MASS SPECTROMETRY

Group2
Asperga
Cantos
Garonita
Yuro

Lee
Ortega
Sarayno

INTRODUCTION
Mass spectrometry is a powerful
analytical technique used to:
Quantify known materials
Identify unknown compounds within a
sample
elucidate the structure and chemical
properties of different molecules

INTRODUCTION
The complete process involves the conversion
of the sample into gaseous ions with or
without fragmentation which are then
characterized by their mass to change ratios
(m/z) and relative abundances

Principle of Mass
Organic molecules are bombarded with
Spectrometry
electron

Converted into Highly energetic positively

charged ions
(Molecular ions or parent ions)
Further break up onto smaller ions (Fragment
ions or Daughter ions)
The formed ions are separated by deflection in
Magnetic field according to their mass and
charge
MASS SPECTRUM

BASIC COMPONENTS
1. Sample inlet
2. Ionization source
For producing gaseous ions from the substance
being studied

3. Mass analyser
For resolving the ions into their characteristics mass
components according to their mass-to-charge- ratio

4. Ion detector
For detecting the ions and recording the relative

SAMPLE
INTRODUCTION
AND IONIZATION

WHY DO WE NEED TO
IONIZED THE SAMPLE?
Ionization is a process of charging a
molecule.
Molecules must be charged in order to
measure them using a mass
spectrometer

ELECTRON IONIZATION
Most common form of ionization used in GC/MS
Requires: source in form of filaments at 70eV

Source:
Molecules
are
bombarded
with high
energy

Molecules
break
into
fragment
s,
according
to its
structure

Used for
qualitative
identificati
on

ATMOSPHERIC PRESSURE
IONIZATION
Most LC/MS ionization techniques are
conducted at atmospheric pressure.
2 types of ionization:
Electrospray ionization (ESI)
Atmospheric pressure chemical ionization (APCI)

3-Matrix
Assisted Laser
Desorption
Ionization
(MALDI)

SURFACE-ENHANCED
LASER DESORPTION
IONIZATION
(SELDI)
Modification
of MALDI surface
a type of affinity capture
property
-Sample of interest is exposed to affinity surface ,
certain analyte will preferentially bind
-Washing to remove excess
-Matrix is added to asset ionization

Uses: analysis of protein mixture with low

sample size (purification&analysis occur on the
same surface

ELECTROSPRAY
IONIZATION (ESI)
Wide mass range
High sensitivity
Applied to wide range of biological macromolecules
in addition to small molecules
Most common ionization source for LC/MS

ELECTROSPRAY
IONIZATION
(ESI)
Involves passing the LC effluent through
a capillary
The energy is transferred to the solvent
droplets
Evaporation of the solvent through heat
and gas
Evaporation of the solvent through heat
and gas
The Coulombic repulsion of like charges

ELECTROSPRAY
IONIZATION (ESI)
The Coulombic repulsion of like charges
Individually charged molecules are drawn into
the MS for mass analysis

THE COLOUMBIC REPULSION OF

LIKE CHARGES LEAD TO THE
EJECTION OF IONS FROM THE
DROPLET

ELECTROSPRAY
IONIZATION (ESI)
Adept at forming
molecules

singly

charged

BUT larger molecules can ALSO be

ionized
Proteins: multiply charged
Can be observed in an instrument with a
relatively small mass range

LARGE MOLECULES CAN BE

OBSERVED IN AN INSTRUMENT
WITH A RELATIVELY SMALL
MASS RANGE

ATMOSPHERIC PRESSURE
CHEMICAL IONIZATION
(APCI)
Similar to ESI
The liquid form LC is introduced directly into the
ionization source

The droplets are NOT charged

The source contains a heated vaporizer
A high voltage is applied to a corona
discharge needle

-generate

electric fields that can

manipulate the charged molecules to
sort them according to their m/z.

