RAPID COMMUNICATIONS IN MASS SPECTROMETRY
Rapid Commun. Mass Spectrom. 12, 519–528 (1998)
Application of Size Exclusion Chromatography
Matrix-assisted Laser Desorption/Ionization
Time-of-flight to the Determination of Molecular
Masses in Polydisperse Polymers
Maurizio S. Montaudo,1 Concetto Puglisi,1 Filippo Samperi1 and Giorgio Montaudo2*
1
Istituto per la Chimica e la Tecnologia dei Materiali Polimerici, Consiglio Nazionale delle Ricerche, Viale A. Doria, 6-95125
Catania, Italy
2
Dipartimento di Scienze Chimiche, Università di Catania, Viale A. Doria, 6-95125 Catania, Italy
The determination of molecular mass (MM) data for polydisperse polymers by size exclusion
chromatography matrix assisted laser desorption/ionization time-of-flight (SEC/MALDI-TOF) involves
the fractionation of samples through an analytical SEC. Selected fractions are then analysed by MALDITOF and the mass spectra of these nearly monodisperse samples allow the determination of Mn and Mw
averages. To test the reliability of the molecular mass estimates by the SEC/MALDI-TOF method, a sample
of polymethylmethacrylate (PMMA), two samples of polydimethylsiloxane (PDMS), and four samples of
copolyesters, all polydisperse, were analysed. The results show that the molecular mass values of PMMA
fractions obtained by MALDI-TOF are coincident with those obtained using the SEC calibration plots
obtained with anionic PMMA standards. In the case of the two polydimethylsiloxanes (PDMS1 and PDMS2:
linear and cyclic, respectively), two slightly differing SEC calibration plots were obtained, reflecting the
different structures of the polymer chains of the two samples. The SEC traces of four copolyesters were
obtained in tetrahydrofuran and CHCl3. Data on MM, MM distribution solvent effects and copolymer
composition are reported. # 1998 John Wiley & Sons, Ltd.
Received 9 February 1998; Revised 24 February 1998; Accepted 26 February 1998
Matrix-assisted laser desorption ionization-time of flight
mass spectrometry (MALDI-TOF), a high sensitivity
technique, allows desorption and ionization of very large
molecules, even in complex mixtures1. However, molecular
mass (MM) estimates provided by MALDI-TOF for
synthetic polymers agree with the values obtained by
conventional techniques only in the case of samples with
narrow molecular mass distribution (MMD).2
To overcome this problem, polydisperse polymers such
as dextrans,3 polyesters4 and polysiloxanes5 have been
fractionated by analytical size exclusion chromatography
(SEC), yielding fractions with very narrow distributions
which, analysed by MALDI-TOF, were found to give mass
spectra with MM values in excellent agreement with those
obtained by conventional techniques.4,5 These findings
opened the way to a new development, and MALDI-TOF is
today widely recognized as an excellent detector in the SEC
fractionation of polydisperse polymer samples.6–11 We have
now furthered our studies on this hyphenated SEC/MALDITOF method, applying it to characterize the molecular
properties of specific synthetic polymers and copolymers.
The MM determination in polydisperse polymer samples
by MALDI-TOF, consists4,5 of the analytical SEC fractio*Correspondence to: G. Montaudo, Dipartimento di Scienze Chimiche,
Università di Catania, Viale A. Doria, 6-95125 Catania, Italy.
Contract/grant sponsor: Italian Ministry for University and for
Scientific and Technological Research (MURST).
Contract/grant sponsor: National Council of Research (CNR, Rome).
Contract/grant sponsor: Progetto Finalizzato Materiali per Tecnologie
Avanzate II (CNR, Rome).
CCC 0951–4198/98/090519–10 $17.50
nation of polydisperse samples to collect numerous fractions per run. Typically, injecting 0.5mg of polymer in the
SEC system and collecting about 100 fractions, the amount
of sample present in each fraction (about 5 mg, on average)
exceeds many times the quantity needed for a MALDI-TOF
spectrum. Selected fractions are then analysed by MALDITOF and the mass spectra of these nearly monodisperse
samples allow the computation of reliable values of Mn and
Mw corresponding to the fractions.
In Fig. 1 is shown an illustrative example of a SEC trace,
together with some MALDI-TOF spectra taken at different
times. The calibrated SEC trace can then be used to compute
average MM and MMD of the unfractionated sample. We
have tested the reliability of the MM estimates thus
obtained, and explored some peculiar aspects of the
MALDI-TOF response to the phenomenon of molecular
association in polymers.
The analysis of copolymers by SEC is a difficult problem,
and much effort has been paid to the task of converting the
SEC traces to molecular masses of copolymers.12–14 The
SEC traces of five copolyesters (Table 1) were obtained in
THF and CHCl3. Data on MM, MMD, solvent effects and
copolymer composition are reported.
