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 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.

Atmospheric Pressure Ionization (and APcI)

Ion Evaporation

Chemical ionization

Atmospheric Pressure Ionization (and APcI)

APCI

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

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 (ESI)

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

Few µl/min

Several kilovolts

320-350 K, 800 torr100 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.

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

Z1

Z2

M/ZP1 P2

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

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 spray1000 V - Taylor-cone/droplet oscillation, more "drops" than spray1100 V - cone/droplet oscillation. approx 50% spray1200 V - cone/droplet oscillation, on the verge of a stable Taylor cone1300 V - stable cone-jet1400 V - cone-jet on the verge of "jumping", slight instability1550 V - multiple cone-jets

What happens when voltage is applied?http://www.newobjective.com/electrospray/spray_anim.html

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’ssurface tension, resulting in a release of ions from dropletsurfaces

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

• 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.

4 easy steps to ESI:

++ -

+-

--+

+-

+

+ ---

++

-+

+ ---

++

-+

- ++

+

- ++

+

- ++

+

- ++

+

- ++

+

- ++

+

- ++

+

Emitter(Ground)

Desolvation

CoulombicExplosion

DesolvatedIons

GlassCapillary-2 to -5 kV

End Plate-2 to -3kV

Electrospray Ionization Process

Liquid flow

3-6KV

0.3-2 cm

106V/m

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)

r

d

CapillaryCounter-electrode

HV

VE = 2V ln( )r r

4d

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.

CapillaryTaylor cone

Surface Tension and Droplet Production

• The cone instability is profoundly influenced by the surface tension () of the fluid.

• The onset voltage (Von) required to initiate charged-droplet emission is related to surface tension by:

Von = 2x105(r)0.5 ln( )r4d

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 flow rate (Vf), and surface tension ().

• 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 (Vf 2)1/3

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 = 642oR3

where o 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  

Peptide sequencing by nano-electrospray mass spectrometry

ESI: Protein analysis  

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

M+17

Concentration and Sensitivity

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.

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

MassRange

Method Highlights

Electron Impact (EI) Relatively small.Volatile.

GC or liquidor solid probe

To 1000Daltons

Hard method.Provides structural info

Chemical Ionization (CI) Relatively small.Volatile.

GC or liquidor solid probe

To 1000Daltons

Soft method. Molecular ion peak [M+H]+

Electrospray (ESI) Peptides/proteins.Non-volatile.

LiquidChromatography

To 200,000Daltons

Soft method. Ions often multiply charged.

Matrix Assisted LaserDesorption (MALDI)

Peptides/proteins.Non-volatile.

Sample mixed insolid matrix

To 500,000Daltons

Soft method.Very high mass range.

Fast Atom Bombardment (FAB) Carbs/peptides.Non-volatile.

Sample mixed inviscous matrix

To 6000Daltons

Soft method, but harder than ESI or MALDI