elucidated by Mass Spectrometry
33
1 Max Planck Institute for Polymer Research ASMS Fall Workshop 2004 Polymer Structure Baltimore Dec. 9-10 Mass Spectrometry Polymer Structure elucidated by Hans Joachim Räder Max Planck Institute for Polymer Research • Structure determination of monodisperse macromolecules – Phenylene dendrimers – Giant polycyclic aromatic hydrocarbons (PAH’s) • Structure determination of polydisperse macromolecules End group determination of homo-polymers – in simple cases (Poly(styrene)) – in difficult cases (Poly(carbonate) and Poly(fluorene)) • Copolymers – PPE-b-PEO Diblock-copolymer – Random copolymers of amino acids Outline ASMS Fall Workshop 2004 Polymer Structure Baltimore Dec. 9-10
Transcript of elucidated by Mass Spectrometry
1
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
ASMS Fall Workshop 2004 Polymer Structure Baltimore Dec. 9-10
Mass Spectrometry
Polymer Structureelucidated by
Hans Joachim Räder
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
• Structure determination of monodisperse macromolecules– Phenylene dendrimers– Giant polycyclic aromatic hydrocarbons (PAH’s)
• Structure determination of polydisperse macromoleculesEnd group determination of homo-polymers– in simple cases (Poly(styrene))– in difficult cases (Poly(carbonate) and Poly(fluorene))
• Copolymers– PPE-b-PEO Diblock-copolymer– Random copolymers of amino acids
Outline
ASMS Fall Workshop 2004 Polymer Structure Baltimore Dec. 9-10
2
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
Analysis of a well defined phenylene dendrimer by MALDI-TOF MS
C474H318
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
MALDI-TOF Analysis of a soluble C474 Dendrimer
a.u.
C474H318
3000 4000 5000 6000 7000 8000 9000m/z
6013
3
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
6000 6005 6010 6015 6020 6025
3000 4500 6000 7500 9000
a.u
.
m/z
simulated
6013 experimental
Measured and Simulated Isotopes of C474H318 -Dendrimer
13 Isotopes(mainly 13C)
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
- 106 H
Synthesis of a C222 - PAH - disc
Reaction Scheme
soluble dendritic precursor insoluble PAH product
Lewis acidin CH2Cl2
oxidative cyclodehydrogenation
4
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
2700 2705 2710 2715 2720 2725 2730
1000
2000
3000
4000
5000
6000
2711
.46
2710
.45
2709
.45
2708
.53
2707
.44
2706
.52
m/z
MALDI-TOF-Mass Spectrum with TCNQ as Matrix
C222H42
2706.33 Characterization Method for Insoluble Samples
Simulated Isotopes
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
matrix solution
analytesolution
homogenous solution homogenous crystal formation
crucial step 1: crucial step 2:(risk of phase separation)
MALDI sample preparation
Commonly mixing dissolved samples and matrix in molar ratios of 1 : 1000
5
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
solid matrix
solidanalyte
mechanical homogenization!?
Solvent-free sample preparation
crucial step 1:crucial step 2 is overcome!No risk of phase separation
Mechanical mixing of solid samples in e.g. a ball mill
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
a
bCyclization
1,2-Phenyl shift
Cyclization
109 C114H30M=1398
Structure Defects
Incomplete Cyclodehydrogenation?
