Mass.pptx instrumentation, principle, theory

1. Mass spectrometry
Presented by : Dr. Vijaya U. Barge
(Vice Principal & Professor)
Pune District Education
Association’s Shankarrao
Ursal College of
Pharmaceutical Sciences &
Research Centre.

2. LearningObjectives:
• Mass spectrometry is sensitive analytical technique with potential
applications in the field of analytical and bio-analytical chemistry.
• This chapter covers all aspects of this versatile technique including
instrumentationdetails and newer avenues being explored.
• Soft ionization techniques are described, including chemical
ionization Cl, electrospray ionization ESI, FAB, MALDI, etc.
• Different types of ionic species are described, such as molecular
ions, isotopic ions,metastable ions, rearrangement ions, etc.
• Recognition of molecular ion peak is an important aspect of mass
spectrometry and this has been described in detail along with
application of nitrogen rule, index of unsaturation, ring rule, etc.
• General rules for fragmentation are described including application
of even electron rule.
• Application of the technique for elucidating molecular formulae of
the compounds is described.
• Mass fragmentation pattern of different chemical classes of
compounds is highlighted.
• Common fragment ions and fragments lost from the molecular ion
have been tabulated at the end of the chapter.

3. Mass spectrophotometry:
Mass spectrometry is an analytical technique
that is used to measure the mass-to-charge
ratio of ions.The results are presented as a mass
spectrum, a plot of intensity as a function of the
mass-to-charge ratio
mass spectrometers can be used to identify unknown
compounds via molecular weight determination

4. Ionisation in mass spectroscopy:
The atom is ionised by knocking one or more
electrons off to give a positive ion. (Mass
spectrometers always work with positive ions).
The particles in the sample (atoms or molecules)
are bombarded with a stream of electrons to
knock one or more electrons out of the sample
particles to make positive ions.

5. • The choice of ionization method depends on the
nature of the sample and the type of information
required from the analysis. So-called 'soft
ionization' methods such as field desorption and
elec- trospray ionization tend to produce mass
spectra with little or no fragment-ion content.

6. Classification according to hard and soft source:
Hard source: It imparts sufficiently energy to analyte
molecule
• Relaxation then involves rupture of bonds producing
fragment ions that have mass to charge ratio less than
that of molecular ion.
Peaks in hard source provide useful information about the
kind of functional groups and thus structural information
about analytes.
E.g. Chemical ionization

7. Soft source:
Little fragmentation.
• Supply accurate information about the molecular
weight of the analyte molecules.
E.g.
1)Matrix Assisted Laser Desorption Ionization
(MALDI)
2) Electron impact ionization

9. • Electron Impact (El-Hard method)
• - small molecules, 1-1000 Daltons, structure
• Fast Atom Bombardment (FAB - Semi-hard) peptides, sugars, up to 6000
Daltons -
• Electrospray lonization (ESI - Soft) peptides, proteins, up to 200,000
Daltons
• Matrix Assisted Laser Desorption (MALDI-Soft) - peptides, proteins, DNA,
up to 500 kD

10. • EI is done by volatilizing a sample directly in the source that is contained
in a vacuum system directly attached to the analyzer.
• The gas phase molecules are bombarded by a beam of electrons formed
by
• heating a filament bias at a negative voltage compared to the source. The
bias voltage is most commonly at -70 volts.
• The electron beam ejects an ion from the gas phase molecule producing a
radical ion.
• This technique is considered a hard ionization technique, because it
causes the
• ion to fragment. EI is also the method that is most commonly used for
GC-MS.
Electron Impact ionization (EI) -

12. Chemical ionization:

13. Generally hydrogen (H2), methane (CH4), isobutane (iso-
C4H10) and
ammonia (NH3) are used as reagent gases in CI mass
spectrometry;
with all these CI gases the compounds form protonated
molecule
ion in their CI spectra.
In CI mass spectrometry the molecules of a
vaporized sample are ionized
by a set of reagent ions (reagent plasma) in a series
of ion-molecule
reactions

14. • Electrospray relies in part on chemistry to generate analyte ions
in solution before the analyte reaches the mass spectrometer.
• The LC eluent is sprayed (nebulized) into a chamber at
atmospheric pressure in the presence of a strong electrostatic
field and heated drying gas. The electrostatic field causes
further dissociation of the analyte molecules. The heated drying
gas causes the solvent in the droplets to evaporate. As the
droplets shrink, the charge concentration in the droplets
increases. Eventually, the repulsive force between ions with like
charges exceeds the cohesive
• forces and ions are ejected (desorbed) into the gas phase.
• These ions are attracted to and pass through a capillary
sampling orifice into themass analyzer

15. Diagram of spray:

16. Atmospheric pressure photoionization:
• Atmospheric pressure photoionization (APPI) for LC/MS is a
relatively new
• technique. As in APCI, a vaporizer converts the LC eluent to
the gas phase.
• A discharge lamp generates photons in a narrow range of
ionization energies.
• The range of energies is carefully chosen to ionize as many
analyte molecules
• as possible while minimizing the ionization of solvent
molecules.
• The resulting ions pass through a capillary sampling orifice
into the mass analyzer.

