Základy interpretace áklady interpretace hmotnostních spekter 1

Transkript

Klepnutím lze upravit styl předlohy podnadpisů.
Projekt vzdělávání Ekotech 2012
Základy interpretace áklady interpretace
hmotnostních spekter 1
Petr ŠIMEK
Š
1
Practical approach
Mass Spectrometry
Ob
Obecné
é principy
i i a základy
ákl d
2
Flow Chart of the Instrumental Analysis
Analytical problem
Analytical problem
Sampling
Sample preparation
Instrumental analysis
Separation Chromatography/Electrophoresis
Detection
Spectroscopy
⇓
Data acquisition/processing
⇓
INFORMATION
⇓
Management
3
Praktická MS a separace malých molekul
MASS SPECTROMETRY (MS)
MASS SPECTROMETRY
Physical method of IONIZATION, SEPARATION
y
,
and DETECTION of molecules according to their MASS m a CHARGE z. i.e. m/z
effective mass
m (12C) = 1.66 x 10‐27 kg
(
)
= 1.602 x 10‐19 C
z (electron)
Energy = 100 kJ/mol = 1.04 eV = 23.9 kcal/mol
1 mol = 6.022 0045 molecules
Hypotetický nositel hmotnosti – Higgsův boson
Hypotetický nositel hmotnosti Higgsův boson
4
The Periodic Table – Groups All Elements, periodická t.
Atomic number = number
of electrons and protons
p
Chemical symbol
Mass
Elements in columns have similar chemical properties
p p
Note biggest mass!
Number of neutrons = mass - Z
Common organic element
Often encountered in MS
Important trace element
5
Mass Scale – Orders of Magnitude
10−31
9.1×10−31 kg
Electron , the lightest elementary particle with a measured nonzero
rest mass.
27 kg Atomic
11.661×10
661 10−27
A
i mass unit
i (amu)
(
) or ddalton
l
(Da)
( )
1.673×10−27 kg Proton (938.3 MeV/c²)
y g atom,, the lightest
g
atom
1.674×10−27 kgg Hydrogen
1.675×10−27 kg Neutron (939.6 MeV/c²)
10−27
yoctogram
y
g
(yg)
2.99×10−26 kg Water molecule (18.015 amu)
1.6×10−25 kg Z boson (91.2 GeV/c²)
3.1×10−25 kg Top quark (173 GeV/c²), the heaviest known elementary particle
25 kg Caffeine molecule (194 amu)
33.2×10
2×10−25
3.45×10−25 kg Lead-208 atom, the heaviest stable isotope known
10−24
zeptogram (zg)
10−22
1.1×10−22 kg Haemoglobin A molecule
10−21
attogram (ag)
6
Mass Spectrometry
Mass Spectrometry
Basic Prerequisites – základní podmínky
• Molecules of the analyte must get an ELECTRIC CHARGE !
Elektrický náboj
• Separation of ions in an analyzer requires high vacuum !
Vakuum
min 10–3 Pa (10‐5) torr ‐ sufficient for mean free path of 1m
• For pumping by rotary pumps, high vacuum delivered by turbomolecular pumps 7
Mass Spectrometry
• Formation of ions is a chemical reaction !
• Destructive method with minimal sample consumption
• Separation method •
History (historie) :
first
after world war II
since 60th
since 1976
1984
1989
2002
applied to separation of isotopes (Thompson, 1913)
applied
to separation of isotopes (Thompson 1913)
developement in petroleum industry
GC‐MS
first LC‐MS
Fenn et al ‐ electrospray
p,
MALDI
Hillenkamp, Karas –
Fenn, Tanaka – Nobel price for protein MS
8
Appearance of the Mass Spectrum
Y i R l i i
Y‐axis Relative intenzity
i
X‐axis Effective mass m/z
Relative Abundance of the Ions in the Spectrum Depends on their Stability !!!
Relativní výskyt iontů v záznamu spektra závisí na jejich stabilitě !!!
