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MALDI-TOF mass spectrometry in the analysis of synthetic polymers

H. J. Rader+ and W. Schrepp*++

+ Max-Planck-Institut fur Polymerforschung, Ackermannweg 10, D-55128 Mainz, FRG+ + Polymer Research Department, BASF AG, D-67056 Ludwigshafen, FRG

Matrix-assisted laser desorption/ionization mass spectrometry has been applied over the past 5 years with increasing successto the analysis of synthetic polymers. Basic principles and instrumental parameters influencing the spectra are described.Examples from different areas of polymer research with emphasis on technically relevant systems elucidate the highly usefulfeatures of this kind of mass spectrometry, i. e. determination of absolute molar masses, high mass range, reasonable massresolution, high sensitivity, relative ease of operation even for polar molecules. Molar mass distributions can be obtained in amass range that is problematic for other mass determining techniques.A detailed characterization of the underlying chemistry can be obtained in many cases by an endgroup analysis. Especiallyin combination with other separation techniques, a comprehensive characterization of chemical heterogeneity can beachieved. Recent developments, e. g. delayed extraction to enhance the mass resolution obtainable, will further increase thefavorable prospects of this technique.

1. Introduction

Some of the important properties of a synthetic polymer,such as toughness, elasticity, and processability, are corre-lated to chemical structure and molecular weight. There-fore determination of molecular weight and the molecularweight distribution is of central interest in polymer analy-sis. Well established conventional methods for this pur-pose are gel permeation chromatography (GPC; synony-mously called size exclusion chromatography (SEC)1)),vapor pressure and membrane osmosimetry, viscometry,light scattering and the analytical ultracentrifuge [1].

Among them, GPC is the most frequently used method,because this technique measures not only an average mole-cular weight value but also the complete polymer distribu-tion. However, GPC is not an absolute method and showsinaccuracies when samples are measured for which nocalibration standards exist. Another limitation of GPCoccurs when the analyte shows adsorption effects on thecolumn material or when the molecules form aggregates insolution [2]. In this way, all the methods of molecularweight determination have their own limited applicationfields and must be chosen according to the physico-chemi-cal properties of the polymer being investigated.

Considering the importance of molecular weight analy-

sis in polymer chemistry and the still existing need for newanalytical methods, especially for problematic compoundssuch as copolymers, dendrimers, rigid rods and moleculeswith non-covalent bonds, it is essential to establish newmethods that provide additional information.

Mass spectrometry seems to be ideally suited for molecu-lar weight analysis because of some unique features inher-ent to the method, such as absolute determination of mole-cular weightindependent of structure, very lowsample con-sumption and short analysis time. Since the development ofsoft ionization techniques such as field desorption (FD),fast atom bombardment (FAB), laser desorption (LD), and252Cf plasma desorption (PD) it has been possible to cover a

molecular weight range of up to a few thousand dalton

[3, 4], which is still quite low for common synthetic poly-mers. A more general application of mass spectrometry hasbeenpossible since the development of electrospray ioniza-tion (ESI) [5] and matrix-assisted laser desorption/ioniza-tion (MALDI) mass spectrometry [6, 7]. Both techniquesare able to produce intact molecular ions of polymers wellabove 100000 Da. ESI mass spectrometry occupies only asmall, but valuable segment in the analysis of syntheticpolymers [8] and it is much better suited for the analysis ofnearly monodisperse biopolymers because of the formationof multiply charged ion distributions, which interfere with

the molecular distribution in the mass spectra of polymers.MALDI-TOF MS (time-of-flight mass spectrometry), how-ever, is applicable to the measurement of polymer distribu-tions, because it mainly produces singly charged species,which in general can be interpreted easily. Some furtheradvantages of the methodare:

i) MALDI-TOF MS allows for the measurement ofextremely high molecular weights with virtually no frag-mentation [9];

ii) in a mass range where single polymer chains areresolved, MALDI-TOF enables determination of repeatingunits and end group compositions [10 12];

iii) in the region below 20000 Da, where other absolute

methods such as osmometric mass analysis might giveinaccurate results, MALDI-TOF mass spectrometry can beused as a supplemental independent absolute method.

