LECTURE 3

Ionization Techniques for Mass Spectrometry

Jack Henion, Ph.D. Emeritus Professor, Analytical Toxicology

Cornell University Ithaca, NY 14850

Lecture 3, Page 1

Contents

• Electron ionization (EI) • Electrospray ionization (ESI)

– Nano ESI

• Atmospheric pressure chemical ionization ( APCI) • Atmospheric pressure photoionization ( APPI) • Matrix assisted laser desorption ionization (MALDI) • LAESI • Direct analysis in real time (DART) • Desorption electrospray ionization (DESI) • Atmospheric sampling analysis probe (ASAP) • And there are many more that may or may not mature.

Lecture 3, Page 2

Many Forms of Ionization have been Reported

• REIMS

• PESI

• LIAD-ESI

• DART

• FAPA

• LAESI/IR-LDESI/MALDESI

• ELDI

• LIAD-ESI

• EI

• APCI

• ESI

• APPI

• ASAP

• MALDI

• DESI

• RADIO

• DAPPI

Lecture 3, Page 3

Electron Ionization (EI) Source (not yet commercially available for LC/MS)

High vacuum

Lecture 3, Page 4

Ionization Processes: Electron Ionization (EI) vs. API

• Electron ionization (EI) forms radical cations as shown below: – M + e- M+. + 2 e-

– The radical cation is very reactive and often is the primary director of the ensuing fragmentation

• API techniques generate (M+1)+ ions as shown below:

• Electrospray or APCI:

• (M+H)+ (M+H)+ • (in solution) (ion evaporation or APCI) (gas-phase ion)

But how does this occur?

70 eV electrons

Lecture 3, Page 5

Positive Ions: Odd-Electron and Even-Electron Ions

Odd Electron Ions

N

HH HN

HH H

EE (8) OE (7)

N

HH HN

HH H

EE (8) EE (8)

Even Electron Ions

H

e2e

OE and EE Ions

H

+

Lecture 3, Page 6

Electrospray Ionization

Lecture 3, Page 7

Electrospray Process

“A method by which ions

present in solution can be

transferred to the gas phase”

Classical or Pure ESI or NSI: Droplet Size influenced purely by electrostatic repulsion

High Flow ESI – Gas Assisted: Droplet size influenced by high

velocity gas & electrostatic repulsion

Courtesy ThermoFinnigan Lecture 3, Page 8

Ink Jet printing

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High-Voltage

Power Supply

electrons electrons

OXIDATION REDUCTION

Taylor Cone

+4 kV

+1 kV

Lecture 3, Page 9

Maximizing Signal. Minimizing Noise.

Agilent Animation of S/N

Lecture 3, Page 10

AB SCIEX “Turbo-V “ Gas Entrainment Source High sensitivity at any flow rate.

4. Controlled Entrainment

Background Reduction

1. Increased Thermal Efficiency

of Heater

3. Promote Turbulent Mixing

Improved Heat and Mass Transfer

Maximize Desolvation

2. Second Heater

Double Thermal Load

Signal Enhancement Flow Rate Dependent

5. Reduced Dispersion of Droplets

Lecture 3, Page 11

• Benefits: Sensitivity across a wider range of

flow rates

–Higher desolvation for >1mL/min flow rates • More heated nitrogen auxiliary gas flow aids

desolvation at high flow rates

–One piece metal needle allows for easy replacement • Available as low flow and regular (high) flow

– Contoured tip for enhanced low flow (micro-spray; 5-25uL) stability • Generates better electrostatic fields enabling spray

stability for lower flow rates

Electrospray Ionization Technology Enhancements: Thermo HESI-II

* Available on all TSQ products Metal Needle Standard

Contoured tip

Higher desolvation

Lecture 3, Page 12

Synapt HDMS System

Waters Z-Spray

Lecture 3, Page 13

Agilent Jet Stream Technology

Courtesy of Dr. Alex Mordehai, Agilent Technologies

Lecture 3, Page 14

Collision Cell

H-SRM Operation – Enhancing Specificity

= only pre-cursor ion transmitted

Lower chemical noise

Lower detection limits

Thermo TSQ Quantum Lecture 3, Page 15

• No Line of sight – enhanced S/N

• 5x better SRM transmission efficiency

• Argon as collision gas for higher sensitivity

Enhancing Detection Limits – 90º Collision Cell Design

Exit lens

assembly

Entrance lens

assembly

Collision cell

housing

Square quadrupole

collision cell

Lecture 3, Page 16

A trace of DMSO to Improve Peptide Ionization Reponse

Jesse G. Meyer, Elizabeth A. Komives, Charge State Coalescence During Electrospray Ionization Improves Peptide Identification by Tandem Mass Spectrometry, J. Am. Soc. Mass Spectrom. (2012) 23:1390Y1399.

