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3 Applications of MALDI- TOF- MS to Synthetic Polymers

3 Applications of MALDI- TOF- MS to Synthetic Polymers

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Determinations of MM in relatively low MM polymer samples (up to a mass

limit of about 10,000 Da) have appeared in the last decades, by a number of

132

133,134

135

soft ionization MS techniques as static SIMS-TOF, LD-FTMS,

DCI,

2

1,2

8

FD, FAB, ESI. The ESI-TOF technique, which for proteins is capable of

providing high MM measurements, in the case of synthetic polymers yields

8

spectra only below 5000 Da, due to chain entanglement.

Before the introduction of MALDI, mass spectrometry was not a widely

applicable method for the determination of MM of synthetic polymers. Since

MALDI-TOF allows desorption and ionization of very large molecules, it is

now possible to perform the direct identification of mass-resolved polymer

chains and the measurement of MM in numerous high polymers. MM values

up to 1.5 million Daltons have been measured for PS monodisperse stan74

dards. MALDI-TOF is therefore unique for the MM and MMD estimation

in synthetic polymers by MS techniques.

Comparison of MALDI-TOF spectra of relatively low-mass polymers with

38,125,126

127

those obtained by other ionization techniques (ESI,

SIMS,125,127 FAB ),

yields MM estimates in general agreement. MALDI-TOF was originally devel3–5,12–15,27,31

oped for proteins (exactly monodisperse polymers),

and its extension to the characterization of synthetic polymers was not straightforward.

Contrary to the case of proteins, synthetic polymers may show a wide range

of molar mass distributions, according to the synthetic method used in their

preparation.

The determination of MM by SEC (which is an indirect method and needs

appropriate standards for the calibration of the SEC traces) is very popular,

and average values of MM and of MMD for all kinds of polymer samples

can be routinely extracted from SEC measurements (Chapter 2). Therefore

it was a logical choice to compare the MM values obtained from MALDI

with those from SEC.

In the MALDI process, ions strike the detector and produce a current that

is a function of the number of ions, so that the detector response is proportional to molar fractions. Therefore, in a mass spectrum the intensity of the

peaks is proportional to the molar amount of each species, and the masses

are displayed on a linear scale.

In contrast, the intensity of the SEC response is proportional to the weight

amount of each species, and the masses are displayed on a logarithmic scale

in SEC traces. The two traces are therefore not directly comparable, and this

has to be taken into account when handling data from the two tech113,114,128,131

niques.

Furthermore, SEC traces are continuous, i.e., the oligomers

contributing to the intensity of the detector signal are not mass resolved,

whereas MALDI-TOF spectra are typically mass-resolved up to about

50–60 kDa. At higher masses MALDI-TOF yields continuous traces, which

is analogous to SEC.

The difference between SEC and MALDI traces does not constitute a problem in computing average molar masses, because the average MM of polymers are calculated from a summation of the abundance of each oligomer,



©2002 CRC Press LLC



and the same equations apply both to the SEC and MALDI-TOF techniques

(Chapter 2).

The most serious problems encountered in achieving correct estimates of

the molar masses of polymers from their MALDI-TOF spectra do have an

instrumental origin. For the quantitative analysis of mass spectra of polymers,

the number of charged adducts revealed by the MS detector must reflect the

number of polymeric chains actually existing in the sample. This requires that

the ionization yield of the various oligomer species present in polymers must

be independent of chain size, and that the MALDI-TOF detector ought to show

a constant response to ions as the mass of the oligomer increases.

However, the detectors currently in use in commercial MALDI-TOF instru118

ments (see Section 10.2.6) do not meet this crucial condition. Mass discrimination effects at high masses are observed, and therefore MALDI spectra of

107

unfractionated polymer samples may not produce the correct MM estimate.

Average MM estimates by MALDI-TOF for narrow disperse polymers were

found to be in good agreement with conventional methods of MM determination,25,34,37,38,44,64,74,107,113–128,131,136–138 but several authors have reported that for

broad distribution polymers MALDI yields false MM values. In the initial

systematic attempt to explore the accuracy of MM estimates obtained with

MALDI-TOF, several polymethylmethacrylate (PMMA), polystyrene (PS),

polyethyleneglycol (PEG) samples with a varying degree of MMD were analyzed.107 Measurements of MALDI-TOF spectra in linear mode were used to

107

estimate the MM and MMD of these polymer samples. The results, reported

in Table 10.1, show that the molar mass estimates provided by MALDI-TOF

measurements agree with the values obtained by conventional techniques

(such as SEC), only in the case of polymer samples with very narrow MMD

(Mw /Mn < 1.10).

