A description of Mass spectrometry and regarding methods and practical uses details discussed in this presentation

1. MASS SPECTROMETRY
PRESENTER
PRIYANKA.S
1

2. Contents
 History
 Introduction
 Principle
 Mass spectrum
 Components of MS
 Sample introduction
Different Ionization
Technique
 Different Mass
analyzers
 Mass detectors
 Applications
 Summary
2

3. History
 JJ Thomson built MS prototype to measure m/z
of electron, awarded Nobel Prize in 1906
 MS concept first put into practice by Francis
Aston, a physicist working in Cambridge England
in 1919 Aston Awarded Nobel Prize in 1922
 In 2002, john fenn & koichi tanaka – Nobel Prize
for development of electrospray and laser
desorption, ionization respectively
3

4. Introduction
 Technique: Separate the ions according to
their mass-to-charge ratio (m/z)
 Designed to measure mass of elements
 Quantitative and qualitative analysis
 Molecular structure determination
(organic and inorganic)
 MS provides high sensitivity with fast
response time to probe chemically
complex particle 4

5. Mass spectrometry
5

6. Principle
6
• Separates gas phase ionized atoms, molecules, and
fragments of molecules
• Separation is based on the difference in mass-to-
charge ratio (m/z)
• m = unified atomic mass units (u)
• z = charge on the ion (may be positive or negative)
• 1 dalton (Da) = 1 u = 1.665402 x 10-27 kg

7. • Analyte molecule can undergo electron
ionization
M + e- → M+ + 2e-
• M+ is the ionized analyte molecule called
molecular ion
• Radical cation is formed by the loss of one
electron
• Computer algorithms are used to deconvolute
m/z values of multiply
• Charged ions into the equivalent mass of singly
charged ion
• Permits easy determination of molecular weight
of analyte
7

8. • Different compounds can be uniquely
identified by their mass
CH3CH2OH
N
OH
HO
-CH2-
-CH2CH-NH2
COOH
HO
HO
Butorphanol L-dopa Ethanol
MW = 327.1 MW = 197.2 MW = 46.1
8

9. • A plot of relative abundance vs m/z
• The most abundant peak is known as the base
peak
• Spectrum shows fragmentation patterns
• The m/z values and the fragmentation pattern
are used to determine the molecular weight and
structure of organic compounds
• Provides the accurate mass of a given isotope
not the weighted average 9
The Mass Spectrum

10. Typical Mass Spectrum
aspirin
Relative Abundance
120 m/z-for singly charged ion this is the mass
10

11. Resolving Power
• The ability of a mass spectrometer to separate
ions of two different m/z values
• Resolving power = M/∆M
• M = mass of one singly charged ion
• ∆M = difference in mass between M and the
next m/z value
11

12. 12

13. Parts of Mass
Spectrometry(LC-MS)
Mass
Spectrometer
Liquid
Chromatograph
Rough pump
UV
Detector
Sampler
Injection port
Column
Solvents
Ion source
Pumps Interface
Computer
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14. Sample input or introduction
14
1.By using probe
-By placing a sample on a probe, then insert
through vacuum lock into the ionization region. the
sample is then vaporized using any desorption
processes
2.By capillary infusion
-this delivers small quantites of sample to the
ionization chamber without disturbing the vaccum
- GC/LC

15. Different Ionization Techniques
• Soft ionization methods
• Semi hard ionization methods
• Hard ionization methods
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16. Different Ionization Methods
• Electro spray Ionization (ESI - Soft)
–peptides, proteins, up to 200,000 Daltons
• Matrix Assisted Laser Desorption (MALDI-Soft)
–peptides, proteins, DNA, up to 500,000 Daltons
• Fast Atom Bombardment (FAB – Semi-hard)
–peptides, sugars, up to 6000 Daltons
• Electron Impact (EI - Hard method)
–small molecules, 1-1000 Daltons, structure
16

17. Electron Impact (EI)
• Commonly used for analysis of organic samples
• Electrons are emitted from a heated tungsten
filament cathode
• Electrons are accelerated towards the anode
with a potential of about 70V
• Suitable for gas chromatography
• Interaction with the high energy electrons causes
ionization of sample molecules and
fragmentation into smaller ions
17

18. 18

19. • Can analyse both +ve and –ve ions (but not
simultaneously)
• Can be connected with LC
• Solutions forced through a needle which is kept
at a high potential (3.5kV)
• Very small droplets are created & they are
charged on their surfaces
• Ions get vaporize in vacuum
• Now the ion is led into the mass anlyzer
• Used to estimate shotgun lipidomics 19
Electrospray Ionization

