MASS SPECTROMETRY

Cristian Ryan A. Argamino, RCh., M. Sc.

INTRODUCTION
DEFINITION

Mass spectrometers use the difference in mass-to-charge ratio (m/z) of

ionized atoms or molecules to separate them.

Mass spectroscopy allows quantitation of atoms or molecules and provides

structural information by the identification of distinctive fragmentation
patterns
APPLICATIONS
Biotechnology: the analysis of proteins, peptides, oligonucleotides
Pharmaceutical: drug discovery, combinatorial chemistry, pharmacokinetics,
drug metabolism
Clinical: neonatal screening, hemoglobin analysis, drug testing
Environmental: PAHs, PCBs, water quality, food contamination
Geological: oil composition
INSTRUMENTATION
General Workflow

Ion Source Mass Ion

Analyzer Transducer

Ionization Sorting Detection

 INSTRUMENTATION

Ionization Techniques
Chemical Ionization (CI) Field Desorption/Field Ionization
(FD/FI)
Atmospheric Pressure Chemical
Ionization (APCI) Matrix Assisted Laser Desorption
Ionization (MALDI)
Electron Impact (EI)
Thermospray Ionization (TI)
Electrospray Ionization (ESI)

Fast Atom Bombardment (FAB)

 INSTRUMENTATION

• Ionization Techniques
• Electron Impact (EI)
 Definition:  Sample introduction
 EI is the oldest and best characterized of all the  heated batch inlet
ionization methods.  heated direct insertion probe
 EI probably is the most common used ionization  gas chromatograph
technique and still is of major importance.  liquid chromatograph (particle-
 The ionization process can either produce a molecular beam interface)
ion which will have the same molecular weight and
elemental composition of the starting analyte, or it
can produce a fragment ion which corresponds to a
smaller piece of the analyte molecule.
 INSTRUMENTATION

• Ionization Techniques
• Electron Impact (EI)
 Benefits  Limitations
 well-understood  sample must be thermally volatile and stable
 can be applied to virtually all volatile compounds  the molecular ion may be weak or absent for
 reproducible mass spectra many compounds.
 structural information
 libraries fragmentation provides of mass spectra can be  Mass range
searched for EI mass spectral "fingerprint"  Low Typically less than 1,000 Da.
 INSTRUMENTATION

• Ionization Techniques
• Electron Impact (EI)

www.mhhe.com
 INSTRUMENTATION

• Ionization Techniques
• Chemical Ionization (CI)

 Definition:  Sample Introduction

 Chemical ionization uses ion-molecule reactions  heated batch inlet
to produce ions from the analyte.  heated direct insertion
probe
 The chemical ionization process begins when a
 gas chromatograph
reagent gas such as methane, isobutane, or
 liquid chromatograph
ammonia is ionized by electron impact. (particle-beam interface)
 Some of the products of these ion-molecule
reactions can react with the analyte molecules to
produce analyte ions.
 INSTRUMENTATION
• Ionization Techniques
• Chemical Ionization (CI)
 Benefits
 often gives molecular
weight information
through molecular-like
ions such as [M+H]+, even
 when EI would not
produce a molecular ion.
 simple mass spectra,
fragmentation reduced
compared to EI
 Limitations  Mass range
 sample must be thermally volatile  Low Typically less
and stable than 1,000 Da
 less fragmentation than EI,
fragment pattern not informative
or reproducible enough for library
search
 results depend on reagent gas
type, reagent gas pressure or
reaction time, and nature of
sample
 INSTRUMENTATION

Ionization Techniques
Chemical Ionization (CI)
 INSTRUMENTATION

• Ionization Techniques
• Electrospray Ionization (ESI)
 Definition:  Sample introduction
 The sample solution is sprayed across a high potential  flow injection
difference (a few kilovolts) from a needle into an orifice  LC/MS
in the interface.  typical flow rates are less than 1
 Heat and gas flows are used to desolvate the ions microliter per minute up to about a
existing in the sample solution. milliliter per minute
 Electrospray ionization can produce multiply charged
ions with the number of charges tending to increase
as the molecular weight increases.
 INSTRUMENTATION
• Ionization Techniques
• Electrospray Ionization (ESI)
 Benefits
 good for charged, polar or basic compounds
 permits the detection of high-mass compounds at
mass-to-charge ratios that are easily determined by
most mass spectrometers (m/z typically less than 2000
to 3000).
 best method for analyzing multiply charged
compounds
 very low chemical background leads to excellent
detection limits
 can control presence or absence of fragmentation by
controlling the interface lens potentials
 compatible with MS/MS methods
Mass range
Low-high Typically less than 200,000 D.
 Limitations
 multiply charged species require
interpretation and mathematical
transformation (can sometimes be
difficult) complementary to APCI.
 No good for uncharged, non-basic, low-
polarity compounds (e.g. steroids)
 very sensitive to contaminants such as
alkali metals or basic compounds
 relatively low ion currents
 relatively complex hardware compared to
other ion sources
 INSTRUMENTATION

