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Anal. Chem. 2000, 72, 1707-1710
A MALDI Sample Preparation Method Suitable for
Insoluble Polymers
Randall Skelton,† Fre´de´ric Dubois, and Renato Zenobi*
Department of Chemistry, Swiss Federal Institute of Technology, CH-8092 Zu¨rich, Switzerland
Polyamides are insoluble or poorly soluble in common
organic solvents, which makes normal sample preparation
for matrix-assisted laser desorption/ ionization (MALDI)
mass spectrometry very difficult. An new analytical protocol for MALDI analysis of polyamides or other insoluble
samples is described. It consists of pressing a pellet from
a solid mixture of the polymer and a matrix, both in the
form of finely ground powder. This sample preparation is
compared with the common dried droplet sample preparation method and found to perform much better, both
in terms of robustness against variation of experimental
parameters and high-mass capability.
Insoluble or poorly soluble polymers pose particular challenges
to analysis by MALDI MS because they do not readily form mixed
polymer/matrix crystals, a requirement for preparing good
MALDI samples. In addition, quantitative information is often
required in mass spectrometric studies of synthetic polymers, to
answer questions about their molecular weight distribution
(MWD) or end group distribution.1-6 Obtaining reliable quantitative information is to a large extent dependent on homogeneous,
high-quality samples but is difficult by MALDI MS of poorly
soluble materials. As noted by Yalcin et al.,7 even slight differences
in the solubilities of matrix and polymer can lead to grave errors
in the measured polymer molecular weight. We describe a new
analytical protocol for MALDI analysis of polyamides which
circumvents solubility problems. It should be easily applicable to
other insoluble samples as well.
The polymers investigated here (Figure 1) were polyamides,
produced either as condensates between 1,6-diaminohexane and
1,4-benzenedicarboxylic acid (resulting in oligomers with a low
molecular mass, up to 1000 Da) or as oligomers of laurin lactame
regulated by either 1,6-diaminohexane or by 1,10-decanedicarboxylic acid, with a somewhat higher molecular mass (2500-4000
† Present address: Department of Chemistry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.
(1) Montaudo, G.; Garozzo, D.; Montaudo, M. S.; Puglisi, C.; Samperi, F.
Macromolecules 1995, 28, 7983-7989.
(2) Lehrle, R. S.; Sarson, D. S. Rapid Commun. Mass Spectrom. 1995, 9, 9192.
(3) Axelsson, J.; Scrivener, E.; Haddleton, D. M.; Derrick, P. J. Macromolecules
1996, 29, 8875-8882.
(4) Martin, K.; Spickermann, J.; Ra¨der, H. J.; Mu
¨ llen, K. Rapid Commun. Mass
Spectrom. 1996, 10, 1471-1474.
(5) Jackson, C.; Larsen, B.; McEwen, C. Anal. Chem. 1996, 68, 1303-1308.
(6) Kassis, C. M.; Desimone, J. M.; Linton, E. W.; Remsen, E. E.; Lange, G.
W.; Friedman, R. M. Rapid Commun. Mass Spectrom. 1997, 11, 11341138.
(7) Yalcin, T.; Dai, Y.; Li, L. J. Am. Soc. Mass Spectrom. 1998, 9, 1303-1310.
10.1021/ac991181u CCC: $19.00
Published on Web 02/05/2000
© 2000 American Chemical Society
Figure 1. General structures of compounds investigated.
Da). The production method had an important influence on the
end groups of the polymers, which included diamine-terminated
(N-N), mixed-terminus (N-H), and diacid-terminated (H-H)
polymers. The samples were partially soluble in trifluoroethanol,
concentrated sulfuric acid, and hot m-cresol; the best known
solvent is hexafluoro-2-propanol. All of these solvents are incompatible with common MALDI matrixes. Only one study has
appeared in the literature8 where the end groups of a polyamide,
nylon-6, were analyzed by MALDI MS. 2-((4-Hydroxyphenyl)azo)benzoic acid was used as the matrix, and it was possible to dissolve
the sample in trifluoroethanol.
It is important to stress that these polyamides were real
industrial products, not polymer standards that are often used in
(8) Montaudo, G.; Montaudo, M. S.; Puglisi, C.; Samperi, F. J. Polym. Sci., Part
A 1996, 34, 439-447.
Analytical Chemistry, Vol. 72, No. 7, April 1, 2000 1707
MALDI research. They had previously been characterized by
other methods. Titration of the carboxyl and amine groups9 had
been used for some of them to obtain a quantitative end group
analysis. If no solubility problems occur, the accuracy of the end
group distribution can reach about 5% with the titration method.