EXPERIMENTAL
Materials
Polymethylmethacrylate (PMMA) MW standards were
supplied by Polymer Lab. A polydisperse PMMA sample
# 1998 John Wiley & Sons, Ltd.
520
SEC/MALDI-TOF FOR POLYDISPERSE POLYMERS
Figure 1. SEC trace of PDMS1 sample in THF. The insets display the MALDI-TOF spectra of selected
fractions.
(PMMA_W1) was supplied by the Aldrich Chem. Co.
Polydimethylsiloxane (PDMS1) was a high molecular mass
sample supplied by the Aldrich Chem. Co. The PDMS2
sample was obtained from a PDMS1 sample equilibrated
with 0.2% (w/w) of NaOH at 250°C for two hours. The
reaction mixture was quenched with HCl (1M in methanol),
washed several times with fresh methanol and dried at 40°C
overnight. Dimethylsuccinate, dimethyladipate, dimethylsebacate and dimethylterephthalate were purchased from
Sigma-Aldrich (Milan Italy), whereas 1,4-butanediol was
purchased from Jansen Chimica. Dimethylterephthalate was
purified by crystallization from n-hexane, whereas the other
reagents were purified by vacuum distillation before use.
Copolyesters synthesis
Copolyesters were synthesized by melt polymerization
starting from stoichiometric amounts of dimethylesters
and 1,4-butanediol in the presence of a mixture of
Zn(COCH3)2 and Sb2O3 (80/20, w/w) as trans-esterification
catalyst. An equimolecular mixture of two, three or four
dimethylesters was reacted with 1,4-butanediol to obtain
Table 1. Structure and properties of the copolyesters analysed
Samplea
PBA/PBSe
PBSu/PBT
PBSu/PBSe
PBSu/PBA/PBSe
PBSu/PBA/PBSe/PBT
Solubility
Feedb
Compositionc
inhd
MSECe
Mwf
Mnf
Mw/Mn
THF, CHCl3
CHCl3
THF, CHCl3
THF, CHCl3
THF, CHCl3
50/50
50/50
50/50
33.3/33.3/33.3
25/25/25/25
47/53
49/51
45/55
33/30/37
23/26/24/27
0.20
0.25
0.19
0.17
0.23
13200
12000
15400
8400
17000
12800
10600
8600
15000
8300
8500
6300
5400
8300
5500
1.51
1.68
1.59
1.81
1.51
a
PBA=polybutyleneadipate, PBSe=polybutylenesebacate, PBSu=polybultylenesuccinate, PBT=polybutyleneterephthalate.
Molecular ratio of monomers in the feed. Polymerization reactions were led to completion.
Composition of the copolymers obtained by NMR analysis.
d
Inherent viscosity values were obtained in THF at 30 0.1°C, for PBSu/PBt sample was obtained in CHCl3.
e
Molecular mass computed using the universal calibration curve obtained in THF (see experimental).
f
Weight average and number average molecular masses computed using the SEC-MALDI method (see experimental).
b
c
Rapid Commun. Mass Spectrom. 12, 519–528 (1998)
# 1998 John Wiley & Sons, Ltd.
SEC/MALDI-TOF FOR POLYDISPERSE POLYMERS
521
Table 2. Average molecular masses and molecular mass distribution determined from the analysis of the MALDI-TOF mass spectra of the
GPC fractions of samples; PBSu/PBSe, PBA/PBSe and PBSu/PBA/PBSe, using THF as a solvent.
Fraction
Ve a
Mpb
Mnc
Mwd
Mw/Mn
PBSu/PBSe
18
16
14
12
10
8
7
5
42.0
40.5
39.1
37.6
36.2
34.8
34.0
32.6
1900
2800
4400
6100
8900
14000
19900
31000
2050
2900
4500
6200
9000
14100
20200
31200
2200
3100
4800
6500
9300
14400
20900
31700
1.07
1.07
1.07
1.05
1.03
1.02
1.03
1.02
PBA/PBSe
20
16
15
12
10
9
7
6
5
4
43.4
40.5
39.8
37.6
36.2
35.5
34.0
33.3
32.6
31.9
1600
3700
4200
8200
11500
14300
19600
23800
29100
35500
1600
3600
4100
8100
11200
14000
19500
24100
29000
35200
1700
3700
4300
8600
11900
14700
20100
24500
29600
35900
1.06
1.06
1.05
1.06
1.06
1.05
1.03
1.02
1.02
1.02
PBSu/PBA/PBSe
19
16
14
12
8
5
3
39.7
38.4
37.4
35.5
34.7
33.3
32.4
1800
2800
3500
5500
10300
15000
21000
2000
2700
3500
5700
10400
14900
21200
2100
2800
3700
5900
10800
15000
21600
1.05
1.04
1.06
1.03
1.04
1.01
1.02
Sample
a
Elution volume at which the fraction was collected.
Most probable molecular weight.