Product mixture with different numbers of hydrogens
6
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
1390 1395 1400 1405 1410 1415
e
d
c
b
a
m/z
Structure Defects
crude powder (product)
product dispersion in TCE
simulation of mixture with
C114H30 : C114H34 : C114H36
1 : 1 : 0.54
simulation of mixture withC114H30 : C114H34 : C114H381 : 1 : 1
simulation of C114H30
measured
best fit
isotopes of pure compound
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
HH
a) b) c)
C114H34(m=1402m/z) C114H36(m=1404m/z) C114H38(m=1406m/z)
Figure 7
Possible side reactions (but unlikely)
Incomplete cyclodehydrogenation reaction
7
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
1340 1360 1380 1400
m=1328
-76 m/z
-74 m/z
-70 m/z
C114H36
C114H34
C114H30
m/z
Fragmentation by In-Source-Decay (ISD)
No selectionof precursorIon possible
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
(a)
(1) arrow type
sample 2
660 680 700 720 740 760
-72
m/z
742
(b) sample 3
880 900 920 940 960 980
-72
m/z
962
(a)
(2) bay type
sample 4
740 760 780 800 820
818
-74
m/z
(b) sample 5
900 920 940 960 980
-74
966
m/z
(a)
(3) arrow & bay type
sample 6
600 620 640 660 680
670-74
-72
m/z
Structure Dependent Differences in Fragmentation Pattern
8
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
H
HH
H
HH
H
H
H H
H
H
H
H
H
H
H
H
hν+3H H
HH
∆m=-75+3=-72 m/za
HH
H
HH
H
H HH
HH
H
H
HH
hν +2H ∆m=-76+2=-74 m/zb
H
H HH
HH
HH
HH
H
HH
H
hν +H
H
H
H
c
∆m=-77+1=-76 m/z
Loss of Cyclic C6 Units and Subsequent RehydrogenationM
ax P
lanc
k In
stitu
te fo
rPol
ymer
Res
earc
h
a
b
c
C114H34M=1402
C114H36M=1404
Rotation
Cyclization
Cyclization
1,2-Phenyl shift
Cyclization
109 C114H30M=1398
Plausible Side Products by Rearrangement Reactions
9
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
Mol Weight =6013.82C474H318
Mol Weight =5759.81C474H66
MW=5814.24C474H120
- 252 H
- 198 H
Cyclodehydrogenation Products of C474 PrecursorM
ax P
lanc
k In
stitu
te fo
rPol
ymer
Res
earc
h
5000 5200 5400 5600 5800 6000 6200 6400 6600 6800 7000
13a2
b)
c)
a)
m/z
a.u.
Spectra of different synthetic cyclodehydrogenation attempts
Multiple chlorination
Best result
Incomplete reaction
Dendrimer (educt)Planarized Disk
Molecular Propeller
10
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
C474H120M=5814
Computer generated visualization “C474 trimer“
Molecular propeller
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
400 600 800 1000 1200 1400 1600 1800 20000,00
0,02
0,04
0,06
0,08
0,10
0,12
0,14
0,16
0,18
364
700
574
a.u.
Wavelength [ nm ]
C474H120M=5814
Solid-state UV-vis Spectrum of “C474 trimer“
11
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
- 252 H
1 2
4a
3b
4b
3a
- 198 H
- 186 H
(fragmentation)
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
400 600 800 1000 1200 1400 1600 1800 2000
700
560
a.u.
λ / nm
565
12
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
• Structure determination of monodisperse macromolecules– Phenylene dendrimers– Giant polycyclic aromatic hydrocarbons (PAH’s)
• Structure determination of polydisperse macromoleculesEnd group determination of homo-polymers– in simple cases (Poly(styrene))– in difficult cases (Poly(carbonate) and Poly(fluorene))
• Copolymers– PPE-b-PEO Diblock-copolymer– Random copolymers of amino acids
Outline
ASMS Fall Workshop 2004 Polymer Structure Baltimore Dec. 9-10
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
100000 200000 300000
0
100
200
300
400
500
Poly(styrene)
M3+=70 000
M 2+=105 000
M +=210 000
a.i.
m /z
MALDI-TOF Spectrum of Poly(styrene) 210,000 Da
Unresolved polymer distribution- No structural information!
