MASS SPECTROMETRY & XRD TECHNIQUES

MASS SPECTROMETRY & XRD
By
K.S.RAJESH
11HF1SO316 C M COLLEGE OF PHARMACY Under the guidance of Mr. PAVAN PHARMACEUTICS REDDY KUMAR Dept OF
Dept OF PHARMACEUTICS
The three essential components, are: 1. The Ion Source : A small sample of compound is ionized, usually to cations by loss of an electron. 2. The Mass Analyzer :The ions are sorted and separated according to their mass and charge. 3. The Detector: The separated ions are then detected and tallied, and the results are displayed on a chart.
ADVANTAGES
Different elements can be uniquely identified by their mass
Different compounds can be uniquely identified by their mass.

L-dopa COOH HO HO -CH2CH-NH2 CH3CH2OH Ethanol
Butorphanol N -CH2OH
HO MW = 327.1 MW = 197.2 MW = 46.1
Analytical method to measure the molecular or atomic weight of samples
Materials present at concentration less than one part per million can be easily detected. It is extremely sensitive and small quantity of sample is sufficient. It is capable of high precision. Proteins can now be sequenced by MS.
BASIC CONSIDERATIONS
SAMPLE:
Must have an obtainable vapour pressure of at least 10-7 mm Hg. IONS: Ions are produced from sample molecules by bombardment of electrons. Electrons must possess sufficient energy of 50-100eV. POSITIVE IONS: Positive ions are abundant than negative ions by a factor of 102-104.
INSTRUMENTATION
PRINCIPLE
Ions kinetic Energy as a function of accelerating voltage (V) and charge (e).
Applied magnetic field Centrifugal force
F=HeV
Combine equations to obtain
10
WORKING
11
Mass Spectrum
Mass spectrum: A plot of the relative abundance of ions versus their massto-charge ratio. Base peak: The most abundant (highest intensity) peak. Molecular ion peak: The peak formed at the highest mass number.
12
100%
43
base peak given 100% abundance

58
50% 71 114
molecular ion
m/e
Isotope peaks - P+1, P+2, etc

Dept OF PHARMACEUTICS 13
MS of METHANE
Molecular ion base peak fragments 1 15 16
14
Sample Introduction
Gas source: sample volatized and purified (by GC) and injected into mass spectrometry. Liquid source: handled similar to gases. Solid source: For metals and semi conductors spark source is used. Organic solids are handled by means of a probe.
ION SOURCE
The ionizing electrons are liberated by thermionic emission from heated tungsten or rhenium filament. TYPES OF ION SOURCE: Electron impact ionization Electron spray ionization Matrix assisted laser desorption ionization Spark ionization Surface ionization
ELECTRON IMPACT IONIZATION: In the ionization chamber, the sample is bombarded with a beam of high-energy electrons.
17
18
ELECTRON SPRAY IONIZATION:

Sample dissolved in polar, volatile buffer and pumped through a stainless steel capillary (70 - 150 mm) at a rate of 10-100 mL/min. Strong voltage (3-4 kV) applied at tip along with flow of nebulizing gas causes the sample to nebulize or aerosolize. Aerosol is directed through regions of higher vacuum until droplets evaporate to near atomic size (still carrying charges)
19
Electron spray ionization:

Pressure = 1 atm Inner tube diam. = 100 um Sample Inlet Nozzle (Lower Voltage)
Partial vacuum
N2
+ + + + ++ ++ + + ++ ++ + + ++ + ++ + ++ + ++ + ++ + ++ + ++
MH+
+ +
+
+
+ + +
Sample in solution N2 gas
+ +
MH2+ MH3+
High voltage applied to metal sheath (~4 kV)
Charged droplets
20
Electron spray ionization:
Sample
+ _
Ionizer
Mass Analyzer
Detector
21
MALDI: Matrix Assisted Laser Desorption Ionization
Sample is mixed with a UV absorbent matrix (sinapinic acid for proteins, 4hydroxycinnaminic acid for peptides) Sample is ionized by bombarding sample with laser light
337 nm UV laser
22
MALDI
Sample plate Laser
1. Sample is mixed with
matrix (X) and dried on plate.
MH+
2. Laser flash ionizes matrix molecules. 3. Sample molecules (M) are ionized by proton transfer: XH+ + M MH+ + X.
Grid (0 V)
+/- 20 kV
23
Types of ions produced during fragmentation in Mass Spectrometer:

1.
2. 3. 4. 5.
6.
7.
Molecular ion Fragment ion Meta Stable ion Isotopic ion Multiple charge ion Negatively charged ion Rearrangement ion
Molecular Ions:
Intensity of molecular ion peak in mass spectrometry depends on molecular ion stability. Molecular ions are stable in electron containing compounds. For the formation of molecular ions 10-5-10-7 mm Hg pressure and 70eV energy is required. Stability order: aromatic conjugated acyclic unbranched branched alcohols
25
Fragmentation of a molecular ion, M, produces a radical and a cation. Only the cation is detected by MS.
A + B+ Radical Cation A+ + B Cation Radical
A-B
Molecular ion (a radical cation)
Fragment Ions:
Homolytic Cleavage
Cleavage due to 1 electron transfer Cleavage due to transfer of 2 electrons
Molecule(M)
Heterolytic cleavage
In homolytic cleavage a pair of electrons move independent of each other In heterolytic cleavage a pair of electrons move in same direction
Homolytic cleavage
CH3CH2 Cl CH3CH2Cl
CH2-CH Cl
CH3CH2+ + Cl.
CH2 CH2++ HCl
H
Heterolytic cleavage
Meta Stable Ions:

Ions
stability Stable Unstable
Meta stable
The position of meta stable ion peak can be obtained by using the formula m mass of daughter ion M mass of molecular ion
Isotopic Ions:
When isotopic ions differ by only one mass unit , the peak obtained is m+1 peak. Other peaks obtained in mass spectrometry is m+2 peak m+3 peak. Carbon, for example, in nature is 98.90% 12C and 1.10% 13C. There are 1.11 atoms of carbon-13 in nature for every 100 atoms of carbon-12.
30
M+2 Peaks
The most common elements giving rise to significant M + 2 peaks are chlorine and bromine.
Chlorine in nature is 75.77% 35Cl and 24.23% 37Cl. A ratio of M to M + 2 of approximately 3:1
31
M+2 Peaks
Bromine in nature is 50.7% 79Br and 49.3% 81Br. A ratio of M to M + 2 of approximately 1:1 indicates the presence of a single bromine atom in a compound
32
Isotopic Abundance
81Br
33
Multiple Charge Ions: In aromatic compounds the molecular ion formed is highly stable.
.+ e M
molecular Ion charged
M+2
doubly charged
M+3
triply
M+2
M+3
Position can be determined by using formula
Position can be determined by using formula
34
Negatively Charged Ions:

e
M-
They are formed in rare case. Do not have any importance in mass spectrometry.
35
Rearrangement ions
Retro
Diels-alder: +
.
CH2
+.
+
CH2 CH2
CH2
McLafferty:
H Z H Z CH2 CH2 Z R Z R
36
DIFFERENT MASS ANALYZERS

Magnetic Sector Analyzer (MSA) Quadrupole Analyzer (Q) Time-of-Flight Analyzer (TOF) Ion Trap Mass Analyzer (QSTAR) Ion Cyclotron Resonance (FT-ICR)
37
Magnetic Sector Analyzer

When a charged particle is subjected to magnetic field it traverses a circular path
38
Quadrupole Analyzer
Four parallel rods or poles through which the ions being separated are passed. Poles have a fixed DC and alternating RF voltages applied to them.
Depending on the produced electric field, only ions of a particular m/e will be focused on the detector, all the other ions will be deflected
39
Quadrupole Analyzer
40
Time-of-Flight (TOF)Analyzer
This type of analyzer measures the time for ions to reach the detector. Small ions reach the detector before large ones. There are two types of TOF analyzers MALDI-TOF Analyzer Q-TOF Analyzer
41
MALDI-TOF ANALYZER
peptide mixture embedded in light absorbing chemicals (matrix) pulsed UV or IR laser (3-4 ns) detector
+ + + + +
vacuum
+
+
strong electric field
Vacc
cloud of protonated peptide molecules
Time Of Flight tube
42
MALDI-TOF ANALYZER
Linear Tim e O f Flight tube
ion source
detector
tim e of flight
Reflector Tim e Of Flight tube