MALDI-TOF mass spectrometry was developed in 1988by Hillenkamp and Karas for the analysis of large biomole-cules [13, 14], but it was not demonstrated until 1992 thatalso synthetic polymers can be analyzed with a molecularweight above 100000 Da [15 17]. There are two main rea-sons for thisdelay: 1) the methods of sample preparation forbiopolymers with water based solvents were not applicableto most synthetic polymers and 2) synthetic polymers arealways polydisperse. The latter is responsible for a worsesignal-to-noise ratio, because the whole signal intensity,

which adds up to a single signal omitting isotope effects

272 Acta Polymer., 49, 272293 i WILEY-VCH Verlag GmbH, D-69451 Weinheim 1998 0323-7648/98/0606-0272$17.50+.50/0

Fax: +49-621-60-922811 Abbreviations are collected in a separate list at the end of the paper.

Feature Article

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Acta Polymer., 49, 272 2 93 (1998) MALDI-TOF mass spectrometry in the analysis of synthetic polymers 273

in the case of a biomolecule, isdivided bythe number ofdif-ferent degrees of polymerization in the case of a syntheticpolymer. Thus, the method had to be developed for syn-thetic polymers especially. During the last five years,MALDI-TOF mass spectrometry has become an important

new tool for the characterization of synthetic polymers andthe interests of biochemists and polymerchemists haveseparated so far that we decided to dedicate this paper espe-cially to the application of MALDI-TOF MS in the analysisof synthetic polymers. Since the number of publications inthis field is rapidly increasing and MALDI-TOF analysiscovers a widespreadrange of applications, this paper shouldbe regarded as an interim report and does not claim comple-teness. In the first part, the relevant fundamental features ofthe method are described without going into theoreticaldetails, representing a guide to measure high molecularweight synthetic polymers. In the second part emphasis islaid on practical examples that demonstrate the scope and

limitations of MALDI-MS at present.

2. Fundamentals of the method

2.1. MALDI process

The matrix-assisted laser desorption process is the keyfeature of the method, because it is responsible for theaccessibility of a very high mass range up to several hun-dred thousand dalton. It works similarly to the laser desorp-tion (LD) method developed earlier [18], which is capable

of producing intact molecular ions only in the low massrange up to a few thousand dalton. Contrary to laser deso-rption, where the analyte is irradiated directly by UV-laserlight, in matrix-assisted laser desorption the analyte isassumed to be homogeneously embedded in a matrix mate-rial, which absorbs the laser energy and is responsible forthe transfer of the analyteinto the gas phase without thermalstress (Fig. 1). The molar mixing ratios between matrix andanalyteareintherangeof500:1to106 :1[9,19].

The matrix has to meet various requirements: 1) highelectronic absorption at the employed laser wavelength(usually 337 nm of a nitrogen laser), 2) good vacuumstability, 3) good solubility in organic solvents that can

also dissolve the analyte, and 4) good miscibility with theanalyte in the solid state [20]. In the analysis of biopoly-mers, where the ionization usually takes place via protontransfer, the matrix also plays the role of the proton source[21, 22], whereas in the analysis of synthetic polymersionization is usually achieved by cation attachment [2325], which is a matrix-independent process. Some com-monly used matrices for synthetic polymers are dithranol(1,8,9-trihydroxyanthracene), DHB (2,5-dihydroxyben-zoic acid), IAA (3-b-indoleacrylic acid), sinapinic acid(3,5-dimethoxy-4-hydroxy cinnamic acid) and 5-chlorosa-licylic acid, to mention only a few. There is no general rule

how to select the ideal matrix for a given polymer. Theselection of a good matrix is still a trial and error processand must be worked out each time a new polymer class isunder investigation.

