LCMS

Introduction

LCMS Group
Shimadzu Europa GmbH
Overview of LCMS
Ionization in LCMS
Vacuumsystem
Ion Optics
Quadrupole Mass Analyzer
Measuring modes
Mass Calibration
Detector
Keypoints for LCMS
Resolution
Signal to Noise
Overview of LCMS
 Overview of LCMS

Ionization- Mass-
Detector
source analyzer
 Overview of LCMS

Ionization source:
Ions are generated at atmospheric pressure

Mass-analyzer:
Ions are transferred from atmospheric pressure into
the vacuum chamber for mass analysis. Here they are
separated by the mass/charge (m/z) ratio.

Detector:
Ions are detected by Electron-Multiplier
 Overview of LCMS

Separation Lens- Mass

Ionization Desolvation Detector
(HPLC) system Analyzer
-Line

Analytes are separated in HPLC

and transferred to the Ionization
Vacuumsystem Control unit
source

Once in the ion source analyte-molecules are ionized to

produce an ion beam of charged molecular ions
PC
After passing the desolvation line, Ions are focused in the
lens-system, separated in m/z-ratio in mass-analyzer and
detected at the detector
Ionization in LCMS
 Ionization Process
What are Ions?

 Ions are positive or negative charged molecules.

Why to use ions and not uncharged molecules in MS?

 Uncharged molecules can not be affected by voltages. Only ions can be

effected by voltage settings. Neutral molecules cannot be measured.

How can molecules be ionized in LCMS?

 Molecules are ionized by using Atmospheric-Pressure-Ionization (API)

What does API mean?

 Molecules get ionized at atmospheric pressure, not in the vacuum like

in the GCMS.
 Ion generation

Why is the ionization done in atmospheric

pressure?

1.Better heat transfer leading to enhanced solvent

evaporation.

2.Better charge-transfer from ESI-pipe to the

solvent. Charge transfer would not occur so
successfully in vacuum.
 Ion generation

Different types of Atmospheric pressure ionization:

• ESI Electrospray ionization

• APCI Atmospheric pressure chemical ionization
• APPI Atmospheric pressure photo ionization
(only available for LCMS-IT-TOF)

The three techniques are different in the mechanism of

ion generation and consequently show different
response to different sample molecules.
 ESI
Electrospray Ionization
 ESI (Electrospray Ionization)
Principle of ESI:
The effect of the electrospray ionization process is to create
charged molecules, which are in gas-phase before they insert
the vacuum. (4-Step-pocedure).

1. ESI-pipe is charged (typically to 4,5 kV)

2. The high-electric field assisted by nebulizing gas creates a mist
of multiple charged droplets
3. During the way to the desolvation line, the droplets reduce their
size by evaporating their solvent. At a special surface/charge-
ratio a “coulomb explosion happens”
4. After the Coulomb explosion fully desolvated ions are created,
which are single or multiple charged
 ESI
To the Analyzer

Drying gas Drying gas

N2 (10-20L/min) N2 (10-20L/min)
Power supply
0,0 kV
Nebulizing gas
N2 (1-1,5 L/min)

4,5 kV
++++ + Coulomb
+ + + +
+ + ++
+++ + Explosion

Nebulizing gas
N2 (1-1,5 L/min)

1. Charge of ESI-pipe
2. Mist creation of multiple charged droplets
3. Minimizing surface/charge-ratio  Coulomb explosion
4. Fully desolvated single and multiple charges ions are created.
 Advantages of ESI

• Very effective to polar compounds

• Can produce multiply charged ions
 Peptide and protein-analysis is possible
• Concentration dependent technique
• Well suited to reverse phase solvents
• Soft ionization method (no fragments)
 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, eg.

concentration of trifluoro acetic acid, sample matrix

• Adduct ion generation must also be accounted for

interpretation and quantification.
 Additives in mobile phase

Most used additives:

1. Formic acid in water: HCOOH  HCOO- + H+

2. Acetic acid in water: H3CCOOH  H3COO- + H+

3. Ammonium formiate NH4+HCOO-  NH4+ + HCOO-

4. Ammonium acetate NH4+H3CCOO-  NH4+ + H3CCOO-

Not so often used additives:

1. Trifluoro acetic acid F3CCOOH  F3COO- + H+

2. Li-salts, Ag-salts (sometimes for polymeric analysis)

 to raise ESI ionization efficiency

Basic compounds:
 ionization works better in positive mode
 add acidic compound

M-NH2 + AH → [M-NH3]+ + A-
Example: A-=HCOO- AH=HCOOH (Formic acid)

Acidic compounds:
 ionization works better in negative mode
 add basic compound

M-COOH + B → [M-COO]- + BH+

Example: B=HCOO- BH=HCOOH (Ammoniumformiate)
 Adducts in LCMS positive mode

Specification-control-sample:
Papavarine:
Measurement mode: Positive

Monoisotopic: M m/z= 339,2

Possible ion-adducts:

Protonated [M+H]+ m/z= 340,2 Δm/z= 1

Sodium-adduct [M+Na]+ m/z= 362,1 (sometimes from glas) Δm/z=23
Potassium-adduct [M+K]+ m/z= 378,1 Δm/z=39
Ammonium-adduct [M+NH4]+ m/z= 357,2 Δm/z=18

Possible solvent-adducts:

Protonated water-adduct [M+H+H2O]+ m/z= 358,2 Δm/z=19

Protonated methanol-adduct [M+H+CH3OH]+ m/z= 372,2 Δm/z=33
Protonated acetonitrile-adduct [M+H+CH3CN]+ m/z= 381,2 Δm/z=42
 Adducts in LCMS negative mode

Specification-control-sample:
para-Nitrophenol:
Measurement mode: Negative

Monoisotopic: M m/z= 139,0

Possible ion-adducts:

Deprotonated [M-H]- m/z= 138,0 Δm/z= -1

Formiate adduct [M+HCOO]- m/z= 184,0 Δm/z=+45
Acetate adduct [M+CH3COO]- m/z= 198,0 Δm/z=+59

Possible solvent-adducts:

In 99,99% not visible in negative mode

 Multiply charged ions with ESI

• Multiply charged ions can be generated with ESI

interface if the chemistry fits, e.g. Peptides and
Proteins
• Mass range of MS (m/z=10-2000) is
mathematically extended up to 100.000 Da
• Acquisition has to be made in profile-mode and a
special deconvolution-software is used for
calculation
 Deconvolution
Int.

ESI mass spectrum of Myoglobin 4.0e6

3.0e6

16953.2
2.0e6

Multiply charged ions

Int. 1.0e6
1131
600e3 1060 1212+14

998 0.0e6
500e3
+13 16800 16900 17000 Mass
1305
943
400e3 +12
1414
893
300e3 Deconvolution
+20 +11
200e3
1542
849

100e3 +10
1696
1064 1136 1418 +9
0e3
750 1000 1250 1500 1750 m/z
 Deconvolution

Molecular weight of a compound : M

A and B are neighboring observed ions (A < B)

A : (n) charged
Intensit

B : (n-1) charged
y

A = (M + (n) x 1,0078) / n
B = (M + (n-1) x 1,0078 / (n-1)

M = (A – 1,0078 ) x (n)
m/z n = (B – 1,0078) / (B-A)

MW of Hydrogen: 1,0078 Da
 Multiply charged ions
Formula of Myoglobin: C769 H1213 N211 O217
M 16.950,5587; [M+H]+ 16.951,5667

[M+9H]9+ 1.884,4034 [M+15H] 15+ 1.131,0452

[M+10H]10+ 1.696,0639 [M+16H] 16+ 1.060,4179
[M+11H]11+ 1.541,9679 [M+17H] 17+ 998,0997
[M+12H]12+ 1.413,5545 [M+18H] 18+ 942,7057
[M+13H]13+ 1.304,8971 [M+19H] 19+ 893,1426
[M+14H]14+ 1.211,7622 [M+20H] 20+ 848,5359
 APCI
Atmospheric Pressure Chemical
Ionization
 APCI mechanism
Principle of APCI:
The Atmospheric Pressure Chemical Ionization process is to create
charged molecules, which are in gas-phase before they insert the vacuum.
(5-Step-pocedure).

1. Vaporizing the HPLC-flow in a heated tube

2. Creation of a heated gas cloud with solvent and analyte-molecules.
3. The heated cloud moves to the charged Corona-needle (up to 5kV) and is
transfered to a plasma status.
4. In the plasma the solvent molecules are singly charged.
5. Afterwards rapidly a charge-transfer between solvent- and analyte
molecules happens  analyte-molecules are singly charged
 APCI-mechanism
Drying and nebulizing gas (N2) To the Analyzer
Solvent (MeOH, ACN, …)
Drying gas
Analyte-molecule
Drying gas N2 (10-20L/min)
N2 (1-1,5 L/min)
Nebulizing gas

N2 (10-20L/min)

0,0 kV
Heating 500ºC Solvent
charge Charge Transfer

++
++
++
++
Heating 500ºC
4,5 kV
N2 (1-1,5 L/min)
Nebulizing gas

Power supply
 APCI: electric field

DL and Heat block

Corona-
Needle-Tip
5kV, 2-10µA

Point to plate discharge

 Charged solvent molecules
Step 3: Creation of charged solvent molecules (primary Ions):

Positive mode:

3 different groups of Ions can be created at the corona needle:

1. Gas-Ions: [N2]+ , [2N2]+ =[N4]+ ,[O2]+ and [NO]+

2. Solvent-Ions: [H3O]+ , [H2O]+ (water) and [CH3CNH]+ (acetonitrile)
3. Buffer-Ions: [NH4]+ (ammoniumacetate)

Negative mode:

3 different groups of Ions can be created at the corona needle:

1. Gas-Ions: [O2]-,[O]- and also adducts [O2]-[O2]n , [O2]-[H2O]n

2. Solvent-Ions: [OH]- (water), [CH3O]- (methanol)
3. Buffer-Ions: [CH3COO]- (ammoniumacetate)
 Positive APCI ionization
- Cascade-reaction time (from N2 to [H3O]+: 1µs (10-6 seconds)

- Proton hydrates [H3O]+ are highly charged

- Proton hydrates will react with sample molecules by Charge-Transfer

and Proton-Transfer to create Analyte-Ions
(Chemical Ionization)

- During the chemical Ionization small clusters could be

created with 4 solvent molecules and one analyte-molecule

- By using drying gas and heat in the desolvation line these clusters are
destroyed
 Reaction-mechanism in APCI

  
N2  e  N 2  2e
 
N 2  2N2  N 4  N2
 
N 4  H 2O  H 2O  2N 2
Positive mode

H 2O   H 2O  H 3O   OH 

2OH  H 2O2
H 2O2  H 2O  H 2

H 3O  M  M  H   H 2O
 
 Negative APCI ionization

- Mechanism different than in positive mode

- Corona-discharge produces electrons which become quickly

thermalized

- By collision with neutral species, they lose energy and are

then captured by electronegative species such as O2 to
form O2- and O-
 Reaction-mechanism in APCI

 
O2  e  O
2

O  n  H 2O  O  H 2O n
 
Negative mode

2 2


4 HO  3O2  2 H 2O
2

O  ( H 2O) n  M  M  H   HO
  
2 2
 Factors affecting APCI response

The parameters controlling the ionization effiziency in positive APCI are:

1.Gas phase basicity of the analyte M and
2.Hydration or solvation of the reagent H3O+ and protonated analyte MH+

H 3O   M  H 2O  MH  166 kcal/mol (700 kJ/mol)

- If the analyte-molecule has a higher gas-phase basicity than water (>166

kcal/mol) , the proton-transfer works exothermic.
- If the analyte-molecule has a lower gas-phase-basicity than water , the proton-
transfer does not work, because it would be an endothermic reaction.
- Rule: Analyte-molecules with a gas-phase-basicity of >200 kcal/mol could be
ionized very well. In this case the proton-Transfer is exothermic.

 APCI is a thermodynamic controlled ionization technique

 Gas phase basicity
Molecule Formula Gas-phase-basicity
[kcal/mol]
Water H2O 166,5

Methanol CH3OH 181,9

Acetonitrile CH3CN 188,0

Tetrahydrofuran C4H8O 198,1

Ammoniumformiat NH4+HCOO- 179,2

Ammoniumacetat NH4+CH3COO 191,2

N-Methylmorpholin C5H7NO 220,0

 Gas phase basicity
Target-molecule: Norbornanacetic-acid
Ionization: APCI, negative mode
Gas-phase-basicity: 191,2 kcal/mol

Solvents/buffer: Ammoniumformiate : 179,2 kcal/mol  no ionization

Ammoniumacetate: 191,2 kcal/mol  very bad ionization
N-Methylmorpholin: 220,0 kcal/mol  good ionization
 Critical factors in APCI

In case of co-elution, APCI creates the weaker acid, that

means, the thermodynamically more stable product-ion
APCI prefers the biggest Δenergy :

Example: H 3O+ (166,5 kcal/mol)

RNH2 (220,0 kcal/mol)
R 3N (230,0 kcal/mol)

 
H 3O  RNH 2  R3 N  R3 NH  H 2O  RNH 2

No ionization, because R3N has a higher gas-phase-basizity (230 instead of

220 kcal/mol)
 Critical factors in APCI

• Solvation plays an important role on ionization

efficiency.
• High temperature APCI often results in a higher
ionization efficiency. Especially with compounds
that have a low gas phase basicity
• High temperature APCI also generates a complex
chemical ionization condition that sometimes
produces fragment ions that are difficult to assign.
• Analytes must be temperature stable
• Not usable with non-volatile buffers!
 Advantages of APCI

• Works with normal HPLC flow rates (up to 2ml/min)

• Minimum suppression of ionization, and good
ionization efficiency for some compounds that are
not pre-ionized in the mobile phase.
• Operates with normal phase solvents
• More tolerant to volatile buffers than ESI
• Soft ionization method, but stronger than ESI
 DUIS
Dual Ion(ization) Source
 DUIS

ESI + APCI => DUIS

DUIS means Dual Ion Source

• High voltages are applied simultaneously

to the nebulizer for ESI probe and the
corona needle.
• Heated drying gas (up to 500 ºC) is used to
assist ionization in the APCI mode.
DUIS

Hot drying gas

for APCI effect
 DUIS

Advantage:
DUIS can be a helpful tool especially for unknown
substances and mixtures as it delivers ESI and APCI
information in one experiment

Disadvantage:
DUIS offers slightly reduced sensitivity compared to
single ESI or APCI. (About 30% loss of sensitivity
dependent on application)
 APPI
Atmospheric Pressure
Photoionization
 APPI-mechanism
Drying and nebulizing gas (N2) To the Analyzer
Solvent (MeOH, ACN, …)
Drying gas
Analyte-molecule
Drying gas N2 (10-20L/min)
N2 (1-1,5 L/min)
Nebulizing gas

