Ionization Sources - II

1. 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)

2. 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

3. FAB

4. 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

5. FAB (nanomole) 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

6. 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

7. MALDI

8. 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

9. MALDI

10. MALDI

11. Matrix Application Structure α-Cyano-4-hydroxycinnamic acid (CCA) peptides 3,5-Dimethoxy-4-hydroxycinnamic acid (sinapinic acid) proteins 2,5 Dihydroxybenzoic acid (DHB) peptides, proteins, polymers, sugars 3-Hydroxypicolinic acid (HPA) oligonucleotides Dithranol (anthralin) polymers UV-MALDI Matrices

12. MALDI (low femtomole) 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

13. 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.

14. ESI

15. 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.

16. 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

17. ESI

18. ESI

19. ESI

20. 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

21. ESI-Multiply Charged Ions M=16953 Δm = 1 amu ; ∆(m/z) ≈ 0.055; z = 18 ; Δ(m/z) ≈ 0.10 Δm = 1 amu z = 10

22. j(m2-mp) z1 = M = z1(m1-mp) (m2-m1) 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

23. 10(1621.3-1.0073) j(m2-mp) = 51.0 z1 = z1 = (m2-m1) (1621.3-1303.8) ESI-Multiply Charged Ions j=10 1303.8 1621.3 M = z1(m1-mp) M = 51.0(1303.8-1.0073) M = 66485

24. ESI-Multiply Charged Ions

25. ESI (low femtomole to zeptomole) 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

26. 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

27. APCI

28. APCI

29. APCI

30. APCI (high femtomole) 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

31. 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

32. Multimode

33. Multimode

34. Multimode