In the PMMA samples analyzed (see Table 10.1), when the polydispersion

index reaches values around 1.10 the Mp (SEC) and Mp (MALDI) values may

differ up to about 20%. At higher dispersions, MALDI spectra failed to yield

reliable MM values, and the MM measured were much lower than those

107

obtained by conventional methods.

Also a number of condensation polymers such as Nylon 6, polycarbonate,

and polyesters were studied (see Table 10.2). These polymers usually possess broad MM distributions, the value of the ratio Mw /Mn being usually

around 2. From the inspection of Table 10.2 it can be seen that MALDI

underestimates both Mw and Mn in the case of condensation polymers and

that the ratio Mw /Mn derived from MALDI spectra of polydisperse polymers

is strongly underestimated, and the MM distribution is much narrower. This

evidence indicates that lighter molecules are preferentially detected in

MALDI-TOF instruments, causing the underestimation of the presence of

larger molecules and limiting the use of MALDI for MM and MMD determinations to “monodisperse” samples.

A great part of the MALDI work published to date has been concerned with

low-mass polymers (<5–10 kDa), thus the value of the conclusions reached in



©2002 CRC Press LLC



TABLE 10.1

Molecular Weight Distribution Data for Polymethylmethacrylates (PMMA),

Polyethyleneglycols (PEG), and Polystyrenes (PS)

Sample



Mp(GPC)



PMMA2400

PMMA3100

PMMA4700

PMMA6540

PMMA9400

PMMA12700

PMMA17000

PMMA29400

PMMA48600

PMMA95000

e

PMMA_W1

PS5050

PS7000

PS9680

PS11600

PS22000

PS30300

PS52000

e

PS_W2

PEG4100

PEG7100

PEG8650

PEG12600

PEG23600



2400

3100

4700

6540

9400

12700

17000

29400

48600

95000

33000

5050

7000

9680

11600

22000

30300

52000

9000

4100

7100

8650

12600

23600



a

b

c



d



e



a



Mp(MALDI)

2100

2700

4200

5200

7500

10400

15000

27000

47000

90000

2200

5100

7020

9600

11300

20800

28000

46000

2000

3900

7420

8610

12790

23710



a



⌬%



b



12.5

12.9

10.6

20.5

20.2

18.1

11.8

8.2

2.1

5.3

94.0

1.0

0.3

0.8

2.6

5.5

7.6

11.5

77.0

4.9

4.3

0.5

1.5

0.1



Mw /Mn

c

(GPC)



Mw /Mn

d

(MALDI)



1.08

1.09

1.10

1.09

1.10

1.08

1.06

1.06

1.05

1.04

2.50

1.05

1.04

1.02

1.03

1.03

1.03

1.03

2.00

1.05

1.03

1.03

1.04

1.06



1.10

1.11

1.08

1.11

1.08

1.10

1.03

1.01

1.01

1.01

1.15

1.02

1.02

1.01

1.02

1.01

1.02

1.01

1.06

1.01

1.02

1.02

1.01

1.02



Most probable molecular weight.

Percent difference between Mp(GPC) and Mp(MALDI).

2

Calculated from the GPC curve using the formulas M n = (ΣmiNi)/(ΣNi), M w = (Σmi Ni)/

(ΣmiNi).

Calculated from the MALDI-TOF MS using the formulas M n = (ΣmiNi)/(ΣNi),

2

M w = (Σmi Ni)/(⌺miNi).

Synthesized by free-radical polymerization.

34,137



these studies on the determination of MM and MMD is somewhat limited.

However, MALDI-TOF studies dealing with the MM determination of higher

44,74,107,123,124,131

mass range polymers have appeared

confirming the above

results.

Great caution is therefore needed in estimating MM and MMD of unfractionated polymers by MALDI-TOF, with accurate estimates being limited to

107

nearly monodisperse polymer samples. Since the great majority of synthetic polymers show a marked polydispersion, this conclusion has raised

some concern about the general applicability of the MALDI-TOF method for

the determination of MM in polymers. This concern, in turn, has stimulated

several studies devised to test these results and to ascertain the reasons why

25,37,38,63,113–127,137

MALDI spectra have shown such a limitation.