MASS
ANALYZE
RS

MASS ANALYZERS
Include:
-Quadrupole
-Ion Trap
-Tandem Mass Spectrometry
-High Resolution MS

QUADRUPOLE
oMost common mass analyzer in use
ois a beam-type analyzer,where mass-to-charge ratios are
scanned during a prescribed time period to form a mass
spectrum
Other Applications
High specificity detectors in Liquid/ Gas
Chromatography-Mass Spectrometry

QUADRUPOLE
How does it work:
4 rods (electrodes)parallel to each other has electric fields with voltages of either + or -
Samples when ionized from the ion source travel in the between the Electrodes
And goes through filtration on the ions which depends on;

Ion Charge
Ion Mass
Electrode Charge
The ions are then detected by the DETECTOR via the exit slit
The results are displayed in a SPECTRA of relative abundance and identified through
their characteristic fragmentation pattern.

QUADRUPOLE MASS
ANALYZER

Principle:
-The

electric field on two sets of diagonally opposed

rods allows only ions of a single selected m/z value
top pass through the analyzer to the detector

2 Techniques:
a.Full scan mass Spectrum
b. Selected Ion Monitoring

QUADRUPOLE MASS
ANALYZER
A. Full Scan Analyzer
All other ions are
deflected into the rods
Rods can be scanned from
low to high mass
Provides more information
than SIM
Preferable for general
unkown screening

B. Selected Ion Monitoring

(SIIM)
Specific mass analyzer
Seleccted to monitor a few
target analytes
Allows for longer dwell time
Higher sensitivity
For target compound
analysis

ION TRAP
Modified quadrupole
Electric field adjusts to selectively destabilize trapped ions
Trap and store ions generated over time
Greater sensitivity
2 Types:
A. Linear Ion Trap
Employs a stopping potential on the end electrodes to confine ions
along the two-dimensional ion axis of the quadrupole

ION TRAP
B. Three-Dimensional Ion trap
Four rods form a three-dimensional sphere

TANDEM MASS
SPECTROMETRY
Used in greater selectivity and lower
detection units.
Linss 3 quadrupoles in series Triple quad
Q1 Scan across a preset m/z range, and select an
ion of interest.
Q2 - Collision Cell
Q3 Product IonAnalyzer

HIGH-RESOLUTION MASS
SPECTROMETRY

Time-Of-Flight ionized light ions/heavy ions travel

through an acceleration area as they are given Kinetic
Energy
Prinicple states that: Given the same kinetic E, lighter ions
travel faster than heavy ions
10 000 50 000 FWMH can be achieved

Thermo Fisher Orbitrap Spectrometer

100 000 250 000 FWMH can be achieved

Note:
HRMS are capable of multi-sampling, specially in drug

TIME-OF-FLIGHT

THERMO FISHER ORBITRAP

SENSITIVITY
HRMS detects mass of very small compounds
of upto .5 Da
Able to exactly measure an unknown
compound with a mass between 0.001 to
0.0001 Da

RESOLUTION
Expressed and results are derived from
Mass of a corr. Compound (Da)
_________________________
Width of corresponding peak
Called Full Width At Half Maximum (FWHM)

DETECTOR

DETECTOR
Electron multiplier
Most common means of detecting ions
A series of dynodes with increasing
potentials are linked
When ions strike the first dynode
surface, electrons are emitted

DETECTOR
Electron multiplier
These electrons are attracted to the next
dynode
A cascade of electrons is formed by the end
of chain of dynodes
Result: Overall signal amplification on
the order of 1 million or greater

ELECTRON MULTIPLIER

APPLICATIONS OF MS IN THE
CHEMICAL LABORATORY

SMALL MOLECULE ANALYSIS

MS spectrometers coupled to GC or LC
identification & quantification of compounds
Also for structural information and molecular
weight determination (high resolution MS)

GC/MS

Widely used for measuring drugs of abuse in urine

toxicology confirmations

Drugs and metabolites

Must be extracted from body fluids
Typically reacted with derivatizing reagents
TO FORM COMPOUNDS=more volatile for the GC process

Computerized libraries + matching algorithms

Compare mass spectral results (unknown substance
obtained) from a sample (reference library)

