1. Atmospheric Pressure Chemical Ionization (APCI)
APCI is an ionization technique using gas-phase ion-molecule
reaction at atmospheric pressure.

2. The nebulizer consists of three concentric tubes, the eluent is pumped through the inner most tube and nebulizer gas and make-up gas through the outer tubes.
The combination of the heat and gas flow desolvates the nebulized droplets, producing dry vapor of solvent and analyte molecules.
The solvent molecules are then ionized by a corona discharge
The results of these reactions produce water cluster ions, H3O+(H2O)n or protonated solvent, such as CH3OH2+ (H2O)n(CH3OH)m with n + m < = 4.

3. These ions enter in gas-phase ion-molecule reactions with an analyte molecules, leading to (solvated) protonated analyte molecules.
Subsequent declustering (removal of solvent molecules from the protonated molecule) takes place when the ions are transferred from the atmospheric-pressure ion source towards the high vacuum of the mass analyzer.
Proton transfer reactions are major process, while other reactions such as adduct formation and charge exchange in positive ion mode or anion attachment and electron capture reactions in negative ion mode are also possible.

4. Atmospheric Pressure Ionization (and APcI)

5. Atmospheric Pressure Ionization (and APcI)
Ion Evaporation
Chemical ionization

6. APCI Analogous to CI For compounds with MW about 1,500 Da
Produce monocharged ions

7. Electrospray ionization (ESI)
Method used to produce gaseous ionized molecules from a liquid solution by creating a fine spray of droplets in the presence of a strong electric field.
Electrospray ionization/mass spectrometry (ESI/MS) which was first described in 1984 (commercial available in 1988), has now become one of the most important techniques for analyzing biomolecules, such as polypeptides, proteins having MW of 100,000 Da or more

8. In the eyes of Fenn…
“Although ESI is now in daily use all over the world, its component processes and mechanisms, especially the dispersion of the sample liquid into charged droplets, and the formation of gas phase ions from those droplets are poorly understood”
Professor John B. Fenn

9. Several kilovolts
Few µl/min
K, 800 torr
100 ml/s

10. Iribarne-Thomson Model:
Charge density increases
Raylaeigh limit (Coulomb repulsion = surface tension)
Coulomb explosion (forms daughter droplets)
Evaporation of daughter droplets

11. Special features of ESI process:
Little fragmentation of large and thermally unstable molecules
Multiple charge
Linear relationship between average charge and molecular weight
Easily coupled to HPLC

12. 21

13. Applications: Assumptions
Determination of MW and charges for each peak (Smith et al. Anal. Chem., 1990, 62, ):
Assumptions
The adjacent peaks of a series differ by only one charge
For proteins, the charging is due to proton attachment to the molecular ion.
This has been an excellent (but not crucial) assumption of nearly all proteins studied to data where alkali attachment contributions are small.
Ionization of only the intact molecule.

14. Z1 Z2
Given these assumptions, eq 1 describes the relationship between a multiply charged ion at m/z P1 with charge z1 and molecular weight M.
M/Z P1 P2
P1Z1 = M + MaZ1 = M Z1 [1]
Assume that the charge carrying species (Ma) is a proton. The molecular weight of a second multiply protonated ion at m/z P2 (where P2 > P1) that is j peaks away from P1 (e.g. j = 1 for two adjacent peaks) is given by
P2(Z1-j) = M (Z1-j) [2]
Equations 1 and 2 can be solved for the charge of P1.
Z1 = j(P )/(P2-P1) [3]
The molecular weight is obtained by taking Z1 as the nearest integer valve.

15. Electrospray

16. What happens when voltage is applied?
These images are frame captures of a PicoTip spraying 5% Acetic acid in 30% MeOH at 200 nl/min by direct infusion from a syringe pump. Each frame differs by an applied voltage of approximately 100 volts. The tip-to-inlet distance was ca. 5 mm V - no spray 1000 V - Taylor-cone/droplet oscillation, more "drops" than spray 1100 V - cone/droplet oscillation. approx 50% spray 1200 V - cone/droplet oscillation, on the verge of a stable Taylor cone 1300 V - stable cone-jet 1400 V - cone-jet on the verge of "jumping", slight instability 1550 V - multiple cone-jets

23. Ionization Mechanisms
Coulomb Fission:
Assumes that the increased charge density, due to solvent evaporation, causes large droplets to divide into smaller droplets eventually leading to single ions.
Ion Evaporation:
Assumes the increased charge density that results from solvent evaporation causes Coulombic repulsion to overcome the liquid’s
surface tension, resulting in a release of ions from dropletsurfaces

24. HOW MANY AMINO-ACIDS?
~ 1 charge per 1000 Da!

25. 4 easy steps to ESI:
Production of charged droplets from electrolyte dissolved in solvent.
Shrinkage of charged droplets by solvent evaporation and droplet disintegration.
Mechanism of gas-phase ion production.
Secondary processes by which gas-phase ions are modified in the atmospheric and ion sampling regions.
Kebarle and Tang, Anal. Chem. 1993, 65, 972A-986A.

