Polymer Analysis by MALDI-TOF MS - · PDF file[ application note ] GPC MALDI polymer analysis...
Transcript of Polymer Analysis by MALDI-TOF MS - · PDF file[ application note ] GPC MALDI polymer analysis...
[ application note ][ application note ]
OV ERV IEW
Molecular weight is an important parameter for synthetic polymers
because it relates directly to their physical properties.
The most commonly used techniques to characterize synthetic poly-
mers, such as osmometry, cryscopy, end-group titration and light
scattering, only yield an average molecular weight and do not yield
any information about chemical structure or chain branching, etc.
Other methods, such as gel permeation chromatography (GPC) and
high performance liquid chromatography (HPLC), separate the oligo-
meric components of the polymer system with limited resolution.
Furthermore, the accuracy of molecular weight values is limited by
the need to calibrate against reference compounds.
Thus these techniques are not suitable for the determination of
absolute molecular weight distributions of the individual components
of the polymer distribution.
MALDI MS has an important advantage in synthetic polymer
analysis: absolute molecular weights of oligomers can be
determined, as opposed to obtaining relative molecular weights
by chromatographic techniques. MALDI polymer analysis permits
accurate determination of molecular weights from narrowly
distributed polymers (polydispersity <1.2).
This application note demonstrates the use of the Waters®
MALDI micro MX™ for polymer characterization. This includes
molecular weight averages (both the number [Mn] and weight [Mw]
averaged molecular weights), polydispersity, mass of repeat units
and end-group mass structure.
INT RODUCT ION
Polymer distributions are typically characterized by Mn and Mw,
which are calculated from the formula:
Mn = (NiMi)/Ni and Mw = (NiMi2)/NiMi
Ni and Mi are the abundance and mass of the ith oligomer,
respectively. For the polystyrene 2000 MALDI MX spectrum
(Figure 1), the molecular weight averages can be calculated directly
from the spectrum: Mn = 2079, Mw = 2232, and polydispersity
(D = MwMn) = 1.07, using programs such as Polymerix
(Sierra Analytics, Modesto, CA, Figure 2 and 3)
The repeating mass unit of 104.063 confirms that the analyte is
a styrene-based polymer. For the peak at m/z = 1934.083, it can
easily calculate that the peak is a PS with 17 repeat styrene units
(mass = 1769.071 Da). The difference of 165.012 mass units
(1934.083 – 1769.071) is made up by the addition of 107.913 and
57.099 with 107.913 is the average mass of the silver atom (from
silver-cationized PS ions). Mass 57.099 is the butyl end group.
In this case, MALDI results provide good confirmation of the
polymer structure. The technique can accurately determine the
masses of oligomers, confirming that MALDI is a useful tool for
end-group analysis as well as product confirmation.
P O LYM E R A NA LYSIS BY MA L D I -T O F -MS
Henry Shion and Nicholas Ellor Waters Corporation, Beverly, MA, USA
m/z1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900 3000 3100 3200 3300 3400 3500 3600
%
0
100 1936.091832.04
1727.96
1623.90
1519.85
1518.831415.77
1414.78
1413.771311.71
1310.71
1207.64
1161.671103.58
1312.71
1517.83
1416.78
1474.86
1622.90
1520.85
1621.90
1578.92
1624.91
1726.96
1725.95
1682.99
1728.97
1831.03
1830.03
1788.05
1833.04
1935.09
1834.02
1892.13
1937.09 2041.15
2040.15
2039.15
1938.10
1996.18
2145.21
2144.22
2042.15
2143.22
2100.24
2249.27
2248.29
2146.23
2247.27
2204.32
2353.35
2352.34
2351.34
2251.27
2308.37
2457.392354.36
2456.39
2455.40
2355.36
2412.43
2561.472458.42
2560.46
2559.44
2459.41
2517.48
2562.45
2665.53
2664.50
2563.46
2663.53
2620.54
2666.53
2769.59
2768.57
2667.53
2767.56
2724.60
2770.57
2873.64
2872.64
2771.59
2871.64
2874.64
2977.72
Figure 1. Polystyrene 2000 MALDI micro MX spectrum.
[ application note ]
EX PERIMENTAL
The major difference between MALDI analysis of proteins/peptides
and synthetic polymers is in the ionization process. For protein/pep-
tide MALDI analysis, most samples are ionized through protonation;
for synthetic polymer MALDI experiments most samples are ionized
by cationization.