9
Element
Rel. Atomic Mass
Isotopic Composition
Standard MW
1H
1.007 825 032 1(4)
99.9885(70)
2D
2.014 101 778 0(4)
( )
0.0115(70)
( )
4He
.002 603 2497(10)
99.999 863(3)
12C
12.000 000 0(0)
98.93(8)
13C
13 003 354 8378(10)
13.003
1 07(8)
1.07(8)
14N
14.003 074 005 2(9)
99.632(7)
15N
15.000 108 898 4(9)
0.368(7)
16O
15 994 914 6221(15)
15.994
99 757(16)
99.757(16)
18O
17.999 160 4(9)
0.205(14)
19F
18.998 403 20(7)
100
18.998 403 2(5)
23N
23Na
22 989 769 67(23)
22.989
100
22 989 770(2)
22.989
31P
30.973 761 51(20)
100
30.973 761(2)
32S
31.972 070 69(12)
94.93(31)
32.065(5)
33S
32.971 458 50(12)
0.76(2)
34S
33.967 866 83(11)
4.29(28)
35Cl
34.968 852 71(4)
75.78(4)
37Cl
36.965 902 60(5)
24.22(4)
39K
38.963 706 9(3)
93.2581(44)
39.0983(1)
40K
38.963 706 9(3)
93.2581(44)
39.0983(1)
41K
40.961 825 97(28)
6.7302(44)
79Br
78.918 3376(20)
50.69(7)
81Br
80.916 291(3)
49.31(7)
Type
1.007 94(7)
M+1
12.0107(8)
M+1
14.0067(2)
M+1
15 9994(3)
15.9994(3)
M+2
M+2
35.453(2)
M+2
M+2
79.904(1)
M+2
Isotopic Abundance
Isotopic Abundance
11
Elements and Isotopes, prvky a izotopy
p p y
py
Chemically pure analyte is a mixture of isotopes
Common net elements
H(D), F, P, I (only a single isotope)
M+1 elements
12C/13C, 14N/15N, M+2 elements
16O/18O, 28Si/30Si
(100:3.4)
32S/34S
(100:4.4)
(100
4 4)
35Cl/37Cl (3:1), 79Br/81Br (1:1)
Combination of elements their distribution is binomic (a+b)n
e.g. for Br2 1:2:1 (a2+2ab+b2), for Cl2 9:6:1(9a2+6ab+3b2) etc.
12
Elements and Isotopes
If M+2 < 3%
M 2 3%
M 2 l
M+2 elements –
t Si,S,Cl,Br not present
Si S Cl B
t
t
Characteristic combinations of isotopic abundances
13
Elements and Isotopes
p
M+1 elements
13C/
/12C
15N/14N
= 1.1%
net signal
g
intensityy is divided byy 1.1
= 0.37% net signal intensity is divided by 0.37
14
Rings plus Double Bonds
plus Double Bonds Rule
(určení počtu cyklů a násobných vazeb) ‐ rdb rule
(určení počtu cyklů a násobných vazeb)
rdb rule
Total number of rings and double bonds in a molecule CxHyNzOn
• R+db = x – ½ y + ½ z + 1
C…Si, H…X, N.…P, O....S
Values 0, 1, 2, 3, 4 ….
Odd‐electron ion (OE)+.
E
Even‐electron ion (EE)+
l t
i (EE)
Values ½, 1 ½, 2 ½, 3 ½…
V
l
½ 1½ 2½ 3½
Consequence: EE ion cannot be cation‐radical ion M+.
C2H5OH+.
C4H11N +.
C6H5CO+
2 ‐ 6 * ½ + ½ * 0 + 1 = 0 ⇒ OE
4 ‐ ½ * 11 + ½ * 1 + 1 = 0 ⇒ OE
7 ‐ ½ * 5 + ½ * 0 + 1 = 5.5 ⇒ EE
15
Rings plus double bonds – rdb rule
Napthalene C10 H8
5 double bonds + 2 cycles
16
The Nitrogen Rule
Dusíkové pravidlo
Nitrogen ‐
Nitrogen ‐
the only element with even mass and odd valence
the only element with even mass and odd valence
If a compound contains odd number of nitrogen atoms,
atoms its molecular ion
will be at odd mass number.
