topic 8 mass spectrometry
TRANSCRIPT
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SKA6014
ADVANCED ANALYTICAL CHEMISTRY
TOPIC 9Mass Spectrometry 1
Azlan Kamari, PhD
Department of ChemistryFaculty of Science and Mathematics
Universiti Pendidikan Sultan Idris
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Mass Spectrometry
Mass Spectrometry (a.k.a. MS or mass spec) a method
of separating and analyzingionsby their mass-to-chargeratio
MS does not involve a specific region of the
electromagnetic spectrum (because it is not directly
interested in the energies of emitted photons, electronicor vibrational transitions, nuclear spin transitions, etc)
Ion
abundance
Up to m/z = 100000!m/z
Ion
IonIon
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General Notes on Atomic and Molecular Mass
Helpful units and conversions:
1 amu = 1 Da = 1/12 the mass of a neutral 12C atom.
1 kDa = 1000 amu
Atomic weights of other elements are defined bycomparison.
Mass-to-charge ratio (m/z): the ratio of the mass of an
ion (m) to its charge (z)
Molecular ion: an ion consisting of essentially the whole
molecule
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Mass Spectrometers
A block diagram of a generic mass spectrometer:
IonizationSource
MassAnalyzer
Detector
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Ionization Sources
Electron Ionization (EI)
Chemical Ionization (CI/APCI) Photo-ionization (APPI)
Electrospray (ESI)
Matrix-assisted Laser Desorption (MALDI)
Field Desorption (FD) Plasma Desorption (PD)
Fast atom bombardment (FAB)
High-temperature Plasma (ICP)
Desorption
Gas Phase
Ionization
SourceMass
AnalyzerDetector
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EI: Electron Ionization/Electron Impact
The electron ionization
(EI) source is designedto produce gaseous
ions for analysis.
EI, which was one of theearliest sources in wide
use for MS, usually
operates on vapors
(such as those elutingfrom a GC)
Heated Incandescent
Tungsten/Rhenium Filament
Accel!
E-
Vaporized
Molecules
70 eV
IonsTo
Mass
Analyzer
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EI: Electron Ionization/Electron Impact
How EI works:
Electrons are emitted froma filament made of
tungsten, rhenium, etc
They are accelerated by a
potential of 70 V
The electrons andmolecules cross (usually at
a right angle) and collide
The ions are primarly
singly-charged, positive
ions, that are extracted by asmall potential (5V) through
a slit
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EI: Electron Ionization/Electron Impact
When electrons hit the molecules undergo
rovibrational excitation (the mass of electrons is toosmall to really move the molecules)
About one in a million molecules undergo the reaction:
M+ e- M
+ + 2e-
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EI: Electron Ionization/Electron Impact
Advantages:
Results in complex mass spectra with fragment ions,
useful for structural identification
Disadvantages: Can produce too much fragmentation, leading to no
molecular ions! (makes structural identification
difficult!)
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CI: Chemical Ionization
Chemical ionization (CI) is a form of gas-phase
chemistry that is softer (less energetic) than EI
Ionization via proton transfer reactions
A gas (ex. methane, isobutane, ammonia) is introduced
into the source at ~1 torr.
Example: CH4 reagent gas
CH4EI
CH4+
CH4+ + CH4
CH5+ + CH3
AH + CH5+ AH2
+ + CH4
Strong acid
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APCI: Atmospheric-Pressure Chemical
Ionization
A form of API (atmospheric pressure ionization) arange of ionization techniques that operate at higher
pressures, outside the vaccuum MS regions.
APCI a form of chemical ionization using the liquideffluent in a spray chamber as the reagent
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APCI: Atmospheric-Pressure Chemical
Ionization The APCI process:
The sample is in a flowing stream of a carrier liquid (or gas)and is nebulized at moderate temperatures.
This stream is flowed past an ionizer which ionizes the carrier
gas/liquid.
63Ni beta-emitters
Corona (electric) discharge needle at several kV
The ionized stream (which can be an LC solvent) acts as the
primary reactant ions, forming secondary ions with the
analytes.
