2
Ion Generation: Atmospheric
Pressure Ionization (API)
2
3
ESI is works very well for polar analytes. APCI and APPI can be used
as complementary techniques for less- or non-polar compounds.
Molecular W
eight
Analyte Polarity
very polarnonpolar
100,000
10,000
1,000 APCI
Electrospray
APPI
Relative Applicability of LC/MS Techniques
3
4
Generation of Ions – ESI Source
The heated drying gas creates a
warm environment in the source.
As the heated gas flows round
the front of the capillary and out
of the spray shield, it acts as a
counter-current gas to the ion
stream, therefore reducing the
flow of neutrals and unwanted
ions into the ion optics.
The glass capillary itself is not
heated.
4
5
Formation of charged droplets at the
needle tip within the spray chamber
Capillary (4
kV)
Nebulizer
GasSample
Solution
Dry Gas
Spray Needle
grounded
Dry Gas
Capillary Cap (4 kV)
Generation of Ions – ESI Source
Nebulization
5
6
Generation of Ions - Desolvation
When the force of the Coulomb repulsion exceeds the surface tension of
the droplet, the droplet explodes, producing charged daughter droplets that
are subject to further evaporation.
EvaporationRayleigh
Limit
Reached
Coulomb
Explosion
Droplet
6
7
Generation of Ions – Ion Evaporation
Evaporation (Multiply Charged)
Ion
Droplet
As solvent evaporates from the droplet, the surface becomes highly
charged. When the field created by the ions at the surface of the droplets
exceeds the surface tension, ions are emitted directly into the gas phase.
7
8
Fundamentals of
Mass Spectrometry:
Mass Resolution
Isotope Patterns
Monoisotopic Mass
Mass Accuracy
8
9
It is a measure of a mass spectrometer’s ability to distinguish two
compounds of nearly equal mass.
600 800 1000 1200 1400 1600 1800 2000 2200 2400 m/z
100
200
300
400
500
600
700
800
900
1000
1100
a.i.
1618 1623 1628 m/z
<-301->
<-273->
“easy” “not so easy”Resolution
What is meant by ‘Resolution’?
9
10
The narrower the FWHM, the higher the resolution.
With the same FWHM, higher masses will have higher
resolution than lower masses.
Resolution = (m/z) / FWHM
FWHM Full Width at Half
Maximum
How do we measure mass resolution?
1521.9743
1522.9772
1521 1522 1523 1524
m/z
10
11
Isotopic Distribution Patterns
1312
100
90
8070
60
50
40
3020
10
0
C1
122121120
100
90
8070
60
50
40
3020
10
0
C10
1,2061,2041,2021,200
100
90
8070
60
50
40
3020
10
0
C100
12,03012,02012,01012,000
100
90
8070
60
50
40
3020
10
0
C1000
Isotopes are atoms with the same number of protons in the nuclei, but with different numbers of neutrons. Only 21 elements have only one stable isotope. All
other elements are mixtures of at least 2 stable isotopes, and the proportions of these isotopes can vary greatly depending on the element. Carbon has 2 stable
isotopes, C-12 and C-13, with natural abundance of 98.892% and 1.108% respectively. As the number of carbons increase in a molecule, the isotopic
distribution pattern will reflect the mass contribution of the isotopes with their extra neutrons.
11
12
pQLYENKPRRPYIL
MW 1672.9
Res. 1’000
1,6811,6791,6771,6751,6731,671
100
90
8070
60
50
40
3020
10
1673.9Average Mass
40
1,6821,6801,6781,6761,6741,672
100
908070
60
50
3020
10
0
Res. 10’000[M+H]+
[M+H+1]+
[M+H+2]+
[M+H+3]+
[M+H+4]+
1672.9 Monoisotopic Mass
Neurotensin
Resolution = (m/z) / FWHM
Average and monoisotopic masses
Lower resolution results in an “average”
-inaccurate determination of peak center
-calculated average mass is inaccurate
12
13
Defining the TOF mass measurement
Mass accuracy can be expressed as a percentage or ppm:
e.g. % mass accuracy =Measurement error
True massx 100%
Very small percentage errors (<0.01%) are expressed as
parts per million or ppm. 0.01% = 100ppm
e.g. ppm =Measurement error
True
mass
x 106
Measured mass: 1296.970
True mass: 1296.685 0.02% error
ppm = 0.001 per thousand
13
Precision and mass accuracy
15
Calibration for ESI-TOF
Good instrument calibration is mandatory for any accurate mass measurement !