N2 (10-20L/min)

Heating 500ºC Solvent

charge Charge Transfer

Heating 500ºC
N2 (1-1,5 L/min)
Nebulizing gas

The APPI works similar to

APCI but the corona needle
is replaced by a UV lamp
 APPI (direct APPI)

Direct APPI
+H

M+H+
M+

UV
(hν=10 or 10.6eV)
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+.
Non-Polar
 APPI (dopant APPI)

Dopant APPI
Molecular Weight +
M+
e-
100,000
M+

+H
M+H+

Dopant M+
UV
Solvent (hν=10 or 10.6eV)
Analyte

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

ions. D+ ionizes analyte M by proton or electron transfer.

Non-Polar
 Energetics for Photoionization

Molecular Weight
100,000 Krypton Lamp 10&10.6eV

Ionization
Solvent Ionization
Potentials(IP)
Potentials(IP)
Anthracene 7.4eV Works without
Toluene 7.4eV Dopant-molecules
Fluoranthene 7.8eV
Acetone 9.5eV
Caffeine 8.0eV
4-Nitrotoluene 9.5eV Works only with
Methanol 10.85eV Dopant-molecules
Acetonitrile 12.19eV
Water 12.61eV
Non-Polar
 Scope of API-MS
Molecular Weight

Molecular Weight ESI

10,000

APCI

1,000 APPI

1000
100

Non-Polar Very polar

Polarity
 Comparison ESI / APCI / APPI

ESI and APCI are well understood

In ESI some solvents work and others do
not
In APCI some molecules work, other do not
APPI offers the potential to examine
molecules that can be separated by LC using
solvent systems that are less appropriate for
typical LC/MS
APPI is less susceptible to ion suppression
than ESI and APCI
Non-Polar
 Comparison ESI / APCI / APPI
ESI APCI APPI (not for
LCMS-2020)
mass-range Up to m/z~100.000 Up to m/z ~1.000 Up to m/z ~1.000
HPLC-Flow 0,001 - 2,0 mL/min 0,05 - 2,0 mL/min ------------------------
polarity of analyte polar Middlepolar-unpolar Unpolar
polarity of solvent polar polar & unpolar polar & unpolar
volatile buffer less tolerant high tolerant high tolerant
nebulizing gas flow <1,5 L/min <4,4 L/min -------------------------
ionization ion evaporation chemical ionization photo ionization
softness of very soft soft soft
ionization

Sample stability Good for many Not good for heat- Not good for heat-
analytes unstable analytes unstable analytes
Vacuumsystem
 Why do we need vacuum?

Why is it neccessary to work in vacuum and not at atmospheric

pressure?

1.At atmospheric pressure too many air-molecules (O2, N2 and Ar) are in
the ion-path. That means, the air-molecules disturb the flow-path of the ions.
In case of collision, the ion-pathway is changed and then they collide with
the electric parts (ion-lenses, octapole, quadrupole). After the collision the
ions are lost.

2.The air-molecules are moving in the system and would create a big noise-
ratio, because some of them hit the detector.

3.In vacuum no charge-transfer between the rods happen. Without vacuum

the system would be destroyed due to electric discharges between the rods.
 Why do we need vacuum?
Without or with unsufficient vacuum

Probability that Ions

reach the detector
is very small

Sufficient and stable vacuum

Probability that Ions

reach the detector
is very high
 Vacuumsystem
The ions are smoothly transferred from atmosphere to vacuum in several
steps.
To achieve this a combination of several turbo molecular pumps or of one
multi-stage turbo pump together with a roughing pump is necessary.
2nd vacuum chamber
1st vacuum chamber 0.2 Pa (No vacuum gauge)
100-200 Pa
Ion gauge 3rd vacuum chamber
Pirani gauge 5e-4 Pa

Side stage
28 m3/hour 155 L/sec Main stage Dual inlet TMP
155 L/sec
Edwards EXT200/200H
Atmospheric
pressure

Leak valve
Rotary Pump
Edwards E2M28
 Dual Inlet TMP

5*10-4 Pa
0,2 Pa
Complete TMP is in use for the
Only half of
analyzer chamber
TMP is in use
for the lens
chamber To roughing pump
Vacuum Pressure Vacuum-Pump-curve

Vacuum Pressure Change in

Pump Down Process

Contribution from gas/water desorption from chamber surface

Pump Limit

Time
Ion Optics
 Ion optics

Ions are transfered from Atmosphere to Vacuum

through the DL (Desolvation line)
Afterwards they are focused and aligned with the help
of an electromagnetic lens system consisting of Q-
Array and Octapole
 Ion optics
Why is it necessary to use ion-optics in MS?
Example: Without Ion-optics
Skimmer

DL

Without Ion-optics only a few Ions can reach the detector (<<0,1%)!
 Ion optics
Why is it necessary to use ion-optics in MS?
Example: With Ion-optics
Skimmer

Q-Array (Focusing)
Octapole (Aligning)

DL

With ion-optics a big amount of ions will reach the analyzer!