Number average molecular weight.
d
Weight average molecular weight.
b
c
two, three and four component copolymers. The reaction
was carried out at 180°C for 2 hours and then for five hours
at 230°C under reduced pressure (1.5 torr) to eliminate the
methanol formed in the reaction. In the following, the
synthesis of poly(butylenadipate-co-butylenesebacate) is
described as an example. 1.71 g (0.0088 mol) of dimethyladipate were placed in a flask together with 2.024 g (0.0088
mol) of dimethylsebacate, with 1.58 g. (0.0175 mol) of 1,4butanediol and with 3.95 mg (0.25% of the diol weight) of
catalyst. The temperature of the mixture was gradually
raised to 180°C and kept at this value for two hours, with
stirring. Thereafter, the pressure was reduced to 1.5 torr and
the temperature was gradually increased up to 230°C and
kept at this value for five hours. To remove the residual
catalyst from the reaction mixture, the crude homopolymers
and copolymers were dissolved in the minimum amount of
CHCl3, filtered and precipitated into methanol. The solid
materials were filtered washed several times with methanol,
dried at 50°C under vacuum and characterized by
viscosimetry, SEC, NMR and MS.
SEC fractionation
The SEC analyses were performed using a Waters 6000A
apparatus equipped with four m-Styragel columns (in the
order 1000, 500, 10 000 and 100 Å pore size) connected in
series, using a Waters R401 differential refractometer.
60 mL of polymer solution (15 mg/mL) were injected and
eluted at a flow rate of 1 mL/min. 50 fractions of 0.24 mL
were collected for PMMA_W1 and PDMS2, whereas 81
fractions were collected for PDMS1 sample (1–61, 0.10 mL;
# 1998 John Wiley & Sons, Ltd.
62–81, 0.30 mL), using THF as solvent. The fractionation of
copolyesters was performed by collecting 20 drops for each
fraction corresponding to 0.26 mL for CHCl3 and 0.46 mL
for THF. For each sample 50 fractions were collected.
MALDI-TOF sample preparation
Indole acrylic acid was used as a matrix for the PMMA
sample. 2-(4-Hydroxyphenylazo)-benzoic acid (HABA)
and 2,4-dihydroxybenzoic acid (DHB) (0.5M THF solution)
were used as matrix for polydimethylsiloxane samples.
HABA (0.1 M in THF/CHCl3, 1:1 mixture) was used as a
matrix for copolyester samples. Samples for MALDI-TOF
analysis were prepared as follows: 0.2 mL of matrix solution
were mixed with about 0.1 mL of each collected fraction,
and 2 mL of the resulting solution were placed on the probe
tip and slowly dried.
MALDI-TOF mass spectra
A Bruker Reflex mass spectrometer was used to obtain the
MALDI-TOF mass spectra of PMMA, and PDMS1 and
PDMS2 samples. The spectrometer is equipped with a
Nitrogen laser (337 nm, 5 ns, a flash analogue-to-digital
converter (time base 4 ns), and with two detectors. The first
detector works in the linear mode, whereas the second
detector is placed at the end of the second flight tube, and
allows the detection of ions in the reflectron mode. The
detection in linear mode was achieved by means of the
HIMAS detector, which provides a very wide dynamic
range, but with a low temporal resolution. This detector is
Rapid Commun. Mass Spectrom. Vol. 12, 519–528 (1998)
522
SEC/MALDI-TOF FOR POLYDISPERSE POLYMERS
very sensitive to high molecular mass ions ( 500 000 Da)
compared to conventional microchannel detectors which
show a sharp drop in response for ions with molecular mass
higher than 15 000 daltons.
The accelerating voltage was 30 kV. The laser irradiance
was slightly above threshold (ca. 106 W/cm2). Ions below
m/z 350 were removed with pulsed deflection, and 100
transients were summed.
The MALDI-TOF mass spectra of the SEC fractions were
processed with the XMASS program from Bruker. The
program uses mass spectral intensities to compute the
quantities known as most-probable molecular mass, number-average molecular mass, weight-average molecular
mass and polydispersity index (denoted as Mp, Mn, Mw
and Mw/Mn, respectively). Data obtained for some selected
PDMS fractions are reported in Table 5.
The MALDI-TOF mass spectra of the five copolyesters in
Table 2 were acquired using a Perseptive Voyager-DE
MALDI-TOF mass spectrometer equipped with delayed
extraction, using an accelerating voltage of 23KV, a grid
voltage from 94 up to 95%, a delay time of 1 000 ns and a
laser intensity 60–70 % of maximum which corresponds to
the minimum intensity to observe the mass spectrum.
Molecular mass calculations
The molecular mass of the unfractionated polymer and
copolymer samples were calculated from the SEC curves by
the Polymer Lab Caliber software using the absolute
calibration curves obtained by plotting log Mw (calculated
from the MALDI-TOF spectra) as a function of the elution
volume of each SEC selected fraction. The molecular mass
distribution data obtained for copolyesters are reported in
Table 1.