13
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
2000 4000 6000 8000 10000 12000 14000
0
2000
4000
6000
8000
10000
a.
i.
m/z
n
MALDI-TOF Spectrum of Poly(styrene) 9,000 Da
Resolved chain length distribution
Representative analysis of individualsignals
Ionized by attachment of Ag+
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
Mass of Repeat Units for Recognition of Polymer Type
8500 8600 8700 88002000
4000
6000
8000
10000
12000
n=83n=81n=80 n=82
104.15104.15104.15
8810870686028498
a.i.
m/z
n
C8H8 = 104.15Styrene unit
14
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
1104.1557
n
Butyl
H
+ 1 (Hydrogen)+ 108 (Ag+)
= 82 (n) * 104.15 (Styrene)+ 57 (Butyl)
8706
Calculation of End Group Molecular Weight
Mass contributions:
End group calculation: 8706 - 108 (Ag+) = 8598 (corrected polymer mass)8598 : 104.15 (styrene unit) = 82.554 (repeat units)82.554 82 styrene repeat units (n)
and decimal place bears end group mass:0.554 * 104.15 = 57.7 (mass of both end groups)
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch Anionic Polymerization of Styrene
Butyl Li + n
Butyl
n-1
+ H
H
Butyl
n
Li
- Li
Cationisation with Ag MALDI-TOFMass Spectrum
Sample History: Way of Synthesis
15
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
34 36 38 40 42 44 46
0,0
0,2
0,4
0,6
0,8
1,0
40.96
K38.96
a.i.
m/z
102 104 106 108 110 112 114
0,0
0,2
0,4
0,6
0,8
1,0 Ag108.90106.90
1650 1655 1660 1665 16700,00,20,40,60,81,0
1657.98
[PS15 + K]+
a.i.
m/z
1720 1725 1730 17350,00,20,40,60,81,0
1725.92
[PS15 + Ag]+
Isotopic Distributions of a Polystyrene Chain ofn = 15 (1619,02 Da) Ionized with Different Cations
Isotopic Pattern
Important for unambiguous interpretation: Knowledge of attached cation!
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
Field Desorption Spectrum of Polystyrene 5 kDa
ionization mechanism:radical cation formation
direct measurement of polymer mass
M.+M2+
M.3+
M4+
16
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
Principle of Field Desorption
+ 8 kV
-5 kV
0,5 – 2 mm
Ion BeamAnalyzer
Activated Tungsten Wirewith Carbon Micro Needles
FD Emitter
Extraction Rods
Field Strength: 108 V/cmIonization by Tunneling Processes
at Needle Tips
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
2 or even 3 unknowns interpretation not straightforward
Expectations from fragmentation analysis: differentiation of chain ends
deduction of MEnd1, MEnd2, n
Investigated system: Polycarbonate - Elucidation of a spectrum with 7 superposed distributions
End group determination of homopolymers in difficult cases
End1 repeat unit End2-[- -]n-
17
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
7 compositions !
End group determination of a polycarbonate sample
n
O O C
OCH3
CH3
C
PCA
0 1000 2000 3000 4000 5000 6000 70000
250
500
750
1000
a.i.
m/z
1400 1500 1600 1700 1800 1900
S2
S1S6
S5S4
S3
S2
S1
S7
S3
S7
S6
S5S4
S3
S2
S1
m/z
Conventional MALDI-TOF mass spectrum of a PCA sample
PCA repeat unit
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch Interpretation of conventional
MALDI-TOF mass spectrum:
MX , n , M Y ????
M=177.2+nx254.29+149.2
O C O C
CH3
CH3
CH3
OO
H3CCH3
CH3
C
CH3
CH3
O C O C
n
O C YOCH3
CH3
X O C
n
M=1.0+nx254.29+149.2
M=1.0+nx254.29+227.3
O C O C
CH3
CH3
O
OHH
CH3
CH3
O C
n
M=nx254.29
CO
CH3
CH3 O
CO
n
O C YOCH3
CH3
X O C
n
O C YOCH3
CH3
X O C
n
O C O C
CH3
CH3
CH3
OCH3
CH3
H O C
n
S1
S2
S3
S4
S5
S6
S7
MX , n , M Y ????
MX , n , M Y ????