If the compound does not contain nitrogen or has even number of
nitrogens,
it
its
it molecular
l
l ion
i is
i att even mass number.
b
Lichá hmotnost = lichý počet atomů dusíku v molekule
Lichá hmotnost = lichý počet atomů dusíku v molekule
17
Ions
•
A charged form of an atom or molecule
•
Formed by adding or subtracting a charged entity (‘ionization’):
– M M ‐ e‐ ‐>> M
M+. Kation
Kation‐radikál
radikál
– M + H+ ‐> MH+ Kation
Electron ionization
Chemical ionization
•
Simplest ion is H+ which is equivalent to a proton
•
Ions are fundamental to mass spectrometry:
Ions
are fundamental to mass spectrometry:
– they can be manipulated with electric and/or magnetic fields. Neutral molecules cannot
– MS measures m/z (mass divided by charge), so 1000++ is the same as 500+
18
Appearance of the Mass Spectrum
Y‐axis Relative intenzityy
X‐axis Effective mass m/z
19
Classification of Ions – Klasifikace iontů
OE+. ions
reactive, not stable, rare in low mass region lichý e‐
most typical decomposition to EE+ and R.
Odd electron ion OE+. → EE+ + R.
OE+. ion is formed from M+. by cleavage of two bonds
(typical for reactions and rearrangements of a g)
ring) EE+ ions
generally more stable, fully paired electrons in sudý e‐
outer shell Even electron ion EE + The ease of their ionization n > π > σ
MH+ is another EE+ ion
( i ld
(typical decomposition to another EE+ and N ii
h EE
dN
(neutral fragment)
OE+. → EE+ + N
+N
20
Which Analytes Are Easily Ionized
Which
Analytes Are Easily Ionized ?
That possessing :
•
non/bond n‐electrons (nevazebný &volný elektronový pár) heteroatomy N,O,S, halogens >
•
π‐conjugated electrons, aromates >
•
π‐nonconjugated electrons (alkenes) >
•
σ‐electrones (alkanes, C‐C bond) >
•
σ‐electrones (alkanes, C‐H bond)
21
MASS (Hmotnost)
• Mass Unit (u) is also called Dalton (Da)
( )
( )
– All mass spectrometers measure m/z and never mass.
• Monoisotopic Molecular Weight
i
i
l l W i h
– Weight of predominant (non average) isotopes.
– eg. : Stearic Acid CH
S
i A id CH3(CH2 )16CO2 H is 284.2715 Da
H i 284 2715 D
• Average Molecular Weight
–A
Average weight of all Isotopes.
i ht f ll I t
– eg. : Stearic Acid CH3(CH2 )16CO2 H is 284.4844 Da
22
MASS
NAPHTHALENE C10H8
128
128
100
50
51
64
0
20
30
(mainlib) Naphthalene
40
102
77
39
50
60
70
80
90
100
110
Monoisotopic Molecular Weight
p
g
128.1735
Average Molecular Weight
128.0626
120
130
140
23
MASS
LEUCINE C5H5N1O2
LEUCINE C
86
100
86
44
OH
O
50
30
NH 2
74
28
41
39
10
20
(mainlib) Leucine
57
46
15 18
0
30
131
40
50
70
60
70
84
80
90
100
110
M
Monoisotopic Molecular Weight
i t i M l l W i ht
131 0946
131. 0946
Average Molecular Weight
131.1747
120
130
140
24
MASS RESOLUTION
MASS RESOLUTION
• Mass Resolution (rozlišení)
Mass Resolution (rozlišení)
– The ability of distinguish two different masses
• Eg: 100.1 Da vs. 100.2 Da
g
R=m/Δm
• Mass Accuracy
Mass Accuracy (přesnost změřené hmotnosti)
(přesnost změřené hmotnosti)
•
•
•
•
•
The ability to obtain the exact or “true” mass of a compound “unit”
“millidalton”
“percentage”
“ppm”
ppm (parts per million)
(parts per million)
25
MASS RESOLUTION
MASS RESOLUTION
•
Mass Resolution/Mass Accuracy
Mass resolution: represents the ability to separate two adjacent
masses. It measures the "sharpness" of the MS peak.
Mass accuracy: indicates the accuracy of the mass information
provided
id d b
by the
h mass spectrometer.