2.2. Sample preparation

Sample preparation is the crucial point in MALDI massspectrometry (see also Sect. 3.6) because it mainly influ-ences the quality of the spectra [26]. Figure 2 shows a typi-cal sample preparation procedure for MALDI measure-ments. Appropriate amounts of matrix and polymer dis-solved in compatible, preferentially identical solvents aremixed to yield a molar ratio of about 1000:1 (matrix:ana-lyte). The metal ions often required for enhanced cationi-zation are added as their organic or inorganic salts, depend-ing on their solubility, and 1 ll of the whole solution isthen applied to a sample holder and allowed to dry. Theresulting homogeneous solid mixture, which ideally con-sists of a thin layer of microcrystals, is then introduced intothe ion source of the mass spectrometer and irradiated by apulsed UV laser.

Fig. 1. Scheme of the matrix-assisted laser desorption/ionizationprocess.

Fig. 2. Sample preparation for MALDI measurements.

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274 Rader, Schrepp Acta Polymer., 49, 272293 (1998)

As mentioned before, synthetic polymers always have adistribution of different chain length (polydispersity), con-trary to biomolecules, which ideally consist of only onesort of molecule with the same molecular weight (depend-ing on mass-range the different isotopes can be separated

by modern MALDI equipment; see Sects. 2.3 and 3.6).This requires even more demanding methods of samplepreparation, because the signal-to-noise ratio decreaseswith increasing number of different mass-to-charge ratiosin polydisperse polymers. Since it is assumed that the dis-tribution of ions in the gas phase is representative of thenumber of ions in the condensed state, polydispersity ofsynthetic polymers also entails the problem of discrimina-tion effects, which may falsify the shape of polymer distri-butions and causes wrong results for the calculation ofaverage molecular weight values. So care must be taken toavoid the loss of sample fractions during sample prepara-tion. Keeping this in mind, sample preparation is of central

importance in MALDI-TOF polymer analysis becausehomogeneous mixtures of matrix and analyte in the solidstate are a prerequisite to avoid mass discriminationalready at this early stage of measurement [27, 28].

Polydispersity also requires some instrumental conse-quences, which will be described in more detail in the fol-lowing section.

2.3. Time-of-flight analyzer

The MALDI method is ideally combined with a time-of-

flight (TOF) mass spectrometer because of the pulsed nat-ure of the desorption laser. Beyond this, the TOF analyzerhas some additional unique characteristics, such as highsensitivity and an in principle unlimited mass range [29],that predestine it for the analysis of large molecules. Sincequestions of mass resolution, sensitivity and ion detectionare discussed in the literature (see e.g. [29]), we shall notgo into numerical detail in this overview. Figure 3 shows atypical set-up of an instrument equipped with a matrixassisted laser desorption ion source.

The ions are produced in most commercial instrumentsby irradiation of the sample with the light of a pulsed nitro-

gen laser. The intensity of the laser beam can be adjustedby a variable attenuator to a value slightly above thresholdfor ion production. The laser beam is focused on thesample surface in the ion source. Once the ions are formedafter a laser pulse, they are accelerated simultaneously by

a static electric field of up to 35 kV. Depending on theirmass-to-charge ratio, they have different velocities whenthey leave the acceleration zone and pass the followingfield-free drift tube with different flight times. The time-of-flight for each ion is then measured by the time differ-ence between the start signal, given by the laser pulse andthe stop signals, caused by the ions impinging on the detec-tor.

According to the simple relationship of Eq. (1), thesquare of the flight time is proportional to the mass-to-charge ratio.

m

z

2 U t2

s21

m = mass of the ion, z = number of charges, U= accelerat-ing voltage, t= ion flight time, s = flight distance.