End group determination of a polycarbonate sample
1400 1500 1600 1700 1800 1900
S2
S1S6
S5S4
S3
S2
S1
S7
S3
S7
S6
S5S4
S3
S2
S1
m/z
18
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
Vacc
Source
Vgate
Detector
Reflector
Drift regionVref
gate off
gate on
400 600 800 1000 1200 14000
2000
4000
6000
8000
10000
a.i.
m/z
400 600 800 1000 1200 14000
2000
4000
6000
8000
10000
a.i.
m/z
Vgate
t(m/z)
Selection of a defined signal for fragment ion analysisM
ax P
lanc
k In
stitu
te fo
rPol
ymer
Res
earc
h
Vgate
Detector
Reflector
Drift region Vref
+ +
5 6 0 5 8 0 6 0 0 6 2 0 6 4 0 6 6 0 6 8 0 7 0 00
2 0 0 0
4 0 0 0
6 0 0 0
8 0 0 0
1 0 0 0 0
a .i.
m /z
Parent ion
PSD fragments
Vacc
Source
Post-source decay
+++
19
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch Main fragmentation of PCA:
0 200 400 600 800 1000
0
2500
5000
7500
10000
12500
∆Mrepeating unit
repeating unit
repeating unit
[M+Li]+
1096.2
904.3
649.6395.5141.1
a.i.
m/z
∆M = MCH3 + MEnd= 15 + MEnd
n
OCO
H3CCH3
CH3
C O O C O C
CH3
CH3
CH3
OCH3
CH3
C
Fragment mass spectrum
C
CH3
CH3
O C
O
O
Li+
Li+
CH3
O
analogue to mechanismdescribed for EI, SSIMS (Literature)C
O
O
- CH3
and -
Polycarbonate: fragmentation mechanism?
MEnd deduced !
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
Result: Complete elucidation of the end group composition
0 1000 2000 3000 4000 5000 6000 70000
250
500
750
1000
a.i.
m/zConventional MALDI-TOF mass spectrum of a PCA sample
1400 1500 1600 1700 1800 1900
S2
S1S6
S5S4
S3
S2
S1
S7
S3
S7
S6
S5S4
S3
S2
S1
m/z
M=59.0+nx254.29+31.0
M=177.2+nx254.29+149.2
O C O CCH3
CH3
CH3
OO
H3CCH3
CH3
C
CH3
CH3
O C O C
n
O C OO
CH3H3CO CH3
CH3
O C O C
n
M=1.0+nx254.29+149.2
O C O C
CH3
CH3
CH3
OCH3
CH3
H O C
n
M=177.2+nx254.29+31.0
M=1.0+nx254.29+227.3
O C O C
CH3
CH3
O
OHH
CH3
CH3
O C
n
O C O CH3
OOH3C
CH3
CH3
C
CH3
CH3
O C O C
n
M=nx254.29
COCH3
CH3 OCO
n
M=1.0+nx254.29+31.0
O C O CH3
OCH3
CH3
H O C
n
S1
S2
S3
S4
S5
S6
S7
20
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
Synthesis of Polyfluorene (PF)
2a: R1=Br; R2=Br2b: R1=Br; R2=H or R1=H; R2=Br2c: R1=H; R2=H2d: R1=R2=no atom (cyclic structure)
2,7-dibromo-9,9-diphenylfluorene
Yamamoto couplingin toluol-DMFBr Br
Poly(9,9-diphenyl-2,7-fluorene)repeating unit: 316 Da
R1 R2
n
1 2
Dr. A. C. Grimsdale
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
1000 2000 3000 4000 5000
500
1000
1500
2000
2500
3000
3500
4000
(a)
[H-( )4-H]+.
[H-( )12-H]+.
[H-( )8-H]+.
[H-( )10-H]+.
[H-( )6-H]+.
a.i.
m/z
800 1000 1200 1400 1600 1800 2000
500
1000
1500
2000
2500
3000
3500
4000
(b)
[Br-( )5-Br]+.
[Br-( )4-Br]+.
[Br-( )3-Br]+.
[H-( )4-H]+. [H-( )5-H]+.
[H-( )3-H]+.
[H-( )6-H]+.
a.i.
m/z
- dithranol- THF
end group analysis!
Solvent-Based MALDI MS of the Soluble Fraction (< 10%) of PF
oligomerization degree ~ n = 15
21
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
end group analysis?