Some polymers, such as polyethylene glycols and polymethylmeth-
acylate form ions with alkali metals (added to the sample or simply
present as an impurity) in the form of MLi+, MNa+, and MK+, etc. Others,
such as polystyrene, undergo a cationization process which preferen-
tially involves transition metal ions, such as silver and copper1. The
actual mechanism of the cationization process remains unclear, but
it appears that as the result of a gas phase collision between cation
and polymer chain, the cation will link itself in some fashion to an
electron-rich site on the polymer chain.
Therefore, it is more challenging to analyze unknown polymer
samples by MALDI MS than unknown peptides and proteins samples
because of the sample preparation issues. Very often different MALDI MS
sample preparation methods (matrices, solvents, adding/removing
salts, etc.) are employed for different types of polymers. Here are
some of the examples from MALDI polymer analysis experiments.
[ application note ]
600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800m/z0
100
%
1027.58
969.54
911.50
853.44
795.40
737.38
677.29619.25
1085.64
1143.67
1201.72
2073.472015.41
1259.78 2014.411898.31
1840.271317.83
1783.20
1375.86 1725.18
1724.14
2189.54
2247.60
2305.64
2363.70
2421.73
2479.82
2537.86
2538.862653.93
2711.02
600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800m/z0
100
%
600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800m/z0
100
%
1027.58
969.54
911.50
853.44
795.40
737.38
677.29619.25
1085.64
1143.67
1201.72
2073.472015.41
1259.78 2014.411898.31
1840.271317.83
1783.20
1375.86 1725.18
1724.14
2189.54
2247.60
2305.64
2363.70
2421.73
2479.82
2537.86
2538.862653.93
2711.02
Figure 5. Polystyrene 19K (5 mg/mL in THF) MALDI micro MX spec-trum. Dithranol (50 mg/mL) was used as the matrix. AgTFA (5 mg/mL) was added to the matrix and sample. Data was acquired in linear mode. All major peaks are silver cationized peaks.
Figure 4. Polypropylene glycol 1000 and 2000 (1 mg/mL) mixture MALDI micro MX spectrum. DHB (10 mg/mL) was used as the matrix. Data was acquired in reflectron mode. All major peaks are sodiated peaks.
Figures 2 and 3. Polymerix (Sierra Analytics) is used for molecular weight distribution measurement for Polystyrene 2000.
10000 12000 14000 16000 18000 20000 22000 24000 26000 28000
%
0
100 18953.818643.418538.718434.2
18330.4
18227.8
18123.4
18019.4
17915.6
17811.0
17604.2
17499.7
17395.5
17292.1
17187.7
16979.7
19474.9
19577.7
19682.1
19786.3
19889.2
19993.9
20201.0
20305.3
20512.3
20617.0
20720.3
20823.4
20928.5
21032.2
21135.7
10000 12000 14000 16000 18000 20000 22000 24000 26000 28000
%
0
100
m/z10000 12000 14000 16000 18000 20000 22000 24000 26000 28000
%
0
100 18953.818643.418538.718434.2
18330.4
18227.8
18123.4
18019.4
17915.6
17811.0
17604.2
17499.7
17395.5
17292.1
17187.7
16979.7
19474.9
19577.7
19682.1
19786.3
19889.2
19993.9
20201.0
20305.3
20512.3
20617.0
20720.3
20823.4
20928.5
21032.2
21135.7
[ application note ]
GPC MALDI polymer analysis
Although MALDI MS has been used widely to provide molecular
weight and structural and compositional information of synthetic
polymers, one limitation of the technique is that it fails to provide
correct molecular-weight values for polydisperse polymers (polydis-
persity, Mw/Mn >1.2).
To overcome this limitation, the output of GPC can be coupled to the
MALDI micro MX system by collecting GPC fractions and perform-
ing MALDI analysis off-line. The average molecular weight of each
fraction is then determined, allowing calibration of the GPC curve
against absolute molecular weight. The calibrated GPC trace can then
be used to compute average molecular weight and molecular-weight
distribution of the unfractionated polymer samples.