20. Electrospray Ionization
20

21. Matrix Assisted Laser Desorption
Ionization (MALDI)
+
+
+
+
-
-
-
+
+
+
+
-
-
--+
+
Analyte
Matrix
Laser
+
+
+
• Absorption of UV radiation by
chromophoric matrix and
ionization of matrix
• Dissociation of matrix, phase
change to super-compressed gas,
charge transfer to analyte
molecule
• Expansion of matrix at
supersonic velocity, analyte
trapped in expanding matrix
plume (explosion/”popping”)
+
+
+ 21

22. 22
MALDI
• Matrix disperses large amounts of energy
absorbed by the laser
• Minimizes fragmentation of the molecule
• Permits analysis of molecular weight over
10,000 Da
• Used for study of polymers, proteins,
peptides

23. Matrix Application
α-Cyano-4-hydroxycinnamic acid
(CCA)
peptides
3,5-Dimethoxy-4-hydroxycinnamic
acid (sinapinic acid)
Proteins
2,5 Dihydroxybenzoic acid (DHB)
peptides,
proteins,
polymers,
sugars
3-Hydroxypicolinic acid (HPA)
oligonucleoti
des
Dithranol (anthralin) polymers
Different matrix and its applications
23

24. FAB
• Fast atom bombardment
• It produces ions when high velocity beam of
atoms impacts the surface of a nonvolatile liquid
(usually glycerol)
• Protonization occurs when analytes on the
surface of vaporized droplets are transferred to
gas state
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25. FAB
25

26. Chemical ionization
• Proton is transferred to or abstracted from a gas
phase analyte by a reagent molecule
• Typical gas reagents are methane, ammonia
isobutane and water
• Source pressure increased to 0.1 torr
• An electron beam ionizes reagent gas and
produces reactive species
• Little fragmentation occurs
• Negative ion electron capture CI for
quantification of drugs 26

27. Atmospheric Pressure Chemical
Ionization (APCI)
• Chemical ionization in an atmospheric pressure
electric discharge is called atmospheric pressure
chemical ionization.
• Modified version of ESI is the Ion Spray Source
• Used for mixtures of nonvolatile high molecular
weight compounds 27

28. 28

29. Different Mass Analyzers
• Quadrupole Analyzer (Q)
– Low (1 amu) resolution, fast, cheap
• Magnetic Sector Analyzer (MSA)
– High resolution, exact mass, original MA
• Time-of-Flight Analyzer (TOF)
– No upper m/z limit, high throughput
• Ion Trap Mass Analyzer (QSTAR)
– Good resolution, all-in-one mass analyzer
• Ion Cyclotron Resonance (FT-ICR)
– Highest resolution, exact mass, costly
29

30. Quadrupole Mass Analyzer
• A quadrupole mass filter consists of four parallel
metal rods with different charges
• Two opposite rods have an applied (+) potential
and the other two rods have a (– )potential
• For given dc and ac voltages, only ions of a
certain mass-to-charge ratio pass through the
quadrupole filter and all other ions are thrown
out of their original path
• m/z range is 1 – 1000 Da
30

31. Quadrupole Mass Analyzer
31

32. Magnetic Sector Analyzer
• Gas phase molecules are ionized by a beam of
high energy electrons
• Electrons may be ejected from molecules
(ionization) or bonds in molecules may rapture
(fragmentation)
• Ions are then accelerated in a field (sector) at a
voltage V
• Sector can have any apex angle (60o and 90o are
common)
• Ions with small masses must travel at a higher
velocity than ions with larger masses
32

33. Magnetic Sector Analyzer
33

34. Time Of FlightAnalyzer (TOF)
• Non Scanning technique where a full spectrum is
obtained as a snapshot
• Principle : A lighter ion travels faster than a
heavier ion provided both have the same kinetic
energy
• DRS operates at a 1 nanosecond scale and
produces accurate mass measurements with low
ppm accuracy
• TOF is inherently a pulsed technique
• Often combined with MALDI
34

