradually increasing U and V during an experiment (i.e.
during a scan), ions with different m/z ratios can be made to
make contact with the rods or can be made to possess
stable trajectories through the quadrupole assembly

•

Only ions possessing stable trajectories through the entire
quadrupole assembly will pass through the quadrupole and
be detected

•

Generally, ions of different m/z ratios are sequentially
transmitted through the quadrupole during a scan to obtain
a mass spectrum

Quadrupoles - modes of operation

There are 2 main modes of quadrupole operation

:

1. Scanning

- the potentials applied to the quadrupole rods are

gradually increased such that ions between a

user defined m/z range sequentially possess

stable trajectories and are detected

2. Selected ion monitoring (SIM)

- The potentials applied to the quadrupole rods are

such that only ions of a specific m/z or a very

narrow range possess stable trajectories

SQ-mass analyzer

Quadrupoles - modes of operation

SIM offers increased sensitivity compared with scanning
experiments as a result of the increased (100%) ‘duty
cycle’ of a SIM experiment and reduced noise

•

in a scanning experiment, the quadrupole is
scanned across a m/z range and therefore ions of a
particular m/z only have stable trajectories for a
fraction of the total scan time

•

in SIM mode, ions of the specified m/z have stable
trajectories 100% of the scan time and therefore
more ions are detected, and hence sensitivity is
increased

Coelution

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

11.0

12.0

13.0

14.0

mi n

0.001

0.001

0.002

0.002

0.003

0.003

0.004

0.004

0.004

0.005

0.005

0.006

0.006

0.007

0.007

0.008

0.009

0.009

0.009

0.010

0.011

0.011

0.012

0.012

0.013

mAU( x1,000)

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

50.0

55.0

60.0

65.0

70.0

75.0

80.0

85.0

90.0

95.0

bar

B .Pre ss .(S ta t us)

A.Pre ss.( S t a tus)

Extr ac t -230nm,4nm ( 1.00)

Flavonoids - PDA chromatogram @230nm

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

11.0

12.0

13.0

14.0

15.0

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

11.0

(x1,000,000)

609.00 (1.13)

579.05 (1.11)

595.00 (1.00)

TIC

1

2

3

4

5

MS chromatogram

TIC

S

I

M

P2

MS Spectra Peak 2 - Naringin

100

150

200

250

300

350

400

450

500

550

600

650

700

750

m/z

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

2.50

2.75

3.00

Inte n.

( x1,000,000)

579.05

615.00

497.20

661.70

270.90

370.80

173.95

676.85

748.85

461.85

795.20

429.85

292.30 316.75

714.90

119.00

406.60

221.90

556.65

Naringin (MW=578)

Difficult Matrix Background
of Plasma Simplified with MS
Detection

0.0

1.0

2.0

3.0

4.0

5.0

6.0

0.0

2.5

5.0

7.5

10.0

(x100,000)

VER:m/z 455.0 (×2)

PRO:m/z 331.9 (×6)

ALB:m/z 266.0 (×60)

PIR:m/z 260.0 (×4)

ANT:m/z 189.0 (×6)

A

LB

V

E

R

P

R

O

P

IR

A

N

T

MS chromatogram

min.

Int.

0.0

1.0

2.0

3.0

4.0

5.0

min.

-2.0

-1.0

0.0

1.0

2.0

3.0

4.0

mAU

(x10)

254nm,4nm (1.00)

6.0

Int.

PDA chromatogram

A

LB

V

E

R

P

R

O

P

IR

A

N

T

ANT:Antipyrine
PIR:Piroxicam
ALB:Albedazole
PRO:Propraolol
VER:Verapamil

High throughput

• Shorten the run time
without
sacrificing the
chromatografic
resolution

• Higher throughput

• Less solvent

UV chromatogram

MS chromatogram

0

5

10

15

20 min

0

5

10

15 min

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

mi
n

1

2

3

4

5

6

Column

: STR ODS-Ⅱ ( 2.0 × 150mm )

Mobile phase : 0.1% formic acid / methanol, gradient
elution
Flow rate : 0.2 mL / min
Ionization mode : ESI (+)

