Atmospheric Pressure Chemical Ionization (APCI)

APCI is an ionization technique using gas-phase ion-molecule

reaction at atmospheric pressure.

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.

 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 atmosphericpressure 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.

Atmospheric Pressure Ionization (and APcI)

Atmospheric Pressure Ionization (and APcI)

Ion Evaporation

Chemical ionization

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

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

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

Several kilovolts

Few l/min

320-350 K, 800 torr

100 ml/s

Iribarne-Thomson Model:

Charge density increases

Raylaeigh limit (Coulomb repulsion = surface tension)
Coulomb explosion (forms daughter droplets)
Evaporation of daughter droplets

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

21

Applications:
Determination of MW and charges for each peak (Smith
et al. Anal. Chem., 1990, 62, 882-899):

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.

Z1

Z2

M/Z
P1

P2

Given these assumptions,

eq 1 describes the
relationship between a
multiply charged ion at
m/z P1 with charge z1 and
molecular weight M.

P1Z1 = M + MaZ1 = M + 1.0079Z1

[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 + 1.0079(Z1-j)

[2]

Equations 1 and 2 can be solved for the charge of P1.

Z1 = j(P2-1.0079)/(P2-P1)
[3]
The molecular weight is obtained by taking Z1 as the nearest integer valve.

Electrospray

What happens when voltage is applied?

http://www.newobjective.com/electrospray/spray_anim.html

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.

900 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

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 liquids
surface tension, resulting in a release of ions
from dropletsurfaces

HOW MANY AMINO-ACIDS?

~ 1 charge per 1000 Da!

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.

Electrospray Ionization Process

3-6KV

End Plate
-2 to -3kV

Desolvation

+
+- +

0.3-2 cm
106V/m

- ++

Liquid
flow

- +
- +--

+
+

-+

+
+- +

+ -

+
+- +

+
+- +

- ++

-+

+ -

+
+- +
+
+- +
+
+- +

Emitter
(Ground)

Coulombic
Explosion

Desolvated
Ions

Glass
Capillary
-2 to -5 kV

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.

Electric Field at Tip (E)

V

4d
2V
E = r ln( r )

Capillary

r
d

HV

Counterelectrode

Taylor Cone
Accumulated charge at surface leads to
destabilization of surface because ions at
surface are drawn toward counterelectrode yet cant escape surface.
Leads to formation of the Taylor cone.

Capillary

Q = 49.3
Taylor cone

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:
Von =

2x105(g

r)0.5 ln(

4d
)
r

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.

Parameters Influencing Droplet Size

The radius (R) of an electrosprayed droplet
depends upon fluid density (r), flow rate (Vf),
and surface tension (g).

R (rVf 2g)1/3
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

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

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.

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)

Ion Sources: OLD ESI DESIGN

ESI Z Spray Source

ESI: Protein analysis

Peptide sequencing by nano-electrospray mass spectrometry

M+17
Electrospray spectrum of horse
myoglobin (mw 16,951.5)

Multiply-charged ion distribution

from +12 to +24 shown at low
resolution.

M+17

The +17 charge state at a resolution

of about 15,000 showing the
resolved isotope peaks.

Concentration and Sensitivity

Limitation of Ion Current

Electrochemical reactions
occur in last few M.
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.

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.

Analyte concentration and ion intensity

Analyte concentration and ion intensity

Atmospheric Pressure Photoionization

Atmospheric Pressure Photoionization

Desorption Electrospray Ionization

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)

Relatively small.
Volatile.

GC or liquid
or solid probe

To 1000
Daltons

Soft method. Molecular ion

peak [M+H]+

Electrospray (ESI)

Peptides/proteins.
Non-volatile.

Liquid
Chromatography

To 200,000
Daltons

Soft method. Ions often

multiply charged.

Matrix Assisted Laser

Desorption (MALDI)

Peptides/proteins.
Non-volatile.

Sample mixed in
solid matrix

To 500,000
Daltons

Soft method.
Very high mass range.

Fast Atom Bombardment (FAB)

Carbs/peptides.
Non-volatile.

Sample mixed in
viscous matrix

To 6000
Daltons

Soft method, but harder than

ESI or MALDI