The molecular mass of the unfractionated copolyester
samples were also calculated with the universal calibration
curve obtained by using a set of 12 PMMA and 14
polystyrene well-characterized samples (purchased from
Polymer Lab) each having a narrow molecular mass
distribution (Mw/Mn < 1.1). We measured the viscosity
() and the elution volume at which the SEC trace shows its
maximum (Ve), and plotted the log(M) versus Ve. The
resulting plot was the universal calibration line for our set of
SEC columns, described by the following equation:
log…M† ˆ 0:016…V e †2 ÿ 1:76V e ‡ 22:7
Molecular mass data (Msec, Table 1) were calculated
from this equation using the measured viscosity of
unfractionated copolymer samples and the elution volume
at the maximum of the SEC curves.
NMR analysis
NMR analyses were performed using a Brucker A-CF 200
spectrometer at room temperature, using deuterated chloroform as solvent and tetramethylsilane as internal standard.
Figure 2. SEC calibration plots of the PMMA samples: & Mw of the
PMMA SEC standards as indicated by the supplier, * Mw of the
PMMA SEC standards obtained by MALDI-TOF spectra, * Mw of the
PMMA_W1 SEC fractions obtained by MALDI-TOF spectra.
RESULTS AND DISCUSSION
Accuracy of SEC/MALDI-TOF
In a set of experiments, we analysed by SEC/MALDI-TOF a
PMMA (PMMA_W1) sample synthesized by free radical
polymerization, with a polydispersity index of 2.5. The
MALDI-TOF mass spectrum of this sample displays a most
probable molecular mass of 2200, which falls well below
the correct value (33 000).
The PMMA_W1 sample was then injected into the SEC
apparatus, and about 50 fractions were collected from the
eluate. These fractions were analysed by MALDI-TOF and
yielded excellent spectra with narrow distributions, up to
high molecular masses, from which the corresponding Mw
could be calculated. The log Mw values of the PMMA_W1
fractions show a linear correlation with the elution volume
of each fraction, and allowed the calibration of the SEC
trace against MM (Fig. 2). The computed MM averages of
PMMA_W1 sample are Mn = 12 000; Mw = 33 000, which
compare well with the values given by the manufacturer:
Mn = 13 000 and Mw = 33 000.
In order to test the accuracy of Mw estimates obtained by
SEC/MALDI-TOF, several well characterized anionic
PMMA samples (SEC standards) were injected in the SEC
apparatus, and the elution volumes (Ve) at the SEC
maximum were plotted against the MM values as shown
in Fig. 2. This independent set of data is nearly coincident
with the SEC calibration curve obtained from the SEC
fractionation of sample PMMA_W1, lending further credibility to the test of accuracy of the hyphenated method
proposed. Data in Fig. 2 show also a reasonable agreement
between the MM values estimated from MALDI-TOF mass
spectra and those determined by conventional techniques
for the anionic PMMA standards.
Aliphatic copolyesters
Viscometry
Inherent viscosities (inh = lnr/C; C = 0.5g/dL) were
measured in a Desreux–Bishoff suspended-level viscometer
at 30 1°C. The solvent was toluene for PDMS samples,
and THF for PMMA. Viscosity values for polydimethylsiloxane were: PDMS1 = 0.22; PDMS2 = 0.12. Copolyester
data are reported in Table 1.
Rapid Commun. Mass Spectrom. 12, 519–528 (1998)
In Table 1 are listed the structure and properties of the
copolyesters studied, which contain butyleneadipate (BA),
butylenesuccinate (BSu), butylenesebacate (BSe) and
butyleneterephthalate (BT) units. These copolyesters have
flexible chains and possess a homogeneous composition,
since they were synthesized by condensation polymerization. The synthesis was performed starting from a mixture of
# 1998 John Wiley & Sons, Ltd.
SEC/MALDI-TOF FOR POLYDISPERSE POLYMERS
523
Table 3. Average molecular masses and molecular mass distribution determined from the analysis of the MALDI-TOF mass spectra of the
GPC fractions of samples: PBSu /PBT; PBA/PBSe and PBSu/PBA/PBSe/PBT, using CHCl3 as a solvent.