©2002 CRC Press LLC



TABLE 10.2

Molecular Weight Distribution Data for Nylon 6, Polycarbonates, and Other Polymers

Sample

Polybutyleneadipate

Polycarbonate

Polycaprolactone

Nylon 6 diamino

Nylon 6 monoamino

Nylon 6 hydrolysis

Nylon 6 dicarboxyl

a

b

c

d

e

f

g



a



Mw



24200

7000

15000

11000

19000



Mw

b

(MALDI)



Mn

c

(NMR)



3020

7930

6740

2250

3080

2070

2770



4000

f

17000

f

10000

g

3000

g

6200

g

6800

g

6400



f



Mn

f

(MALDI)



⌬%



2090

6470

4690

2020

2320

1850

2120



47.8

61.9

53.1

48.5

62.6

72.8

66.7



d



e



Mw/ Mn



1.42



2.33

2.42

1.62

2.97



107



b



Mw/ Mn

1.45

1.23

1.44

1.11

1.32

1.12

1.30



Calculated from intrinsic viscosity using appropriate k and a values.

Calculated from the MALDI-TOF mass spectrum.

Calculated from proton NMR or from end-group analysis.

Percent difference between Mn(GPC) and Mn(MALDI).

Calculated using viscosity and NMR data.

Calculated from proton NMR.

Calculated from end-group analysis.



10.3.1.1 Polymers with a Narrow MM Distribution

Samples with a narrow MM distribution are obtained in various ways. The

139

most used method is anionic polymerization, and this synthetic route can

polymerize a large number of monomers. This includes styrene and substituted

styrenes (p-methyl, 2,4,6 trimethyl, p-methoxy, α-methyl), nitrogen-containing

compounds (vinylpiridine), acrylonitrile and substituted acrylonitriles

(methacrylonitrile), a large number of alkylmethacrylates, heterocylic monomers such as ethylene oxide, propylene oxide, isobutylene oxide, ethylene

sulfide and substituted ethylene sulfides, heterocylic dimers as glycolide and

lactide, propiolactone and higher lactones, hexamethylcyclotrisiloxane and

octamethylcyclotetrasiloxane along with dienes (butadiene, isoprene, piperylene, phenylbutadiene).

The theory predicts that the MM distribution of samples obtained by

anionic polymerization follows the Poisson distribution, which is extremely

narrow, as discussed in Chapter 2 of this book. Monodisperse polyelectrolytes such as poly(styrenesulfonic acid), poly(styrenecarboxlic acid), and

poly(acrylic acid) can be synthesized from samples polymerized by anionic

139

polymerization. Fractionation also affords samples with a narrow MM

distribution, but it requires substantial quantities of solvents and therefore

140

it is less frequently adopted for samples that do not dissolve in water.

Controlled radical (or “living”) polymerization is a relatively new field

which is rapidly developing. Samples obtained by this technique display a

narrow MM distribution with a polydispersion index lower than 1.2, and

139

often lower than 1.1.

A large number of authors have recorded MALDI-TOF mass spectra of

samples obtained by anionic polymerization and compared the values for



©2002 CRC Press LLC



200

180



M+

156970



160



counts/1000



140

120

100



M++



Membrane Osmometry Mn= 153,600

Viscometry

Mv= 159770

Light Scattering

Mw=15818

(SEC)

Mn= 152,350

Mw= 156050

MALDI

Mn = 152350

Mw= 156450



79160



80

60

40



2M+

312530



20

0



m/z

FIGURE 10.2

MALDI-TOF mass spectrum of a polystyrene sample.



Mn and Mw obtained by MALDI with the values obtained by osmometry,

viscometry, light scattering, and SEC. In general there is good agreement with

MALDI-TOF MM estimates, due to the fact that mass discrimination effects

are negligible and instrumental limitations become less severe. Figure 10.2

shows the MALDI-TOF mass spectrum of a polystyrene sample obtained by

anionic polymerization (initiator n-butyl lithium, termination by methanol).117

The peak due to singly charged ions is at 157 kDa, whereas the peak due

to doubly charged ions is at 79 kDa and the peak due to dimeric ions is at

312 kDa. The figure also reports the values for Mn and Mw obtained by

conventional methods. It can be seen that the values for Mn and Mw obtained

by MALDI agree with the latter.

Figure 10.3a shows the MALDI-TOF mass spectrum of a PMMA sample

obtained by anionic polymerization (initiator cumyl lithium, termination by

methanol).107 It can be seen that the peak due to singly charged ions is at 48 kDa

and that the peak due to dimeric ions is at 94 kDa. The figure also gives the

values for Mn and Mw obtained by viscometry and SEC, which agree with

the values obtained by MALDI.

Figure 10.3b reports the MALDI-TOF mass spectrum of a PMMA sample

107

obtained with the same initiator, but using a higher [M]/[I] ratio. It can

be seen that the spectrum is mass-resolved and that the values for Mn and

Mw , namely Mn = 8600 and Mw = 9600, are in excellent agreement with the

values obtained by viscometry, light scattering, and SEC.

