Hyphenated Techniques

GC-MS
LC-MS
Dr. Tabassum Khan
Oct 2019
Hyphenated techniques (HTs)
• HTs couple a separation technique with a spectroscopic detection
technique.
• The advantage of HTs is the additional dimensionality of the data
obtained.
• Most HTs require an interface between the separation technique and
the detector which is decisive for their performance as the main
problem here is the adaptation of one set of parameters i.e
chromatographic running conditions to the operating conditions of
the second technique. The interface provides this adaptability to the
HTs.
Applications
• Analysis of food, pesticides, environmental samples, biological samples, natural products,
forensic samples and pharmaceutical samples.
• GC-MS is widely used for detection of organic pollutants in the environment; forensic
sample analysis; narcotics and steroid analysis (antidoping studies); food, beverages,
aromatic oils and perfume analysis (perfume industry) and drug analysis wherever
applicable.
• LC-MS for pharmacokinetic and biodistribution studies of drugs and other samples.
• LC-MS for metabolite profiling of biological samples (pre clinical and clinical studies).
• In drug discovery and development- LC-MS is used in drug development as it allows quick
MW confirmation and structure identification. These features speed up the process of
generating, testing, and validating a discovery starting from a vast array of products with
potential application. LC-MS applications for drug development are highly automated
methods used for peptide mapping, glycoprotein mapping, lipodomics, natural products
dereplication, bioaffinity screening, in vivo drug screening, metabolic stability screening,
metabolite identification, impurity identification, quantitative bioanalysis and quality
control.
Applications
• Proteomics
LC-MS is used in proteomics as a method to detect and identify the
components of a complex mixture.
Protein identification in complex biological samples by peptide mass
fingerprinting.
(Peptide mass fingerprinting is an analytical technique in which the
unknown protein is first cleaved into smaller peptides, whose absolute
masses can be accurately measured with a MS such as ESI-TOF or
MALDI-TOF.
GC MS
• After separation in the GC column, analyte species have to be transported to the
mass spectrometer to be ionised, mass filtered and detected.
• The column outlet needs to be connected to the ion source of the mass
spectrometer and different strategies had been implemented, all of which need
to fulfill the following conditions:
• Analyte must not condense in the interface.
• Analyte must not decompose before entering the mass spectrometer ion
source.
• The gas load (dictated by the mobile phase gas flow rate) entering the
ion source must be within the pumping capacity of the mass
spectrometer.
• Coupling of GC to MS requires the introduction of the analytes at
atmospheric pressure into the high vacuum of the mass analyser (10 -3 to 10-6
Pa).
GC-MS instrument
GC-MS Coupling
GC-MS interfaces
Interfaces based on:
• Enrichment of analyte in the carrier gas by eliminating carrier molecules. In this
way enough gas can be introduced into the ion source with total gas flows
compatible with the pumping capacity of the system.
E.gs- Jet interface, permselective interface and molecular effusion
interface used with packed GC columns.
• Flow splitting- In this no analyte enrichment takes place and is useful when
sensitivity is not a critical factor. It can be performed at the exit of the gas
chromatograph allowing the diverted gas to be directed to a parallel detector, or
at the interface itself such as in the open slit interface. Here the GC column exit
is situated close to the capillary restrictor entrance in an open connector. The
restrictor samples the effluent from the GC column exit and the excess column
flow is removed from the connector by helium. This interface allows one to
work with a wide range of column flows without any interface modification.
Jet separator
Jet separator
• In this, the GC flow is introduced into an evacuated chamber through a
restricted capillary. At the capillary tip, a supersonic expanding jet of
analyte and the carrier gas molecules is formed and its core area
sampled into the mass spectrometer.
• In an expanding jet, high molecular mass compounds are concentrated
in the core flow while the lighter and more diffusive carrier molecules
are dispersed away in part through collisions.
• Thus sampling of the core flow produces an enrichment of the analyte.
• The jet interface is very versatile, inert and sufficiently efficient.