A rough estimation of the average molecular weight can be
obtained from these data, by using the relationship 2/Mn )
n(COOH) + n(NH2), where n(COOH) is the concentration of the
acid end groups in mol/g, n(NH2) is the concentration of the
amine end groups in mol/g, and Mn is the number-average
molecular weight. Molecular weight distributions were also
available from gel permeation chromatography (GPC) data. For
the higher polyamide oligomers, GPC was only possible after
chemical derivatization,10 which leaves some doubt as to the
accuracy of the GPC data. Proton NMR spectroscopy has also
been proposed for molecular weight and quantitative end group
determination,11 by making use of the slight difference in chemical
shift of terminal hexanamine and acid groups compared to those
inside an extended chain. However, this method does not yield
very accurate data. Solubility problems can hamper analysis both
by GPC and by NMR spectroscopy.
MALDI mass spectrometry has been shown to yield very useful
information for polymer analysis,5,12-17 provided that a good sample
can be prepared. We investigated experimental protocols for the
detection and quantitative analysis of polyamide oligomers by
MALDI MS. Efforts to circumvent problems associated with the
poor solubility of these samples included work with liquid and
slurry samples and ultimately led to the discovery of a new solid/
solid sample preparation for polymers that are insoluble in MALDIcompatible solvents. To judge the usefulness of a given analytical
protocol, we observed how sensitive it was to changes in
composition (e.g., matrix-to-analyte ratio) and experimental parameters (e.g., desorption laser power).
EXPERIMENTAL SECTION
Materials. All polyamide samples were donated by EMS
CHEMIE (Domat-Ems, Switzerland). Solid MALDI matrixes
included 4-hydroxy-R-cyanocinnamic acid (HCCA), 2,5-dihydroxybenzoic acid (DHB), 3-aminoquinoline (all from Fluka), 3,5dimethoxy-4-hydroxycinnamic acid, and dithranol (from Aldrich).
They were used without further purification.
Instrumentation. MALDI time-of-flight (TOF) experiments
were performed on a home-built 2 m linear TOF mass spectrometer.18 The total acceleration potential was +25 kV. Delayed
extraction was generally used, with delay times of 150-700 ns,
(9) Roerdink, E.; Warnier, J. M. M. Polymer 1985, 26, 1582.
(10) Jacobi, E.; Schuttenberg, H.; Schulz, R. C. Macromol. Chem. Rapid Commun.
1980, 1, 397-402.
(11) Shit, S. C.; Maiti, S. Eur. Polym. J. 1986, 22, 1001-1008.
(12) Bahr, U.; Deppe, A.; Karas, M.; Hillenkamp, F.; Giessmann, U. Anal. Chem.
1992, 64, 2866-2869.
(13) Montaudo, G.; Montaudo, M. S.; Puglisi, C.; Samperi, F. Macromolecules
1995, 28, 4562-4569.
(14) Montaudo, G.; Scamporrino, E.; Vitalini, D.; Mineo, P. Rapid Commun. Mass
Spectrom. 1996, 10, 1551-1559.
(15) Chaudhary, A. K.; Critchley, G.; Diaf, A.; Beckmann, E. J.; Russell, A. J.
Macromolecules 1996, 29, 2213-2221.
(16) Belu, A. M.; De Simone, J. M.; Linton, R. W.; Lange, G. W.; Friedman, R.
M. J. Am. Soc. Mass Spectrom. 1996, 7, 11-24.
(17) Ra¨der, H. J.; Schrepp, W. Acta Polym. 1998, 49, 272-293.
(18) Dubois, F.; Knochenmuss, R.; Steenvoorden, R. J. J. M.; Breuker, K.; Zenobi,
R. Eur. Mass Spectrom. 1996, 2/3, 167-172.
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Analytical Chemistry, Vol. 72, No. 7, April 1, 2000
depending on the mass range investigated. Desorption was
performed using a nitrogen laser (model VSL-337ND-T, Laser
Science Inc.), incident at ca. 60° to the surface normal. The energy
was generally slightly above threshold, in the range 10-20 µJ, as
measured with a pyroelectric detector (model ED 100, Gentec,
Quebec, Canada). The irradiance at the sample surface was
estimated to be in the 106-107 W/cm2 range. Spectra were
obtained in the positive mode, by averaging 50-100 single shots.
Preparations. (a) Crystalline MALDI Samples. Initially,
samples were prepared with crystalline MALDI matrixes. A layer
of matrix solution was applied to the sample target, dried in air
or under vacuum, and covered by a second layer of sample
solution. The layered sample preparation technique was necessary
because the matrix and the sample solutions were generally
immiscible. Often, the addition of the sample solution onto the
dried matrix was observed to disrupt the matrix microcrystals.