Figure 10.4 reports the MALDI-TOF mass spectrum of a PEG sample

obtained by ring-opening polymerization (initiator potassium ethanolate, ter141

mination by ethanol). The comparison between the values for Mn and Mw

©2002 CRC Press LLC



(a)



counts



1500



Mv=47400



SEC



Mn=47000

Mw=49000



MALDI



Mn=48000

Mw=50000



1000



500



0



30000



60000



m/z



4000



counts



Viscometry



3000



120000

(b)



Viscometry



Mv=8600



Light Scattering

SEC



Mw=9000

Mn=8200

Mw=8900



MALDI



2000



90000



Mn=8600

Mw=9200



1000

0



4000



8000



12000



m/z



16000



20000



FIGURE 10.3

MALDI-TOF mass spectrum of PMMA that possesses a narrow MMD: (a) sample at 49 kDa,

(b) sample at 9.4 kDa.



obtained by MALDI, namely Mn = 7000 and Mw = 7200, and the values

obtained by osmometry, viscometry, light scattering, and SEC is satisfactory.

Lactones can be polymerized by ring-opening polymerization. MALDITOF spectra of anionic poly(lactic acid) (PLA) display negligible mass discrimination effects. Values of MM measured by MALDI turn out to be in

142

good agreement with those obtained by conventional methods.

Polybutadiene (PB) and polyisoprene (PI) samples obtained by anionic

polymerization are employed as primary MM standard for SEC calibration.

143

Yalcin et al. has recorded MALDI-TOF spectra of anionic PB and PI. As an

example, Figure 10.5 shows the MALDI-TOF mass spectrum of a polyisoprene sample with Mn = 19300. Yalcin et al. also analyzed PB6000, PI10500,

and PB22000, and compared the MALDI method with conventional methods

143

for measuring Mn and Mw values, finding a reasonable agreement.

©2002 CRC Press LLC



FIGURE 10.4

MALDI-TOF mass spectrum of PEG7100.



FIGURE 10.5

MALDI-TOF mass spectrum of polyisoprene 19300. (Adaped by permission from Ref. 143)



©2002 CRC Press LLC



Light Scattering Mw=50000

MALDI Mw=50000



Light Scattering Mw=94000

MALDI

Mw=90000



49589.8



counts



89442.5



25517.4



30000



60000



m/z



90000



120000



FIGURE 10.6

MALDI-TOF mass spectrum of SEC fractions of dextran. (Reprinted with permission from Ref.

140, Copyright 1995 John Wiley & Sons Ltd)



Garozzo et al. injected in a preparative SEC apparatus a polydisperse

polymer (dextran) and analyzed the collected fractions by light scattering

140

and by MALDI-TOF. Figure 10.6 shows the MALDI-TOF mass spectrum

of two dextran samples obtained by this method. The analysis of the spectra

yielded Mw = 50000 for the sample on the left and Mw = 90000 for the sample

on the right, and these values compare well with the light scattering values

(Mw = 50000; Mw = 94000).

Figure 10.7 shows the MALDI-TOF mass spectrum of a sample obtained by

144

ring-opening polymerization of a disubstituted cyclopropane. Figure 10.7

also reports the values for Mn and Mw obtained by various conventional techniques, among which is NMR (NMR gives reliable results in the case of low

MM polymers, as the present one) and the analysis of the MALDI spectrum.

Block copolymers produced by anionic polymerization do possess a very

narrow MM distribution (Chapter 2). Wilczek-Vera et al. recorded the MALDITOF mass spectrum of a block copolymer containing units of α-methylstyrene

145

and units of styrene. MALDI peak intensities were inserted in the equations

which define Mn and Mw and the result was Mn = 4273, Mw = 4411, in fair

agreement with the molar mass averages obtained by SEC (Mn = 4190, Mw =

145

4510).

Figure 10.8 reports the MALDI-TOF mass spectrum of a St-MMA block

146

copolymer. The peak due to singly charged ions is at 27000 Da, whereas the

peak due to dimeric ions is at 54000 Da. The SEC analysis yielded Mn = 26000,



©2002 CRC Press LLC



FIGURE 10.7

MALDI-TOF mass spectrum of a polymer obtained from a disubstituted cyclopropane. (Reprinted with permission from Ref. 144, Copyright 2000 American Chemical Society)

100

90



27000



80



PMMA-block-PS

SEC Mn=26000 Mw=27500

MALDI Mw=26400, Mw=27300



counts/100



70

60

50

40



55000



30

20

10

0

0



30000



60000



90000



120000



m/z



FIGURE 10.8

MALDI-TOF mass spectrum of a PMMA-block-PS copolymer sample obtained by anionic

polymerization.