ion source
detector reflector
tim e of flight
43
Q-TOF ANALYZER
NANO SPRAY TIP MCP DETECTOR
PUSHER
HEXAPOLE
HEXAPOLE COLLISION CELL
TOF
REFLECTRON
QUADRUPOLE
SKIMMER ION SOURCE
HEXAPOLE
44
Ion Trap Analyzer
Ion traps are ion trapping devices that make use of a three-dimensional quadrupole field to trap and massanalyze ions
45
Ion Cyclotron Resonance

Uses powerful magnet (5-10 Tesla) to create a miniature cyclotron Fourier Transform approach allows many ion masses to be determined simultaneously (efficient) Has higher mass resolution than any other MS analyzer available
46
DETECTORS
Early detectors used photographic methods, this is called Mass Spectrograph. Todays detectors produce electronic signals by secondary electronic emission when struck by an ion. Types of detectors used are: Faraday cup Electron multiplier Photographic detection
Faraday collector
Electron multiplier
48
FRAGMENTATION RULES
1. 2. 3.
Intensity of M.+ is Larger for linear chain than for branched compound Intensity of M.+ decrease with Increasing M.W. Cleavage is favored at branching.
R R CH R
Loss of Largest Subst. Is most favored
49
50
Branched alkanes
H3C H3C H3C H3C H3C CH3 CH3
MW=170
51
FRAGMENTATION RULES
4. Aromatic Rings, Double bond, Cyclic structures stabilize M.+
52
FRAGMENTATION RULES
5. a) Saturated Rings lose a Alkyl Chain (case of branching)
R
+.
-R
+
.
b) Unsaturated Rings Retro-DielsCH2 Alder . + . CH2 +

+
CH2 CH2
53
FRAGMENTATION RULES
6. Alkyl substituted aromatic Compounds Cleave in b Resonance Stabilized Tropylium ion
R C CH CH2
-R
.
CH
CH2
+
FRAGMENTATION RULES
7. C-C Next to Heteroatom cleave leaving the charge on the Heteroatom
- [RCH2]
R
+
CH2
CH2
H2C
H2C
55
FRAGMENTATION RULES
8. Cleavage of small neutral molecules (CO2, CO, olefins, H2O .) Result often from McLafferty rearrangement. McLafferty Rearrangement: Intra molecular migration of hydrogen from e- rich centre to e- deficit centre followed by cleavage at position resulting in elimination of an alkene.
McLAFFERTY REARRANGEMENT
x
O C Y CH2
CH2 CH2 Y
O C CH2
H CH2 CH2
- CH2=CH2
Y H, R, OH, NR2
O C Y
x
CH2
Ion Stabilized by resonance

FRAGMENTATION RULES
Nitrogen Rule: If a compound has zero or an even number of nitrogen atoms, its molecular ion will have an even m/z value. an odd number of nitrogen atoms, its molecular ion will have an odd m/z value. Ring Rule: The number of unsaturated site, R is equal to the number of double bonds and/or rings in the molecule. Ring rule for molecule CaHbNcOd .
SPECIFIC FRAGMENTATION RULES

Alkanes: Fragmentation point indicates the weakest linkage in a compound. Fragmentation tends to occur in the middle of unbranched chains rather than at the ends. In case of branched alkanes the branching point is most preferred fragment point.
Straight Chain Alkanes
60
Branched Chain Alkanes
61
ALKENES
Alkenes characteristically show a strong molecular ion peak. Alkenes prefer to fragment at position to double bond. o cleave to form resonance-stabilized allylic cations
+ [CH2 =CHCH2 CH2 CH3 ] CH2 =CHCH2 + +
CH2 CH3
62
ALKENES
63
ALKYNES
Alkynes typically show a strong molecular ion peak. cleave readily to form the resonance stabilized propargyl cation or substituted propargyl cations.
HC C=CH2
3-Propynyl cation HC C-CH + 2 (Propargyl cation)
64
ALKYNES
65
Alcohols
One of the most common fragmentation patterns of alcohols is loss of H2O to give a peak which corresponds to M-18. Another common pattern is loss of an alkyl group from the carbon bearing the OH to give a resonance-stabilized R' oxonium ion and an alkyl radical. + + +
R + A radical
R C O H R" Molecular ion (a radical cation)
R' -C H O R' -C= O- H R" R" A reson ance-stabilized oxonium ion

