Lecture 3:
Ionization Techniques – Part II
CU- Boulder
CHEM 5181
Mass Spectrometry & Chromatography
J. Kimmel
Fall 2006
Announcements
• HW 2 due Thursday
• Journal Skim 3 due Thursday
• Exam next Tuesday
– Clicker review in class on Thursday
• Please email 2 questions with answers to Joel by noon on
Wednesday
• We are keeping original presentation schedule
Science (Journal) Presentations
Date
Presenter
Oct 9
Coburn
Oct 16
--------------
Oct 23
Thalman
Oct 30
Axson & Craven
Nov 27
Robinson
Dec 4
Tienes
Electrospray Ionization
Atmospheric pressure ionization
Enables MS detection of large, non-volatile
molecules (e.g., proteins) with no
fragmentation (→Nobel Prize 2002)
Search “ESI-MS” = 13,000 articles
Fenn’s 1985 A Chem paper cited 845 times
Liquid elutes through a high voltage tip
Coulombic explosions yield a continuous
mist of bare, gas-phase ions (positive or
negative)
Conveniently coupled to liquid separations
Characterized by multiply charged ions
Newobjective.com
Electrospray Mechanism
•An electrolytic analyte solution is pushed
through the conductive end of capillary (id
10-100 um) at very low flow rate (0.1-10
uL/min) held a few mm from the entrance of
the MS
•High potential (2-4 kV) induces a strong
electric field (106 – 107 Vm-1)
•For positive field, cations will move
towards the liquid surface and anions will
move towards the conductive tip.
•Repulsions between adjacent cations
combined with the pull of the cations towards
the grounded MS inlet cause the surface to
expand into a so-called ‘Taylor cone.’
Gomez & Tang, Phys Fluids, 1994, 6:404–414
ESI Mech (con’t)
Balance induced E field and surface tension of
liquid
Tip of the cone elongates into a filament, which
breaks up and emits a stream of charged droplets
towards the inlet of the mass spectrometer.
Evaporation of solvent from the droplets increases
the charge density.
At the ‘Rayleigh limit,’ repulsion between cations
equal surface tension, causing ‘Coulombic
explosions’ that produce even finer droplets.
This process of evaporation and explosion repeats
until fully desolvated ions are released.
The release of ions occurs either by repeated
fission events until total evaporation of the solvent
(Charge Residue Model) or by direct ion emission
from a charged droplet (Ion Evaporation Model).
Gomez & Tang, Phys Fluids, 1994, 6:404–414
ESI Mass Spectrum
High charge states make m/z
practicle for most mass analyzer
types.
z can be determined by isotope
distibution or sequence of peaks
(see section 1.8.1 of De Hoffmann
and HW #2)
ESI-MS of Cytochrome C, ~12,360 Da
From Fig 13-18 Lambert
ESI Source Design
ESI source must:
1.
2.
3.
On 1.
•
•
On 2.
•
•
•
On 3.
•
•
Move ions from solution to the gas phase
Transfer the gas-phase ions from atmospheric
pressure to vacuum
Yield ion beam with maximum current and
minimum kinetic energy distribution
Stable spray requires user optimization
High flow rates may require nebulizing gas to form
droplets
Heated drying gas + capillary encourage
desolvation, and limits solvent analyte-adduct
formation during expansion
Pumping speed places practical limit on size of
entrance aperature
Transfer of ions between stages of decreasing
pressure can result in a total ion loss on the
order of four to five orders of magnitude
Harnessing expansion
Constant Velocity = high E distribution
From de Hoffmann
For discussion, see: “ESI Source Design and Dynamic
Range Considerations,” A. P. Bruins, in “Electrospray
Ionization Mass Spectrometry,” R. B. Cole, 1997.
Controlled Current Electrolytic Flow Cell
From: Cech and Enke, Mass Spec
Rev, 20, 362, 2001.
•Electrical circuit to sustain ESI current : (+) Terminal to tip, to counter electrode,
to (-) Terminal.
•Electrolysis at electrodes maintains the charge balance to allow continuous
production of charged droplets.
•In order to supply demanded current, potential at electrode/solution interface has
value permitting the oxidation process characterized by lowest oxidation potential in
solution.
