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Chromatogr./Mass Spec. Coupling
Chapter 7 Chromatography/Mass Spectroscopy:
Coupling
1.
GC/MS
Column outlet in GC: atmospheric pressure
Ionization source: in the range of 2 to 10-5 Torr.
1.1 General requirements of interfaces
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


An adequate pressure drop
Maximize the throughput of sample while maintaining a gas
flow rate compatible with the source operating pressure.
Low dead volume at the column exit.
Remain the chemical constitution of the sample.
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1.2 Capillary column
Capillary column flow rates of 1-2 ml/min are
compatible with most modern MS.
F1
Direct coupling or
Open split coupling
F2
F4 F3
•Connect to second detector
•Easy change of GC column
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1.3 Interface for high gas flow (packed column)
High column flow rate (20-60 ml/min)
Interface requirements:
 Provide a pressure drop between column and the
MS source on the order of 104-106.
 Reduce the volumetric flow of gas into the MS
without dismissing the mass flow of the sample by
the same amount.
 Must retain the integrity of the sample eluting from
the column in terms of the separation obtained and
its chemical constitution.
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Molecular separator:
The performance of any type of molecular separator is
characterized in terms of its separation factor
(enrichment) N and separator yield (efficiency) Y.
Y = (WMS/WGC) x 100
WMS : the amount of sample entering the MS
WGC : the amount of sample entering the interface or
from GC
Separator yield represents the ability of the device to
allow organic material to pass into the source of the
MS.
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The separation factor N is defined as the ratio
of analyte concentration in the sample entering
the MS and the concentration from GC.
WMS VGC
Y VGC
N
(
)(
)(
)
100 VMS
WGC VMS
VGC is the volume of carrier gas entering the
separator.
VMS is the volume of the carrier gas entering
the MS.
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 Effusion separator
–
–
The sample is enriched in the carrier gas reaching the mass
spectrometer.
Effusion rates are different between sample and carrier gas
F = 1/(MW)1/2
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 Jet separator

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Most popular separator for use with packed column
Relies on the differential diffusion of the lighter carrier gas
molecules away from a jet created by passing the effluent
stream from the GC into a small vacuum chamber.
During this expansion the lighter helium gas molecules rapidly
diffuse away from the core of the jet which becomes enriched
in the heavier molecules.
Removes about 90% of carrier gas. About 60% of the sample
reach the MS
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Advanced Analytical Chemistry – CHM 6157
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 Membrane separator
 The silicone membrane separator works on the
principle of differential permeability for the
transmission of organic solutes compared to carrier
molecules.
 The transmission ability of organic molecules is
much higher than those for carrier gas (two orders
of magnitude).
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LC/MS
Brief history of LC/MS
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–
–
Early 70’s: research on on-line LC and MS started
1977: 1st commercial LC/MS interface (moving-belt interface)
1980: 2nd commercial LC/MS interface (based on a modification of restricted capillary
inlet interface, DLI, direct liquid introduction).
1983: thermospray interface (breakthrough).
1985 and 1986: frit-FAB and continuous-flow FAB.
From 1988: several commercial adaptations of the MAGIC (monodisperse aerosol
generation interface). The particle-beam interface most closely resembles the MAGIC.
1988: electrospray interface (major breakthrough) commercial availability was
archived by the observation of multiply-charged ions from peptides and proteins. This
made the electrospray interface to one of the most popular and powerful methods for
LC/MS.
Following the early research efforts in the mid 1970’s of the group of Horning, the
potential use of APCI in LC/MS continued to be investigated.
–
–
–
–
–
Further explorations
–
Currently, API based LC/MS interfaces, i.e., electrospray and APCI, are the most
widely approaches, while other interfaces like particle-beam, thermospray and
continues-flow FAB are also used to a more limited extent.
New efforts including:
–
•
•
•
•
hyperthermal surface ionization in (particle-beam) LC-MS
On-line LC/MS using matrix-assisted laser desorption/ionization
Sonic spray interface
???????
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Coupling LC to MS

High gas volume
Typical flow rate for LC are 0.5-5 ml/min which translated into
gas flow rate in the range 100-300 ml/min.

Special ion sources
LC is often selected for the separation of nonvolatile and
thermally unstable compounds. Therefore it requires alternative
ionization methods.

Complex matrix
The mobile liquid phases used in LC range from low boiling
organic solvents to aqueous mixtures, modified with a variety of
acids, bases and organic and inorganic salts to buffer them and
improve chromatographic performance.
Interface? Ionization source? or Both?
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2.1 Direct liquid introduction (DLI)
 In the DLI approach, a small portion of the eluent
from the LC is fed into the MS ion source via a
capillary inlet and the vaporized solvent becomes a
CI reactant gas.
 A solvent jet is formed by passing 10-40 μl/min of
LC eluent through a laser-drilled pinhole (2-5 μm in
diameter) in a replaceable diaphram. To prevent
premature evaporation of the solvent, the tip of the
interface is water-cooled. This jet then passes
through a desolvation chamber where the droplets
are vaporized, and the vapor enters the MS ion
source.
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DLI Limitations:
Only volatile solvents and volatile buffers can be used
(ammonium acetate and ammonium formate). The use of
phosphate and sulfate buffers should be avoided.
Limited structure information due to the CI source
Low flow rate (10-40 μl/min)
Limited sample capacity
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2.2 Moving belt interface (MBI)
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–
–
–
Deposition of the column eluent
Removal of solvent
Sputtering of the sample into ion source
Clean-up
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MBI Advantages:
–
–
–
Compatible with normal HPLC column flow
rate and solvents
Free choice of EI, CI and FAB ion sources.
Free choice of reactant gas in CI.
MBI Limitations:
–
–
–
Fairly complex
Adsorption/decomposition of sample on the
surface of the belt.
Memory effects
Question: Do you have to use different HPLC flow rates between using
volatile non-polar solvent or water containing mobile phase?
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2.3
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Continuous-flow FAB (Fast-atom bombardment)
Samples in a condensed state, often in a glycerol solution matrix
(reduce lattice energy), are ionized by bombardment with energetic
(several keV) xenon or argon atoms. Mainly for polar highmolecular-weight species
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Continuous-flow FAB (Cont’d)
2.3
Formation of high energy atoms