Solvent-Free MALDI MS of Insoluble Polymer Fraction (> 90%) of PF
2000 4000 6000 8000 10000
0
100
200
300
400
500
600
700
800
(a)
[H-( )20-H]+.
[H-( )15-H]+.
[H-( )10-H]+.
[H-( )7-H]+.
a.i.
m/z
1800 1900 2000 2100 2200 2300 2400 2500 2600
0
100
200
300
400
500
600
700
800
(b)
-69
-69-69
[H-( )8-H]+.
[H-( )6-H]+.[H-( )7-H]+.
a.i.
m/z
dithranol matrix
?
oligomerization degree ~ n = 25
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
1500 2000 2500 3000 3500
200
400
600
800
a.i.
m/z
[H-( )5-H]+.
[H-( )7-H]+.
[H-( )9-H]+.
[H-( )11-H]+.
(b)
(a)
0
200
400
600
800
[H-( )5-H]+.
-69-69-69-69-69-69-69
[H-( )11
-H]+.
[H-( )9-H]+.
[H-( )7-H]+.
successful suppression of fragmentation& independence of matrix choice!
- insoluble fraction- solvent-free- minimum
laser power- TCNQ
- insoluble fraction- solvent-free- minimum
laser power- dithranol
Influence of the MALDI Matrix
22
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
2000 4000 6000 8000 10000
0
500
1000
1500
2000
2500
3000
3500
(a)
[H-( )25-H]+.[H-( )
20-H]+.
[H-( )15-H]+.
[H-( )10
-H]+.
[H-( )7-H]+.
a.i.
m/z
1800 1900 2000 2100 2200 2300 2400 2500 2600
0
500
1000
1500
2000
2500
3000
3500
-77-77-77
-69 -69 -69
-77-77
(b)
-77
-69-69-69
[H-( )8-H]+.[H-( )7-H]+.[H-( )6-H]+.
a.i.
m/z
- TCNQ- solvent-free
fragmentation of the phenyl side-chain:ISD - 77 DaPSD - 69 Da ?
Insoluble Fraction of PF at High Laser PowerM
ax P
lanc
k In
stitu
te fo
rPol
ymer
Res
earc
h
Unequivocal Determination of ISD- and PSD-Fragmentation
500 1000 1500 20000
2000
4000
6000
8000
10000
m/z
(a) [H-( )7-H]+.
a.i.
1900 2000 2100 22000
2000
4000
6000
8000
10000
a.i.
m/z
-77-77
-77
(b)[H-( )7-H]+.
- insoluble fraction - TCNQ- solvent-free- high laser power
PSD-fragmentation of the phenyl side-chain:-77 Da (calibrated) & -69 Da (not calibrated)!
PSD-MALDI MS:
23
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
• Structure determination of monodisperse macromolecules– Phenylene dendrimers– Giant polycyclic aromatic hydrocarbons (PAH’s)
• Structure determination of polydisperse macromoleculesEnd group determination of homo-polymers– in simple cases (Poly(styrene))– in difficult cases (Poly(carbonate) and Poly(fluorene))
• Copolymers– PPE-b-PEO Diblock-copolymer– Random copolymers of amino acids
Outline
ASMS Fall Workshop 2004 Polymer Structure Baltimore Dec. 9-10
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
MALDI-Fragmentation MS?
combinations of analytical methods (reactivity ratios, NMR)
averaged properties of different chain lengths
Traditional Copolymer Characterization:
Allows direct information of
Copolymer distribution and composition
MALDI-MS Copolymer Characterization:
„random“- and „block“-polymer sequenz determination
Information about the primary structure?