26
MASS RESOLUTION
MASS RESOLUTION
Resolution calculation:
R=m/Δm
27
MASS RESOLUTION
MASS RESOLUTION
N i l it
Nominal unit resolution
l ti
Hi h
High resolution l ti
28
MASS RESOLUTION
MASS RESOLUTION
High resolution (R= 6000)
(R
Nominal resolution (R=1000)
(R
1000)
29
What type of information can be obtained by Mass Spectrometry ?
by Mass Spectrometry ?
Q li i
Qualitative
–
–
–
–
Molecular weight
Elemental Composition
Elemental Composition
Structural Information
Chromatographic Peak Purity
Quantitative
– Trace Level Analyses
30
Components of a Mass Spectrometer
Components of a Mass Spectrometer
Mass Sorting
Ionization/ desorption
Detection
+
Ion Detection
Analyzer
Source
Form ions
(charged
molecules))
Sort Ions by Weight
Detect
D
t t
ions
100
75
Inlet
•
•
•
Solid
Liquid
Vapor
Sample Introduction
Method to vaporize sample
50
25
0
1330
1340
1350
Data Analysis
31
Current Mass Spectrometric Working Range
Amount [Moles]
Number of Molecules
Weight
g
[grams]
Peptide
p
1000.4
Reserpine
p
608.3
Peptide
p
1000.4
Reserpine
p
608.3
1 ng
1 pmol
1.64 pmol
6.02 x 1011
1012
1 pg
1 fmol
1.64 fmol
6.02 x 108
109
10 fg
10 attomol
16.4 attomol
6.02 x 106
107
1 fg
1 attomol
1.64 attomol
6.02 x 105
106
100
100ag
100
zeptomol
164 zeptomol
t
l
6 02 x 104
6.02
105
1 mol = mol weight x
1 mol = mol. weight x gram
gram
1 nmol = mol. weight x nanogram
1 pmol = mol. weight x pikogram
1 pmol mol. weight x pikogram
1 fmol = mol. weight x femtogram
32
Ionization
T h i
Techniques
Electron
I
Impact
t
(EI)
Hard, Fragments
Chemical
I i ti
Ionization
(CI)
Fast Atom
B
Bombardment
b d
t
(FAB)
API
MALDI
Soft, Intact
33
ELECTRON IONIZATION
Lenses
Analyzer
e- beam
Vacuum
system (10-5
to 10-6 Torr)
Heated filament
Ionization by electron beam
– M + e‐ ‐> M+. + 2e‐
– Very energetic; M+. will typically fragment
– Fragments can distinguish structure
d
h
– Must be able to get sample vapour into source
– Good for thermally stable analytes with low
Good for thermally stable analytes with low polarity
34
Chemical Ionization
Chemical Ionization
1 Torr reagent gas,
e g CH4
e.g.
Analyzer
e- beam
Vacuum
system (10-5
to 10-6 Torr)
Heated filament
Reagent gas ionized by electron beam
– CH4 + e‐ ‐> CH4+ + 2e‐
– CH4+ + CH4 ‐> CH5+ + CH3
– CH5+ + M ‐> [M+H]
+ M > [M+H]+ + CH
+ CH4
– Mild; MH+ doesn’t fragment as much as with EI
– Good for thermally stable analytes with low
ood o e a y s ab e a a y es
o po
polarity
a y
35
APCI (Atmospheric Pressure Ionization)
ionizace za atmosférického tlaku
Heated Nebulizer:
Atmospheric Pressure Chemical Ionisation (APCI)
corona discharge needle
polar to unpolar and thermally stable compounds
36
APCI
Heated Nebulizer:
corona discharge
di h
needle
dl
37
APCI
Heated Nebulizer:
38
APCI
Heated Nebulizer:
39
APCI
Heated Nebulizer:
40
APCI
Atmospheric Pressure Chemical Ionization
Features:
• Entire mobile phase and sample vaporized into the
gas phase (with heat), then ionized
• Accommodates high LC flow from (0.1 - 2 mL/min)
• Uses heat (400–500 °C) & nebulizer gas to vaporize HPLC eluent and “steam distill” sample into the gas phase for APCI
• Temperature setting is not critical
• Detection limits are very good
•
Limitations:
Li
it ti
– Thermally labile analytes may degrade when vaporized
41
APCI Inlet
•
•
•
•
•
Suitable for polar, thermally stable compounds