LC/MS
ADVANTAGES
Offers a number of
advantages over GC
Less extensive
extraction procedures
Derivatization is rarely
used (saving time &
expense)
Polar and heat-labile
compounds fare better

Measures low-level and

mixed-polarity analytes
Vitamin D
Testosterone
Immunosupressant drugs

Detects multiple analytes

in one run
Panel of drugs
Series of metabolites

ADVANTAGES
Free from antibody interferences
Has its own type interference
Ion suppression
this effect is seen when a co-eluting chemical
in the sample prevents a compound of
interest from being ionized
Reducing & eliminating its signals

LC/MS
DISADVANTAGES
Chromatography in LC can be less robust
Wider peaks and more variable retention times
Potentially requiring maintenance (more frequent)

Less reproducible mass spectra

Requires highly skilled operators
Is not nearly as automated as immunoassay
instruments

GC/MS & LC/MS

Drawback:
Long run times asso. w/the chromatographic separation
Not an issue for low-volume testing
medium- or high-volume testing
remains a significant challenge

Ultra Performance Liquid Chromatography

(UPLC) or Multiplexing
Both the column and the pumps are robust enough to
handle very high pressures (>600 bar)
Faster flow rates (reduces the run time)

HPLC/UPLC
For very high volume testing
Pumps can be connected to a single mass
spectrometer (multiplexing)
It works because analyte peaks are only
typically ~5-30 seconds in width
In theory: only time the LC flow needs to be in-line
with the mass spectrometer
LC separations require much longer gradients &
takes several minutes

MULTIPLEXING
LC pumps
Staggered so that once the peak of interest has
been analyzed by the mass spectrometer, the flow
is diverted to waste
Placed in-line with mass spectrometer and the
cycle repeates

MULTIPLEXING
Can reduce the run time by a factor of the
number of LC pumps
Is well suited for high volume targeted
analysis but not for untargeted screening

MASS SPECTROMETRY IN
PROTEOMICS AND PATHOGEN
IDENTIFICATION

GENOMICS
-uses the known sequences of the entire
human genome for determining the role of
genetics in certain human disease.

PROTEOMICS
is the investigation of the protein products
encoded by genes.

Proteins from samples of normal

individual are compared with those
derived form patients with disease
being studied.

CHROMATOGRAPHIC TECHNIQUES
used to separate complex mixtures
before being analyzed on Mass
Spectrometer

Digestion
TRYPSIN
Separation
HPLC
two-dimensional electrophoresis
Goal: to separate proteins into individual
spots or bands

MALDI-TOF AND SELDI-TOF

MALDI-TOF
matrix-assisted laser desorption
ionization time-of-light
used for analysis of biomolecules
(peptides, proteins)

PROCESS:
protein samples are mixed with an appropriate
matrix solvent
spotted onto a stainless steel plate
solvent is dried
plate is introduced into the vacuum system of
MALDI-TOF analyzer

Laser pulse irradiates the sample

Desorption and ionization of matrix and
sample
Mass spectral range (>500 Da)
ionization of low-molecular weight matrix can be
distinguished from high-molecular weight peptides
and proteins
does not interfere with assay of protein

very large proteins

Pretreated with TRYPSIN

-cleaves peptide bonds after lysine and arginine

- lower-molecular-weight fragments
detection limit
10 (-15) to 10 (-18) mol

SELDI-TOF
Surface-enhanced laser desorption
ionization time-of-light
modification lf MALDI-TOF
proteins are directly captured on a
chromatigraphic biochip without the need of
samome preparation.

PATHOGEN IDENTIFICATION
High resolution MS
can be used for pathogen identification
in microbiology labs

Abbott PLEX-ID
polymerase chain
reaction
- PCR is used to
amplify DNA from the
unknown pathogen

-ESI-TOF MS is used ro
calculate exact masses of
these PCR-generated DNA
fragments
purine and pyrimidine
nucleotide bases can be
determined.

- can be used directly

from patient specimen.
- no need for organisms
to be cultured.