1
Quattro Premier Z-SprayTM Ion Source
ElectrosprayProbe
ProbeTiltStage
Vacuum &OperateLights
NebulizerGas
DesolvationGas
Capillary
DesolvationHeater
Quattro Premier Z-SprayTM Ion Source
Exhaust Trap
Isolation Valve
Sample Cone
Electrospray Probe
Source Block
2
Triple Quadrupole Instruments
MS1MS1 Collision CellCollision Cell MS2MS2
MS1 is used as a mass selector and allows ions of a particular mass to pass into the collision cell
In the collision cell, the ions from MS1 collide with Ar atoms and fragment into daughter (product) ions
MS2 is used as a mass selector and allows daughter ions of a particular mass to pass on to the detector
In a triple quadrupole or tandem mass spectrometer, MS1 and MS2 are mass analyzers that filter ions.
In a triple quadrupole or tandem mass spectrometer, MS1 and MS2 are mass analyzers that filter ions.
Quattro Premier DetectorIonsIons
MS 2MS 2
ElectronsElectrons
PhotonsPhotons
Cylindrical Conversion
Dynode
Cylindrical Conversion
Dynode
PMTPhotomultiplier TubePMTPhotomultiplier Tube
PhosphorPhosphor
3
Electrospray -Liquid is sprayed out of a capillary tube to which a high voltage is applied to form a spray of charged droplets.
Atmospheric Pressure Chemical Ionization (APcI) -Liquid is passed through a heated tube (fused silica capillary).The liquid is evaporated to produce gas phase molecules.
A high voltage is applied to a corona pin near the exit of the tube and the molecules are ionized when they pass through a cloud of ionized nitrogen atoms produced by the corona pin.
Electrospray Electrospray --Liquid is sprayed out of a capillary tube to which a high voltagLiquid is sprayed out of a capillary tube to which a high voltage e is applied to form a spray of charged droplets.is applied to form a spray of charged droplets.
Atmospheric Pressure Chemical Ionization (Atmospheric Pressure Chemical Ionization (APcIAPcI) ) --Liquid is passed through a heated tube (fused silica capillary).Liquid is passed through a heated tube (fused silica capillary).The liquid is evaporated to produce gas phase molecules. The liquid is evaporated to produce gas phase molecules.
A high voltage is applied to a corona pin near the exit of the A high voltage is applied to a corona pin near the exit of the tube and the molecules are ionized when they pass through a tube and the molecules are ionized when they pass through a cloud of ionized nitrogen atoms produced by the corona pin.cloud of ionized nitrogen atoms produced by the corona pin.
Atmospheric Pressure Ionization (API)
3L263L26
High Voltage Power Supply
+
2.5-4.0 kV
Counter Electrode+ -
Example of Positive Electrospray
Electrospray Ionisation
4
- --Liquid
ElectrosprayProbe Tip
+ + + +
High Voltage
+ + + +
++ +
++ -
++ +
+
++
+
++ - +
++
++
--
++ +
++-- +
+ +
++ -- +
+ +
++ -- +
++--
-+- ---+ ---+
-+-- -+-- + -+-
- -+-
-+- - +--
-
+
Droplet Formation in Positive Ion Electrospray
Taylor Cone
More Negative Ionsthan Positive Ions
More Positive Ionsthan Negative Ions
PositivelyChargedDroplets
The electrospray droplets carry positive charges away from the capillary tube.
To balance this flow of positive charges, electrons flow out of the capillary tube.
These electrons come from negative ions close to the surface of the capillary wall via an electrochemical oxidation reaction. Electrospray can be thought of as an electrochemical process.
Example of reaction that can occur at the capillary wall:
2 OH- H2O + O + 2e-
Electrospray – Electrochemical Processes
5
+
+
+
+
++
+
+
+
+
++
+
++
+
+
+
+
+ +
SolventEvaporation
+++
+
+
+
CoulombicFission
+
+
+
Electrospray Droplet Undergoing Fission
Charge resides on the surface of the droplet.
Solvent evaporates from the droplet and the droplet shrinks until the charge density on the surface reaches a point where the repulsive force between charges exceeds the liquid surface tension that holds the drop together.
At that point, the drop fissions and a set of small droplets areexpelled from the main droplet.
Rough Sketch of Photo fromP. Kebarle and L. Tang, Analytical Chemistry, 64, 972 A (1993)
It is estimated, that in the fissioning process a charged droplet willlose on the order of 15% of its charge but as little as 2% of its mass.
Actual Droplet Undergoing Coulumbic Fission
6
Electrospray tends to work best with solutions that have a high percentage of organic solvents such as acetonitrile or methanol, though the solution cannot be totally organic. The solution must have some aqueous content.