Alcohols
67
Aldehydes and Ketones
Characteristic fragmentation patterns are a-cleavage O McLafferty rearrangement+ +

+
a-cleavage m/z 128
m/z 43 O + m/z 113
CH3
McLafferty re arrangement
H +
Mole cular ion m/z 114
m/z 58
Aldehydes and Ketones
69
Carboxylic Acids
Characteristic fragmentation patterns are a-cleavage to give the ion [CO2H]+ with m/z 45. McLaffertya-cleavage rearrangement O
OH Mole cular ion m/z 88
H O OH Mole cular ion m/z 88
O= C-O- H m/z 45
McLafferty re arrangement +
O OH
m/z 60
70
Carboxylic Acids
71
Esters
a-cleavage and McLafferty rearrangement

O OCH 3 + a-cleavage
O +
OCH 3
+ m/z 71 +
O + OCH 3 m/z 59 + O OCH 3 m/z 74

O OCH 3
McLafferty re arrangement
H +
Aromatic Hydrocarbons
Most alkyl benzenes show a fragment ion of m/z 91. H

+
CH3 Toluene radical cation
- H
H
+
H H
H Tropylium cation (m/z 91) H
73
Amines
The most characteristic fragmentation pattern of 1, 2, and 3 aliphatic amines is b-cleavage.

CH3 b-cleavage CH3 CH3 - CH- CH 2 + + CH2 = NH2 m/z 30
CH3 - CH- CH 2 -CH 2 -NH2
74
AROMATIC ETHER
O
x
O
R
- R
-CO
C5H5
Cleavage in b of aromatic ring

O
m/z 93
- CH2=CH2
H Rearrangement
H H
m/z 94
O H
x
75
Aliphatic Ether
Cleavage of C-C next At Oxygen Loss of biggest fragment
CH3 CH2 OCH2 CH2 CH2 CH3

m/z 59
CH3 CH2 O+ =CH2 CH3 CH2 O CH2+

Resolution
Resolution: A measure of how well a mass spectrometer separates ions of different mass. Low resolution: Refers to instruments capable of separating only ions that differ in nominal mass; that is ions that differ by at least 1 or more atomic mass units (amu). High resolution: Refers to instruments capable of separating ions that differ in mass by as little as 0.0001 amu.
R=m/m m is the mass of the first peak m is the mass difference between two adjacent peaks that are just resolved
78
Monoisotopic mass
Monoisotopic mass corresponds to lowest mass peak
When the isotopes are clearly resolved the monoisotopic mass is used as it is the most accurate measurement.
79
Average mass
Average mass corresponds to the centroid of the unresolved peak cluster
When the isotopes are not resolved, the centroid of the envelope corresponds to the weighted average of all the the isotope peaks in the cluster, which is the same as the Dept OF PHARMACEUTICS 80 average or chemical mass.
Different Types of MS
GC-MS - Gas Chromatography MS separates volatile compounds in gas column and IDs by mass LC-MS - Liquid Chromatography MS separates delicate compounds in HPLC column and IDs by mass MS-MS - Tandem Mass Spectrometry separates compound fragments by magnetic field and IDs by mass LC/LC-MS/MS-Tandem LC and Tandem MS Separates by HPLC, IDs by mass and AA sequence Dept OF PHARMACEUTICS 81
LC-MS..
Study protein complexes without gel electrophoresis
Peptides all bind to cation exchange column Successive elution with increasing salt gradients separates peptides by are Peptides charge separated by hydrophobicit y on reverse phase column
82
LC-MS...
83
GC-MS
A mixture of compounds is separated by gas chromatography, then identified by mass spectrometry.
84
Tandem MS- MS/MS