17. Fast Atom Bombardment (FAB) -
• FAB is a technique that was popular in
• the 80's to early 90's because it was the first technique that
allowed
• ionization of non-volatile compounds that could be done
simply.
• It was done by bombarding a sample in a vacuum with a
beam of atoms,
• typically Ar or Xe, accelerated to Kilovolt energies. The
sample was
• typically mixed in a matrix. The two most common
matrixes were glycerol
• and 3 Nitro-benzoic acid.
• It was developed by Michael Barber at the University of
Manchester in 1980.

18. Electrospray mass spectrometry (ESI-MS)
• - Liquid containing analyte is forced through a steel capillary at
high voltage to electrostatically disperse analyte. Charge
imparted from rapidly evaporating liquid. If the sample has
functional groups that readily accept H+ .(such as amide and
amino groups found in peptides and proteins) then positive ion
detection is used-PROTEINS
• If a sample has functional groups that readily lose a proton
(such as carboxylic acids and hydroxyls as found in nucleic
acids and sugars) then negative ion detection is used-DNA

19. Matrix-assisted laser desorption ionization (MALDI)
• - Analyte (protein) is mixed with large excess of matrix (small
organic molecule)
• Irradiated with short pulse of laser light. Wavelength of laser is
the same as absorbance max of matrix.
• Sample is ionized by bombarding sample with laser light
• Sample is mixed with a UV absorbant matrix
• Light wavelength matches that of absorbance maximum of
matrix so that the matrix transfers some of its energy to the
analyte (leads to ion sputtering)

20. Mass Analyzers
Although in theory many type of mass analyzer could be used for
LC/MS, four types are used most often
•Quadrupole
• Time-of-flight
• Ion trap
• Fourier transform-ion cyclotron resonance
(FT-ICR or FT-MS)

21. Quadrupole
• A quadrupole mass analyzer consists of four parallel rods arranged
in a square. The analyte ions are directed down the center of the
square
• Voltages applied to the rods generate electromagnetic fields.
These fields determine which mass-to-charge ratio of ions can
pass through the filter at a given time
• Quadrupoles tend to be the simplest and least expensive
mass analyzers.

22. Diagram of quadruple

23. Quadrupole mass analyzers can operate in
two modes:
• Scanning (scan) mode
• Selected ion monitoring (SIM) mode
In scan mode, the mass analyzer monitors a range of
mass-to-charge ratios.
In SIM mode, the mass analyzer monitors only a few
mass to-charge ratios.

24. SIM mode is significantly more sensitive than scan
mode but provides
information about fewer ions.
Scan mode is typically used for qualitative
analyses or for quantitation when all analyte masses
are not known in
advance.

25. Time-of-flight
• In a time-of-flight (TOF) mass analyzer, a uniform electromagnetic
force is applied
• to all ions at the same time, causing them to accelerate down a flight
tube.
• Lighter ions travel faster and arrive at the detector first, so the
mass-to-charge
• ratios of the ions are determined by their arrival times. Time-off
light mass
• analyzers have a wide mass range and can be very accurate in
their mass measurements.

26. Metastable ion
• Metastable ion in mass spectrometry, An ion which is formed
with sufficient excitation to dissociate spontaneously during its
flight from the ion source to the detector.
• Fragment of a parent ion will give rise to a new ion (daughter)
plus either a neutral molecule or a radical. M1 + M2 + + non
charged particle .An intermediate situation is possible; M1 +
may decompose to M2 + while being accelerated.
• The resultant daughter ion M2 + will not be recorded at either
M1 or M2, but at a position M* as a rather broad, poorly
focused peak. Such an ion is called a metastable ion.

27. Significance of Metastable lons:
• Metastable ions are useful in helping to establish
fragments routes.
• Metastable ion peak can also be used to distinguish
between fragmentation Processes, which occur in
few microseconds

28. 1. Astronomy: analysis of astronomical components of the solar system
2. Electronics: analysis of microchips
3. Environmental: detection of toxic chemical, monitoring of nuclear
facilities, analysis of petroleum products, etc.
4. Forensics: toxicology, trace metals, biological materials, etc.
5. Medical: drug abuse diagnosis, analysis of pharmaceuticals and
products of genetic engineering
6. Military: mobile mass spectrometers are used to detect liquid chemica
warfare agents
Applications
Many disciplines use mass spectroscopy for chemical
identification.