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.