The ions are formed at AP in this process, and are sent into the
vaccuum
In the vaccuum, a free-jet expansion occurs to form a Mach
disk and strong adiabatic cooling occurs.
Cooling promotes the stability of analyte ions (soft
ionization)
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APCI: Chemical Ionization
APCI (diagram from Agilent)
Diagram from Agilent Technologies
760 torr
10-6 torr
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APCI: Chemical Ionization
An APCI mass spectrum:
Diagram from Agilent Technologies
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Electrospray Ionization (ESI)
The ESI process:
Electrospray ionization (ESI) is accomplished by flowing a
solution through an electrically-conductive capillary held at high
voltage (several keV DC).
The capillary faces a grid/plate held at 0 VDC.
The solution flows out of the capillary and feels the voltage
charges build up on nebulized droplets, which then begin to
evaporate
Coulombic explosions occur when the repulsion of the charges
overcomes the surface tension of the solution (holding the drop
together) known as the Rayleigh limit.
Depending on whose theory you believe
the analyte ion is eventually the only ion left
orthe analyte ion is evaporated from a small enough droplet
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Electrospray Ionization (ESI)
A picture of two ideas for the electrospray process:
Diagram from John B. Fenn (Nobel Lecture), 2002Picture from http://www.newobjective.com/electrospray/electrospray.html
Noteions which are
surface-active will be
preferentially ionized
this can lead to ion
suppression!
http://www.newobjective.com/electrospray/electrospray.htmlhttp://www.newobjective.com/electrospray/electrospray.html -
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Electrospray Ionization (ESI)
An ESI source:
Diagram from Agilent Technologies
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Typical ESI Spectra
An ESI mass spectrum:
Diagram from Agilent Technologies
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Typical ESI Spectra
An ESI Mass Spectrum of a 14.4 kDa enzyme:
Diagram from http://www.nd.edu/~masspec/ions.html
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ESI and APCI
ESI and APCI complementary techniques:
Figure from Agilent Instruments
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ESI and APCI
ESI and APCIcomplementary techniques:
ESI APCI
Very soft ionization
can ionize thermally
labile samples
Some sample volatility
needed (nebulizer)
Ions formed in solution Ions formed in gas
phase
Singly- and multiply-charged ions [M+H]+
Singly-charged ions,[M+H]+ and [M-H]-
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Atmospheric Phase Photo-ionization
APPI can ionize things that ESI and APCI cant:
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Atmospheric Phase Photo-ionization
APPI can ionize things that ESI and APCI cant:
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Comparison of Ionization Methods
How to choose an ionization technique:
Figure from Agilent Instruments
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MALDI: Matrix-Assisted Laser
Desorption/Ionization
A method for desorbing
a sample with a laser,
while preventing thermal
degradation
A sample is mixed with a
radiation-absorbing
matrix used to help it
ionize
MALDI is mostly used for
large biomolecules and
polymers.
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MALDI: Matrix Effects The role of the matrix
Must absorb strongly at the laser wavelength
The analyte should preferably not absorb at this wavelength
Common matrices include nicotinic acid and many other
organic acids
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MALDI at Atmospheric Pressure
Advantages: fast, easy and sensitive
Disadvantages: no LC, matrix still needed
S. Moyer and R. Cotter, Atmospheric Pressure MALDI, Anal. Chem., 74, 468A-476A (2002)
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FAB: Fast Atom Bombardment
A soft ionization technique
Often used for polar, higher-mwt, thermally labile molecules
(masses up to 10 kDa) that are thermally labile.
Samples are atomized by bombardment with ~keV range Ar or
Xe atoms.
The atom beam is produced via an electron exchange process
from an ion gun.