1



and the composition (determined by H-NMR) turned out to be almost equimo146

lar. This implies that the styrene and MMA blocks have both Mn = 13000.

It can be concluded that the MALDI method for measuring Mn and Mw

values in polymers and copolymers gives a good agreement with conventional methods.

©2002 CRC Press LLC



10.3.1.2 Polymers with a Broad MM Distribution

When the sample possesses a broad MM distribution, in order to calculate

the MM it is necessary to consider the intensities of peaks spanning over a

wide range of mass numbers. This constitutes a serious problem in MALDITOF MS, since mass discrimination is likely to occur.

Before analyzing the effect of mass discrimination in the MM determination, it is interesting here to consider two special cases where MALDI-TOF

measurements of wide distribution polymers are useful not for estimating

the MM averages, but for detecting the MMD shape.

The first case occurs when a light-sensitive molecule initiates the polymerization process and the light source is pulsed, such as in the rotating sector

147

148

polymerization or in pulsed-laser polymerization (PLP). In this case, the

resulting polymer follows a peculiar MMD, since as the chain length (n)

increases, the molar fraction of chains of size n, denoted by I(n), grows and

147

falls. More specifically, it falls exponentially, then it grows, reaches a maximum, and then falls again. I(n) possesses a point of inflection, at mass Minf,

which is given by:

Minf = Φ[I(n)]



(10.3)



where Φ is an expression which involves the second derivative of I(n) with

respect to “n.” By plotting the oligomer’s abundance against its mass, and

taking the second derivative, the point at which the second derivative

changes from negative to positive is the point of inflection. Minf, is important

in assessing the rate constant of termination and the rate constant of chain

transfer.44, 147–152

Figure 10.9 reports the MALDI-TOF mass spectrum of a polymer obtained

by pulsed-laser polymerization that displays a series of peaks ranging from

148

3000 Da to 12000 Da. The MMD resulting from the MS was plotted, and

the point of inflection of the MMD (Eq. 10.3) was determined. The data allow

one to determine the rate constant of termination and the rate constant of

148

chain transfer. The method based on MS has now become a standard.

The second case occurs when the polymeric sample possesses a large fraction

of cyclic oligomers. The presence of cyclic oligomers is often noticed in polymers, although the abundance of cyclics decreases drastically as the mass

grows, and cyclic oligomers with n > 50 are very rare. Several MS techniques

153

have been used to detect cyclics in polymeric samples (Chapters 2 and 7).

MALDI can be used as well, since the mass range of cyclic oligomers is

relatively limited, and therefore mass discrimination is not severe. Figure 10.10

154

reports the MALDI-TOF mass spectrum of a polylactic acid (PLA) sample.

In the region 1300–2600 Da, peaks due to cyclics (°) are stronger than peaks

due to linear (×) oligomers, whereas in the region 2600–4000 Da peaks due to

cyclics are weaker. Remarkably, cyclic oligomers up to mass 4000 are indeed

seen, corresponding to chains with a size up to n = 70 and even higher, showing

154

an unusually extended range of PLA cyclics.

©2002 CRC Press LLC



0



FIGURE 10.9

MALDI-TOF mass spectrum of a PMMA obtained by pulsed laser polymerization. The arrow

indicates the position of the inflection point Minf. (Reprinted with permission from Ref. 11,

Copyright 1993 American Chemical Society)

o



2000



o



o



o



1600



o



o

o



1200



X



X



X



X



X



X



o

X



o



o



X



800



o



o

X



X



X



X



X



X



X



400

1000



1300



800



X



1400

X



X



1500

X



X



X



1600



X



1700



X



X



X



1800



X



X



1900



X



X



2000



X



X



2100



2200



2300



2400



X



600

400

2200



2400



2600



2800



3000



3200



3400



3600



3800



4000



FIGURE 10.10

MALDI-TOF mass spectrum of poly(lactic acid). In the region 1300–2600 peaks due to cyclic

are stronger than peaks due to linear, whereas in the region 2600–4000 peaks due to cyclic are

weaker.



10.3.2



Effect of Mass Discrimination in the MM Determination



All the quantitative applications of MS to polymers, regardless of the ionization method utilized to produce ions, are based on the assumption that there

is a quantitative correspondence between the relative abundance of the species to be analyzed and the relative intensity of MS peaks (see Chapter 2).

©2002 CRC Press LLC



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