Here is an example of GPC MALDI analysis for Poly-(DL-Lactide-
co-glycolide) (PLG). OLG polymers are biocompatible and biodegrad-
able polyesters used in drug delivery. Molecular weight data for
these polymers is of importance as it relates to performance char-
acteristics. PLG polymers are usually highly polydisperse (Pd >1.5),
thus present a challenge for MALDI analysis. Figures 7 and 8 show
MALDI data obtained before and after GPC (Waters Styragel® HR2 4.6
x 300 mm on a Waters 616 pump with 600S controller) separation.
m/z10000 12000 14000 16000 18000 20000 22000 24000 26000 28000
%
0
17571.616481.9
16283.3
16184.1
15389.5
15193.4
14895.8
14695.4
14398.1
14201.6
14195.4
19065.0
19458.3
19658.1
20348.4
20944.8
21248.3
21637.9
22929.7
m/z10000 12000 14000 16000 18000 20000 22000 24000 26000 28000
%
0 m/z10000 12000 14000 16000 18000 20000 22000 24000 26000 28000
0
100 17571.616481.9
16283.3
16184.1
15389.5
15193.4
14895.8
14695.4
14398.1
14201.6
14195.4
19065.0
19458.3
19658.1
20348.4
20944.8
21248.3
21637.9
22929.7
800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500 2600 2700m/z0
100
%
772.6464
830.5659
844.5173
888.4003
902.3625
920.3138
960.2321
976.1414
1062.04031135.7813
1193.66851480.9788 1727.4363
1000 2000 3000 4000 5000 6000 7000 8000 9000 10000m/z0
100
%
0
100
%
0
100
%
0
100
%
0
100
%
2010.21264584.06354238.5762
4093.07593805.06303777.0925
2466.8064
4785.46295607.9390
5795.4712
1349.4900
3834.21883271.67241364.0499
3241.77031571.73602766.4451
4325.4932
4482.85064801.1978
921.7535
929.77831130.4019
1146.4146
1355.93543241.77951579.7407 3618.0776
929.9463
2228.8044930.9546
2070.59421170.7274
2447.03642750.3337
3142.6060
1854.31261519.81802056.62622186.8171
2316.96702461.1458
Figure 8. Poly-(DL-Lactide-co-glycolide) (1 mg/mL) MALDI micro MX spectrum after GPC separation. Dithranol (10 mg/mL) was used as the matrix. Data was acquired in reflectron mode. All major peaks are sodiated peaks. The molecular weight distribution of the polymer sample can be calculated by combining the spectra.
Figure 7. Poly-(DL-Lactide-co-glycolide) (1 mg/mL) MALDI micro MX spectrum before GPC separation. Dithranol (10 mg/mL) was used as the matrix. Data was acquired in reflectron mode. All major peaks are sodiated peaks. The spectrum does not reflect the real molecular distribution of the polymer sample.
Figure 6. Polymethyl methacrylate 20K (1 mg/mL) MALDI micro MX spectrum. Dithranol (25 mg/mL) was used as the matrix. Data was acquired in linear mode. All major peaks are sodiated peaks.
[ application note ]
MALDI analysis of polyamidoamine (PAMAM) dendrimers
PAMAM dendrimers represent an exciting new class of macromo-
lecular architecture called “dense star” polymers. Unlike classical
polymers, dendrimers have a high degree of molecular uniformity,
narrow molecular weight distribution, specific size and shape char-
acteristics, and a highly-functionalized terminal surface. The
manufacturing process is a series of repetitive steps starting with
a central initiator core. Each subsequent growth step represents a
new generation of polymer with a larger molecular diameter, twice
the number of reactive surface sites, and approximately double the
molecular weight of the preceding generation.
First discovered in the early 1980s by Dr. Donald A. Tomalia2 at
Dow Chemical, these polymers were called dendrimers to describe
their tree-like branching structure. Figure 8 shows the polymerization
process of dendrimers.
Because of the structure, dendrimer samples tend to grasp as much
salt as possible from the surroundings; thus better results are obtained
by desalting the samples before mixing with the matrix. The common
matrix used for dendrimer samples is DHB (2,5-dihydroxybenzoic acid).
Figure 9 is a MALDI spectrum for polyamindoamine 3 (PAMAM3,
third generation). Fragmentations from the branches of the intact
dendrimer molecule are often observed in MALDI spectrum.
Post acceleration detector (PAD)
Higher molecular weight samples have less sensitivity in MALDI
experiments. This is especially true when the molecular weights
(distributions) of polymer samples are more than 50K Dalton.
Therefore, a post acceleration detector (PAD) is equipped for all MALDI
micro MX, to increase sensitivity for higher molecular weight analytes.
Figures 10 and 11 show two examples for polystyrene 190K and 410K,
using PAD on the MALDI micro MX for the improvement of sensitivity.