Xee-
Xe+ + 2e-
Advantages:
Rapid sample heating reduced fragmentation
A glycerol solution matrix is often used to make it easier to
vaporize ions
Xe+accel
Xe+ (high KE)
Xe+ (high KE) + Xe Xe (high KE) + Xe+
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SIMS: Secondary Ion MS
Focused Ion Beam3He+, 16O+, 40Ar+
Beam energy 5 to 20 keV
Beam diameter 0.3 to 5 mm
Beam Hits Target
A small % of the target material is sputtered off and enters the
gas phase as ions (usually positive)
Advantages:
Imaging of ions (characteristic masses) on a surface or in
biological specimens
Surface analysis using beam penetration depth/angle
Can be used for bothatomicandmolecularanalysis
Sensitive to low levels, picogram, femtogram and lower
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Desorption Electrospray: DESI and DART
Desorption-
electrospray
ionization(DESI)
A new technique
for desorbingions using
supersonic jets
of solvents
(charged like inelectrospray)
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Inductively Coupled Plasma (ICP)
The inductively-coupled
plasma serves as anatomization and
ionization source (two-
in-one!) for elemental
studies.
Photo by Steve Kvech, http://www.cee.vt.edu/program_areas/environmental/teach/smprimer/icpms/icpms.htm#Argon%20Plasma/Sample%20Ionization
See optical electroniclecture for more details
Solution flow rates up to:
50-100 mL/min
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Mass Analyzers - Outline
Sector Mass Analyzers (Magnetic and Electrostatic)
Quadrupole Analyzers
Ion Traps
Ion Cyclotron Resonance
Time-of-Flight
and many more.
Ionization
Source
Mass
AnalyzerDetector
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Properties of Mass Analyzers
Resolution (R):
R = m/m
m = mass difference of two adjacent resolved peaks
(typically
m = mass of first peak or average
Example: R= 500 (low resolution)
resolves m/z=50 and 50.1, and m/z=500 and 501
Example: R= 150000 (high resolution)
resolves m/z=50 and 50.0003, and m/z=500 and
500.0033
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Sector Mass Analyzers
Basic Features
A sector: a geometrical construction
that has two arcs inside of one
another.
(Technically, a pie slice!)
Types:
Magnetic
Electrostatic Combination (e.g. double-focusing)
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Magnetic Sector Mass Analyzers
Ion kinetic energy:
V
erB
z
m
2
22
2
2
1
mvzeVT
BzeVFm
rmvFc
2
mc FF
Forces:
Only ions with equal
forces will pass:
Therefore:
Where:
Tis kinetic energyzis charge on ion
e is electron charge (1.60 x 10-19 C)
B is magnetic field (T)
v is velocity (m/s)
Vis the accelerating voltage
m is the mass
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Electrostatic Sector Mass Analyzers
2
v
reV
z
m
Therefore:
Ion kinetic energy:
221 mvzeVT
eVFm
rmvFc
2
Mc FF
Forces:
Only ions with equal
forces will pass:V
can be varied to bring ions ofdifferent KE (and different m/z
ratio to the exit)
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Double-Focusing Sector Mass Analyzers
If a batch of ions of equal
m/z but with differentkinetic energies enters a
magnetic sector
instrument, this will result
in a spread-out beam
Soution: minimize
directional and energy
differences between ions
of the same m/z.
Example of a double-
focusing MS: the Nier-
Johnson geometry
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Double-Focusing Sector Mass Analyzers
Another design, the Mattauch-Herzog geometry
This geometry is analogous to CCD-based opticalelectronic spectroscopy systems, while Nier-Johnson
instruments are similar in nature to traditional scanning
monochromator spectrometers.
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Time-Of-Flight (TOF) Mass Analyzers
The principle of Time-of-flight mass analysis:
A batch of ions is introduced into a chamber by anpulse of accelerating current.
This chamber has no fields, and is a drift tube
Since the ions have the same kinetic energy, their
velocities vary inversely with their mass during theirdrift.