Since the accelerating voltage and the length of the drifttube are known, the mass-to-charge ratio can be calculatedsolely from Eq. (1). However, in practice more exactvalues for the mass scale can be obtained from the empiri-cal Eq. (2) because of uncertainties in the determination ofthe flight time. This uncertainty is due to a short delay inion formation after the laser pulse, so that the real startingtime of the ions is not identical to the time of the laserpulse, which provides the starting signal for the measure-

ment of flight time [30].m

z a t2 b 2

m = mass of the ion, z = number of charges, t = ion flighttime, a, b = constants.

The constants a and b in Eq. (2) are measured by theflight times of two ions with known masses, which areused for calibration.

The accelerated ions can be detected in two differentways, the linear and the reflection mode. The main differ-ences between the two detection methods are higher resol-

Fig. 3. Reflection time-of-flight mass spectrometer.

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ution in the reflection mode and higher sensitivity for lar-ger molecules in the linear mode. The higher resolution inthe reflection mode is achieved by a reflecting field at theend of the flight tube with somewhat higher potential andthe same polarity as the accelerating voltage. Ions with thesame mass-to-charge ratio but different velocities, whichcause peak broadening in the linear detection mode, can betime-focused with the reflector, because the faster ions getdeeper into the reflection field and have to travel a longerflight path [31]. It should be mentioned that the resolution

of MALDI-TOF MS is mainly restricted by the ionizationprocess, rather than by instrumental limits, because theions have a certain time span of formation, a spatial distri-bution and a kinetic energy spread. In reflection mode theresolution is typically around 1000 (FWHM) whereasdetection in linear mode has a resolution of around 300(FWHM). Therefore the linear mode has a very high sensi-tivity (detection efficiency about 50%) and requires only avery short ion lifetime of about 1 ls (acceleration time)[32] for the detection of the molecular ions even in thecase of a metastable decay on the flight path. Other typesof mass spectrometers, for example sector field instru-ments, require ion lifetimes of about 100 ls for the detec-

tion of molecular ions. In the linear mode of a time-of-flight instrument, however, the fragments of moleculesthat decompose after the acceleration zone have still nearlythe same velocity as the intact molecular ions and causesignals at the same flight time with a slight increase inpeak width [32]. Since molecules of high molecular weighttend to decompose during the flight time, because of thehigher energy required for their desorption, this is also animportant feature of the MALDI-TOF method, whichenables the measurement of very high molecular weights.

In the reflection mode of a time-of-flight instrument,ions that undergo fragmentation cannot be detected at their

correct molecular weight. With a special technique, how-ever, the reflection mode can be used to analyze meta-stables. By stepping down the reflection potential, all thefragments of a selected ion can be analyzed. The precursor

ion is selected after the acceleration zone by a gating sys-tem, which deflects all the unwanted ions perpendicular tothe normal flight path [33]. Analysis of metastables is ofspecial interest in the case of peptides, where informationon the sequence of amino acids is readily obtained [34,35]. In analysis of synthetic polymers, investigation ofmetastables can also be useful to achieve structure infor-mation for single polymer chains.

Both detection methods, the linear mode and the reflec-tion mode, have advantages and drawbacks and must be

chosen according to the information of interest. In the caseof measuring polymer distributions, especially with highmolecular weights above 10000 Da, the linear detector isused preferentially, because the detection is independentof a metastable decay as long as special detectors are usedthat are insensitive to oversaturation [36, 37]. Detection inthe reflection mode is used preferentially in the case ofdetermining exact molecular weights of individual poly-mer chains, which is necessary for end group determina-tion or identification of side products.

Beyond the improvement in resolution enabled by appli-cation of a reflection field, there is a further significantimprovement in resolution by introduction of time lag

focusing or delayed extraction [38, 39]. This principlewas first applied to MALDI-MS by Spengler et al. [40]and can be used in both linear and reflection modes, withan improvement in resolution by a factor of 10 in bothcases. Whereas the reflection field compensates for theinitial energy spread of the created ions, delayed extractioncan also compensate for the time spread in ion formation.