Gas atoms are first ionized from an ion source, or gun.
These ions are then passed through an electric field.
After acceleration, the fast moving ions pass into a chamber
containing further gas atoms and collision of ions and atoms leads to
charge exchange. This is called a resonance electron exchange
reaction.
Xe.+ (fast) + Xe →
Xe.+ + Xe (fast)
The fast atoms formed in this process remain most of the original
kinetic energy of the fast ions and carry on in the original
direction. The lower energy ions from the exchange are readily
removed by an electrostate deflector.
Advanced Analytical Chemistry – CHM 6157
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2.3 Continuous-flow FAB (Cont’d)
Advantages:
Greatly increased the range of compounds
amenable to mass spectral analysis to include
ionic compounds, polar compounds and
thermally labile compounds such as quaternary
ammonium salts, peptide and carbohydrates.
Limitation:
Column flow rates are restricted to about 5-10 μl/min.
Advanced Analytical Chemistry – CHM 6157
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2.4 Particle-beam Interface (Monodisperse
aerosol generating interface for
chromatography, MAGIC)
(Momentum separator)
Perpendicular
Small uniform dropsmonodisperse, ~14 um
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Particle-beam Interface (Cont’d)
Steps involved in Particle-beam interface
• Eluent is pumped into the desolvation chamber through a
small orifice to form a liquid jet. This jet breaks up
spontaneously into uniform drops with perpendicular flow
of helium.
• The solvent rapidly evaporates from the drops and the
analyte present in the drops forms a solid residue, thus
becoming a high velocity particle beam.
• The analyte beam, helium and solvent vapor passes into a
momentum separator, which is very similar in concept to
the jet separator developed for packed column GC-MS.
• After leaving the momentum separator the analyte particles
enter the ion source where they are flash vaporized and
ionized by CI or EI.
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2.4 Particle-beam Interface
Advantage:
– EI and CI are available
– Independent operation of LC and MS
Disadvantages:
– Flow-rate 0.1 - 0.6 ml/min
– Limited to volatile compounds (Since flash
vaporization of the analytes in the source is part
of the ion formation process)
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2.5 Thermospray interface (TSP)
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Thermospray ionization (Cont’d)
Two basic processes:
 The generation of a fine mist of charged droplets from a solution containing the
analyte.
 Vaporization of the solvent to give ions of the analyte
Advanced Analytical Chemistry – CHM 6157
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Thermospray terms:
• Thermospray interface: a piece of
hardware
• Thermospray vaporization: a nebulization
technique.
• Thermospray (buffer) ionization: an
ionization technique.
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Thermospray ionization:
– Ion-evaporation
– Buffer ionization or solvent-mediated CI (ionmolecule reactions)
– Filament ionization
– Discharge ionization
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Ion Evaporation
• Nonvolatile molecules are preferentially
retained in the droplets
• The droplets are either positively or negatively
charged as a result of continuous solvent
evaporation from droplets.
• The droplets are broken down by Rayleigh
instabilities in a high local field strength
• Evaporation continues from the droplets
• Finally, the ions are sampled by the sampling
cone and mass analyzed.
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Solvent-mediated Chemical Ionization
• When thermospray ionization is considered as a solventmediated method, analyte ionization is due to gas-phase
ion-molecule reactions between analyte molecules and
reagent gas ions.
• Requires volatile buffer, such as ammonium acetate and
ammonium formate. The buffer can be present during
the chromatographic separation, or added post-column.
• Spray droplets emerging into the jet chamber will
contain a negative or positive charge, and as they
evaporate in the vacuum, ions will be formed which are
characteristic of the salt, the solvent, and any sample that
is present in the eluent.
• Sample ions formed in this process are usually molecular
adduct ions, e.g MH+, MNH4+, MOAc- ions etc, and
fragmentation is observed only for very sensitive
compounds.
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Positive-ion mode:
An analyte molecule M is protonated by a protonated solvent molecule
SH+:
M + SH+ → MH+ + S
The proton affinity of M should be larger than that of S. When the proton
affinity of the analyte molecule is roughly equal to or up to ca. 30 kJ/mol
below that of the reagent gas an adduct ion MSH+ is formed:
M + SH+ → MSH+
Negative-ion mode:
A proton is abstracted from the analyte molecule in the gas phase by the
deprotonated solvent molecules [S-H]-:
M + [S-H]- → [M-H]- + S
Another important process in negative-ion formation is anion attachment
or adduct formation:
M + A- → MA-
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Thermospray ionization/interface
Advantages:
– Flow-rate: 1-2 ml/min
– Commercially available interface for most
of the common quadrupole and magnetic
sector MS
Disadvantages:
– For thermally stable compounds
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2.6 Atmospheric Pressure Chemical Ionization
(APCI)
desolvation
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–
–
–
–
–
–
–
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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.
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2.7 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.
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Several kilovolts
Few µl/min
320-350 K, 800 torr
100 ml/s
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Iribarne-Thomson Model:




Charge density increases
Raylaeigh limit (Coulomb repulsion = surface tension)
Coulomb explosion (forms daughter droplets)
Evaporation of daughter droplets
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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
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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.
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Z1
Z2
M/Z
P1
P2
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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.
Advanced Analytical Chemistry – CHM 6157
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Chromatogr./Mass Spec. Coupling