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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)

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ionization sources ii
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)
  • 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
  • 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



  • 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
maldi matrix assisted laser desorption
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
  • Choice of matrix based on empirical evidence
  • 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
uv maldi matrices




α-Cyano-4-hydroxycinnamic acid



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


2,5 Dihydroxybenzoic acid (DHB)

peptides, proteins, polymers, sugars

3-Hydroxypicolinic acid (HPA)


Dithranol (anthralin)


UV-MALDI Matrices

(low femtomole)


  • 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
esi electrospray ionization
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.
  • 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 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 multiply charged ions
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 ions1
ESI-Multiply Charged Ions


Δm = 1 amu ; ∆(m/z) ≈ 0.055; z = 18

; Δ(m/z) ≈ 0.10

Δm = 1 amu

z = 10

esi multiply charged ions2


z1 =

M = z1(m1-mp)


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
esi multiply charged ions3



= 51.0

z1 =

z1 =



ESI-Multiply Charged Ions




M = z1(m1-mp)

M = 51.0(1303.8-1.0073)

M = 66485


(low femtomole to zeptomole)


  • 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
apci atmospheric pressure ci
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

(high femtomole)


  • Parent Ion
  • Insensitive to Salts
  • Interface to HPLC
  • Can use Normal Phase Solvents
  • Handles High Flow Rates
  • Disadvantages
  • Need Volatile Sample
  • Need Thermal Stability
  • 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