Structural Elucidation of Synthetic Copolymers
24
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
Compositional information of resolved molecular weight distributions are not unequivocal
Interpretation of mass spectra often uncleardue to signal overlapping caused by :
- unfavorable monomer masses e.g. MMonomer1 ~ k x MMonomer2
- low resolution due to high mass range or instrumental limitation
Composition of a diblock copolymer
End1 Monomer1 Spacer-[- -]n- -[- Monomer2 -]m- End2
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
n
mH3C O CH2 CH2 O
O
Hexyl
Hexyl
Poly(ethylene oxide) = PEO
Poly(para-phenyleneethynylene) = PPE
PEO-b-PPE
+
n
Hexyl
Hexyl
O
HOH3C O CH2 CH2 OHm
diblock copolymer synthesis
Mcopo = MEnd1 + m x MMonomer1 + MSpacer + n x MMonomer2 + MEnd2
= 15 + m x 44 + 120 + n x 268 + 101
Formation of a PPE-b-PEO diblock copolymer
25
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch Investigation of each block, before reaction:
Agreement with expected PEO and PPE blocks
400 600 800 1000 1200 14000
2000
4000
6000
8000
10000
m=2
2
m=1
0
m=2
0
m=2
5
m=1
2
m=1
8
m=1
5
a.i.
m/z
MALDI-TOF mass spectrum of PEO block
H3C O CH2 CH2 OHm
1000 1500 2000 2500 30000
2000
4000
6000
8000
n=8n=
7n=6
n=5
n=3
n=4
a.i.
m/z
n
Hexyl
Hexyl
O
HO
MALDI-TOF mass spectrum of PPE block
Composition of a PPE-b-PEO diblock copolymer
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400
500
1000
1500
2000
2500
268=MMonomer2MMonomer1=44a.i.
m/z2100 2200 2300 2400
500
1000
1500
2000
2500
2231=Mcopo(n=5;m=14) + K+
2227=Mcopo(n=4;m=20) + K+
4444
∆M=8
a.i.
m/z
Mcopo = MEnd1 + m x MMonomer1 + MSpacer + n x MMonomer2 + MEnd2
= 15 + m x 44 + 120 + n x 268 + 101
Need of additional data fragmentation of copolymer
Composition of a PPE-b-PEO diblock copolymer
MALDI-TOF mass spectrum of PPE-b-PEO (low resolution)
1900 2000 2100 2200
500
1000
1500
2000
2500
2007=Mcopo(n=4;m=15) + K+
4444
∆M=5a.i.
m/z
pure signal
overlapping signals
26
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
Most intense fragment peak Deduction of m, n
0 500 1000 1500 20000
2000
4000
6000
8000
Loss of alkyl side chains
Fragmentation of PEO block
PPE-b-PEO (n=4;m=15)
[M+Li]+
CH
3-[-O
-CH
2-CH
2-]15
a.i.
m/z
1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400
500
1000
1500
2000
2500
a.i.
m/z
MALDI-TOF mass spectrum of PPE-b-PEO
Fragment mass spectrum
selection and fragmentation
Composition of a PPE-b-PEO diblock copolymer
pure signal
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch Information in case of superposition
Proof of 2 different structures !
1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400
500
1000
1500
2000
2500
a.i.
m/z
0 500 1000 1500 20000
2000
4000
6000
8000
PPE-b-PEO(n=4;m=20
andn=5;m=14)
CH
3-[-O
-CH
2-CH
2-]14
CH
3-[-O
-CH
2-CH
2-]20
a.i.
m/z
Fragment mass spectrum
MALDI-TOF mass spectrum of PPE-b-PEO
Composition of a PPE-b-PEO diblock copolymer
2100 2200 2300 2400
500
1000
1500
2000
2500
2231=Mcopo(n=5;m=14) + K+
2227=Mcopo(n=4;m=20) + K+
4444
∆M=8
a.i.
m/z
Selection andFragmentation
overlapping signals
27
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
1500 2000 2500 30000
1000
2000
3000
4000
5000
n=7n=6
n=5
n=4
n=3
a.i.