(b) Solid/Solid Pressed Samples. The new solid/solid
sample preparation is analogous to making a crystalline KBr
sample for infrared analysis. The polymer samples were ground
with a mortar and pestle to fine powders. Solid samples were then
mixed with one of the common MALDI matrixes, for example,
DHB, 3-aminoquinoline (Fluka), or dithranol (Aldrich), and the
mixtures were ground together in the mortar. Other materials
such as KBr, cobalt powder, and graphite were also tried, but
without success. A variety of matrix-to-polymer mixing ratios were
used, ranging from 1:10 to 10:1 (by weight). Empirically it was
observed that mixing ratios between 1:1 and 1:5 provided optimal
conditions for desorption/ionization of both the low molecular
weight samples and the higher molecular weight polyamide
oligomers. These are unusually low mixing ratios compared to
those of standard MALDI. Increasing the matrix content only
served to increase the intensity of the matrix peaks, which
obscured the peaks of the low-mass oligomers present in both
samples. Finally, a few milligrams of the finely ground mixture
was pressed with 1.3 × 105 N/cm2 (10 tons) for approximately 2
min using a hydraulic press to yield a flat, thin (<1 mm), solid
sample. This sample was placed on the MALDI probe tip with
common double-sided tape and introduced into the instrument
source.
RESULTS AND DISCUSSION
Using the crystalline matrix preparation, a strong dependence
of the mass spectrum on the matrix was found. In each case, the
ratio involving N-N, N-H, and H-H oligomers changed. Thus,
the intensity of mass peaks in these samples cannot simply be
considered as proportional to the molar amount of each species
in the sample. In addition, the molecular weight distribution
showed a skewed distribution where peaks of lower mass
oligomers predominated in the spectrum. This trend was observed
in the analysis of all low molecular weight polyamide samples.
The average polymer molecular weight and end group distribution
calculated from such data did not agree well with similar data
determined by conventional means. We also found an effect when
varying the matrix-to-analyte molar ratio. With matrix:polymer
mixing ratios of 100:1, reasonably stable ion signals with bellshapedsalthough asymmetricsdistributions were obtained. At
mixing ratios of 10:1, insufficient ionization of the polymer was
observed with a lower signal-to-noise ratio and poor reproducibility
from one laser shot to the next. As the mixing ratio was increased
Figure 2. MALDI mass spectra of a low molecular weight polyamide
sample using solid/solid preparation. Mixing ratios (matrix:analyte, by
weight): (a) 10:1; (b) 5:1; (c) 1:1; (d) 0.2:1. Matrix: 3-aminoquinoline.
Asterisks denote peaks derived from the matrix.
to 1000:1, the MWD skewed and peaks of the lower mass
oligomers became more prevalent in the mass spectrum. Considerably more laser irradiance was required to create ions for
the 10:1 molar ratio than for the 1000:1 molar ratio. However, the
oligomer distributions did not shift to lower mass for higher laser
energies, opposite to what would be expected if this were due to
fragmentation.4 The absence of fragmentation in the MALDI MS
analysis of these polyamides was further confirmed by a collisioninduced dissociation experiment on a single oligomer peak that
was isolated on an FTICR mass spectrometer. Therefore, the shift
in the mass distribution occurs because higher laser pulse
energies allow desorption/ionization of oligomers with larger m/z
values, which tends to shift the oligomer distribution up in mass.
Using the solid/solid sample preparation, it was observed that
the mass distribution was much more consistent, even if the
mixing ratio was varied by a factor of 50 (Figure 2). The
reproducibility of the spectra themselves was much better than
the data obtained from crystalline matrix samples. Compared to
the case of crystallized or “dried droplet” samples, less shot-toshot variability and less change in shape and oligomer distribution
were observed, although they were not eliminated completely. No
problems with sample charging or decreased resolution due to
the thick insulating sample were encountered. Because of the
inherent sample thickness for such pressed disks, over 5000 laser
shots could be taken at a single sample position with little change
in ion current. We also observed that the relative signal intensities
of H-H, N-N, and N-H terminated oligomers remained fairly
constant. With a calibration measurement that determines the
Figure 3. MALDI mass spectra of (a) an acid-regulated and (b) an
amine-regulated laurin lactame polyamide. Insets: expoanded views
of the m/z 1100-1750 range. Solid/solid sample preparation used
3-aminoquinoline as the matrix (ca. 2:1 matrix:sample ratio (by
weight)).
relative response of diacid-, diamine-, and mixed-terminus oligomers, it would therefore be possible to determine the end group
distribution quantitatively. This was not attempted, however,
because of the lack of suitable standards.