N

+

R

1

R

2

R

3

R

4

N

+

CH

3

O

H

O

CH

3

CH

3

H

O

CH

3

O

H

CH

3

M

W

R

1

R

2

R

3

R

4

M

agnoflorine M

W

341

Berberine

335

-O

-C

H

2

-O

-

O

CH

3

O

CH

3

E

piberberine

335

O

C

H

3

O

CH

3

-O

-CH

2

-O

-

C

optisine

319

-O

-C

H

2

-O

-

-O

-CH

2

-O

-

Jateorrhizine

337

O

H

O

CH

3

O

CH

3

O

CH

3

Palm

atine

351

O

C

H

3

O

CH

3

O

CH

3

O

CH

3

Qualitative Analysis of
Alkaloids

in Coptis Rhizome

Total Ion Chromatogram

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

min

Intensity

TIC

TIC

336.00

352.00

338

.00

.00

320.00

342.00

1: Magnoflorine

2:
Coptisine

3:
Epiberberine

4:
Jateorrhizine

5: Berberine

6: Palmatine

1

3

4

2

5

6

Mass Chromatograms

100

200

300

400

m/z

0e3

100e3

Int.

342.2

297.1

138.9 201.4

371.4413.6 479.6

Magnoflorine

100

200

300

400

m/z

0e3

50e3

Int.

352.2

139.1 196.3

393.4

335.6

279.6

Palmatine

100

200

300

400

m/z

0e3

100e3

200e3

Int.

338.1

323.4

196.1

465.4

117.1

283.1

Jateorrhizine

100

200

300

400

m/z

0e3

250e3

500e3

750e3

Int.

320.2

447.4

336.1

214.1

158.1

391.3

Coptisine

100

200

300

400

m/z

0e3

250e3

Int.

336.2

322.2

158.2 214.1

464.6

413.5

Epiberberine

100

200

300

400

m/z

0e3

25e3

Int.

336.2

452.2

177.0

371.4

324.6

261.0

416.9

Berberine

Mass Spectrum

Triple quad. MS analyzer

Triple Quadrupole MS

CID cell

Collision Induced Dissociation

Functions of the rod assemblies in
different scan modes

Note:
Scan

a

:

Full scan or transmission of selected ions

Pass all ions

b

or fragments:

Pass ions or fragments within a wide range of mass-

to-charge ratios

Fragment ions

c

:

Collisions with argon gas cause ions to fragment

Set

d

:

Set to pass ions of a single mass-to-charge ratio or set of mass-to-charge ratios

Product Ion Scan

Precursor Ion Scan

Neutral loss scan

SIM and SRM

MS1

Set

MS2

Set

CID

Collision Cell
RF only + Ar

M3

+

M5

+

M1

+

M2

+

M3

+

M4

+

M5

+

M1

+

M2

+

M3

+

M4

+

M3

+

m3

+

m2

+

m2

+

m1

+

SRM – Selected Reaction Monitoring

SIM – Selected Ion Monitoring

Feature of SRM

Better signal to noise ratio than SIM. However, the
absolute sensitivity of sample is poorer than SIM.
Can increase confidence by using MS/MS
fragmentation information.

Time of Flight MS analyzer

detector

reflectron

TOF

defined starting point

Reflector Time of Flight (TOF)
MS separation

+

+

+

+

+

io

n d

ete

cto

r

Linear

time-of-flight mass spectrometer

Reflector

time-of-flight mass spectrometer

m

1

= m

2

v

1

< v

2

l

mass range up to 350 kDa

l

high sensitivity

l

low resolution

l

mass range up to 5000

Da

l

low sensitivity

l

high resolution

Linear and Reflector modes

Reflector compensates for initial variation in kinetic energy,
improving resolving power and mass accuracy.

ESI-TOF

Agilent ESI MSD TOF

Bruker MicrO-TOF

ESI-TOF schematic

detector

reflectron

TOF

Pusher

electrode

Ion stream

ESI-TOF

Positive features :

•

High mass range

•

High resolution: Possible to obtain more than 10.000 FWHM resolution

•

High scan speed

•

High mass accuracy

Limitations :

•

No MS/MS

•

no structural information

•

limited suitable for quantitation: The dynamic range is limited due

to
pulsed mode of operation and small dynamic range of detector
electronics.