m/z
Reconstruction of complete copolymer composition
Copolymer Composition of PPE-b-PEO diblock copolymer
n
mH3C O CH2 CH2 O
O
Hexyl
Hexyl
Poly(ethylene oxide) = PEO
Poly(para-phenyleneethynylene) = PPE
PEO-b-PPE
+
n
Hexyl
Hexyl
O
HOH3C O CH2 CH2 OHm
diblock copolymer synthesis
m = 17
m = 19
m = 18
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
Allows direct information on copolymer distribution and compositionby direct measurement of individual copolymer amounts due to resolved isotopic distributions
2100 2150 2200 2250 2300 2350 24000
1000
2000
3000
4000
5000
n=5
; m=1
7
n=5
; m=1
6
n=5
; m=1
5
n=5
; m=1
4
n=5
; m=1
3
n=4
; m=2
3
n=4
; m=2
2
n=4
; m=2
1
n=4
; m=2
0
n=4
; m=1
9
n=5
; m=1
2n=
4 ; m
=18
a.i.
m/z
1500 2000 2500 30000
1000
2000
3000
4000
5000
n=7n=6
n=5
n=4
n=3
a.i.
m/z
MALDI-TOF mass spectrum of PPE-b-PEO
Determination of Copolymer Composition at High Resolution
28
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
Sequence Determination of Synthetic Polymers
amino acid monomer units
no given primary structure
polydisperse
protected side-chains
NH
HN
O
R1 ONH
R2
x y
H
Monomer Units: 2
Copolymer Types:
A B A B B A “random”
A A A B B B “block”
“Copolymer”:
Characteristical structural elements of natural polypeptides and industrial polymers
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
Synthetic Polypeptides
NH
HN
O
ONH
NH
O
O
OO
x y
H
n-C6H13NH2DMF, RT-CO2
HNO
O
O
BnO
O
HNO
O
O
ZHN
+x y
Reactivity: Glu ≅ Lys
„Random“ Copolymers: n-Hex-NH-(Glu)x-st-(Lys)y-H
n-Hex-NH-(Glu)x-bl-(Lys)y-H
n-Hex-NH-(Lys)y-bl-(Glu)x-H
Block-Copolymers:
Glu Lys
29
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
1000 2000 3000 4000 5000 6000 7000 8000 9000 10000
MALDI-TOF of „Random“ n-Hex-NH-(Glu)x-st-(Lys)y-H
1000 1200 1400 1600 1800 2000 2200 2400
m/z
Inte
nsity
(a.i.
) n=x+y
m/z
X = Y = 0.50
n=4
n=5n=6
n=7n=8
n=9
1200 1250 1300 1350 1400
n=5
(Glu)4(Lys)1
(Glu)3(Lys)2
(Glu)2(Lys)3
(Glu)1(Lys)4
Copolymer composition:
Degree of oligomerization:
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
Theory of Probabilities: Monitoring with MALDI MS?
75:25
50:50
25:75
Identical Reactivitiesof the Monomer Units:
1 : 5 : 10 : 10 : 5 : 1
(Glu)5
(Glu)4(Lys)1
(Glu)3(Lys)2
(Glu)2(Lys)3(Glu)1(Lys)4 (Lys)5
1 : 15 : 90 : 270 : 405 : 243
243 : 405 : 270 : 90 : 15 : 1
M = 219 M = 262
30
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
MALDI-TOF of „Random“ Pentamers
1200 1250 1300 1350 1400
(Glu)5
(Glu)4(Lys)1(Glu)3(Lys)2
(Glu)2(Lys)3(Glu)1(Lys)4 (Lys)5
m/z
Inte
nsity
(a.i.
)Feed-
composition:Glu : Lys
75:25
50:50
25:75
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
Theory of Probabilities for Sequenz Composition
Fragment Spectra
5 Equal Probabilities for Sequenz Composition of
n-Hex-NH-(Glu)4-st-(Lys)1-H!