separation and identification of compounds in complex mixtures - Uses two or more mass analyzers/filters separated by a collision cell filled with Argon or Xenon - induce fragmentation and mass analyze the fragment ions.
Mixture of ions Single ion
MS-1 MS-2
Fragments
Ion source
Applications of Mass Spectrometry
Pharmaceutical analysis Analysis of mixture Component analysis Gas analysis Isotope abundance measurement Thermodynamic studies Ionization potential measurement
86
Applications of Mass Spectrometry

Biomolecule characterization Proteins and peptides Oligonucleotides Environmental analysis Pesticides on foods Soil and groundwater contamination Forensic analysis/clinical
87
Protein Identification
Peptide Mass Fingerprinting (PMF)
88
Peptide Mass Fingerprinting
Used to identify protein spots on gels or protein peaks from an HPLC run Depends of the fact that if a peptide is cut up or fragmented, the resulting fragments (and resulting masses) are enough to identify the protein
Requires a database of known sequences

Uses software to compare observed masses with masses calculated from
90
PROBLEM
Bank President
Who robbed the bank?
Biologist What protein was isolated?
91
GATHER EVIDENCE Mass Spectrometrist

Police Officer
1. Interview witnesses 2. Dust for fingerprints
1. Interview biologist who isolated the protein 2. Cleave protein to obtain peptide mixture
enzyme
3. Analyze peptide mixture by MS to obtain peptide molecular masses!
92
DATABASE SEARCH
Police Officer
Height: 57
Weight: 160 lbs Gender: male Age: 35-40
Mass Spectrometrist
Approx. molecular weight: 30,000
Origin: bovine liver Peptide mass list from MS analysis: 975.4832, 1112.5368, 632.3147, 803.4134, 764.3892
Fingerprints
search
search
PEPTIDE MASS DATABASE OF KNOWN PROTEINS
DATABASE OF KNOWN FELONS
DATABASE SEARCH RESULTS Police Officer Identifies the robber Antony J. Felon Mass Spectrometrist
Identifies the protein
bovine carbonic anhydrase
94
ISOTOPE-CODED AFFINITY TAG (ICAT): a quantitative method

Chemically reactive group: forms a covalent bond to the protein or peptide Isotope-labeled linker: heavy or light, depending on which isotope is used Affinity tag: enables the protein or peptide bearing an ICAT to be isolated by affinity chromatography in a single step
Label protein samples with heavy and light reagent Reagent contains affinity tag and heavy or light isotopes
95
Example of an ICAT Reagent

Biotin Affinity tag: Binds tightly to streptavidinagarose resin
O
NH NH
Reactive group: Indoacetamide-reactive group will bind to Cysteine Linker: Heavy version will have deuterium at * Light version will have hydrogen's at * H
* *
H N S O
O O
O
*
N I O
96
The ICAT Reagent
97
How ICAT works?

Lyse & Label
Affinity isolation on streptavidin beads

Quantification MS Identification MS/MS
Light
100 MIX
100
Heavy
Proteolysis (ie trypsin)

0
0 550 570 m/z

590
200
400 m/z
600
98
A modern radiograph of a hand
99
SOURCE OF X-RAY
Target
X-rays
Beam of electrons
100
Target Metal Mo
Of Ka radiation () 0.71
Cu
Co Fe Cr
1.54
1.79 1.94 2.29
101
TYPES OF X-RAY
X-rays are produced when high energy electrons bombard with target molecule. Vacuum
Knocked out electron from inner shell
Energy levels
E L3 E L2 E L1
L3
L2
L1
K
EK Nucleus
Characteristic x-rays
102
103
Generally two types of x-rays are formed Continous radiation Characteristicaradiation K
Intensity
White radiation
Kb
Characteristic radiation due to energy transitions in the atom
0.2
0.6
1.0
1.4
Wavelength ()
104
X RAY DIFFRACTION
The atoms or ions are arranged in the regular places in a crystal. When x-rays are incident on crystal they are diffracted from the atoms or ions, they may have constructive interference or a destructive interference.
105
BRAGGS EQUATION
Braggs Law:
2dsin = n
= X-ray wavelength
= X-ray incident angle