Figure 4 schematically shows the principles of opera-tion. In contrast to the normal MALDI-TOF measure-ments, where the ions are desorbed directly into the accel-eration field, in delayed extraction the acceleration field ispulsed on after a time delay of typically a few hundred

nanoseconds (50600 ns) after a laser pulse. In this waythe initial desorption and ion formation process is sepa-rated from the ion acceleration step. Thus the time windowrequired for ion formation does not contribute to peak

Fig. 4. Ion source design for resolution enhancement by delayed extraction. Theaccelerating voltage V1 is applied to the sample holder. The potential V2 of the firstextraction grid is set to V1 during the delay time. After the delay V2 is set to a slightlylower potential to extract the ions in the direction of the second extraction grid, which

is connected to earth.

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broadening. Another possible benefit of this experimentalprocedure is that during the delay most of the neutral spe-cies created during desorption are allowed to dissipate andpumped away so that ionneutral collisions are minimized.The ion source used for delayed extraction consists in prin-

ciple of three electrodes. The first electrode is the sampleholder with a voltage V1. The second electrode is the firstgrid and has a voltage of V2. The third electrode is con-nected to earth. For a short time after a laser pulse, the vol-tage difference between V1 and V2 is set to 0. After thisdelay, the voltage between V1 and V2 is increased by only afraction of the complete acceleration voltage, and thus theions leave the first acceleration stage. On entering the sec-ond stage, the ions are subjected to the main accelerationfield and attain their final velocity. When the potential dif-ference in the first stage is switched on, the slower ionshave a greater distance from the first grid than the fasterions and are thus accelerated by a higher field gradient. By

optimizing the potential difference between V1 and V2,values can be found where all ions with the same mass-to-charge ratio but different initial velocities arrive at thedetector at the same time. With this technique it is possibleto achieve resolutions of up to 10 000 ((FWHM; i.e. singlemass resolution can be obtained up to 7000 Da) [41].

2.4. Interpretation of the mass spectra of synthetic

polymers

Mass spectra of synthetic polymers can provide a varietyof information in a mass range where single polymer

chains are resolved. Among this information, the most fun-damental are the mass of the constituent repeating unit, theend group and the average molecular weight data Mn and

Mw. As an example we describe the anionic polymerizationof styrene (Fig. 5).

Figure 6 shows the MALDI-TOF mass spectrum2 of apoly(styrene) polymer distribution with the most probablepeak (Mp) at 8800 Da. Each peak in this spectrum is repre-sentative of a different degree of polymerization. Thepeak-to-peak distance amounts to 104.15 Da and reflects

the mass of a poly(styrene) repeat unit. Since differentpolymers have different masses typical of their constituentrepeating units, the peak-to-peak distance can in principlebe used to identify an unknown sample. With the absolutemasses of each signal of the polymer distribution, the endgroup can be calculated. Therefore knowledge about theionization process is a prerequisite. In our example theions were produced by silver cationization. Each signal ofthe measured polymer distribution has therefore to be cor-rected by the mass of silver. Hence, the correct molecularweight of the molecule that gives rise to the peak at 8706Da (Fig. 7) is therefore 8598 Da.

This mass is composed of a number n of repeating units

with a molecular weight of 104.15 Da and the masses ofthe two end groups. The number n is determined by thefraction 8598 Da/104.15 Da = 82.554. The masses of bothend groups are then given by multiplying the residualvalue 0.554 by 104.15 Da = 57.7 Da. From this, togetherwith the knowledge of the starting and the terminatingreaction of anionically polymerized poly(styrene), themost probable end groups in our case are a butyl group anda hydrogen atom. The butyl end group results from theinitiation reaction with butyllithium and the hydrogen isintroduced by protonation of the living polymers withmethanol, which is the termination reaction.

Besides the information obtained from resolved polymerchains, MALDI-TOF MS also provides data for the char-acterization of the polymer distribution. The averagemolecular weight values Mn and Mw and the polydispersityD can be calculated by the following equations:

2 The authors use either a Bruker REFLEX spectrometer (H.J.R.) in the linear or reflection mode or a Bruker BIFLEX (W.S.) spectrometer withHIMASTU detector.