• It has the disadvantages of reduced efficiency with more volatile
compounds and potential plugging problems at the capillary restrictor.
Permselective membrane interface
• This interface is made of a silicone-rubber membrane that transmits
organic non-polar molecules and acts as a barrier for non-organic
carrier gases.
• Despite being a very effective enrichment procedure, it suffers from
discrimination effects with more polar analytes.
Bieman concentrator or Molecular effusion
interface
Bieman concentrator or Molecular effusion interface
• This interface is based on the molecular filtering of the gas effluent
by means of a porous glass frit.
• The column effluent passes through a fritted tube situated in a a
vacuum chamber.
• Small molecules traverse the microscopic pores in the tube walls and
are evacuated while high molecular mass molecules are transferred to
the ion source.
• The main drawbacks of this interface are the high dead volume added
and its high surface area.
Direct introduction
Direct introduction
• Capillary columns use optimum flow rates of 1-2 ml/min instead of
10-20 ml/min used with packed columns, allowing all the effluent to
be directed to the mass spectrometer.
• This is done through a direct coupling where the column exit is
introduced into the ion source without a capillary restriction.
• This direct coupling provides a simpler and more inert procedure for
the GC-MS connection and is extensively used in analytical labs.
LC-MS
The problem of introducing liquid flow rates of upto 2mL/min into the
vacuum of the MS requires the use of a suitable interface. The
differentially pumped vacuum system of a MS can tolerate the
introduction of only about 50 nL/min of liquid mobile phase. Potential
approaches to overcome this limitation are:
• Enlargement of the pumping capacity of the MS vacuum system.
• Solvent elimination prior to introduction into the vacuum system.
• Splitting the effluent stream at the cost of sensitivity.
• Use of micro-LC columns that allow efficient operation at substantially
lower flow rates.
One or combination of these strategies is used in LC-MS interfaces.
LC-MS system
LC-MS
LC-MS interfaces
1. Transport systems
Moving wire and moving belt interface
2. Molecular separators
• Membrane separator interface
• Particle beam interface
3. Direct introduction interface
• Thermospray interface
• ESI interface
• FRIT-FAB/ Flow-FAB interface
• APCI interface
Moving belt interface
Moving wire and belt interface
• The moving wire interface used a 0.12 mm dia SS wire as the
transportation surface. The heating for solvent removal and flash
evaporation of the remaining solid analyte inside the mass spectrometer
was achieved by passing an electric current through the wire. The low
surface area of the wire allowed the deposition of just 1% (10 µl/min of
the total eluate from a conventional column.
• In the moving belt interface, the wire was substituted by a SS or
polyimide ribbon with a larger deposition surface area. The effluent
solution is deposited on the moving belt from which the solvent
evaporates; the belt then passes into the MS, carrying with it the analyte
molecules which are then ionized by SIMS, FAB or LD.
• This permits 30-50 fold more sample and the use of pneumatic or heated
nebulizers, in a vacuum chamber optimizes sample deposition.
Molecular separators
Molecular separators are based on the analyte enrichment devices
developed for GC-MS.
• Membrane separator interface- It is based on the silicone rubber
membrane separator used in GC-MS. This was based on a flash
evaporator chamber coupled to a polymeric dimethyl siloxane
membrane that selectively transports nonpolar molecules to the mass
spectrometer.
• Particle beam interface- It is a momentum separator and is a useful
interface applicable to a wide range of molecules.
Particle beam interface
Particle beam interface
• In this interface, the analyte molecules are separated form the solvent molecules
by a momentum separator.
• The volatile solvent molecules are stripped from the sample and lost in a process
similar to that used in the early jet separators used in GC-MS.
• The jet separator eliminates volatile solvents to transport the analyte in the form
of solid micro-aggregates to the ion source of the MS. The heavier sample
molecules enter the MS and can be ionized by the standard methods of EI,PICI or
NICI.
• The possibility of obtaining gas-phase-like EI spectra which can be library
searched is the most valuable advantage of this interface.