35. MALDI-TOF Mass Spectrometry
35

36. MS – MS (TANDEM MS)INSTRUMENTS
• Employs two or more stages of mass analyzers
• Example is two quadrupoles coupled in series
• First analyzer selects ion (precursor ion) and
second analyzer selects the fragments of the
precursor ion
• Used to obtain more information about the
structure of fragment ions
• Fragment ions may be dissociated into lighter
fragment ions or converted into heavier ions by
reaction with neutral molecule 36

37. Ion Trap
• A device in which gaseous ions are formed
and/or stored for periods of time
Two commercial types
• Quadrupole Ion Trap (QIT)
&
• Ion-Cyclotron Resonance Trap (ICR)
37

38. • Quadrupole Ion Trap (QIT)
-Also called Paul Ion Trap
- Uses a quadrupole field to separate ions
-A3D field is created using a ring-shaped electrode
between two end cap electrodes
-Afixed frequency RF voltage is applied to the ring
electrode
- The end cap electrodes are either grounded or
under
RF or DC voltage
38

39. Mass Detectors
Electron Multiplier (Dynode) 39

40. Two types of ion detectors
A) Faraday collector - long life, stable, for signals > 2-3e6 cps
B) Electron multiplier - limited life, linearity issues, high-precision,
signals < 2e6 cps
40

41. 41
Faraday Cup
- A metal or carbon cup serves to capture ions and
store the charge
- Cup shape decreases loss of electrons
- Least expensive detector for ions
- Has long response time

42. Clinical Applications
• GC – MS :
 Pharmaceuticals :
o Separation and identification of degradation
products
o Molecular weight of drugs
o Analysis of resins fats and waxes
 Agriculture Industry :
o Identification of agrochemicals
o Quantitative determination , Rapid Screening and
conformational analysis of pesticides in food
samples
42

43. Clinical Applications GC MS
Food Industry :
oAnalysis of pesticides , residual solvents , separation
and identification of carbohydrates , lipids, proteins ,
colourants and preservatives
Analyze dairy products for ketones , aldehydes ,fatty
acids and milk sugars
Clinical Toxicology :
oDrug Screening – caffeine , codine , methadone ,
oIdentification and confirmation of Underivatized drug 43

44. Clinical Applications GC MS
Textiles :
oTo detect and estimate banned amines in dyes on
garments
Security Check :
oTo detect explosives in airport baggages
Sports :
oDoping control Agency – Detection of anabolic
steroids like ethylstrenol , methanedienone , methyl
testosterone
44

45. Astrochemistry :
oTwo GC-MS were sent to mars as part of Viking
program
oOne GCMS is positioned in Titan saturns largest
moon
oTo analyse Gerasimenko comet in 2014
45
GC-MS

46. Clinical Applications LC MS
• LC – MS : (More difficult to interface than GC MS)
Medical Applications :
• Confirmation of inborn errors of metabolism
• Carnitine / Acyl Carnitine and amino acid analysis
 Pharmaceutical Applications :
• Rapid chromatography of benzodiazepines
• Identification of bile acid metabolite
Biochemical Applications :
• Rapid protein identification using capillary LC/MS/MS
and database searching 46

47. Clinical Applications LC MS
Clinical Applications:
• High-sensitivity detection of trimipramine and
thioridazine
Food Applications:
• Identification of aflatoxins in food
• Determination of vitamin D3 in poultry feed
supplement
In virology
• Post translational modifications of viral proteins 47

48. Environmental Applications:
• Detection of phenylurea herbicides
• Detection of low levels of carbaryl in food
 Forensic Applications:
• illegal substances, toxic agents,explosives, Drugs
of abuse
48
Clinical Applications LC MS

49. Mass spectrometry is a very powerful method to
analyse the structure of organic compounds, but suffers
from following limitations :
• Compounds cannot be characterised without clean samples
• This technique does not have the ability to provide sensitive
and selective analysis of complex mixture
• For big molecules like peptides spectra are very complex and
very difficult to interpret
• Expensive , requires highly trained technicians
49

50. Summary
• Separating the ions according to their mass-to-
charge ratio (m/z)
• The m/z values and the fragmentation pattern
are used to determine the molecular weight and
structure of organic compounds
• Major components-ionisation source, a mass
analyser & an ion detector
• MS gives us structure of the of the compound as
well as the molecular weight
50

51. References
• Tietz - Fundamentals of clinical biochemistry
• Principles and techniques of biochemistry and
Molecular biology - Wilson and Walker
• Principles and techniques of biophysical
chemistry – Upadhyay
• Stryer – biochemistry 5th edition
• Web References and Images
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52. Thank You
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