The „ideal“ calibrant must have:
• known elemental composition
• no interferences (ion suppression, isobaric interferences) with the analytes
The „ideal“ calibrant nice to have:
• should cover the desired mass range
• ionizable both in positive and negative mode
• well soluble in common HPLC solvents and stable in solution
• not „sticky“ to surfaces (memory effects)
16
External calibration: Calibrant is injected separately from sample.
+ no interference between analyte and calibration standard masses.
- Higher demands on control of laboratory environment (e.g. temperature) compared to
internal calibration mode.
Internal calibration: Calibrant is introduced simultaneously with the sample.
+ Better mass accuracy due to identical instrumental conditions.
- Interference between sample and internal standards possible
A: ion suppression
B: isobaric interferences
Calibration for ESI-TOF
17
A. Injection of standard via 6-port-valve at the beginning/end of the LC-MS run
+ no suppression effects or isobaric interference possible
- slight time offset between calibrant and analyte spectra
B. Introduction of calibrant with second ESI sprayer (continuous or discontinuous)
+ calibration standard present in every spectrum
- suppression effects possible, isobaric interference
C. Add calibration standard via T-piece post-column
+ calibration standard present in every spectrum
- suppression effects possible, isobaric interference
D. calibration based on compounds with known elemental composition in the
analysis
Instrumental solutions
Calibration – micro and standard LC
18
Valve position “Waste”
LC flow directly enters the micrOTOF
The valve can be switched via time segments in the acquisition method.
Injection of calibrant via 6-port valve
Valve position “Source”
Calibrant is introduced into the micrOTOF
autosamplercolumn
ESI-TOF
waste
syringe pump
Cal.Std. Flow 2-10 µl/min
pump
autosamplercolumn
ESI-TOF
waste
syringe pump
Cal.Std. Flow 2-10 µl/min
pump
Configuration for External Calibration
19
Determining Multiple Charge States
Example: m1 = 1000, isotope m2 = 1001
Charge (z) = 1 1000/1, 1001/1 m/z = 1000, 1001
Charge (z) = 2 1000/2, 1001/2 m/z = 500, 500.5
Charge (z) = 3 1000/3, 1001/3 m/z = 333.33, 333.66
The m/z difference is incremental to the charge.
Charge 2 = 0.5
Charge 3 = 0.333
Charge 4 = 0.25
2+3+
1+
19
20
Ion Generation:
Interfaces
20
21
Positive Ion Mode Negative Ion Mode
Formation of protonated molecular ions Formation of deprotonated species
M + HA � [M+H]+ + A- M + B � [M-H]- + BH +
Example: Example:
�
R
NRR
+H
R
NR RHA A-+++ �
O
COR -
O
COR H
B: + B-H+
Analytes with a basic character, like for instance compounds carrying amino groups, are
generally ionized in positive mode. The sample molecule picks up a proton from the more
acidic solvent.
Analytes that are more acidic might to be ionized in negative ion mode (e.f. carboxylic or
sulfonic acids). The molecule looses a proton to a base in solution and becomes negatively
charged.
A mean for the basic or acidic character of a sample is its pKa value.
Electrospray Chemistry
Sample Chemistry
21
22
Cluster Formation
In addition to the formation of adducts clustering of ions and solvent molecules can be observed,
e.g. [M+Na+CH3CN]+, [M+H+MeOH]+, …
For higher concentrated samples very often clusters of the analyte molecules themselves are
formed:
[2M+H]+, [2M+Na]+ , [2M-H]-, [2M+Na-2H]- , …
Common observation:
the dimer often is formed predominatly as [2M+Na]+ even if the monomer prefers formation of
[M+H]+
Electrospray Chemistry
Adducts and Clusters
22
23
Electrospray Solvents and Buffers
Electrospray requires polar solvents. Commonly used are water, methanol, acetonitrile and iso-
propanol
The pH of the solvent has a major effect on analytes which are ionic in solution:
Acidic pH (<7.0; 5 preferred) for positive ions
Basic pH (>7.0; 9 preferred) for negative ions
Acidic additives for positive ion mode:
●Formic acid, 0.1-1.0%
●Acetic acid, 0.1-1.0%
●Trifluoroacetic Acid ≤ 0.05%
The TFA anion forms ion pairs with positive analyte ions and thus leads to signal
suppression! But TFA concentrations of ≤ 0.05% are acceptable.
Basic additives favour the negative ion mode:
● Ammonium hydroxyde (pH 10-11)
23
24
Advantages ☺☺☺☺
• Soft ionization method, providing molecular ions, e.g. M+H +, M+Na+
• Suited for a wide range of moderate to high polarity compounds
• Extended mass range for multiply charged analytes, e.g. proteins,
oligonucleotides
• Very sensitive interface for LC-MS coupling.