Fraction
Ve a
Mpb
Mnc
Mwd
Mw/Mn
PBSu/PBT
32
30
28
26
24
22
20
18
36.6
35.9
35.2
34.6
33.9
33.2
32.6
31.9
2400
3100
4500
6100
7900
11100
13900
19000
2300
3100
4500
6000
8000
11000
13600
18500
2400
3200
4700
6400
8300
11500
14100
19400
1.04
1.03
1.04
1.07
1.04
1.04
1.04
1.05
PBA/PBSe
28
26
24
22
20
18
16
14
12
10
8
6
4
2
35.4
34.9
34.4
33.8
33.3
32.8
32.2
31.7
31.2
30.7
30.1
29.6
29.1
28.5
2500
2900
3400
4000
4800
5800
7000
8400
10300
12200
14700
17800
22000
27500
3000
3350
3950
4600
5150
5900
7200
8400
10300
12200
14700
17800
22000
27500
3250
3500
4150
4800
5300
6050
7300
8700
10600
12500
15100
18300
23400
28200
1.08
1.05
1.05
1.04
1.03
1.02
1.02
1.04
1.02
1.02
1.02
1.02
1.02
1.02
PBSu/PBA/PBSe/PBT
29
27
25
23
21
18
16
14
35.6
34.9
34.2
33.6
32.9
31.9
31.3
30.6
3500
4800
6200
8200
11800
18000
23600
32500
3200
4800
6800
8500
11800
18500
24000
32900
3300
4900
7100
8800
12200
19000
24500
33600
1.03
1.02
1.04
1.04
1.03
1.03
1.02
1.02
Sample
a
Elution volume at which the fraction was collected
Most probable molecular weight
c
Number average molecular weight
d
Weight average molecular weight.
b
the methyl esters, producing random copolymers. All the
polymerization reactions were conducted to completion,
and therefore the copolyester compositions are given by the
ratio of monomers in the feed, as checked by NMR analysis.
As reported in Table 1, all copolyesters are soluble in THF
and/or in CHCl3, and SEC fractionation was performed in
Figure 3. Calibration lines for SEC traces of: (*) PBA\PBSe, (D)
PBSu\PBT, (&) PBSu\PBA\PBSe\PBT in CHCl3; (*) PBA\PBSe,
(&) PBSe\PBSu, (!) PBSu\PBA\PBSe in THF.
# 1998 John Wiley & Sons, Ltd.
these solvents. The fractions collected were then used for
the MALDI-TOF analysis.
In Tables 2 and 3 are reported the average molecular
masses and molecular mass distributions obtained by the
analysis of the MALDI-TOF spectra of selected SEC
fractions of each copolymer. These data were used to build
absolute calibration plots (log M versus Ve) corresponding
to the polymers investigated, as reported in Fig. 3. From the
inspection of Fig. 3 it can be seen that the calibration lines in
chloroform are shifted by 3–4 mL with respect to the
calibration lines in THF. These calibration lines have been
used to calibrate the SEC Ve values against absolute
molecular masses, and to compute average molecular
masses and molecular mass distributions. The results of
these calculations (Mw, Mn and MMD=Mw/Mn) are reported
in Table 1, together with the estimates computed from the
SEC universal calibration curve for the weight average
molecular mass. As expected, the universal calibration
method provides only rough estimates of the weight average
molecular mass, with errors that can amount to a factor of
two (Table 1).
Average Mn values for the copolymers in Table 1 indicate
conversions in the range of 0.97, and therefore the
dispersions (Mn/Mw) measured (1.5–1.8) appear lower than
expected. This observation could be due to purification
procedures that strip the low masses from the crude sample.
Rapid Commun. Mass Spectrom. Vol. 12, 519–528 (1998)
524
SEC/MALDI-TOF FOR POLYDISPERSE POLYMERS
Figure 4. MALDI-TOF mass spectrum of the fraction 26 of copolymer PBA\PBSe.
Figure 4 reports the MALDI-TOF mass spectrum of
fraction 26 of copolymer PBA\PBSe. It displays a large
number of peaks which belong to the same mass series.
They have been assigned to open chain oligomers
terminated by methylester on both ends, which are
cationized with a lithium ion. The detailed peak assignments
and intensities are reported in Table 4. The most intense
peaks are in the region 2000–5000. The MM calculation for
this fraction (see Table 3) gives Mn = 3350 and Mw = 3500.
Figure 5. SEC calibration plots for PDMS. MALDI-TOF molecular
masses versus elution volume (Ve) of each SEC fractions. *PDMS1;
!PDMS2.
Rapid Commun. Mass Spectrom. 12, 519–528 (1998)
Figure 6. MALDI-TOF mass spectra, obtained in linear mode, of four
selected PDMS1a fractions: (a) fraction 26, (b) fraction 17, (c) fraction
8, (d) fraction 6.
# 1998 John Wiley & Sons, Ltd.
SEC/MALDI-TOF FOR POLYDISPERSE POLYMERS
Table 4. Experimental and calculated relative amounts of
PBA\PBSe lithiated oligomersa observed in the MALDI
spectrum of fraction 26.
