Advanced Analytical Chemistry

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International University10/9/2006
Chapter 8 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
• 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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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International University9/25/2008
Chapter 7 Chromatogr./Mass Spec. Coupling

• 1.2 Capillary column
• Capillary column flow rates of 1-2 ml/min are
  compatible with most modern MS.

• Connect to second detector
• Easy change of GC column

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/9/2006 Chapter 7 Chromatogr./Mass Spec.
Coupling

• 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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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/9/2006 Chapter 7 Chromatogr./Mass Spec.
Coupling

• 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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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/9/2006 Chapter 7 Chromatogr./Mass Spec.
Coupling

• The separation factor N is defined as the ratio
  of analyte concentration in the sample entering
  the MS and the concentration from GC.
• 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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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/9/2006 Chapter 7 Chromatogr./Mass Spec.
Coupling

• 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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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/9/2006 Chapter 7 Chromatogr./Mass Spec.
Coupling

• Jet separator
• 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 Y.
CAI Florida International UniversityUpdated on
10/9/2006 Chapter 7 Chromatogr./Mass Spec.
Coupling
9
Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/9/2006 Chapter 7 Chromatogr./Mass Spec.
Coupling

• 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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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/9/2006 Chapter 7 Chromatogr./Mass Spec.
Coupling

• 2. LC/MS
• Brief history of LC/MS
• Early 70s 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
  1970s 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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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/9/2006 Chapter 7 Chromatogr./Mass Spec.
Coupling

• 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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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/9/2006 Chapter 7 Chromatogr./Mass Spec.
Coupling

• 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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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/9/2006 Chapter 7 Chromatogr./Mass Spec.
Coupling

• 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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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/9/2006 Chapter 7 Chromatogr./Mass Spec.
Coupling

• 2.2 Moving belt interface (MBI)
• Deposition of the column eluent
• Removal of solvent
• Sputtering of the sample into ion source
• Clean-up

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/9/2006 Chapter 7 Chromatogr./Mass Spec.
Coupling

• 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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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/9/2006 Chapter 7 Chromatogr./Mass Spec.
Coupling

• 2.3 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
high-molecular-weight species
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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/9/2006 Chapter 7 Chromatogr./Mass Spec.
Coupling

• 2.3 Continuous-flow FAB (Contd)
• 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.

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/9/2006 Chapter 7 Chromatogr./Mass Spec.
Coupling

• 2.3 Continuous-flow FAB (Contd)
• 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.

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/9/2006 Chapter 7 Chromatogr./Mass Spec.
Coupling

• 2.4 Particle-beam Interface (Monodisperse aerosol
  generating interface for chromatography, MAGIC)

(Momentum separator)
Perpendicular
Small uniform drops- monodisperse, 14 um
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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/9/2006 Chapter 7 Chromatogr./Mass Spec.
Coupling

• 2.4 Particle-beam Interface (Contd)
• 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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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/9/2006 Chapter 7 Chromatogr./Mass Spec.
Coupling

• 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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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/9/2006 Chapter 7 Chromatogr./Mass Spec.
Coupling

• 2.5 Thermospray interface (TSP)

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/9/2006 Chapter 7 Chromatogr./Mass Spec.
Coupling

• Thermospray ionization (Contd)

• 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

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/9/2006 Chapter 7 Chromatogr./Mass Spec.
Coupling

• Thermospray terms
• Thermospray interface a piece of hardware
• Thermospray vaporization a nebulization
  technique.
• Thermospray (buffer) ionization an ionization
  technique.

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/9/2006 Chapter 7 Chromatogr./Mass Spec.
Coupling

• Thermospray ionization
• Ion-evaporation
• Buffer ionization or solvent-mediated CI
  (ion-molecule reactions)
• Filament ionization
• Discharge ionization

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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/9/2006 Chapter 7 Chromatogr./Mass Spec.
Coupling

• 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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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/9/2006 Chapter 7 Chromatogr./Mass Spec.
Coupling

• Solvent-mediated Chemical Ionization
• When thermospray ionization is considered as a
  solvent-mediated 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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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/9/2006 Chapter 7 Chromatogr./Mass Spec.
Coupling

• 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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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/9/2006 Chapter 7 Chromatogr./Mass Spec.
Coupling

• 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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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/9/2006 Chapter 7 Chromatogr./Mass Spec.
Coupling

• 2.6 Atmospheric Pressure Chemical Ionization
  (APCI)

desolvation
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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/9/2006 Chapter 7 Chromatogr./Mass Spec.
Coupling

• 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 lt 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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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/9/2006 Chapter 7 Chromatogr./Mass Spec.
Coupling

• 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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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/9/2006 Chapter 7 Chromatogr./Mass Spec.
Coupling

Several kilovolts
Few µl/min
320-350 K, 800 torr 100 ml/s
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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/9/2006 Chapter 7 Chromatogr./Mass Spec.
Coupling

• 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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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/9/2006 Chapter 7 Chromatogr./Mass Spec.
Coupling

• 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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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/9/2006 Chapter 7 Chromatogr./Mass Spec.
Coupling
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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/9/2006 Chapter 7 Chromatogr./Mass Spec.
Coupling

• 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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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/9/2006 Chapter 7 Chromatogr./Mass Spec.
Coupling

• 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 gt 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.
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Advanced Analytical Chemistry CHM 6157 Y.
CAI Florida International UniversityUpdated on
10/9/2006 Chapter 7 Chromatogr./Mass Spec.
Coupling