• Ionization Techniques
• Electrospray Ionization (ESI)
 INSTRUMENTATION

• Ionization Techniques
• Atmospheric Pressure Chemical Ionization (APCI)

 Definition:  Sample introduction

 Similar interface to that used for ESI.  same as for electrospray
 In APCI, a corona discharge is used to ionize the analyte ionization
in the atmospheric pressure region.
 The gas-phase ionization in APCI is more effective than
ESI for analyzing less-polar species.
 ESI and APCI are complementary methods.
 INSTRUMENTATION

• Ionization Techniques
• Atmospheric Pressure Chemical Ionization (APCI)

 Benefits  Limitations  Mass range

 good for less-polar compounds  complementary to  Low-moderate Typically
 excellent LC/MS interface ESI. less than 2000 D.
 compatible with MS/MS
methods
 INSTRUMENTATION

• Ionization Techniques
• Atmospheric Pressure Chemical Ionization (APCI)

www.chem.pitt.edu
 INSTRUMENTATION

• Ionization Techniques
• Matrix Assisted Laser Desorption Ionization (MALDI)
 Definition:  Sample introduction
 The analyte is dissolved in a solution containing an  direct insertion probe
excess of a matrix such as sinapinic acid or  continuous-flow
dihydroxybenzoic acid that has a chromophore that introduction
absorbs at the laser wavelength
 A small amount of this solution is placed on the laser
target.
 The matrix absorbs the energy from the laser pulse
and produces a plasma that results in vaporization
and ionization of the analyte.
 INSTRUMENTATION

• Ionization Techniques
• Matrix Assisted Laser Desorption Ionization (MALDI)

 Benefits  Limitations
 rapid and convenient molecular weight  MS/MS difficult
determination  requires a mass analyzer that is compatible
with pulsed ionization techniques
 not easily compatible with LC/MS
 Mass range
 Very high Typically less than 500,000 Da.
 INSTRUMENTATION

• Ionization Techniques
• Matrix Assisted Laser Desorption Ionization (MALDI)
INSTRUMENTATION
• Comparison of Methods

Ionization Analytes Sample Mass Limits Method

Method Introductio Type
n
CI Small GC, liquid or 103 Soft Method
volatiles solid probe
APCI Small LC or 2 x 103 Soft Method
volatiles syringe
(less polar
species)
INSTRUMENTATION
• Comparison of Methods

Ionization Analytes Sample Mass Limits Method

Method Introductio Type
n
EI Small GC, liquid or 103 Hard
volatiles solid probe Method;
Structural
Info

ESI Peptides, LC or 2 x 105 Soft

proteins, syringe Method;
nonvolatiles Multiply
charged
ions
INSTRUMENTATION
• Comparison of Methods

Ionization Analytes Sample Mass Limits Method

Method Introductio Type
n
MALDI Peptides, In solid 5 x 105 Soft Method
Proteins, matrix
Nucleotides
 INSTRUMENTATION
• Mass Analyzers
• Quadrupoles
 Definition:  Applications of a quadrupole mass
 The quadrupole mass filter consists of four parallel analyzer can be summarized as:
rods of hyperbolic or circular cross section arranged  Majority of benchtop GC/MS and
symmetrically to a z-axis LC/MS systems.
 A voltage made up of a dc component U and a radio-  Triple quadrupole MS/MS systems
frequency (r.f.) component Vocos(t) is applied to  Sector / quadrupole hybrid MS/MS
adjacent rods. systems.
 Ions injected into the filter with a very small
accelerating voltage, typically 10-20 V, are made to
oscillate in the x and y directions by this field.

Source: MS_Nijmegen
 INSTRUMENTATION
• Mass Analyzers
• Quadrupoles

 Benefits of a quadrupole mass analyzer can be
summarized as:
 Classical mass spectra
 Good reproducibility
 Relatively small and low-cost systems
 Low-energy collision-induced dissociation (CID)
MS/MS spectra in triple quadrupole and hybrid
mass spectrometers have an efficient conversion of
precursor to product
 Limitations of a quadrupole mass analyzer can be
summarized as:
 Limited resolution
 Peak heights are variable as a function of the mass
(mass discrimination).
 The peak height versus response must be 'tuned'.
 Not well suited for pulsed ionisation techniques.
 Low energy collision-induced dissociation (CID) MS/MS
spectra in triple quadrupole and hybrid mass
spectrometers depend strongly on energy, collision gas,
pressure and other factors.