Oligomerb
10-mers
A7B3
A6B4
A5B5
A4B6
A3B7
A2B8
A1B9
B10
11-mers
A8B3
A7B4
A6B5
A5B6
A4B7
A3B8
A2B9
A1B10
12-mers
A9B3
A8B4
A7B5
A6B6
A5B7
A4B8
A3B9
A2B10
13-mers
A10B3
A9B4
A8B5
A7B6
A6B7
A5B8
A4B9
A3B10
A2B11
14-mers
A11B3
A10B4
A9B5
A8B6
A7B7
A6B8
A5B9
A4B10
A3B11
15-mers
A12B3
A11B4
A10B5
A9B6
A8B7
A7B8
A6B9
A5B10
A4B11
16-mers
A12B4
A11B5
A10B6
A9B7
A8B8
A7B9
A6B10
A5B11
A4B12
m/zc
Iexped
Icalce
2407
2463
2519
2575
2631
2687
2743
2799
290
570
780
810
500
240
50
10
398
653
766
642
376
147
34
3
2607
2663
2719
2775
2831
2887
2943
2999
330
710
1010
1020
770
430
160
40
377
709
970
975
715
372
131
27
2807
2863
2919
2975
3031
3087
3143
3199
280
620
940
1090
930
620
280
100
273
576
901
1057
930
606
284
91
3007
3063
3119
3175
3231
3287
3343
3399
3455
180
410
740
960
1010
720
410
160
40
167
393
692
927
952
744
436
186
54
3207
3263
3319
3375
3431
3487
3543
3599
3655
90
250
490
800
880
820
540
300
110
97
250
490
739
867
791
557
297
116
3407
3463
3519
3575
3631
3687
3743
3799
3855
30
120
380
630
760
760
580
390
140
52
148
319
536
707
737
605
387
189
3664
3720
3776
3832
3888
3944
4000
4056
4112
30
210
400
620
640
610
440
240
100
84
197
364
533
626
587
438
257
116
# 1998 John Wiley & Sons, Ltd.
Oligomerb
17-mers
A13B4
A12B5
A11B6
A10B7
A9B8
A8B9
A7B10
A6B11
A5B12
18-mers
A12B6
A11B7
A10B8
A9B9
A8B10
A7B11
A6B12
A5B13
19-mers
A13B6
A12B7
A11B8
A10B9
A9B10
A8B11
A7B12
A6B13
A5B14
20-mers
A14B6
A13B7
A12B8
A11B9
A10B10
A9B11
A8B12
A7B13
A6B14
21-mers
A15B6
A14B7
A13B8
A12B9
A11B10
A10B11
A9B12
A8B13
A7B14
A6B15
22-mers
A16B6
A15B7
A14B8
A13B9
A12B10
A11B11
A10B12
A9B13
A8B14
A7B15
525
m/zc
Iexped
Icalce
3864
3920
3976
4032
4088
4144
4200
4256
4312
10
120
270
430
560
660
460
260
140
47
120
242
291
509
538
459
314
170
4176
4232
4288
4344
4400
4456
4512
4568
160
310
460
590
460
330
200
80
157
276
396
465
446
349
220
110
4376
4432
4488
4544
4600
4656
4712
4768
4824
130
220
360
450
480
430
250
130
50
107
204
320
413
440
387
279
164
77
4576
4632
4688
4744
4800
4856
4912
4968
5024
40
150
250
400
420
400
280
170
80
64
133
226
318
373
364
296
198
108
4777
4833
4889
4945
5001
5057
5113
5169
5225
5281
10
80
170
290
320
360
290
210
130
60
38
84
154
235
301
323
292
220
137
70
4977
5033
5089
5145
5201
5257
5313
5369
5425
5481
10
40
100
180
260
300
270
240
160
90
21
51
100
164
227
266
264
221
155
91
AF = 9%f
The lithiated molecular ions of the oligomers observed correspond to the
follow structure:
H3 COÿCO…CH2 †4 COO…CH2 †4 O ÿ
ÿmÿ
ÿ CO…CH2 †8 COO…CH2 †4 O ÿ
ÿmÿ OCH3 ::::Li‡
a
b
A = butylene adipate, B = butylene sebacate; c Observed m/z values after
calibration procedure17;d Intensity of the MLi‡ ions in the MALDI-TOF
spectrum; e Intensities calculated using MACO4 program15; f AF = agreement
factor between experimental and calculated MALDI-TOF spectrum.15
Rapid Commun. Mass Spectrom. Vol. 12, 519–528 (1998)
526
SEC/MALDI-TOF FOR POLYDISPERSE POLYMERS
Table 5. Molecular mass distribution data of the SEC fraction of commercial and equilibrated PDMS, obtained by the analysis of MALDITOF mass spectra.