Quadrupole Mass Analyzer
 Quadrupole Mass Analyzer

Mass analyser:
Single stage quadrupole

Separates ions of the

same mass-to-charge
ratio (m/z) from all
other ions in a mixture
using RF voltage
overlaid with DC voltage
Affect of DC-voltage

For small m/z-ratio:

Molecules have high speed

Near no effect of DC-voltage

For bigger m/z-ratio:

Molecules are slower

Bigger effect of DC-voltage
Ions get dischaged at the rods

For big m/z-ratios:

Molecules are very slow

Very strong effect of DC-voltage
Ions get dischaged at the rods.
Affect of RF-voltage (no DC)

For small m/z-ratio:

Biggest effect of RF-voltage

 Amplitude is very high

For bigger m/z-ratio:

smaller effect of RF-voltage

Amplitude is smaller

For big m/z-ratios:

Less effect of RF-voltage

 Very small amplitude
Low RF- and high DC-voltage

For small m/z-ratio:

Near no effect of DC-voltage
High effect of RF-voltage
Ion reach the detector
 DC and RF are both OK

For bigger m/z-ratio:

Big effect of DC-Voltage
smaller effect of RF-voltage
Amplitude is smaller
 DC-voltage is too high

For big m/z-ratios:

Big effect of DC-voltage
Less effect of RF-voltage
Very small amplitude
DC-voltage is too high
High RF- and low DC-voltage

For small m/z-ratio:

No effect of DC-voltage
Very big effect of RF-voltage
Amplitude is too big

For bigger m/z-ratio:

Big effect of DC-voltage
Big effect of RF-voltage
Amplitude and DC are too big

For big m/z-ratios:

Big effect of DC-voltage
Less effect of RF-voltage
Ion will reach the detector
DC and RF are both OK
 High- and low pass-filter
Low RF- and low DC-voltage High RF- and high DC-voltage
n
Transmissio

n
Transmissio
Ions can reach No Ions can reach the
the detector detector

m/z-ratio m/z-ratio

Low RF- and High DC-voltage High RF- and Low DC-voltage
n
Transmissio

n
Transmissio

Ions can reach Ions can reach

the detector the detector

m/z-ratio m/z-ratio
 High- and low pass-filter

n
Transmissio

m/z-ratio

Stable flight-way for

defined m/z-ratios

When RF- and DC-voltage is set at the quadrupole, so that only one kind of m/z-
ratio can pass the Quadrupole, the ions have only a transmission in a small triangle.

This is a scheme for the combination of high-pass-filter and the low-pass-filter in

quadrupole-technique.
 Quadrupole-voltage-settings

+[U+V cos(ωt)]
+
-++
U V
0
-+- -+ -
-

-
++
+ -[U+V cos(ωt)]

0
U

V
-
 Quadrupole and Mathieu-diagram

DC-Voltage
RF unstable
DC stable

RF stable
DC unstable

RF stable
DC stable

RF-Voltage

Stable trajectory for the ions  Ions can reach detector.

 Quadrupole and Mathieu-diagram

DC-Voltage
RF unstable
DC stable
RF stable
DC unstable

RF stable
DC stable

RF-Voltage

Unstable trajectory for the ions  Ions cannot reach detector.

 Quadrupole and Mathieu-diagram

DC-Voltage
RF unstable
DC stable

RF stable
DC unstable

RF stable
DC stable

RF-Voltage

Unstable trajectory for the ions  Ions cannot reach detector.

 Mathieu-diagram
In stable RF- and DC-voltage-field:
 Where is the focused voltage (measurement)-range?
 Measurement- or voltage-range is in the red tip
DC-Voltage

RF unstable
DC stable
RF stable
DC unstable

RF stable
DC stable

RF-Voltage
A-value Mathieu-diagram

z = charge state of ion [ ]

U = DC Voltage [V = kg m² s-³ A-1]
V = RF Voltage [V]
m = mass of the ion [kg]
r = inner radius of quadrupole-field [m]
ω = 2*π*f [s-1]
f = frequency [s-1]
A = Parameter A [ ]
Q-value Q = Parameter Q [ ]

Fixed values:
Used Voltages:
r = 3,9 mm
m/z = 10 U = 2,685 V V = 16 V
f = 1,2 MHz
m/z = 1000 U = 268,5 V V = 1600 V
e = 1,602 *10-19 As
m/z = 2000 U = 537,0 V V = 3200 V
m = Da * 1,66*10-19 Kg
 Mathieu-diagram
Calculation of the Peak-tip
A-value

A = 0,240
Q = 0,714

Q-value
Why is the A- and Q-value so important?
For each m/z-ratio the A/Q-ratio is same!

A/Q = 2U/V i.e.: A and Q are fixed values!