•

As m/z value becomes large, it becomes difficult to discriminate

between times of arrival at the detector.

Ion trap MS analyzer

Quadrupole Ion Trap

•Uses a combination of

DC and RF fields to
trap ions

•Ions are sequentially

ejected by scanning
the RF voltage

Linear Trap

• Essentially a quadrupole with end-caps

• Advantage: Larger ion storage capacity, leading to better dynamic range

Ions in

(from ESI)

3D Trap

End caps

Ions out

to detector

Ring electrode
(~V)

Insulated

spacer

He gas

1x10

-3

Torr

Trap filling

Ion traps

Ion trap

By ramping the RF voltage, or by applying supplementary

voltages on the end cap electrodes,
or by combination of both, it is possible to:

•

destabilize the ions and eject them progressively from the

trap (mass analysis)

•

keep only one ion in the trap, fragment it by inducing

vibrations, and observe the fragments (MS/MS

experiment)

•

repeat the last operation a few times to progressively

fragment the ions (MSn experiment)

Quadrupole Ion Traps

End cap electrodes

Ring electrode

Benefits

• High sensitivity

• Multi-stage mass spectrometry (MS

n

analogous to FTICR experiments)

• Compact mass analyzer

1. Low ion mass cut of

2. space-charge efects: too many ions in the trap distort
the electric fields, leading to significantly impaired
performance.

Quadrupole Ion Traps

Multistage fragmentation

MS

4

Experiment

Positive features :

• MSn experiment, multiple levels of fragmentation is possible.

• Instrument for identifying an unknown chemical from a finger print of fragments

• Suitable for structure elucidation about complicated compounds

• Suitable for the detection and quantitation of very subtle target
compounds in complicated matrix.

Limitations :

• The resolution and the performance in an ion trap are depending upon the
charge density of the ions in the trap. If too many ions at the same time in an ion
trap, the electrical fields are destorted by inductive effects. Also, collision
between the ions may occur, leading to unexpected dissociation or chemical
reactions. In this case the spectra and the quantitation will be impacted.

• Low resolution and mass accuracy

• narrow dynamic range of quantitation

Ion Trap

MSn

Q-TOF MS analyzer

Q-TOF

5x10

-7

Torr

Liner

Ion mirror

Ion

detecto

r

Pusher

Puller

Q

1

q

2

MS1 mass

selection

CID

Ion focus

&

transfer

q

0

N

2

ESI

spray tip

Roughing

pump

Turbo

pump

High

vacuum

High

vacuum

High

vacuum

Collision gas

MS2 mass

analysis

Ion

formatio

n

Q-TOF

•

The Q-TOF is a tandem mass spectrometer

(MS/MS) with two analysers

•

the first being a quadrupole analyser that is

used as an ion guide in MS mode, but as a

resolving analyser in MS/MS mode.

•

the second analyser is a reflectron time-of-flight

analyser placed orthogonally to the quadrupole.

•

the final detector is a microchannel plate

detector for high sensitivity.

Q-TOF mass analyser

Positive features :

• Proteome analysis (Mainly for qualitative analysis)

• Popular instruments for high-quality small molecule
work

• Required ions can be selected easily and efficiently
and very accurately.

• Accurate mass measurement

Limitations :

• No MSn capability

• Accurate mass mainly only in MS

• Mass calibration drifts over time

Orbitrap Technology

Image Current Detection
in Orbitrap

From Alexander Makarov’s 2008 ASMS Award Address

• 3D electric field trapping

• No need for magnet

• Easy access

• Final detection device

Orbitrap

TOF

•Simultaneous
excitation

FTICR

• Confined ion trajectory

• Image current detection

• Fourier transform data
conversion

Unique to Orbitrap

• 3D electric field trapping

• No need for magnet

• Easy access

• Final detection device

Orbitrap function

Orbitrap Discovery (Since ASMS 2007)

Orbitrap XL (Since ASMS 2007)

Orbitrap cycle time

Orbitrap Cycle time

Orbitrap Exactive

(2008)

Orbitrap mass analyser

Positive features :

• high mass accuracy

• very high resolution

• popular instruments

• easy to use software

Limitations :

• slow data acquisition or much lower performance

• Accurate mass mainly only in MS for faster analysis

• price

FT-ICR-MS

Technology

Fourier Transform Ion Cyclotron Resonance
(FT-ICR)

•Ions trapped and
measured in ultrahigh vacuum
inside a superconducting magnet.