Mpa
Fractions
Mnb
Mwc
Mw/Mn
VE d
300000
274000
227000
206000
157000
151000
134000
106500
81500
54000
45000
18000
9800
5800
1.01
1.03
1.02
1.04
1.05
1.06
1.05
1.06
1.06
1.06
1.05
1.09
1.09
1.11
20.71
21.01
21.32
21.72
22.12
22.53
22.73
23.03
23.64
24.24
24.64
26.88
28.10
29.63
213000
176000
127000
89600
92200
57600
48800
37500
31500
26600
22500
18800
14800
12100
10900
8500
7230
5830
4880
4100
3150
1.019
1.023
1.024
1.016
1.055
1.014
1.012
1.008
1.016
1.019
1.023
1.033
1.035
1.025
1.068
1.006
1.018
1.012
1.017
1.025
1.016
22.22
22.70
23.18
23.66
23.90
24.38
24.86
25.34
25.82
26.30
26.78
27.26
27.74
28.22
28.70
29.18
29.66
30.14
30.62
31.10
31.58
PDMS1
15
18
21
25
29
33
35
38
44
50
54
66
70
75
300000
275000
230000
190000
155000
140000
128000
100000
78000
49000
42000
16000
6300
3300
296000
267000
222000
198500
150000
142000
127500
100500
77000
51000
43000
16500
9000
5200
3
5
7
9
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
42
211000
170000
122000
87000
90000
56000
47000
35000
30000
26500
22000
17600
15000
11500
9000
8300
7100
5700
5000
4400
3100
209000
172000
124000
88200
87400
56800
48200
37200
31000
26100
22000
18200
14300
11800
10200
8450
7100
5760
4800
4000
3100
PDMS2
a
most probable molecular mass
Mn=SniMi/Sni
Mw=SniMi2/SniMi
d
VE=elution volume of each fraction
b
c
The chain statistic analysis of the mass spectra of
copolymers allows us to determine the composition and
the sequence distribution of comonomer units.15 The
relative intensities of the mass peaks depend on the
copolymer composition and on the type of distribution
along the chain. Therefore, assuming a theoretical distribution model and fitting the calculated oligomer abundances
with the experimental peak intensities, the copolymer
composition can be determined. A computer program
(MACO4) is able to perform these calculations.15 In order
to determine the composition, the mass spectral peak
intensities in Fig. 4 were given as input to MACO4 program
assuming a Bernoullian distribution of units along the
copolymer chains. The program performed a minimization
which converged quickly towards a sharp minimum. The
agreement factor (AF) was 9.4%. The results of the
calculations are given in Table 4 together with the
experimental spectral intensities. From these data the
distribution of monomers in PBA/PBSe copolymer is
deduced to be random with a composition of 46/54 in
Rapid Commun. Mass Spectrom. 12, 519–528 (1998)
favour of sebacate, which compare well with a ratio of 47/53
obtained by NMR (Table 1).
Polydimethylsiloxanes
The SEC calibration curves of linear and cyclic polydimethyl siloxanes against MM have been reported,16 and it
has been shown that cyclic oligomers are eluted at slightly
higher volumes with respect to linear oligomers of the same
Mw, due to the smaller hydrodynamic volume (the ratio
(Mcycle/Mlinear)Ve is 1.24). The detection of this subtle
difference appeared to be an interesting test for the accuracy
of the SEC/MALDI-TOF method. Therefore, we analysed
two PDMS samples, PDMS1 and PDMS2. PDMS1 is a
polydispersed linear polymer, showing a bimodal distribution (Fig. 1), whereas PDMS2 was obtained by partial
alkaline hydrolysis of sample PDMS1 with NaOH, leading
to a lower MM and to a narrower dispersion. Besides
lowering the molecular mass, the alkali induces also a ringchain equilibration process and end-to-end cyclizations into
# 1998 John Wiley & Sons, Ltd.
SEC/MALDI-TOF FOR POLYDISPERSE POLYMERS
527
Figure 7. Reflectron MALDI-TOF mass spectra of two PDMS1 fractions: (a) fraction 70 and (b) fraction 78.
the polymer,16 thus producing a polydisperse cyclic
polymer. SEC fractionation of the two samples afforded a
number of fractions which were analysed by MALDI-TOF,
allowing the determination of their MM values (Table 5).
Figure 5 reports log (Mw) versus elution volumes for
PDMS1 and PDMS2 fractions. Remarkably, owing to the
different structures of the two samples (linear and cyclic
chains), two slightly different SEC calibration lines are
observed (Fig. 5).
In a duplicate experiment, the PDMS1 sample was
fractionated by SEC, and the elution trace was identical to
the previous one. However, the single SEC fractions were
# 1998 John Wiley & Sons, Ltd.
analysed by MALDI-TOF six weeks after the actual
fractionation experiment. During this time, the solvent
slowly evaporated and the fractions remained for a
relatively long time in the solid state. These fractions gave
excellent MALDI-TOF spectra, and four of these are shown
in Fig. 6 (a–d). Fraction 6 (Ve = 21.22) gave a Mw value of
424 000 Da; fraction 8, (Ve = 21.80) 346 000 Da; fraction
17, (Ve = 24.38) 131 000 Da; and fraction 26, (Ve = 31.22)
42 000 Da.