That offers: V/U = 2Q/A  working line

 Mathieu-diagram

e.g. m/z~2000
m/z~400
m/z~100
m/z~1000

DC-Voltage Working Line

RF-Voltage
DC-Voltage
Why does mass-peaks have a width?

Increasing m/z

RF-Voltage
Intensity

m/z-range
 Mathieu-diagram and Resolution
Inten. (x1,000,000)
Int. Max = 2.4 mio
DC-Voltage

609.2
609,2

1,50
Unit Resolution
R~2M
1,25

1,00

0,75
610.2
0,50 610,2

0,25
611.2
611,2

0,00
608,5 609,0 609,5 610,0 610,5 611,0 611,5 612,0 612,5 613,0 613,5 m/z

Inten. (x100,000)
609,3
Int. Max = 1.2 mio
609.3
7,0

6,5

6,0 High Resolution

R >2M
5,5

5,0

4,5

610.3
4,0

3,5

3,0
610,3
2,5

611.3
2,0

1,5

1,0

0,5
611,3
612.3
0,0
609,0 609,5 610,0 610,5 611,0 611,5 612,0 612,5 613,0 613,5 m/z

RF-Voltage
7,0
Inten. (x1,000,000)
Int. Max = 11.0 mio
609,1

6,0

609.1
low
Goodworking
Bad workingline:
line: 5,0

4,0
Resolution
 Resolution is < ~ 2M
> 3,0
R <2M
 Intensity
very goodisspecificity
fine
good 2,0

1,0

 No
good
Intensity
specificity
specificity
is worse 0,0
607,0 607,5 608,0 608,5 609,0 609,5 610,0 610,5 611,0 611,5 612,0 612,5 613,0 m/z
Measuring modes
 Scan-function
1. Scan
2.
7.
3.
4.
5.
6.
DC-Voltage

Scanspeed 15000 Da/s

mass-range 10-2000 (=15000/1990)
 ~7,5 Scans per second (~7 Scans/s)

Time-line: length 1 sec

RF-Voltage
m/z-range

Time-range
1 sec
Intensit
Scanspeed

Intensit
y

y
time time
m/

m/
z

z
time time

As faster the Scanspeed:

1. as more datapoints per second,
2. as more ramps per second
 Scan-Mode (theoretically)

Intensit
m/
z

y
time
Time

z
m/
Intensit
y

Time
 Scan-Mode 15000Da/s
m/
z

m/z=10-2000 Da

Time
ZOOM
m/

time
z

nt

I SD
rem e
I SD

eas u
M

Time
ement

ement
ement

time m/z=100-110 Da

time
IS IS IS
time

Measur

Measur
IS
m/

D D D
Measur

D
z
 Mass-range and Scan-speed
Scanspeed without ISD: 15000 Da/s Scanspeed without ISD: 15000 Da/s
m/z: 10 Da – 2000 Da m/z: 100 Da–110 Da
Minimum Event time: 0,1375 s Minimum Event time: 0,0055 s

Calculation of Inter-Scan-Delay (ISD): Calculation of Inter-Scan-Delay (ISD):

1000ms 1000ms
 5ms  36,36ms  5ms  909,1ms
137,5ms 5,5ms
Number of Inter-Scan-Delays (ISD): Number of Inter-Scan-Delays (ISD):
36,36ms 909,1ms
 7,27  181,2
5ms 5ms
In 1s the system uses 7,27 times the ISD In 1s the system uses 181,2 times the ISD
 Systems ramps 7,27 times!  Systems ramps 181,2 times!

1990 Da x 7,27 = 14471,28 Da 10 Da x 181,2 = 1812 Da

Scanspeed with ISD: Scanspeed with ISD:

 14471 Da per second! 1812 Da per second!
 Statistics in Scan mode

Mass-range 10 - 2000 Da 100-110 Da

Scanspeed 15000 Da /s 15000 Da /s

Inter-Scan-Delay 36,4 ms  3,64 % 909,1 ms  90,91 %

Measurement-time 963,6 ms  96,36 % 90,9 ms  9,09 %

Time per Da ~0,05 ms ~0,05 ms

Datapoints Each 137,5ms one point Each 5,5ms one point

Conclusion: as bigger the mass-range as more measurement-time

as bigger the mass-range as less the number of points per peak
 Scanspeed
15,000 Da/s means 66.67 µs/Da (=1/15,000).

