[ 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.