•This process determines the total # of ions that can be produced per unit time
ESI: Concentration Dependence
•
It is the excess charge in final droplets that imparts charge to gas-phase ions. (See
Fig 1.24 in De Hoffmann)
•
ESI is sensitive to concentration, not flow. Because limiting current, IM, is dependent
on oxidation process at tip.
•
ESI response can vary significantly among different analytes that have identical
concentrations
•
For a system with one analyte, spray current for will depend on its concentration
and a analyte-specific rate constant. IA = kA[A]
•
For system of two analytes, A and B, IT = IM = IA + IB And, currents proportional to relative
desorption rates and signal responses are coupled. Complicates quantification.
(See section 1.8.4 of de Hoffmann)
•
For any system, dynamic range limited at high end (~1 mM) by:
– Limited amount of excess charge
– Limited space on droplet surface
– Ion suppression
•
Consider separations prior to ESI to maximize sensitivity
Nanospray
•10 – 100 nl/min flow rate with fine spray tip
•Flow rate and droplets 100-1000 times smaller
than conventional ESI
•Large proportion of analyte available for
desorption from surface. 2-3 time higher ion
current than ESI at a given concentration
•Smaller tip close to orifice: narrow dispersion of
droplets yields better transfer in MS
•Orders of magnitude (2+) improvement in
efficiency (analyte detected / analyte sprayed)
•At these flow rates, ESI becomes “mass flux
sensitive”
•Longer analysis times -- better SNR and/or more
options in MS experiment
New Objective SilicaTips. Tip
i.d. range from 5 to 30 um.
Flow rate: 20 to 1000 nL/min
See: Wilm and Mann, A. Chem., 68, 1-8, 1996.
EI and CI are methods for molecular analysis
of gas phase sample
APCI and ESI: molecular analysis of liquid
phase
Now, desorption techniques that allow
molecular analysis from static matrix
Fast Atom Bombardment (FAB) and Secondary Ion
Mass Spectrometry (SIMS)
From Watson
Desorption techniques.
Analyze the ions emitted when a surface is irradiated with
an energetic beam of neutrals (FAB) or ions (SIMS).
Producing Primary Beam
From de Hoffmann
SIMS primary ions are
produced, e.g., as Cs atoms
vaporize through a porous
tungsten plug.
From SIMS Tutorial:
http://www.eaglabs.com/enUS/references/tutorial/simstheo/caistheo.html
Primary Beam of neutrals
produced by ionizing and
accelerating compound into
charge exchange collision with
neutral. e.g.:
Ar+rapid + Arslow → Ar+slow + Arrapid
Static SIMS
• Low current (10-10 A cm2) of keV primary ions (Ar+, Cs+ ..) impact the
solid analyte surface
• Low probability of area being struck by mulitiple ions; less than 1/10 of
atomic monolayer consumed
• Primary ion beam focused to less than 1 um enables high resolution
mapping – Often pulsed beam + TOFMS
• Sensitive technique for the ID of organic molecules
• Spectra show high abundance of protonated or cationized molecular
ions.
• Yields depend on substrate and primary beam; as high as 0.1 ions
per incident ion. Elemental yields vary over many orders of magnitude.
FAB and liquid-SIMS
•
Sample is dissolved in non-volatile liquid matrix and bombarded with
beam of neutrals (FAB) or ions
•
Shock wave ejects ions and molecules from solution. Generally eject ions
that already exist in solution.
•
Presence of charge (SIMS vs FAB) has little effect on the desorption
process. Neutrals used out of convenience for coupling to some instruments.
•
Use of liquid allows high primary ion currents while maintaining molecular
ions
– Solution presents mobile, constantly renewed surface to beam
•
Disadvantage: substantial background of matrix (e.g., Glycerol) ions and
matrix adducts.
•
Flow FAB:
– Continuous flow of liquid into mass spectrometer at rate of 1 – 20 uL/min
– Allows more use of more volatile solvent (eg, water, methanol, or
acetonitrile)
– Can be coupled to separation
Matrix Assisted Laser Desorption Ionization (MALDI)
•
Analyte molecules are embedded in a
crystalline matrix composed of a low molecular
weight organic species.