Page 17

APPI

Atmospheric Pressure Photoionization

Lecture 3, Page 18

APPI source configuration employing a VUV light source and orthogonal spray geometry.

Qualitative diagram representing

ionization of nonpolar compounds

by APPI

APPI Source and Compound Strike Zone

APPI

Electrospray

Ionization

Polarity Very PolarNon-Polar

Mo

lec

ula

r W

eig

ht

100

1,000

10,000

100,000

APCI

Lecture 3, Page 19

Direct APPI

M + hv M+

M+ + S MH+ + S[-H]

Dopant APPI

D + hv D+

D+ + M MH+ + D[-H]

D+ + M M+ + D

Analyte molecule M is ionized to a molecular

ion M+. (If analyte ionization potential is

below photon energy)

In the presence of protic solvents, M+ may

abstract a hydrogen atom to form MH+.

A photoionizable dopant is delivered in large

concentration to yield many D+ ions.

D+ ionizes analyte M by proton or electron

transfer.

This is PI-initiated APCI.

Direct vs. Dopant APPI

Lecture 3, Page 20

Benefits of APPI

– Ionizes a relatively wide range of compounds

(e.g., non-polars, electronegative cpds, etc.)

– Predominantly parent ion signal (minimum

fragmentation)

– Low noise source (minimum solvent signal)

– Minimum ion suppression

– Large linear dynamic range

– Works for positive and negative ionization

Lecture 3, Page 21

APPI Is Best For

• Environmental – PAHs – Steroids – Pesticides

• Food Safety – PAHs – Pesticides – Fish Oil Lipids – Vitamins

• Petroleum/Oils/Biofuels – Petroleum – Biofuels – Triacylglycerol Lipids

• Pharmaceuticals/Small Molecules – Steroids – Metabolites (especially lacking functional groups) – Chiral Separations (i.e., normal phase LC)

R2 = 0.9999

0.E+00

5.E+05

1.E+06

2.E+06

2.E+06

0 250 500 750 1000 1250

Inj. Amount (ng)

Peak A

rea

R2 = 0.9995

0

200

400

0 0.1 0.2 0.3

>5-decade linear

response on

the ZQ

Analysis of Lipids: Diarachidin(1:1 Isooctane:IPA @ 100 uL/min)

R2 = 0.9999

0.E+00

5.E+05

1.E+06

2.E+06

2.E+06

0 250 500 750 1000 1250

Inj. Amount (ng)

Peak A

rea

R2 = 0.9995

0

200

400

0 0.1 0.2 0.3

>5-decade linear

response on

the ZQ

Analysis of Lipids: Diarachidin(1:1 Isooctane:IPA @ 100 uL/min)

50 100 150 200 250 300 350 400

Ion m ass

0

100

%

0

100

%

[M+H]+

315.17

APPI

ESI

Progesterone

50 100 150 200 250 300 350 400

Ion m ass

0

100

%

0

100

%

[M+H]+

315.17

APPI

ESI

Progesterone

Dibenzo[a,h]anthracene,

278 > 250

6.1 - 100,000 pg

R2 = 0.9994

0.0E+00

2.0E+04

4.0E+04

6.0E+04

8.0E+04

1.0E+05

1.2E+05

0 20000 40000 60000 80000 100000

Injection Amount (pg)

Peak A

rea

>104 linear response

Lecture 3, Page 22

APPI Very Resistant to Ion/Matrix Suppression

min 0 0.5 1 1.5 2 2.5 3 3.5

APPI TIC Full Scan - Rat Plasma Extract

APPI

APCI

ESI

Determination of ion suppression susceptibility for

APPI, APCI, and ESI by post-column addition and

detection of fluphenazine while running an LC/MS

chromatogram of rat plasma.

APPI

APCI

ESI

Average deviation

74%

57%

23%

Median deviation

68%

52%

22%

Ratio of signal for FIA/MS vs. LC/MS

Measurement performed on a 29-compound library of unknown purity

These results show minimal ion

suppression by APPI. APCI shows

less ion suppression than ESI, but

APCI signal is noisier than by APPI

Lecture 3, Page 23