• Robust and low maintenance
• Interface for routine and automated use
Disadvantages ����
• Solution chemistry influences ionization process
Ion suppression/Matrix effect:
Quantification is challenge for co-elution; need appropriate internal
standards. Stable-isotopic labeled internal standards are optimal.
• Adduct ions (other than M+H) possible with some analytes, no unambiguous
ionization for unknown compounds
• For higher concentrations saturation effects limit the linear range
ESI – Advantages and Disadvantages
24
25
Basics and Theory
25
26
Dry Gas Heater Dual Ion Funnel
Analytical
Quadrupole
Collision
Cell
Orthogonal Accelerator Detector
Reflectron
Flight Tube
Glass
Capillary
Collision
Gas Supply
API Spray Chamber
Sprayer
Hexapole
Functional Overview
ESI-Qq-oTOF
26
Driving forces from source to detector: Pressure gradient
Potential gradient
27
micrOTOF-Q: MS
No isolation in Q :
RF only
Low collision
energy: no
CID
Dry Gas Heater
Dual Ion Funnel
AnalyticalQuadrupole
Collision Cell
Orthogonal Accelerator Detector
Reflectron
Flight Tube
Glass Capillary
Collision Gas Supply
API Spray Chamber
Sprayer
Hexapole
27
28
isolation in Q
high collision
energy: CID
Dry Gas Heater
Dual Ion Funnel
AnalyticalQuadrupole
Collision Cell
Orthogonal Accelerator Detector
Reflectron
Flight Tube
Glass Capillary
Collision Gas Supply
API Spray Chamber
Sprayer
Hexapole
micrOTOF-Q: MS/MS
28
29
micrOTOF-Q ISCID
High voltage step between
funnels: CID in medium
pressure region
Dry Gas Heater
Dual Ion Funnel
AnalyticalQuadrupole
Collision Cell
Orthogonal Accelerator Detector
Reflectron
Flight Tube
Glass Capillary
Collision Gas Supply
API Spray Chamber
Sprayer
Hexapole
29
30
338.340
609.280
0.0
0.5
1.0
1.5
5x10
Intens.
200 400 600 m/z
195.065
397.212
609.281
0.00
0.25
0.50
0.75
1.00
4x10
Intens.
200 400 600 m/z
397.213
0
2000
4000
6000
8000
Intens.
100 200 300 400 500 600 m/z
174.092
227.119
365.187
397.211
0
250
500
750
1000
1250
Intens.
50 100 150 200 250 300 350 400 450 m/z
ISCID
Isolation
Fragmentation
Reserpine
MS3 by In Source CID and CID (q)
30
Quadrupole
31
Mass Filter !
• RF and DC
• RF: Responsible for ion transmission (ALL ions)
• DC: select specific m/z
+
+
- -
+
32
Think of the TOF operation as a drag race between vehicles of different sizes, but all
having identical engines:
• “Start line” = orthogonal accelerator; “Finish line” = TOF detector
• Just as all vehicles have the same engine (i.e., horsepower), all ions are pulsed up
the flight tube with the same kinetic energy.
• Since m = 2E/v2 (E = ½ m·v2), the smaller vehicles/ions will reach the finish
line/detector before the larger ones.
START FINISH
Principle of the TOF Mass AnalyzerTOF: The Great Race
32
33
Separation of Ions by m/z – TOF Assembly
Orthogonal Accelerator
The ion ‘packages’ enter the field free time-of-
flight tube.
In the flight tube they are separated because
of their different velocities resulting in
different flight times.
As all ions have the same kinetic energy they
travel with a velocity that is inversely
proportional to their m/z.
Mass-to-charge ratios are determined by
measuring the time that ions take to move
from the orthogonal acceleration to the
detector.
Field
-free drift reg
ion
33
34
The aim of an electrostatic reflector, also called
reflectron is to improve mass resolution. It creates a
retarding field that acts as an ion mirror by deflecting
the ions and sending them back through the flight
tube.
The reflector corrects the energy dispersion of ions
with the same m/z ratio. Indeed, ions with more
kinetic energy will penetrate the reflectron more
deeply and will spend more time in the reflectron.
Thus they reach the detector at the same time as
slower ions of the same m/z.
Ions receive kinetic energy from electric field.
E = 1/2mv2
Resolution of Ions – TOF Assembly
m1 = m2, but E1 < E2Orthogonal Detector
accelerator
34
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