•
Dried mixture is struck with a short, intense laser
pulse having a wavelength that is strongly
absorbed by the matrix (often UV).
•
Rapid heating of matrix causes sublimation and
expansion into gas phase. Intact analyte
molecules carried with little internal energy.
•
Most widely accepted ionization mechanism is gasphase proton transfer.
Image from: Univ. of Bristol
•
Efficient, “soft,” and relatively universal
(wavelength independent of analyte). Matrix
isolates analyte molecules, preventing clusters.
•
Allows analysis of large (100s of kDa) intact
biopolymers (2002 Nobel Prize)
http://www.chm.bris.ac.uk/ms/theory/maldi-ionisation.html
Example of MALDI Data
•Spectra contain mostly
single-charged ions.
•Fragmentation due to
excess energy imparted on
analyte during DI process is
possible (Prompt, Fast, or
Post Source)
•Optimized conditions
determined empirically.
From De Hoffmann
MALDI-TOF Mass Spectrometer
From Barker
MALDI conveniently coupled to ToF-MS
1. Pulsed source, pulsed analyzer
2. Large m/z, large mass range
(See lab #1, HW #3, and next lecture)
Atmospheric Pressure Desorption Ionization
2000: AP-MALDI is first atmospheric pressure ionization technique for
condensed-phase analyte. Burlingame et al, A Chem, 652, 2000
2004: Desorption Electrospray Ionization (DESI) Cooks et al, Science, 2004, 471-473
2005: Direct Analysis in Real Time (DART) Cody et al, A. Chem, 2005, 2297.
2005 – Present: Rapid development, application, and evolution of these
methods (e.g., MS conferences may have multiple sessions dedicated to DESI
and DART)
** These methods would make for interesting journal presentation
Desorption Electrospray Ionization
Image from: Cooks et al, Science, 2004, 471-473
Photos from:
http://www.prosolia.com/index.html
Advance: (DESI and DART) Analysis in free ambient environment, sample potentially
subject to arbitrarily chosen processes during MS analysis.
•Applicable to small molecule organics and large biomolecules in solids, liquids, and adsorbed
gases.
•Natural products in plant material
•High throughput of pharmaceuticals
•Biological tissue and fluids
•Forensics / Public safety
•Typically no pre-treatment. Sample is sprayed with an electrically charged aqueous mist.
Released ions are transported through air to MS interface (which may be long transfer line).
Desorption Electrospray Ionization
•Solution can be tuned to make process selective.
•Relative movement of sample and spray enables
spatial resolution down to 50 um, for imaging of
surfaces
•The momentum-transfer collisions of DI in vacuum
are not applicable, as projectiles have low kinetic
E.
Two mechanisms:
ESI-like: DESI produces multiply charged ions from
biological macromolecules. Ionization believed to
occur through formation of charged droplets, after
liquid spreads across surface.
APCI-like: For molecules that cannot be analyzed by
ESI (eg, non-polar compounds) see singly charged
species, indicative of charge transfer between liquid
and surface or ion-molecule reactions. Requires
potential (2kV) between sprayer and surface.
Image from: Cooks et al, Science, 2004, 471-473
Summary of DI Techniques
From Lambert
Elemental Analysis Notes
• Need to decompose analyte into vaporphase ATOMS
– E.g. to determine isotope ratios
• Souces are also used by optical
spectroscopy
– MS tends to produce less complex signals,
and can be more sensitive
Thermal Ionization
•One of the earliest ionization techniques
for MS. Remains the most precise and
accurate method in MS to determine isotope
ratios of solid samples.
•Sample may be gas phase, or deposited
on filament surface
•One or more filaments are heated to high T
by passage of a current.
•Electron transfer between atom and
filament produces intense, stable beams of
positive and/or negative atomic ions.
•Multiply charged ions are not observed, and
clusters are rare
•Multiple filaments allows separate control
of vaporization and ionization parameters
Figure from: Inghram and Chupka,
Rev Sci Inst, 24, 518, 1953
Thermal Ionization (cont’d)
Ion yield range from 1 to 10% and depend on electron affinity of the analyte, the work
function of the filament material, and the temperature of the filament. Work function
may depend on treatment of filament surface. (See Heumann et al, Analyst, 120, 1291, 1995)
Positive Ions: compounds with low first ionization potentials; high work function
filaments
Negative ions: High electron affinities; low work function filaments
Thermal ionization cavity source offers order of
magnitudes improvements in efficiency.