Usually, Molecular Weight < 1000 amu Probe is heated to facilitate vaporization Requires nebulizer and auxiliary gas
R
Requires corona discharge needle to produce ionization (APCI)
i
di h
dl t
d
i i ti (APCI)
42
Electrospray ionization - ESI
Electric field on the electrospray needle
43
Electrospray ionization - ESI
Droplet desintegration in time
N
number of droplets
R
droplet diameter [um]
Δt evaporation time [μs]
44
ESI
• Features:
• Hot drying gas improves evaporation of charged droplets
Hot drying gas improves evaporation of charged droplets
• no thermal degradation
• better sensitivity at higher flow rates
• tolerates higher aqueous solvent compositions
• Improved performance over a wide range of LC flow rates without splitting (5µL/min to 1mL/min)
without splitting (5µL/min to 1mL/min)
• Detection limits are very good
45
Application range of ESI, APCI and PI
ESI
46
MALDI ionizace
i i
Matrix
i Assisted
i
d Laser Desorption
i
Ionization
Desorpce analytů laserem v matrici
47
MALDI:
Matrix Assisted Laser Desorption Ionization
Laser
Sample plate
1. Sample (A) is mixed with excess
matrix (M) and dried
on a MALDI plate.
hν
2. Laser flash ionizes matrix
molecules.
3. Sample molecules are ionized by
proton transfer from matrix:
AH+
MH+ + A Æ M + AH+.
Variable Gro
Ground
nd
+20 kV
Grid
Grid
48
MALDI Sample Preparation
MALDI preparation protocol:
f Dried droplet preparation: matrix solution
mixed with analyte solution on the metal target
and dried
f Matrices are e.g.
2.5-dihydroxy
2
5 dihydroxy benzoic acid (DHB) or
4-hydroxy-α-cyanocinnamic acid (CHCA)
Beavis, Chaudhary, Chait, Org. Mass Spectrom. 1992, 27, 156.
T i l DHB d
Typical
dried
i dd
droplet
l t preparation
ti on a
steel target
Strupat,
p , Karas,, Hillenkamp,
p, 1991,, 111,, 89.
49
Desorption: Plume (chochol)
Desorpce analytů v mikrosekundové sekvenci
LIF images of the expansion of both light matrix (3-HPA) and heavy
bimolecule ((dye-tagged
y
gg DNase I p
protein, narrow jjet)) during
g the MALDI p
process
Puretzky, Geohegan, Chem. Phys. Lett. 1998, 286, 425.
50
Matrix Selection
Látky vhodné do matrice pro laserovou desorpci α-Cyano-4-hydroxy-cinnamic
y
y
y
acid
(CHCA)
P tid 10kD
Peptides<10kDa
Sinapinic Acid
Proteins >10kDa
2,5-Dihydroxybenzoic acid (DHB)
Neutral Carbohydrates,
Synthetic Polymers
Proteins,
“Super DHB”
Glycosylated proteins
3-Hydroxypicolinic acid
Oligonucleotides
HABA
Proteins
Proteins,
Oligosaccharides
51
MALDI Sample Plate Options
Terčíky na vzorky pro MALDI 96 x2 Close External Calibration
with hydrophobic surface
Gold 100 well
400 well
with hydrophobic surface
52
Unique MALDI Features
Unique MALDI Features
z
Soft ionization
analyze biomolecules intact
z
z
Sample
p in solid state
Ionization and transfer of analytes of the broad mass
range
g
analyze a wide variety of biomolecules (> 300 KDa)
z
z
High ionization efficiency and thus sensitivity (low
fmole)
Fast
53
Ionization methods : ESI vs. MALDI
Ionization methods :
ESI vs MALDI
MALDI vers. ESI porovnání • ESI Advantages
ESI Advantages
• Direct coupling of LC to MS
• Fast – lots of MS and MS/MS
/
• Accepted MS/MS ionization mode
• ESI LIMITATIONS
• Limited time for MS/MS (limited depth of analysis)
• Miss high MW peptides
i hi h
id
• more difficult than MALDI?