Each peak in the MALDI mass spectra can be readily assigned.
The major peaks in the spectra displayed in Figure 2 are due to
protonated mixed-terminus (N-H) linear oligomers. Minor peaks
are due to cationized signals, as well as protonated (H-H) and
(N-N) oligomers. In principle, mixed-terminus oligomers, at least
above a certain size, have the possibility of cyclizing during
synthesis by losing water. While the linear forms of the mixedterminus oligomers of the low molecular polyamide samples have
nominal m/z values of 510, 756, 1002, 1248, 1494, ..., the cyclic
oligomers should appear at m/z values 18 mass units lower, i.e.,
at m/z 492, 738, 984, 1230, 1476, .... They should be easily
detectable by MALDI mass spectrometry because they can be
protonated (or cationized), for example on the amide nitrogen.
Our data were inspected for signs of cyclic oligomers, but signals
at m/z values corresponding to cyclic oligomers were absent or
Analytical Chemistry, Vol. 72, No. 7, April 1, 2000
1709
very small. There was also no indication for the occurrence of
cyclic oligomers in the higher molecular weight samples (see
below).
Figure 3 shows that the solid/solid sample preparation
described above is also well suited for the analysis of higher
polyamide oligomers. These compounds appear in the mass
spectrum in their protonated forms, with peaks extending up to
m/z 8000. It is also immediately apparent from the data (insets)
that there is a predominance of diamine-terminus peaks for the
amine-regulated sample vs a predominance of diacid-terminus
peaks for the acid-regulated sample. This is fully consistent with
the end group determination by titration (data not shown). Peaks
for mixed-terminus oligomers appear below m/z ∼2000 in the
mass spectra of both samples. As in the case of the low molecular
weight samples, it would be possible to determine the end group
distribution quantitatively with a suitable calibration measurement
for the relative ion yield of the N-N, H-H, and N-H terminated
oligomers.
Minor peaks that were not expected appear in the mass spectra
and remain fairly strong up to high mass. They form series at
m/z 1119, 1316, 1513, 1710, ... in Figure 3a and at m/z 1218, 1415,
1612, ... as well as m/z 1240, 1437, 1634, ... in Figure 3b. The
mass difference of 22 Da in the last two series suggests that these
are due to the protonated and sodiated forms of the same
compounds. The repetition unit is always 197 Da, identical to the
spacing between different oligomers of the main series, a strong
indication that all of the minor signals are all derived from the
polyamide. They could be either impurities or side products, for
example polymers with different end groups, or they may be
fragments. Within the accuracy of our mass determination, the
mass difference of ∼99 Da from the next higher intact oligomer
in the case of the acid-regulated sample could be explained with
loss of a neutral •(CH4)4COOH unit (101 Da) or the corresponding
lactone radical (100 Da). Accordingly, the mass difference of ∼84
Da may be due to the loss of a neutral •(CH2)5NH2 unit (86 Da)
1710
Analytical Chemistry, Vol. 72, No. 7, April 1, 2000
or the corresponding cyclic secondary amine radical (85 Da).
However, such losses are not common, particularly in MALDI
mass spectrometry. Therefore, the origin of these signals is
presently uncertain.
In conclusion, we show the importance of sample preparation
in acquiring MALDI MS data for polyamides. These materials are
difficult to handle by standard MALDI sample preparation
methods because of their poor solubility in common solvents. We
found that a solid/solid sample preparation gave spectra that are
reproducible, show good signal-to noise ratios, and are not
sensitive to changes in sample composition. An important advantage of this sample preparation method is that it is applicable to
completely insoluble materials; it should be applicable to many
other substances besides polyamides. A dependence of the
MALDI spectra on laser irradiance still exists: higher laser pulse
energies allow desorption/ionization of oligomers with larger m/z
values. At the laser pulse energies used, fragmentation was not
observed to be important for this class of compounds. We also
found that cyclic polyamide oligomers were absent in these
samples. Finally, we demonstrate that it is possible to obtain end
group and molecular weight information for high-mass polyamides. The quantitative determination of the end group distribution has to await the calibration of the MALDI response by use of
suitable standard compounds.
ACKNOWLEDGMENT
This study was made possible through a collaboration with
EMS CHEMIE. We thank W. Tobisch and C. Guckel for providing
samples, for giving us access to complementary analytical data,
and for helpful discussions.
Received for review
December 16, 1999.
AC991181U
October
13,
1999.
Accepted