8000 9000 10000 11000 12000 13000 14000 15000 16000 17000 18000 19000m/z0
100
%
14159.6
14095.213687.1
13637.5
13578.3
10955.3
10892.4
10440.9
9520.8
9471.78673.3
8218.5 9009.4
10386.9
10327.2
12716.3
12257.2
12193.3
11756.6
12775.9
13177.9
14214.6
14277.4
14321.8
14787.315571.0 18274.717282.716794.615983.0 18014.1 18830.7
8000 9000 10000 11000 12000 13000 14000 15000 16000 17000 18000 19000m/z0
100
%
8000 9000 10000 11000 12000 13000 14000 15000 16000 17000 18000 19000m/z0
100
%
14159.6
14095.213687.1
13637.5
13578.3
10955.3
10892.4
10440.9
9520.8
9471.78673.3
8218.5 9009.4
10386.9
10327.2
14159.6
14095.213687.1
13637.5
13578.3
10955.3
10892.4
10440.9
9520.8
9471.78673.3
8218.5 9009.4
10386.9
10327.2
12716.3
12257.2
12193.3
11756.6
12775.9
13177.9
14214.6
14277.4
14321.8
14787.315571.0 18274.717282.716794.615983.0 18014.1 18830.7
m/z50000 75000 100000 125000 150000 175000 200000 225000 250000 275000 300000 325000 350000 375000 400000 425000 450000
%
0
100 x4188837
94962
6232846439
376294
m/z50000 75000 100000 125000 150000 175000 200000 225000 250000 275000 300000 325000 350000
m/z50000 75000 100000 125000 150000 175000 200000 225000 250000 275000 300000 325000 350000 375000 400000 425000 450000
%
0
100
375000 400000 425000 450000
%
0
100 x4188837
94962
6232846439
376294
Figure 10. Polystyrene 190K (5 mg/mL) MALDI micro MX spectrum. All-trans retinoic acid (75 mg/mL) was used as the matrix. AgTFA (5 mg/mL) was added to the sample. Data was acquired in linear mode with PAD on. All major peaks (including dimer at m/z of 380K and doubly charged peak at m/z of 95K) are silver cationized peaks.
Figure 9. Polyamidoamine 3 (PAMAM3) (1 mg/mL) dendrimer MALDI micro MX spectrum. DHB (10 mg/mL) was used as the matrix. Data was acquired in linear mode. All major peaks are sodiated peaks.
Figure 8. Polymerization of polyamidoamine (PAMAM) dendrimer.
[ application note ]
Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com
CONCLUSION
Excellent data acquired from the MALDI micro MX have been
shown for polar polymers such as polypropylene glycol; non-polar
polymer, such as polystyrene; poly-dispersed polymer, such as
poly-(DL-lactide-co-glycolide); dendrimer, such as polyamidoamine;
and higher mass polymers such as polystyrene 189K and 410K. This
application note has demonstrated that MALDI micro MX is an ideal
instrument for polymer analysis.
ACKNOWLEDGMENT
Thanks to Marten Snel (Waters MS Technologies Centre, Market
Development Proteomics, Manchester, UK) for constructive discus-
sions and Martin Palmer (Waters MS Technologies Centre, Validation,
Manchester, UK) for PMMA data.
REFERENCES
1. Karas, M., Hillenkamp, F. Anal. Chem. 60, 2299, 1988.
2. Dow Patent 4,289,872 (published 1981, filed 1979).
Waters and Styragel are registered trademarks of Waters Corporation. MALDI micro MX and The Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.
©2007 Waters Corporation. Produced in the U.S.A. February 2007 720002100EN
Figure 11. Polystyrene 410K (75 mg/mL) MALDI micro MX spectrum. All-trans retinoic acid (75 mg/mL) was used as the matrix. AgTFA (5 mg/mL) was added to the sample. Data was acquired in linear mode with PAD on. All major peaks (including doubly charged peak at m/z of 205K and triply charged peak at m/z of 137K) are silver cationized peaks.
m/z100000 150000 200000 250000 300000 350000 400000 450000 500000 550000 600000 650000 700000 750000 800000 850000
%
0
100 200111
132263
87796
171290
402081
260304
317722290826
337957570215524625
453457
660288
721234 802479 856602
m/z100000 150000 200000 250000 300000 350000 400000 450000 500000 550000 600000 650000 700000 750000 800000 850000
%
m/z100000 150000 200000 250000 300000 350000 400000 450000 500000 550000 600000 650000 700000 750000 800000 850000
%
0
100
0
100 200111
132263
87796
171290
402081
260304
317722290826
337957570215524625
453457
660288
721234 802479 856602
Waters MALDI micro MX.