26. Electrospray Ionization Process+ -
Emitter
(Ground)
Desolvation
Coulombic
Explosion
Desolvated
Ions
Glass
Capillary
-2 to -5 kV
End Plate
-2 to -3kV
3-6KV
0.3-2 cm
106V/m
Liquid flow

27. Production of Charged Droplets
Voltage difference between the emitter and counter-electrode establishes an electric field (E  106 V/m). For positive ion mode:
Emitter grounded, counter-electrode biased –ve (2-6 kV)
Emitter biased +ve (2-6 kV), counter-electrode usually +ve a few V.
Liquid flowing through capillary is conductive.

28. Electric Field at Tip (E)2V
E =
ln( ) r r V
Counter-
electrode
Capillary r d HV

29. Taylor Cone
Accumulated charge at surface leads to destabilization of surface because ions at surface are drawn toward counter-electrode yet can’t escape surface.
Leads to formation of the Taylor cone.
Q = 49.3
Capillary
Taylor cone

30. Surface Tension and Droplet Production
The cone instability is profoundly influenced by the surface tension (g) of the fluid.
The onset voltage (Von) required to initiate charged-droplet emission is related to surface tension by: 4d
Von = 2x105(g r)0.5
ln( ) r

31. Thus…
Onset voltage is higher for liquids of higher surface tension. 4kV for water, 2.2 kV for methanol
Relative ranking:
iPrOH < MeOH < AcCN < DMSO < H2O
The higher the voltage, the increased probability of electrical discharge (esp. in negative ion mode)! Corona discharge also increases with decreasing pressure, so this is why ESI is done at atmospheric pressure.

32. Parameters Influencing Droplet Size
The radius (R) of an electrosprayed droplet depends upon fluid density (r), flow rate (Vf), and surface tension (g).
Thus, higher Vf result in larger initial droplet sizes. Larger droplet sizes lead to lower ionization efficiency because the droplets are not so close in size to the Rayleigh limit
R  (rVf 2g)1/3

33. Droplet “Shrinkage”
Now that the charged droplets have been released from the capillary, they are accelerated toward the counter-electrode.
Shrinkage of the droplets results as a combination of two factors:
Solvent evaporation
Droplet disintegration by Coulombic explosions

34. Rayleigh Limit
When charge Q becomes sufficient to overcome the surface tension which holds the droplet together, Coulombic explosions begin:
Q2 = 64p2eogR3
where eo is the permitivity of vacuum.

35. ESI Advantages Soft-ionization technique Controllable fragmentation
Readily coupled to liquid separations
Produces intact non-covalent complexes
Multiple-charging of analyte
Capable of ionizing large molecules (to MDa)

36. Ion Sources: OLD ESI DESIGN  

37. ESI “Z” Spray Source  

38. ESI: Protein analysis  
Peptide sequencing by nano-electrospray mass spectrometry

39. M+17
Electrospray spectrum of horse myoglobin (mw 16,951.5)
Multiply-charged ion distribution from +12 to +24 shown at low resolution.
The +17 charge state at a resolution of about 15,000 showing the resolved isotope peaks.
M+17

40. Concentration and Sensitivity

41. Limitation of Ion Current
Electrochemical reactions occur in last few μM.
Ions extracted per unit of time to the MS is limited by the current produced by the oxidation or reduction process at the probe tip.

42. ESI is a constant-current electrochemical cell
Too many ions from salts will decrease the abundance of sample ion
If too diluted or at very low flow, ion flow from capillary will be insufficient. Oxidation or reduction of solvent or sample will occur, producing radical ions.

43. Analyte concentration and ion intensity

44. Analyte concentration and ion intensity

45. Atmospheric Pressure Photoionization

46. Atmospheric Pressure Photoionization

47. Desorption Electrospray Ionization

49. Other ionization techniques
Ionization Method
Typical Analytes
Sample Introduction
Mass
Range
Method Highlights
Electron Impact (EI)
Relatively small.
Volatile.
GC or liquid
or solid probe
To 1000
Daltons
Hard method.
Provides structural info
Chemical Ionization (CI)
Soft method. Molecular ion peak [M+H]+
Electrospray (ESI)
Peptides/proteins.
Non-volatile.
Liquid
Chromatography
To 200,000
Soft method. Ions often multiply charged.
Matrix Assisted Laser
Desorption (MALDI)
Sample mixed in
solid matrix
To 500,000
Soft method.
Very high mass range.
Fast Atom Bombardment (FAB)
Carbs/peptides.
viscous matrix
To 6000
Soft method, but harder than ESI or MALDI