Solutions must have some ions in it for electrospray to work. Fortunately most solutions that have an aqueous component will have some ionic species such as hydronium/hydroxyl ions and sodium ions.
Electrospray - Solutions
Electrospray Ions
Positive Electrospray Ions are produced by the addition to a molecule of a positively ion (e.g H+, NH4+, Na+). These positively charged ions that are added are often referred to as ‘adducts’.
N N CH3O
CH3
H
C H3
O
OHC H 3
CH 3
C H3
O
OC H 3
CH 3
Negative Electrospray Ions are most often produced by the removal of a proton (hydrogen ion) from a molecule.
+ H+
+ H+
Lidocaine
Ibuprofen
N N CH3O
CH3
H
+
H
7
Electrospray and Ions in Solution
Electrospray is a solution process. Molecules that have a greater tendency to ionize in solution will tend to have stronger electrospray signals.
This is why certain additives to mobile phases in LC/MS analyses can enhance electrospray signals.
An example of this is addition of an acid (e.g. formic acid) to the mobile phase in positive electrosprayLC/MS analyses. This can often result in a strongerelectrospray signal by aiding in the protonation of analytes in solution.
ElectrosprayElectrospray is a solution process. Molecules that have is a solution process. Molecules that have a greater tendency toa greater tendency to ionizeionize in solution will tend to have in solution will tend to have strongerstronger electrosprayelectrospray signals.signals.
This is why certain additives to mobile phases in This is why certain additives to mobile phases in LC/MS analyses can enhanceLC/MS analyses can enhance electrosprayelectrospray signals. signals.
An example of this is addition of an acid (e.g. formic An example of this is addition of an acid (e.g. formic acid) to the mobile phase in positiveacid) to the mobile phase in positive electrosprayelectrosprayLC/MS analyses. This can often result in a strongerLC/MS analyses. This can often result in a strongerelectrosprayelectrospray signal by aiding in the signal by aiding in the protonationprotonation of of analytesanalytes in solution.in solution.
O
OH
OH OH
O
OH
O
OH
OH
OH
O
OOH
OH
OHO
O
OH
OH
O
O
OH
OH
OH
O
O
OH
OHOH
O
OOH
OH
OH O
O
OH
OH
OH
O
ß-Cyclodextrin is a ring of 7Glucose Units
1 2
3
4
5
6
7
Oxygen Linkages are Numbered
ß-Cyclodextrin - Electrospray Example
8
Quattro micro™
1120 1130 1140 1150 1160 1170 1180m/z0
100
%
0
100
%
BetaCyDex_5 1 (10.018) 2: Scan ES+ 6.92e61152.38
1135.37
1136.44 1153.39
BetaCyDex_5 1 (9.983) 1: Scan ES+ 6.36e61157.36
1173.361158.37
Cone Voltage=45 V
Cone Voltage=140 V
(M+Na)+(M+K)+
(M+H)+
(M+NH4)+
10 µg/mL ß-Cyclodextrin in 20/80 Acn/20 mM NH4 Acetate pH=4 in Water infused at 10 µL/min
Positive Ion Electrospray of ß-Cyclodextrin
9
Samples Analyzed in ES mode
Typical ES Positive Ion SamplesPeptides and proteinsSmall polar moleculesDrugs and their metabolitesEnvironmental contaminantsDye compoundsSome organometallicsSmall saccharides
Typical ES Negative Ion SamplesSome proteinsSome drug metabolites (e.g. conjugates)OligonucleotidesSome saccharides and polysaccharides
Typical ES Positive Ion SamplesTypical ES Positive Ion SamplesPeptides and proteinsPeptides and proteinsSmall polar moleculesSmall polar moleculesDrugs and their metabolitesDrugs and their metabolitesEnvironmental contaminantsEnvironmental contaminantsDye compoundsDye compoundsSome Some organometallicsorganometallicsSmall Small saccharidessaccharides
Typical ES Negative Ion SamplesTypical ES Negative Ion SamplesSome proteinsSome proteinsSome drug metabolites (e.g. conjugates)Some drug metabolites (e.g. conjugates)OligonucleotidesOligonucleotidesSome Some saccharidessaccharides and polysaccharidesand polysaccharides
10
Low molecular weight (<1000 Da)
Singly charged species
Fragmentation, even at low cone voltages
Mobile phase can be non-polar (normal-phase chromatography)
Low molecular weight (<1000 Low molecular weight (<1000 DaDa))
Singly charged speciesSingly charged species
Fragmentation, even at low cone voltagesFragmentation, even at low cone voltages
Mobile phase can be nonMobile phase can be non--polar polar (normal(normal--phase chromatography)phase chromatography)
Atmospheric Pressure ChemicalIonization (APcI)
APCI Probe Design
Heater
Heater
Plasma Discharge(Vaporized Sample exits the probe and
is ionized here)
Fused Silica Capillary (Sample flows through)
NebulizingGas
DesolvationGas
Corona Pin(Voltage Applied)Support or
Sheath Gas
11
APCI Ionization
• Higher temperature, more aggressive ionization.