Notes:
Typical flight times are 1-30 us Lighter ions arrive at the detector first
2
21 mvT
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Time-Of-Flight (TOF) Mass Analyzers
Delayed extraction anything you can do to
tighten the KE spread will help a TOFinstrument
m/zis mass-to-charge ratio of the ion
Eis the extraction pulse potential (V)
s is the length of flight tube over which E is applied
dis the length of field free drift zone
tis the measured time-of-flight of the ion
zeEsmvT 221
2
2
v
eEs
z
m
2
2
d
t
eEsz
m
Ti Of Fli ht (TOF) M A l
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Time-Of-Flight (TOF) Mass Analyzers The reflectrona method of compensating for different ion KEs
Figure from http://www.abrf.org/ABRFNews/1997/June1997/jun97lennon.html
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Time-Of-Flight (TOF) Mass Analyzers
The reflectrona method of compensating for different ion KEs
Figure from http://www.abrf.org/ABRFNews/1997/June1997/jun97lennon.html
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Quadrupole Mass Analyzers
The quadrupole (named for its electrical structure) is one of the
simplest and most effective mass spectrometers.
Quadrupole Mass Analyzers
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Quadrupole Mass Analyzers
How a quadrupole works:
Most important points:
It is easier for an applied AC field to deflecta light ion than a heavier ion
Conversely, it is easier for an AC field to
stabilize a light ion
Using this knowledge a combined AC/DC
potential is applied to the rods. Via the DC,the ion is attracted to one set of rods and
repelled by the other
The DC serves to stabilize heavy ions in one
direction (high pass filter). The AC serves to
stabilize light ions in the other direction (lowpass filter).
The ion must pass through the quadrupole to
make it to the detector
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Quadrupole Mass Analyzers
Another view and the concept
of the mass scan
Images from http://www.jic.bbsrc.ac.uk/SERVICES/metabolomics/lcms/single1.htm
Light ion:
(ex. m/z = 100)
Dragged by AC
Heavy ion:
(ex. m/z = 500)
Dragged by DC
Just right:
Dragged by both,
But equally balanced
I T M A l
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Ion Trap Mass Analyzers
Ion trap: a device for
trapping ions and confiningthem for extended periods
using EM fields
Used as mass analyzers because
they can trap ions and eject themto a detector based on their mass.
Theory is based on Mattieus work
on 2nd order linear differential
equations (in the 1860s), and onWolfgang Pauls Nobel Prize
winning implementations
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Ion Trap Mass Analyzers
The stability region of an
ion trap based ondifferential equations
22
0
8
mr
eU
az
22
0
4
mr
eVqz
)cos(0 tVU
Most ITMS systems dont
use DC (U), i.e. only qzis
controlled
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Ion Trap Mass Analyzers
Layout of an ion trap mass analyzer:
Diagram courtesy of M. Olsen, GlaxoSmithKline
+
Main RF
Ring
Endcap
Lenses
Octopole
Optimized Asymptote Angle
End Cap
Shutter
Focus
Electron Multiplier
Conversion Dinode
Low Amplitude Dipole Field
(1/3 frequency of main RF)
++
++
++
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Ion Trap Mass Analyzers
The BrukerEsquire ESI
ITMS - a typical
ion-trap LC-MS
system:
I C l t R
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Ion Cyclotron Resonance
FT-ICR:a FT-based mass spectral method that offers
higher S/N, better sensitivity andhigh resolution
Also contains a form of ion trap, but one in which ion
cyclotron resonance occurs.
When an ion travels through a strong magnetic field, itstarts circulating in a plane perpendicular to the field
with an angular frequency c:
m
zeB
r
vc
Ion Cyclotron Resonance
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Ion Cyclotron Resonance
How ICR works:
The ions are circulated in a field
An RF field is applied to match the cyclotron frequency of the ionsthis field brings them into phase coherence(forming ion packets)!
The image current is produced as these little packets of ions get
near the plates. The frequency of the image current is characteristic
of the ion packets m/z ratio.
http://www-methods.ch.cam.ac.uk/meth/ms/theory/fticr.html
Ion Cyclotron Resonance and Magnetic Field
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Ion Cyclotron Resonance and Magnetic Field
Parallels between NMR/EPR and ICR:
B
B= q B
m
=
B
Picture courtesy Prof. Alan Marshall, FSU/NHMFL
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The OrbitrapTM:A Hybrid Trap Between IT
and ICR
The Orbitrap is a recently developed electrostatic iontrap with FT/MS read-out of image current, coupled
with MS/MS
Advantages
Ease of use
Resolving power (superior to TOF)
Precision and accuracy
Versatility, dynamic range
A lower-resolution, more economical ICR
LTQ O bit h ti
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LTQ Orbitrap schematic
API Ion source Linear Ion Trap C-Trap
Orbitrap
Finnigan LTQ Linear Ion Trap
Differential pumping
Differential pumping
Image/animation from Thermo Electron Inc. See A. Makarov et al.,Anal. Chem.2006,78, 2113-2120.