• The advantage over the moving belt interface is there is no complicated
mechanical device to deal with and it is not necessary to desorb the analyte from
the belt surface.
• Sensitivity upto 10-12 g, flow rates upto 1 ml/min and mass range upto 1000amu.
Direct introduction interfaces
• This includes those interfaces that introduce the column eluate into
the ion source without prior enrichment.
• In this interface, the column eluate is split into a small fraction and
about less than 1 µl/min is allowed to enter the desolvation chamber
through an orifice orr diaphragm where it forms a liquid jet, followed
by disintegration into small droplets.
• The larger part of the solvent is separated by evaporation and
subsequently ionized in the ion source either by EI or CI.
Thermospray interface
• In this interface, the eluate from the column is vaporized and a portion of the
vapour (about 1%) is transferred to the mass spectrometer and the rest of the
vapour is pumped to waste.
• It comprises of a heated probe, a desolvation chamber, an ion extraction skimmer
and a vacuum line.
• The probe is made of a heated capillary tube connecting the column and the ion
source. The eluent flowing through the hot capillary is partially evaporated so that
an ultrasonic spray of vapor and charged microdroplets is obtained at the probe
exit.
• Ions present in the source are transferred into the MS through the ion cone
aperture while the main portion of the residual vapour is captured by the vacuum
line and purged by a rotary pump.
• The different gas conductances of the skimmer and the vacuum line allows the
introduction of flows as high as 1-2 ml/min. Spectra produced is like CI
• Sensitivity upto 10-9 g, flow rates upto 1-2 ml/min and mass range upto 2000amu.
Flow-FAB interface
• In this interface, the column eluate is mixed with a FAB matrix (generally 5%
aqueous glycerol) is continuously deposited on the tip of a FAB probe permanently
situated inside the ion source.
• The liquid mixture is transported to the tip through a fused silica capillary coaxial
to a modified FAB probe.
• The FAB matrix prevents the liquid evaporation inside the capillary and the
mixture can be submitted to atom bombardment in the FAB probe target. The
analyte sample in the matrix is bombarded by fast atoms (Xe or Cs) from the FAB
gun resulting in ionization of the analyte. This is soft ionization producing limited
fragmentation.
• Two main interfaces have been developed- the FRIT-FAB and the continuous flow
FAB.
• Sensitivity upto 10-12 g, flow rates 1-5 µl/min and mass range upto 2000amu or
more. Most suitable for lipophilic molecules.
Electrospray interface
• This interface consists of a capillary SS needle connected to the grounded
side of a high voltage source and situated in a chamber (a metal coated glass
cup) that acted as a counter electrode.
• The liquid sample is introduced into the chamber through the capillary
producing an electrically induced spray of charged microdroplets at the exit.
• Ions in these droplets enter the gas phase through evaporative process and
captured through a glass capillary restrictor and conducted into the MS.
• To promote droplet desolvation, the ionization chamber is continuously
supplied with a countercurrent flow of dry nitrogen.
• Soft ionization and spectra can be simple containing molecular ion only or
fragmentation can be induced by varying the cone voltage.
• Sensitivity upto 10-9 g, flow rates upto 1 ml/min and best at 200 µl/min and
mass range upto 200000 amu. Most suitable for polar molecules.
ESI
APCI interface
• This consists of an atmospheric pressure vaporization chamber in
which 1-2 µl of sample is injected through a septum.
• A heated pneumatic nebulizer probe is used for nebulization and a
high voltage needle to produce a corona discharge responsible for
inducing solvent ionization.
• A hot carrier gas, nitrogen is introduced into the chamber to help
vaporization (desolvation) and transport of the analyte.
• Ionization is analogous to CI, with the corona discharge producing
ions such as H3O+ and N2+, which promotes ionization of the sample.
• Sensitivity upto 10-9 g, flow rates upto 0.2-2 ml/min and mass range
upto 2000 amu. Most suitable for medium to low polarity molecules.
APCI