d = lattice spacing of atomic planes Dept OF PHARMACEUTICS crystal) 106 (assuming a
107
APPLICATIONS
Phase
Composition of a Sample: determine the relative amounts of phases in a mixture by referencing the relative peak intensities. Unit cell lattice parameters and Bravais lattice symmetry. Crystal Structure Texture/Orientation
108
XRD can be used to investigate the crystal environment of particular atoms Ta displaying cubic
symmetry in the mineral microlite.
Ta showing hexagonal symmetry in the mineral calciotantite.

109
REFERENCES
B.K.SHARMA Pg:
S-844 to S-922 A.H BECKETT & J.B.STENLAKE Pg: 78-81, 474, 477. Gurdeep R Chatwal Pg:2.272-2.302 Mass spectrometry instrumentation, interpretation and application by Rolf Ekman, Jerzy silberring.
Chem-805
Identification of organic and inorganic compounds by spectroscopy
110
111

MASS SPECTROMETRY & XRD TECHNIQUES

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MASS SPECTROMETRY & XRD

Different elements can be uniquely identified by their mass

Different compounds can be uniquely identified by their mass.

HO MW = 327.1 MW = 197.2 MW = 46.1

Analytical method to measure the molecular or atomic weight of samples

Applied magnetic field Centrifugal force

Combine equations to obtain

base peak given 100% abundance

Isotope peaks - P+1, P+2, etc

ELECTRON SPRAY IONIZATION:

Electron spray ionization:

Sample in solution N2 gas

High voltage applied to metal sheath (~4 kV)

Electron spray ionization:

MALDI: Matrix Assisted Laser Desorption Ionization

matrix (X) and dried on plate.

Types of ions produced during fragmentation in Mass Spectrometer:

Molecular ion (a radical cation)

Cleavage due to 1 electron transfer Cleavage due to transfer of 2 electrons

CH2 CH2++ HCl

Meta Stable Ions:

Position can be determined by using formula

Position can be determined by using formula

Negatively Charged Ions:

DIFFERENT MASS ANALYZERS

Magnetic Sector Analyzer

strong electric field

cloud of protonated peptide molecules

Time Of Flight tube

Reflector Tim e Of Flight tube

HEXAPOLE COLLISION CELL

Ion Trap Analyzer

Ion Cyclotron Resonance

Loss of Largest Subst. Is most favored

b) Unsaturated Rings Retro-DielsCH2 Alder . + . CH2 +

Ion Stabilized by resonance

SPECIFIC FRAGMENTATION RULES

Straight Chain Alkanes

Branched Chain Alkanes

+ [CH2 =CHCH2 CH2 CH3 ] CH2 =CHCH2 + +

3-Propynyl cation HC C-CH + 2 (Propargyl cation)

R C O H R" Molecular ion (a radical cation)

R' -C H O R' -C= O- H R" R" A reson ance-stabilized oxonium ion

Aldehydes and Ketones

Characteristic fragmentation patterns are a-cleavage O McLafferty rearrangement+ +

a-cleavage m/z 128

m/z 43 O + m/z 113

Mole cular ion m/z 114

Aldehydes and Ketones

a-cleavage and McLafferty rearrangement

O + OCH 3 m/z 59 + O OCH 3 m/z 74

Mole cular ion m/z 102

Mole cular ion m/z 102

Most alkyl benzenes show a fragment ion of m/z 91. H

CH3 Toluene radical cation

H Tropylium cation (m/z 91) H

The most characteristic fragmentation pattern of 1, 2, and 3 aliphatic amines is b-cleavage.

CH3 - CH- CH 2 -CH 2 -NH2

Cleavage in b of aromatic ring

Cleavage of C-C next At Oxygen Loss of biggest fragment

CH3 CH2 OCH2 CH2 CH2 CH3

CH3 CH2 O+ =CH2 CH3 CH2 O CH2+