ionization sources - ii
DESCRIPTION
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) - PowerPoint PPT PresentationTRANSCRIPT
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