• Solvent molecules are in the gas phase.
• Ionization takes place in the plasma.
• Goal of the nitrogen is to evaporate solvent
expelled from fused silica.
• May be more sensitive than electrospray with
some non-polar molecules
12
APcI Ions
In positive ion APcI, ions similar to those formed in positive ion electrospray are formed. For example: (M+H)+ or (M+Na)+
In negative ion APcI, the (M-H)- ion formed in negative ion electrospray is also produced.
Also in negative ion APcI, free electrons are formed. Certain types of molecules can pick up one of the free electrons produced by the corona pin and become negatively charged without a change in mass. This process is sometimes referred to as “M+•” or “M plus dot”.
Non Polar
100,000
1000
0
Mol
ecul
ar W
eigh
t
APcI
Polar
EI
ESP
APCI versus ElectrosprayAPCI versus Electrospray
13
APcI Electrospray
Ionization Gas Phase Process Solution Phase Process
Probe Fused Silica Capillary Stainless Steel Capillary
Potential Applied to Corona Pin Applied to Capillary
Process Probe heater vaporizes Spray of charged dropletsthe liquid. produced.
All molecules are Liquid is evaporated fromnow in the gas phase. the droplets.
Corona pin produces Then droplets split intonitrogen ions. smaller droplets.
Molecules are ionized When the droplets getwhen they collide small enough, ions enter with the nitrogen ions. the gas phase.
APCI versus ElectrosprayAPCI versus Electrospray
APcI Electrospray
Fragments More vigorous ionization. ‘Gentler’ ionization.More fragments produced. Less fragments produced.
Sample Types Low MW<1000 Small & Large MoleculesCan be less polar. Tend to be more polar.
Charges Usually Singly Charged. May be Multiplied Charged.
Flow Rates 0.2 - 2 mL/min 0.001 - 1 mL/min
Temperatures Source ~ 120-140 °C Infusion:Source ~ 80 °CProbe ~ 450-550 °C Desolvation ~ 120°C
HPLC: Source ~ 120 °CDesolvation ~ 350 °C
APCI versus Electrospray (continued)APCI versus Electrospray (continued)
14
Liquid
Nebulizer Gas
Nebulizer Gas
Desolvation Gas
Desolvation Gas
Electrospray ‘Plume’
Electrospray Probe Tip
Nitrogen
Nitrogen
Heater Wires
Heater Wires
Desolvation Gas Flow
15
Quattro Ultima and Quattro LC: Sample Cone and Cone Gas Nozzle
Cone Gas
Plume ofIons,
Clusters,and Stuff
Ions with Fewer Clusters which yields better S/N.
Less Stuff Collectson the Orifice Cone
ESP or APCI Probe
N2 N2
16
MS1 Scan
MS1 CollisionCell (No Argon)
MS2
m1
m2
m3
RF
10 -100V
ScanningRF (+ DC)
Cone Approx
20-100V
Cone Approx
20-100V
Ions Produced by ESP or APCIIons Produced by ESP or APCI
Fragments from Collisions along with
‘Unfragmented’ Ions
Fragments from Collisions along with
‘Unfragmented’ Ions
N2N2
N2N2 N2N2
Ions which are accelerated by the cone voltage, collide with Nitrogen molecules
Ions which are accelerated by the cone voltage, collide with Nitrogen molecules
+ + + + + + ++
Cone Voltage Fragmentation
17
700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900m/z0
100
%
943
893
849
808
771
738
707
998
1060
1131
1212
1305
1414
1542
1696
17
15
14
13
1211
10 91885
18
1619
20
21
22
23
24
Example of Multiply Charged Ion
Electrospray Spectra ofHorse Myoglobin
m/z and Charge States Shown
MS1 Scan (Review)
MS1 CollisionCell (No Argon)
MS2
m1
m2
m3
RF
10 -100V
ScanningRF (+ DC)
MS1 Scans are used to obtain Mass Spectra
18
SIR (Selected Ion Recording)
MS1 CollisionCell (No Argon)
MS2
m1
m2
m3
RF (+ DC) RF
10 -100V
Fixed
SIR’s are used to monitor selected analyte(s)
10 ng/mL Thiamethoxam and Metabolite Quattro micro™
7.50 8.00 8.50 9.00 9.50 10.00Time0
100
%
0
100
%
Thia_1G07_007 Sb (2,1.00 ); Sm (Mn, 2x3) SIR of 2 Channels ES+ 291.8
1.07e4
Thia_1G07_007 Sb (2,1.00 ); Sm (Mn, 2x3) SIR of 2 Channels ES+ 249.8
1.61e4
SIR Example
ThiamethoxamMW = 291
MetaboliteMW = 250
SIR of (M+H)m/z = 292
SIR of (M+H)m/z = 251
19
MS/MS Modes
MS1Collision
Cell (w/Argon) MS2
MS1 is used as a mass selector and allows ions of a particular mass to pass into the collision cell
In the collision cell, the ions from MS1 collide with Ar atoms and fragment into daughter (product) ions.