A comparison with the Mw curve in Fig. 5 (circles) shows
that the MALDI-TOF spectra of these fractions display MM
values roughly doubled with respect to fresh fractions which
Rapid Commun. Mass Spectrom. Vol. 12, 519–528 (1998)
528
SEC/MALDI-TOF FOR POLYDISPERSE POLYMERS
eluted at the same volumes in SEC analysis. This effect is
not easily explained, except by assuming the occurrence of
molecular association of PDMS chains in the condensed
state. This association does not need to be complete in order
to get doubled MM values by the MALDI-TOF technique.
Dimeric, or even higher, polymer species have been often
observed in the MALDI-TOF spectra of PMMA, polystyrenes and polyethyleneglycoles.2,17,18 It is not surprising,
therefore, that in the presence of a molecular association
effect the dimer peak becomes the most intense in the
MALDI-TOF spectrum, justifying the observed doubling of
MM.
The MALDI-TOF spectra of SEC fractions, containing
the lower mass molecular species, show these oligomers as
mass-resolved signals, allowing the assignment of each
peak to a specific oligomer and also the identification of the
end groups.
Figure 7(a) shows the MALDI-TOF mass spectrum of a
low molecular mass PDMS1 fraction in the reflected mode,
showing peaks in the region 5–10 kDa. After accurate mass
calibration, peaks in Fig. 7(a) were assigned to a single mass
series, i.e. linear PDMS oligomers terminated by trimethylsilyl groups. In Fig. 7(b) is reported the spectrum of a lower
MM fraction of PDMS1 (1.7–6.0 kDa), which shows instead
the presence of two mass series having different MMD
values. The most intense peak series in Fig. 7(b)
corresponds to linear PDMS oligomers terminated by
trimethylsilyl groups, whereas the other mass series was
assigned to cyclic dimethylsiloxane oligomers.
Acknowledgements
Partial financial support from the Italian Ministry for University and
for Scientific and Technological Research (MURST), from Progetto
Finalizzato Materiali per Tecnologie Avanzate II (CNR, Rome), and
Rapid Commun. Mass Spectrom. 12, 519–528 (1998)
from the National Council of Research (CNR, Rome) is gratefully
acknowledged.
REFERENCES
1. U. Bahr, A. Deppe, M. Karas, F. Hillenkamp and U. Giessman,
Anal. Chem. 64, 2866 (1992).
2. M. S. Montaudo, G. Montaudo, C. Puglisi and F. Samperi, Rapid
Commun. Mass Spectrom. 9, 453 (1995).
3. D. Garozzo, G. Impallomeni, E. Spina, L. Sturiale and G. Zanetti,
Rapid Commun. Mass Spectrom. 9, 937 (1995).
4. G. Montaudo, D. Garozzo, M. S. Montaudo, C. Puglisi and F.
Samperi, Macromolecules 28, 7983 (1995).
5. G. Montaudo, M. S. Montaudo, C. Puglisi and F. Samperi, Rapid
Commun. Mass Spectrom. 9, 1158 (1995).
6. C. F. Kassis, J. M. DeSimone, R. W. Linton, E. E. Remsen, G. W.
Langer and R. M. Friedman, Rapid Commun. Mass Spectrom. 11,
1134 (1997).
7. M. W. F. Nielen and S. Malucha, Rapid Commun. Mass Spectrom.
11, 1194 (1997).
8. J. Dwyer and D. Botten, Internat. Lab. January, 13A (1997).
9. X. Fei and K. K. Murray, Anal. Chem. 68, 3555 (1996).
10. G. Montaudo, Polym.Prep. (A.C.S., Div. Polym. Chem.) 36, 290
(1996).
11. P. O. Danis, D. A. Saucy and F. J. Huby, Polym. Prep. (A.C.S., Div.
Polym. Chem.) 36, 311 (1996).
12. M. Netopilik, M. Bohdanecky and P. Kratochvil, Macromolecules
29, 6023 (1996).
13. W. Radke, P. F. W. Simon and A. H. E. Muller, Macromolecules
29, 4926 (1996).
14. M. D. Zammit and T. P. Davis, Polymer 38, 4455 (1997).
15. G. Montaudo and M. S. Montaudo, Macromolecules 25, 4264
(1992).
16. P. V. Wright and M. S. Beevers, in Cyclic Polymers, J.A. Semlyen
(Ed.) Elsevier (1986), p.109.
17. G. Montaudo, M. S. Montaudo, C. Puglisi and F. Samperi, Anal.
Chem. 66, 4366 (1994);Rapid Commun. Mass Spectrom. 8, 981,
1011 (1994).
18. G. Montaudo, M. S. Montaudo, C. Puglisi and F. Samperi,
Macromolecules 28, 4562 (1995).
# 1998 John Wiley & Sons, Ltd.