Ionization Sources - II

• EI and CI have limitations– Both require a volatile sample– Samples must be thermally stable– Neither lends itself to LC/MS analysis

• Other techniques have been developed– FAB (Fast Atom Bombardment)– MALDI (Matrix Assisted Laser Desorption)– ESI (Electrospray)– APCI (Atmospheric Pressure CI)

FAB

• Sample is dissolved in a non-volatile liquid matrix– Glycerol and m-Nitrobenzyl alcohol are

common matrices

• A high energy (5kV) beam of neutral atoms (typically Ar or Xe) is focused onto the sample droplet

• Dissolved Ions and Molecules are ejected into the gas phase for analysis

FAB

FAB

• For Organic Molecules M+H and M+Na ions are typically observed

• M+H ions typically fragment more than M+Na ions

• Salts such as NaI can be added to the matrix to induce M+Na formation

FAB

Advantages

• Stable Molecular Ion

• High Mass Compounds (10,000 amu)

• Thermally Labile Compounds (R.T.)

Disadvantages• No Fragment Library• Solubility in Matrix

(MNBA, Glycerol)• Quantitation Difficult• Needs Highly Skilled

Operator• Not amenable to

automation• Relatively Low

Sensitivity

(nanomole)

MALDIMatrix Assisted Laser Desorption

• Sample dissolved in a solid matrix• Typically mixed in solution• Small droplet applied to target and dried

• A wide variety of matrices exist• Choose based on hydrophobic/hydrophilic

character of sample• Also based on laser absorbance (usually UV)

• An ionization agent is often added• Agent must bind to the sample• TFA and its Na+ Ag+ salts are common

MALDI

MALDI

• Choice of matrix based on empirical evidence

• http://polymers.msel.nist.gov/maldirecipes/maldi.html

• Typically singly charged ions observed• Some matrix adducts/cluster ions• Difficult to analyze low MW compounds

due to matrix background• Typically used for MW 500-500,000

MALDI

MALDI

OOMe

CH3

()

UV-MALDI MatricesMatrix Application Structure

α-Cyano-4-hydroxycinnamic acid(CCA)

peptides

OHNC

OHO

3,5-Dimethoxy-4-hydroxycinnamic acid (sinapinic acid)

proteins HO

H3CO

H3CO

O

OH

2,5 Dihydroxybenzoic acid (DHB) peptides, proteins, polymers, sugars

O OH

OH

HO

3-Hydroxypicolinic acid (HPA) oligonucleotidesN

OH

OH

O

Dithranol (anthralin) polymers

OOH OH

MALDI

Advantages

• Parent Ion

• High Mass Compounds (>100,000 amu)

• Thermally Labile Compounds (R.T.)

• Easy to Operate

• Easily Automated

Disadvantages• No Fragment Library• Wide variety of matrices• Quantitation Difficult• Matrix Background

(low femtomole)

ESIElectrospray Ionization

• Sample dissolved in a polar solvent• Solution flows into a strong electric field

(3-6 kV potential) • Electric field induces a spray of highly

charged droplets (charges at surface)• As droplets shrink, repulsion increases

until they break into smaller droplets• In small enough droplets, surface

charges can be desorbed into the gas phase.

ESI

ESI

• Ions formed via charge-residue or ion-evaporation

• Molecules form M+H+ or M-H- ions– Large molecules: 1 charge / 1000 amu– Small molecules: Usually singly charged

• Molecules with no acid/base groups– Can form adduct ions with Na+ K+ NH4

+ Cl-

OAc-, etc.– Salts may be added or already present in

sample.

ESI

• ESI ions formed at high pressure must be transferred into high vacuum

• Differential pumping is needed to move ions through small openings while maintaining low pressures

• Ions become super-cooled by expansion. Solvent can recondense– Two methods to reduce cluster formation

• High temperature transfer tube• Heated counter-current flow of N2

ESI

ESI

ESI

ESI-Multiply Charged Ions

• Large Molecules produce an envelope of charge states

• Deconvolution must be done to determine the charge states if isotopic resolution is not possible

• Typically, MS data systems use software to deconvolute automatically

ESI-Multiply Charged Ions

Δm = 1 amuΔ(m/z) ≈ 0.10

Δm = 1 amu ; ∆(m/z) ≈ 0.055; z = 18

z = 10

M=16953

ESI-Multiply Charged Ions

• Consider (M+zH)z+

– z1m1 = M + z1mp (m1 = measured m/z)

• Consider a peak of m/z=m2 which is (j-1) charge states away from peak m1

– m2(z1-j) = M + (z1-j)mp

z1 =j(m2-mp)

(m2-m1)M = z1(m1-mp)

ESI-Multiply Charged Ions

z1 =j(m2-mp)

(m2-m1)M = z1(m1-mp)

1303.8 1621.3j=10

z1 =10(1621.3-1.0073)

(1621.3-1303.8)= 51.0 M = 51.0(1303.8-1.0073)

M = 66485

ESI-Multiply Charged Ions

ESI

Advantages• Parent Ion• High Mass Compounds

(>100,000 amu)• Thermally Labile

Compounds (<0º C)• Easy to Operate• Interface to HPLC• Zeptomole sensitivity

with nanospray

Disadvantages• No Fragmentation• Need Polar Sample• Need Solubility in Polar

Solvent (MeOH, ACN, H2O, Acetone are best)

• Sensitive to Salts• Supression

(low femtomole to zeptomole)

APCIAtmospheric Pressure CI

• Sample solution flows into a pneumatic nebulizer

• Droplets of sample/solvent are vaporized in a quartz heater

• Vapor passes by a region of corona discharge where electrons ionize N2 gas and solvent (protonated solvent molecules predominate)

• Protonated solvent reacts with sample

APCI

APCI

APCI

APCI

Advantages

• Parent Ion

• Insensitive to Salts

• Interface to HPLC

• Can use Normal Phase Solvents

• Handles High Flow Rates

Disadvantages• Need Volatile Sample• Need Thermal Stability

(high femtomole)

Multimode

• Most instruments use dedicated ESI and APCI sources– samples must be run twice to obtain both

spectra

• Some vendors offer sources which rapidly switch between ESI and APCI– duty cycle/sensitivity are lost, especially

when coupled with fast chromatography

• Agilent has developed a source which ionizes by ESI and APCI without switching

Multimode

Multimode

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Multimode