Source: MS_Nijmegen
INSTRUMENTATION
• Mass Analyzers
• Quadrupoles

Source: MS_Nijmegen
 INSTRUMENTATION
• Mass Analyzers
• Time-of-Flight (TOF)
 Definition:  Applications
 In a time-of-flight (TOF) mass spectrometer, ions are  Almost all MALDI (matrix assisted laser
separated on basis of the time t needed to travel a ionization) systems
path L.  Very fast GC/MS systems

Source: MS_Nijmegen
 INSTRUMENTATION
• Mass Analyzers
• Time-of-Flight (TOF)

 Limitations of a time-of-flight (TOF) mass analyzer can be
summarized as:
 Requires pulsed ionization methods or beam switching
 Fast digitizers used in TOF may have limited dynamic
range
 Limited precursor-ion selectivity for most MS/MS
experiments
 Benefits of a time-of-flight (TOF) mass analyzer can
be summarized as:
 Fastest available MS analyzer
 Well suited for pulsed ionization methods
(majority of MALDI mass spectrometers is
equipped with TOF)
 High ion transfer
 MS/MS information from post-source decay
 Highest practical mass range of all MS
analyzers.

Source: MS_Nijmegen
INSTRUMENTATION
• Mass Analyzers
• Time-of-Flight (TOF)
 INSTRUMENTATION
• Mass Analyzers
• Ion Trap (IT)
 Definition:  Applications
 The ion trap contains three cylindrically symmetrical  Benchtop GC/MS, LC/MS and MS/MS systems
electrodes  Target compound screening
 The use of an r.f. voltage causes rapid reversals of the  Ion chemistry
field direction so the ions are alternately accelerated
and decelerated in the axial (z)direction and vice versa
in the radial direction.
 The long storage times used in the ion-trap cause ion-
molecule reactions like those in chemical ionization at
much lower reagent gas pressure then commonly used
in conventional high pressure sources.

Source: MS_Nijmegen
INSTRUMENTATION
• Mass Analyzers
• Ion Trap
 INSTRUMENTATION
• Mass Analyzers
• Ion Trap (IT)

 Limitations of an ion trap mass analyzer can be summarized  Benefits of a time-of-flight (TOF) mass analyzer can
as: be summarized as:
 Poor Quantitation  High sensitivity
 Very poor dynamic range (sometimes compensated by  Multi-stage mass spectrometry (analogues to
auto-ranging) FTICR)
 Subject to space-charge effects and ion-molecule  Compact mass analyzer
reactions
 Collision energy not well defined in CID MS/MS
 Many parameters comprise the experiment sequence
that defines the quality of the mass spectrum (e.g.
Excitation, trapping, detection conditions)

Source: MS_Nijmegen
INSTRUMENTATION
• Detectors
• Faraday Cup
 Definition:
 The Faraday cup (FC or FEC, for Faraday electrometer
cup) is very simple in concept.

 Coupled with modern electronics, the Faraday cup is

singularly useful in producing high precision
measurements in isotope ratio mass spectrometers.

 Array detectors (which can include arrays of

miniaturized Faraday cups) are used not only as
detectors for dispersive-beam instruments, but also as
developmental tools for characterizing the position and
cross section of a beam of ions as it traverses an
instrument. Source:
www.spectroscopyonline.com
INSTRUMENTATION
• Detectors
• Electron Mutiplier
 Definition:
 An electron multiplier is used to detect the presence of
ion signals emerging from the mass analyzer of a mass
spectrometer.

 It is essentially the “eyes” of the instrument

 The task of the electron multiplier is to detect every

ion of the selected mass passed by the mass filter.

 How efficiently the electron multiplier carries out this

task represents a potentially limiting factor on the
overall system sensitivity.
Source:
www.chromspec.com
INSTRUMENTATION
INSTRUMENTATION
MASS SPECTRUM
• MASS SPECTRUM
• A mass spectrum will usually be presented as a vertical bar graph, in which each bar
represents an ion having a specific mass-to-charge ratio (m/z) and the length of the
bar indicates the relative abundance of the ion.
• The most intense ion is assigned an abundance of 100

Source: MS_MSU
References:
Van Galen, P. (2005). Mass Spectrometry: A Guide to Novel Users.
Nijmegen University. Nijmegen, Netherlands.
Lee, S. (2005). Modern Mass Spectrometry. The MacMillan Group.
Princeton University. Princeton, New Jersey.