• MALDI Advantages
MALDI Advantages
• Sample in solid state
• Not time‐limited for MS or MS/MS
• Analysis can be faster or slower than separation
• Sophisticated workflows
Sophisticated workflows
• Fast – lots of MS and MS/MS
• MALDI LIMITATIONS
MALDI LIMITATIONS
• Offline coupling of LC?
• Miss low MW peptides
• Conv. MALDI not ideal for ID of unknown proteins
54
Combined Desorption and Electrospray ionization= DESI
•
•
Ambientní ionizační technika, kombinace elektrospreje a desorpce
Cooks et al, 2004, Science 2004: 306, 471‐473.
•
Nabité mikrokapky tvořené elektrosprejem z mobilní fáze slouží jako projektily pronikající do povrchové vrstvy, předávají náboj (heterogenous charge transfer mechanism) a desorbují vznikající ionty, které jsou přenášeny pomocí vnějšího el. pole (droplet pick‐up mechanism) do vstupního otvoru MS
N í tř b žád á t i
Není třeba žádná matrice, matrici tvoří vzorek sám
t i i t ří
k á
Komercializace , Prosolia Inc., •
•
55
Desorption Electrospray ionization = DESI p
p y
Velké molekuly – mechanismus ionizace podobný ESI, vznikají mnohonásobně nabité ionty
p y
Malé molekuly – přenos náboje mezi molekulou analytu na povrchu vzorku a iontem
rozpouštědla v (a) kapalné nebo (b) plynné fázi
DESI MS prstu 50 min po orální aplikaci 10 mg antidepresiva/antialergika Claritin
(účinná látka loratadine, MH+ = 383.3)
Mobilní fáze: MeOH-voda
p y
Malé molekuly
DESI MS povrchu ibiškového květu
• MP = EtOH‐water
MP EtOH t 50:50
50 50
Detekováno:
• směs esenciálních olejů
• carotenoids
• antioxidanty typu
4h d
4-hydroxy-3,5-di-tert-butyl-p-cresol
3 5 di t t b t l
l
(m/z MH+ = 219)
Ionizace přímo též v plynné fázi mezi analytem a iontem rozpouštědla nebo částic ve vzorku Praktická MS a separace malých molekul
DART = direct analysis
y in real time
•
•
•
•
Ionizace analytů na povrchu vzorku excitovaným plynem (He, N2)
Ambientní ionizace na rozdíl od DESI nevyžaduje ESI sprejovanou kapalinu
Ambientní ionizace, na rozdíl od DESI nevyžaduje ESI sprejovanou kapalinu
Cody BC, Laramee JA, US Patent 6,949,741; 27/09/2005, Komercializace Jeol, IonSense
DART scheme
DART MS
DART‐MS
Direct analysis in real time
• Není třeba žádná příprava vzorku nebo separace
• Analyt je přímo ionizován na povrchu vzorku
• Kapalina nanášena na skleněnou tyčinku a ionizována • Těkavá látka vnášená přímo do přiváděného ionizovaného plynu
• Na rozdíl od DESI nevznikají adukty s ionty kovů a ionizace probíhá bez rozpouštědel
• Desopce je omezena na malé molekuly s minimální těkavostí
• Chernetsova E, Morlock E, Mass Spectrom Rev 2011, 30, 875‐883.
Výbušniny, toxiny léčiva, drogy, polární i málo polární sloučeniny, cukry, Výbušniny
toxiny léčiva drogy polární i málo polární sloučeniny cukry
potravinářské ……
DART MS
DART‐MS
Direct analysis in real time
• Není třeba žádná příprava vzorku nebo separace
Pří ý vstup
Přímý
t vzorku
k z otevřeného
t ř éh laboratorního
l b
t
íh prostoru
t
Forenzní DART‐MS analýza 5 různých druhů inkoustu
DART‐MS
Jones R et al, J Forensic Sci 2006: 51, 915
Praktické náměty
O
OH
?
NH2
Uvažte, jak probíhá ionizace vašich „oblíbených“ analytů
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