n-Hex-NH-Glu-Glu-Glu-Glu-Lys-Hn-Hex-NH-Glu-Glu-Glu-Lys-Glu-Hn-Hex-NH-Glu-Glu-Lys-Glu-Glu-Hn-Hex-NH-Glu-Lys-Glu-Glu-Glu-Hn-Hex-NH-Lys-Glu-Glu-Glu-Glu-H
n-Hex-NH-Glu4n-Hex-NH-Glu3Lys
Lys-HGlu-H
n-Hex-NH-Glu3n-Hex-NH-Glu2Lys
Lys-Glu-HGlu2-H
n-Hex-NH-Glu2n-Hex-NH-Glu1Lys
Lys-Glu2-HGlu3-H
n-Hex-NH-Glu1n-Hex-NH-Lys1
Lys-Glu3-HGlu4-H
n-Hex-NH Lys-Glu4-H243 : 405 : 270 : 90 : 15 : 1
5:0
4:1
3:2
2:3
1:4
0:5
10 Equal Probabilities
31
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
Inte
nsity
(a.i.
)
(Glu)4H(Glu)3(Lys)H(Glu)2(Lys)H
(Glu)3H(Glu)(Lys)H
(Glu)2H
n-Hex-NH-(Glu)4(Lys)-H
(Glu)4(Lys)H
0 200 400 600 800 1000 1200 m/z
n-Hex-NH-(Glu)4-st-(Lys)1-H
(Glu)H
(Lys)H
PSD Fragment Ion-Analysis of a „Random“ Pentamer
n-Hex-NH-Glu4n-Hex-NH-Glu3Lys
Lys-HGlu-H
n-Hex-NH-Glu3n-Hex-NH-Glu2Lys
Lys-Glu-HGlu2-H
n-Hex-NH-Glu2n-Hex-NH-Glu1Lys
Lys-Glu2-HGlu3-H
n-Hex-NH-Glu1n-Hex-NH-Lys1
Lys-Glu3-HGlu4-H
n-Hex-NH Lys-Glu4-H- 44
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
Sequenz analysis of a Block-Copeptid
n-Hex-NH-Lys-Lys-Lys-Lys-Glu-Hn-Hex-NH-Lys-Lys-Lys-Glu-Lys-Hn-Hex-NH-Lys-Lys-Glu-Lys-Lys-Hn-Hex-NH-Lys-Glu-Lys-Lys-Lys-Hn-Hex-NH-Glu-Lys-Lys-Lys-Lys-H
n-Hex-NH-(Glu)1-st-(Lys)4-HTheory of Probabilities:
n-Hex-NH-Lys-Lys-Lys-Lys-Glu-H
n-Hex-NH-(Lys)4-bl-(Glu)1-HPre-determined Sequenz:
Glu-H
Lys-Glu-H
Lys2-Glu-H
Lys3-Glu-H
Lys4-Glu-H
Glu-H Lys-HLys-Glu-H Lys2-H
Lys2-Glu-H Lys3-HLys3-Glu-H Lys4-H
Lys4-Glu-H
Characteristical differences!
32
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 1 2 0 0 1 4 0 0
Sequenz Analysis of a “Block”-Copolymer
Lys4H
Lys3H
LysHLys2H
“random”
“block”
n-Hex-NH-Lys-Lys-Lys-Lys-Glu-H
n-Hex-NH-Lys2n-Hex-NH-Lys3
n-Hex-NH-Lys
Lys4GluH
Inte
nsity
(a.i.
)
m/z
Characteristical differences for the „block“ peptide: lack of N-terminated Glu and Lysn!
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
NH
HN
O
ONH
NH
O
O
OO
x y
H
RN
O
OH
RN
CH 2
HCO 2H
RNH
CH 3
R'H 2C
O
O//
ONH
OO
HR'' ONH
R'' O
Qualitative Interpretation of Fragmentation
- CO2- 44
- 44 fragments: Lys: McLafferty rearrangement-108 fragments: Lys, Glu: AminolysisC-terminated fragments: Lys- 28 fragments: Glu: different backbone cleveage
Lys Glu - BnOH- 108
Glu
Lys, Glu
Lys
Glu
33
Max
Pla
nck
Inst
itute
forP
olym
er R
esea
rch
Acknowledgement
Laurence Przybilla 1
Sarah Trimpin
Sabine Kummer
Kai Martin
Kimihiro Yoshimura
Jochen Spickermann
Ali Pouhanipour
Stefan Türk
Coworkers