Time of Flight in the Rod Region [µs]

Pre-Rod Quadrupole Rod 400
m/z Time of Flight
350 100 77.0 µs
300 1000 243.3 µs
2000 344.1 µs
250
200
150

Time of Flight in the Rod Region 100

50
m/z
0
0 500 1000 1500 2000

Traditional approch: Sensitivity is lost

DC-Voltage
if acquisition time per Da < time of flight in the rods
Novel approch:
1. Changing Prerod-Bias voltage during a
scan to accelerate bigger ions!
2. Changing the GAIN-voltage (slope) on the
Quadrupole (open Quad for bigger Ions)
RF-Voltage
Scan- & Profile-mode

Profile-mode:
- Peak-shape in MS-spectra
- Necessary for deconvolution
- Bigger datafiles (x5 – x10)
- more information than Scan-mode

Scan-mode:
- Also named as centroid-mode
- Used as standard
- Deconvolution not possible
- Smaller datafiles than Profile-mode
 Voltage
SIM-Mode (Single Ion Monitoring)
m/z=1600

m/z=609

m/z=82

time

z
m/
m/z=1600
Intensit

m/z=609
y

m/z=82

time
 SIM-Mode
Theory (no Inter-Scan-Delay)
Voltage

Time
Praxis (with Inter-Scan-Delay – fix: 5ms)
Voltage

Voltage

Time Time
Inter-Scan-Delay: 5ms Inter-Scan-Delay: 5ms
Measurement-time: 5ms Measurement-time: 1ms
 SIM-Mode
Voltage

100 Da/s

500 ms measurement-time
500 ms Inter Scan Delay

50% measurement-time
Inter-Scan-Delay: 5ms Time
Measurement-time: 5ms
Voltage

166 Da/s

167 ms measurement time

833 ms Inter Scan Delay

 16,6% measurement time

Inter-Scan-Delay: 5ms Time
Measurement-time: 1ms
 Statistics in SIM mode

Mass-range 5 ms measurement time 1 ms measurement time

Number of SIM 3 SIMs 3 SIMs

Inter-Scan-Delay 5 ms  50 % 5 ms  83,4 %

Measurement-time 5 ms  50 % 1 ms  16,6 %

Time per Da 5 ms 1 ms

Datapoints Each 30 ms one point Each 18 ms one point

As more data points per peak, as worse the statistic per data point
Mass Calibration
 Mass calibration of Quadropole MS

• comparison of real and theoretical

mass values following adjustment using
known mass values of a calibrant
• automatic calibration (AutoTuning)
with PEG 200, 600, 1000 + Raffinose
• manually extended calibration
e.g. for high masses (>1500) PPG
 Mass calibration of MS
After installation of a new system:
 Mass-Spectrometer does not know at which voltage which mass reaches the
detector
DC-Voltage

m/z
RF-Voltage Voltage

The MS is measuring at the working

The MS has a calibration line, but the
line and creates a m/z to voltage
system does not know which voltage
diagram
belongs to which mass!
 Mass calibration of MS
Substances with a known m/z-ratio is now used to fix the height of the calibration
curve

PPG (m/z>1500)

Raffinose Dimer(m/z =1007)

m/z

PEG 1000
PEG 600
Raffinose (m/z=503)

PEG 200

Voltage
Detector
 Detector

LCMS uses a Secondary

Electron Multiplier (SEM)
with a Conversion Dynode
in front
 Ion detection – positive Ions

-10 kV
NEGATIVE!
 Ion detection – negative Ions

+10 kV
POSITIVE!
 Ion detection

- After passing the mass-analyzer (quadrupole)

the ions are focused for the detector.
- The number of ions at each m/z value is then
´detected´ by converting the ions current into a
signal by amplifying each ion.
Number of ions

As more ions hit the detector,

as higher the signal

Signal-Intensity
 Detector voltage and amplification

108
amplification

106

104

-1,2 -1,6 -2,0 -2,2 -2,4

Detector voltage applied to E.M. (kV)

! Detector voltage and amplification is not linear !

Keypoints for LCMS
 Mobile phases and concentrations
Major mobile phases:
- water, methanol, acetonitrile (THF for polymeric compounds)

Major additives in mobile phase:

- acids: formic acid, acetic acid (between 0,1 % and 1,0 %)
Trifluoroacetic acid (max 0,1%)
- salts: Ammoniumformiate, Ammoniumacetate (between 5 and 10 mmol/L)
Lithium-salts (max. 10 mmol)
- bases: Ammoniumhydroxide (for negative mode, max pH10)

Possible other organic solvents:

DMSO, DMF, THF, acetone, ester, chloroform, benzene, hexane

Basically there will be no problem even if other organic solvents are

contained, as long as the major mobile phase components are present.
(However, ionization efficiency may decrease with the increase in the
content of other organic solvents.)
 Mobile phases and LC/MS
NO-GOs in LCMS
for mobile phase:

Buffers: Ion-pair reagents:Perfluoro carboxylic acid (C2 - C8)

Dibutylamine
Triethylamine
Triethylammonium-hydroxide
Phosphates
Non-volatile buffers NEVER in APCI
Acids: Trifluoroaceticacid (TFA) in high concentration

Basics: High pH-values (pH > 10)

Inorganic acids: Sulfor-, Phosphorous- , hydrochloric- or nitric-acid
Detergents: NEVER

if they are used: do not expect long term stability

TFA in high concentration
NH3 in high concentration
 Why not to use special additives?
Ion pair reagents: sometimes for peptide and Protein-analysis it is used.
BUT: - is very difficult to clean the system after using
(>4 month and 10 litre organic solvents)
- without cleaning the IPR will catch all ions and
target-compounds are not ionized, only
signals from IPR are detectable (ion
suppression)