MS2 is used as a mass selector and allows daughter ions of a particular mass to pass on to the detector
In the collision cell, a potential is applied (typically 5-40 eV) to control the energy of the collisions between the ions and Ar atoms.
Quattro micro MS-MSQuattro micro MS-MSLow energy collisions (simple fragmentation pathways)
Collision gas of choice is Argon
Collision gas pressure is normally fixed while the collision energy is used to alter the degree of fragmentation
Sodium and potassium adducts are normally too stable to fragment using low energy collisions
Low energy collisions (simple fragmentation pathways)
Collision gas of choice is Argon
Collision gas pressure is normally fixed while the collision energy is used to alter the degree of fragmentation
Sodium and potassium adducts are normally too stable to fragment using low energy collisions
20
Daughter Ion Scan
MS1 CollisionCell (w/Argon)
MS2
m1
5-40 eV Scanning
1 V
Fixed
1 V m2
m1
m3
Determines Collision Induced Dissociation (CID) produced daughter ions of a particular parent ion
MRM (Multiple Reaction Monitoring)
MS1 CollisionCell (w/Argon)
MS2
m1
5-40 eV Fixed
1V
Fixed
1V
mx
MRM’s are used to monitor selected analyte(s) via their daughter ions
21
0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70Time0
100
%
MixIso_1G14_022 SIR of 1 Channel ES+ TIC
5.95e61.31
OH
O
O
O
OH
O
Fenbufen
Ketoprofen
Both have a MW of 254
Ion Chromatograms from SIR’s of m/z=255
FenbufenKetoprofen
From a Sample that is60 ng/mL Ketoprofen60 ng/mL Fenbufen
Fenbufen ??Ketoprofen
From a Sample that is60 ng/mL Ketoprofen
6 ng/mL Fenbufen
0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70Time0
100
%
MixIso_1G14_023 SIR of 1 Channel ES+ TIC
6.03e61.31
Comparing MRM and SIR - Example 1
0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70Time0
100
%
MixIso_1G14_023 SIR of 1 Channel ES+ TIC
6.03e61.31
Ion Chromatograms from SIR and MRM Analyses
of a Sample that is60 ng/mL Ketoprofen
6 ng/mL Fenbufen
From SIR of m/ z= 255
0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70Time0
100
%
0
100
%
MixIso_1G14_024 MRM of 2 Channels ES+ 255.25 > 209.2
1.43e61.31
MixIso_1G14_024 MRM of 2 Channels ES+ 255.25 > 237.2
8.06e41.42
Fenbufen
Ketoprofen
From MRM of m/z= 255 > 209
From MRM of m/z= 255 > 237
Comparing MRM and SIR - Example 1 (cont.)
22
++CID
CID
++
DifferentNeutralFragments
Different CompoundsThat Are Somewhat Similar In Structure
SameChargedFragment
Parent Ion Scan
+
+Parent Ion Scans can be used to detect those compounds whose molecular ions produce the same charge fragment.
Consider a class of compounds that are similar in structure:
PAR (Parent Ion Scan)
MS1 CollisionCell (w/Argon)
MS2
m2
5-40 eV Fixed
3V
Scanning
1V
m3
m1
Find ions that will produce via CID, daughter ions with a particular m/z
23
++CID
CID
+
SameNeutralFragment
Different CompoundsThat Are Somewhat Similar In Structure
DifferentChargedFragments
+
Constant Neutral Loss
+
+Constant Neutral Loss Scans can be used to detect those compounds whose molecular ions produce the same neutral fragment.
Constant Neutral Loss (CNL) Scan
MS1 CollisionCell (w/Argon)
MS2
m2
5-40 eV Scanning
1V
Scanning
1Vm1 - offsetm1
m2 - offset
Q1 and Q2 scan together. m/z of Q2 is m/z of Q1 minus an offset.
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