LTQ Orbitrap Operation Principle
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LTQ Orbitrap Operation Principle
1. Ions are stored in the Linear Trap
2. . are axially ejected
3. . and trapped in the C-trap4. . they are squeezed into a small cloud and injected into the Orbitrap
5. . where they are electrostatically trapped, while rotating around the central electrode
and performing axial oscillation
The oscillating ions induce an image current into the two
outer halves of the orbitrap, which can be detected using
a differential amplifier
Ions of only one mass generate a sine
wave signal
Image/animation from Thermo Electron Inc. See A. Makarov et al.,Anal. Chem.2006,78, 2113-2120.
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The axial oscillation frequency follows the formula
Where = oscillation frequencyk = instrumental constant
m/z = mass-to-charge ratio
zm
k
/
Frequencies and Masses
Many ions in the Orbitrap generate a complex
signal whose frequencies are determined using a
Fourier Transformation
Image/animation from Thermo Electron Inc. See A. Makarov et al.,Anal. Chem.2006,78, 2113-2120.
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Multiple-Stage MS: MS-MS, and MSn
Also known as Tandem MS or MSn
Mass
Analyzer
Mass
Analyzer
Multiple quadrupoles are very common (e.g. triple-quad or
QQQ systems, EB for double-focusing, Q-TOF for quad time-
of-flight)
Why tandem MS? Because of the possibility of doing CIDcollisionally induced dissociation. Ions are allowed to collide
with a background gas (He) for several millliseconds, prior to
analysis. Allows for MSn experiments in an ion trap.
Comparison of Mass Analyzers
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Comparison of Mass Analyzers
A brief overview of the properties of common mass
analyzers
Analyzer Cost Scan speed Resolution
Double-
focusing
High Slow High
Quadrupole Low Medium Low-medium
Trap Low Medium Medium
TOF Medium Medium Medium-high
ICR High Fast High
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Detectors for Mass Spectrometry
Electron multipliers: like a
photomultiplier tube. Ions strike asurface, cause electron emission. Each
successive impact releases more
electrons.
Faraday Cups: Ions striking a cup cause
charge to flow across a load. The
potential across the load is monitored.
Ionization
Source
Mass
AnalyzerDetector
Figure from D. W. Koppenaal, et al.;Anal. Chem., 77; 2005, 418A-427A.
Detectors: Electron Multipliers
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Detectors: Electron Multipliers
Electron multiplier (EM): most common design in current
use
High gain (107), low noise, good dynamic range (104-106) Several designs:
Figure from D. W. Koppenaal, et al.;Anal. Chem., 77; 2005, 418A-427A.
D t t Oth
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Detectors: Others
Super-conducting tunner junction high mass
range, used with MALDI Can detect fmol of 150 kDa proteins
Can measure both energy and arrival time (2D MS
plots of m/z vs. kinetic energy)
Focal-plane array detectors/CCD
Like in electronic spectroscopy, much more
challenging to design for ion detection
Would combine well with mini-traps or other smallMS systems
Figure from D. W. Koppenaal, et al.;Anal. Chem., 77; 2005, 418A-427A.
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MS-Chromatography Interfaces
GC-MS: gas eluent from a column is piped directly to the
MS source
LC-MS: the ionization methods themselves serve as
interfaces techniques like ESI, APCI and APPI work on
liquid phase samples. The methods are generally
tolerant to RP LC solvents and some NP solvents.
Some buffers can quench ionization of analytes though:
Bad: Phosphate leaves a solid upon evaporation.
Also ionizes preferentially. Bad: any other non-volatile additives are also bad
Good: TFA, ammonium acetate, formic acid
Good: lower concentrations,