Non-volatile buffers: clogs the DL and in each case APCI-pipe,

sometimes also the ESI-pipe.
In ESI it inhibits the formation of fine droplets

Phosphates: are not volatile, will create clogings and ion suppression

Trifluoracetic acid: - In negative mode it is long-time stable, makes ghost-

peaks and creates ion suppression
- in high-concentration: It reacts with the Aluminium-
interface-chamber
Basic phases: At a higher pH-range (pH>10) it reacts with the interface-
chamber
 Why not to use special additives?
Inorganic acids: - They are in normal way very strong, reaction with interface
chamber and
- some are not volatile, could create chlorides, phosphates
and sulphates together with the analyte.
Detergents: Long time ion-suppression

Polarity of solvent

A polar solvent is essential

ESI : A solvent which dissolves polar and ionic compounds is required

APCI : A protonic solvent is necessary to generate reactant ions, which can

transfer proton to analyte molecule
 Mobile phases and LC/MS
APCI-solvents:
Under reverse phase column conditions,
methanol/water achieves higher ionization efficiency than
acetonitrile/water, due to
the fact that the gas-phase basicity of
H2O/MeOH is less than H2O/ACN

ESI-solvents:
A mobile phase containing organic solvent is
preferable for promoting ion vaporization and formation of
fine charged droplets in atmospheric
pressure.
Under normal reverse phase column conditions,
acetonitrile/water achieves higher ionization efficiency
than methanol/water. The pH also has a great effect.
 Mobile phases and LC/MS

If you need to use nonvolatile buffer:

• use the lowest amount possible

• use µLC, a small flow
• try to split away the part of the solvent containing salts
(e.g. with a FCV-20AH)
• Try to use not a close position of ESI-pipe to DL
• clean/exchange DL often
• Be careful of ESI-pipe clogging
• With APCI you cannot use nonvolatile buffer
 Troubleshooting

• Fragmentation (Q-Array-Voltage)
• DL-temperature
• APCI-temperature
• DL-voltage
• avoid frequent auto tuning.
calibrate every 3 - 6 month.
• Methanol, Isopropanol, water and
! Formic acid ! for cleaning
 Troubleshooting

• adducts with ESI (Na, K, solvent etc.)

• -COOH and -NO2 groups prefer to be
measured in negative mode
• -NH2 and similar likes to add protons
=> positive mode
• avoid nonvolatile buffer
• acetic- and formic acid as helping reagents
 Quantification

• dynamic range around 1e3 - 1e4

• detection limit typically 2 - 5 pg
• correlation coefficient typically
<5% seldom <2%
• often higher standard deviation as UV
• dynamic range smaller as UV
 Quantification

• often detection limit superior to UV

• internal standards: deuterated compounds
• partly better „resolution” due to MS detection
=> shorter analysis times
 MS/MS with Single Quad

It is possible to use simulated MS/MS function for certain

compounds using CID (Collision Induced Dissociation).
Resolution & Peak Width

FWHM = Full Width

at Half Maximum
or Δm
 Resolution Definition

R = Resolution
m = mass of the 1. peak
Δm = detectable mass-difference
FWHM = 0,7 R=m/z / FWHM

50% Peakheight

10% Peakheight
 Resolution Quadrupole

Quadrupole has Unit-Resolution:

R = 2M
 two peaks with 1Da difference and an
FWHM = 0,7 overlap at 10% Peakheight are
separated.
 Same FWHM over complete mass-
range
 Resolution increases with m/z ratio
 Resolution IT-TOF

Example: Resolution IT-TOF at m/z=1000

 two peaks with 0,1 Da difference and

an overlap at 10% Peakheight are
FWHM = 0,1 separated.
 Different FWHM over complete mass-
range
 Resolution is rather stabil over
complete mass-range, but better than with
quadrupole-technology
 Resolution

Mass-range Quadrupole IT-TOF

R=142 (0,7 FWHM)
R~5000
m/z = 100
Δm = 0,7 Da Δm = 0,05

R=1429 (0,7 FWHM) R=10000

m/z = 1000
Δm = 0,7 Da Δm = 0,1 Da

R=2857 (0,7 FWHM) R~12000

m/z = 2000
Δm = 0,7 Da Δm = 0,17 Da
 Resolution and Peakshape
m/z = 1000 and 1001  1Da difference between the two peaks

FWHM = 0.1 FWHM = 0.2 FWHM = 0.4 FWHM = 0.5 FWHM = 0.7

Quadrupole-range

FWHM = 1,0 FWHM = 1,4 FWHM = 1,6 FWHM = 1,8

Definition of S/N-Ratio
Typical value for a laboratory:

LOD = Limit of Detection

 3 σ (Signal is 3x higher than noise)

LOQ = Limit of quantification

 10 σ (Signal is 10x higher than noise)
 3 sigma(σ) for S/N
max

+3 RMS

Average

-3 RMS

min

max min
NOISE 
2
RMS = Root-Mean-Square Deviation = σ
 That´s it

Questions?