B

0

Detect

+

+

+

++ +

+

+

+

R

C

Excite

+

+

+ + + +

+

+

+

z

m

1





Comparison

Instrument

ESI-TOF

QqQ

Ion Trap Q-q-TOF LCMS-IT-

TOF

Orbitrap

/
FT MS

MS function

MS

MS/M

S

MSn

MS/MS

MS

n

MS

n

Mass accuracy

MS

3 ppm

0,0

1

5

100

ppm
0,

1

5

100

ppm

0,

1

5

3 ppm

0,0

1

5

5 ppm

0,0

1

5

*

< 1-2

ppm

0,00

1

5

*

Mass
resolution

10,000

-

40.000

4,000

(Delta:

0.5

amu)

4,000

(Delta:

0.5

amu)

10,000

-

60.000

10,000

60,000/

>100,00

0

Quantification

+

+++

++

+

+

++

Dynamic

Range

+++

++++

+++

++

++

+++

exact number

* also in MS

n

mode

Hybrid/Tandem Instruments

•Combine (1) ion selection, (2) ion dissociation, and (3) mass analyzer
devices

•Quadrupoles and ion traps good for selective isolation of
precursor ions and for fragmentation (required for MSMS - Topic of
Lecture 2)

•Reflectron TOF, FT-ICR, and OrbiTrap have higher mass accuracy
and resolving power (high mass accuracy is good for identification
– Lecture 3)

Ion Dissociation

•Collision Induced Dissociation (CID or Collision Activated
Dissociation (CAD)

ion traps: off-resonance excitation
rf-only multi-poles: higher kinetic energy (HCD) and
cascaded CID
TOF/TOF: single collision

•Electron capture dissociation (ECD) and Electron transfer
dissociation (ETD)

ECD: FTICR, reagent: electron
ETD: ion traps, reagent: free radical anion

Other important factors to consider: how product ions are
collected and detected

Data Dependent Acquisition

•Data Dependent Scans

MSMS based on intensity ranking of precursor ions

•Dynamic Exclusion

Precursor m/z of previous MSMS are memorized and no
MSMS done on them during a defined time period

•Automatic Gain Control (AGC, unique to ion trap)

Control how many ions are scanned – to achieve signal/noise
ratio and to minimize space charge effect

Detector

Digitally recording ion
arrival

While there is an exact instant when each ion

strikes the detector, it is difficult to transfer this
perfectly into the digital world.
There are two basic approaches used to
translate a detector signal into a digital
measurement:

- the analog-to-digital converter (ADC)
- time-to-digital (TDC)

It is well known TDC has dead-time problem

TDC vs ADC

TDC Vender

Model

Waters

LCT Premier
Q-TOF

Premier

Applied Bio

systems

QSTAR XL

AD

C

Vender

Model

Agilent

LC/MSD TOF

Bruker Daltonics

microTOF
microTOF Q

Shimadzu

LCMS-IT-TOF

TDC dead time causes shift to
shorter arrival times for higher
signal levels.

Ions per transient as a function of
sample amount, showing TDC
limitations.

Questions ????

Comparison of Analyzer Types

 

Ion Trap/
Quadrupol
e

TOF

OrbiTrap

FT-ICR

Sensitivity

+++

++

*

to +++

++

*

+

*

Mass
Accuracy

+

**

++

+++

+++

**

Resolving
Power

+**

++

+++

++++

**

Dynamic
Range

+ to +++**

++

++

++**

Upper m/z

+

++++

+++

++

*Sensitivity lowered due to losing ions on way to analyzer, rather than
inherent sensitivity.
**Can be improved by scanning narrower mass range or slower.