Metal tube is heated by high energy electron
bombardment.
As the sample evaporates inside the crucible,
gaseous analyte atoms are produced which
interact with the inner surface of the crucible
walls to produce positive ions through surface
ionization.
From de Hoffmann
Spark Ionization Source
•Most transition metals difficult to ionize thermally.
Spark developed by Dempster to extend MS
analysis to metals.
•Pulsed 1-MHz rf-voltage of several kilovolts
produces discharges between two rod electrodes.
•Sample may serve as one electrode, or be
mixed with carbon and placed in cup electrode
•Ionization occurs within plasma.
From de Hoffmann
•Singly and multiply charged atomic ions, polymer
ions, and heterogeneous compounds
•Intensity of major constituent peaks decreases
with charge number.
•Approximately equal sensitivity for all
elements. Useful for analysis of trace impurities
(DLs in ppb range).
• ↓ Ions have very wide energy distribution and the
spark is subject to random fluctuations in intensity
From Dempster, Rev Sci Inst, 7, 46, 1936
Glow Discharge Source
From King, Teng, and Steiner, J. Mass Spec., 30, 1061, 1995.
•Produces singly charged atomic cations. Used primarily for bulk metal analysis.
•Discharge between cathode (sample) and anode in Ar at low pressure (ca 0.1-10 Torr; 5 mm
gap, 1 kV, 1-2 mA)
•Sputtering: Ions and electrons from plasma accelerate toward electodes. Ar+ attacks cathode
surface, releasing M (neutral) and M+
•80% of potential drop occurs in non-luminous “dark space” (cathode fall) extending 1 mean free
path from cathode. M+ produced by sputtering cannot escape this region. Effectively decoupling
atomization and ionization steps and reducing matrix effects on ionization.
•Collisions and ionization abound in “Negative Glow.” Neutral M ionized by multiple
pathways.
GD: Collisional Ion Formation Processes
ee-
M0
M+
Electron Ionization
e-
eAr
M0
M+
Penning Ionization
M
Ar
Within Negative Glow, electron ionization and Penning
Ionization account for 90% of the observed M+.
Penning Ionization
A* + BC
A + BC+ + e-
OR
A* + BC
A + B+ + C + e-
From: Faubert et al, IJMS, 1993, 69-77
Electron transfer reaction
Occurs if ionization potential of BC is less than
excited state energy of A*
Inductively Coupled Plasma
•ICP source was originally developed for Atomic
Emission Spectroscopy of solutions.
•ICP-MS is capable of trace multielement analysis of
solids or liquids, often at the part per trillion level.
•Can detect all elements except F, Ne, and He.
Ionization efficiency >= 90% for 54 elements.
Hardware:
3 concentric quartz tubes. Argon flowing through each
at atmospheric pressure.
Cooled induction coil surrounds the top of largest tube
– powered by Rf Generator.
An Initial Tesla sparks ionizes flowing argon.
Rf field accelerates these ions, leading to collisions
and more ions. Equilibrium between ionization and
recombination.
Plasma reaches 10 000 K.
Photo from:
http://ewr.cee.vt.edu/environmental/teach/smprimer/icpms/i
cpms.htm
ICP Source
From de Hoffmann
•Sample (vapor or droplet) carried into plasma through central tube.
•High T desolvates, vaporizes, atomize, and ionize sample – Close to 100%
efficiency.
•As for other atmospheric pressure ionization sources, transfer to MS requires
differentially pumped interface.
ICP-MS vs ICP-OES
•
MS much simpler than the
optical emission spectra Most
heavy elements exhibit
hundreds of emission lines, but
they have only 1-10 natural
isotopes in the mass spectrum.
•
ICP-OES suffers from many
overlapping spectral
interferences from other
elements and a very high
background emission from the
plasma itself, limiting detection
limits.
•
Detection limits of ICP-MS are
three orders of magnitude
better than ICP-OES.
Comparison of Molecular Ionization Techniques
Desorption Ionization Summary
From Lambert