Fig. 5. Anionic polymerization of styrene initiated by butyllithium and terminated byquenching with methanol. The obtained polymer is mixed with a silver salt prior to measure-ment to enable ionization by silver-cation attachment.

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Mn = R (Ni Mi) /Mi (3)

Mw = R (Ni Mi2) / (Ni Mi) (4)

D = Mw/Mn (5)

Mn = number average molecular weight, Mw = weight aver-age molecular weight, Ni = signal intensity at point i, Mi =mass at point i, D = polydispersity.

The calculation ofNi requires a numerical integration of

the polymer signal and necessitates a quantitative evalua-tion of the complete polymer distribution. Quantitativeanalysis by mass spectrometry, however, is not self-evi-dent and it has been shown that MALDI-TOF mass spec-trometry can be considered as an absolute method only formolecular weight distributions with low polydispersities.At values below D = 1.1 MALDI-TOF is probably themost exact polymer analytical method to date [42],whereas at polydispersities D A 1.1 MALDI-TOF resultsshow increasing deviations from values obtained by con-ventional methods such as SEC [43]. In order to obtainabsolute values also for broad polymer distributions,MALDI-TOF mass spectrometry has to be combined with

SEC fractionation [44, 45]. By separating the SEC outputof polymers with high polydispersities into small fractions,again samples with narrow polymer distributions areobtained, which can be measured exactly by MALDI massspectrometry. The obtained absolute molecular weightvalues can then be used to calibrate the SEC curve. HenceMALDI is used as an absolute detector for SEC in thiscase.

3. Applications of MALDI-MS to synthetic polymers

To our knowledge the first attempts to apply MALDI-MS to synthetic polymers with high molecular weight dateback to 1992 [15, 24, 27]. The early results on PMMA andPS had been very encouraging as molecular weights up to

some hundred kDa became detectable, greatly enlargingthe possibilities of mass spectroscopy in general withrespect to the molecular weight range attainable.

In the following driven by this fact and the commercialdevelopment of instrumentation various types of poly-mers have been investigated by this technique up to now.A collection of results without claiming completeness is shown in Table 1.

From Table 1 it can be inferred that

a) the number of publications for the different polymerclasses varies greatly (the well-characterized standard sys-tems PMMA and PEG for example have attracted manyresearchers to do basic-principles work);

b) although a great variety of matrices are described inthe literature most of the polymer work has been doneusing a handful of matrices (DHB, dithranol, HABA, IAA,THAP);

c) the annotations indicate several distinct areas of inter-est as well as parameters influencing the mass spectra.Some of these points are: influence of the polydispersity of the material under

investigation influence of experimental settings (linear mass analyzeror reflector; acceleration voltage; detector system andso on)

Fig. 6. MALDI-TOF mass spectrum of anionically polymerizedpoly(styrene) with Mn = 9000 Da (Polymer Standard Service,Mainz, Germany). Matrix: dithranol, cationizing agent: silvertri-fluoroacetate, solvent: THF.

Fig. 7. Top: Expanded region of Fig. 6. The peaks are labeled withtheir measured molecular weights and the corresponding number ofrepeating units. The interpeak distances reflect the mass of the con-stituent repeating units. Bottom: Demonstration of end group ana-

lysis by calculation of a representative peak of the polymer distribu-tion.

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Table 1. Overview of MALDI experiments described in the literature.

Substance Mol. weight Solvent Matrix/solvent Remarks Ref.

PMMA 95 000 THF HABA/THF variation of polydispersity [43]

18 000 THF DHB/THF det. of cyclic oligomers [46]10 000 THF DHB/THF comparison to SEC [47]15 100 THF DHB/THB comparison to SEC [48]20 and 50mer acetone IAA, H ABA,

DHB/CHCl3, acetoneno mass discrimination observed [49]

4 100 methanol DHB/methanol influence of pH [50]140000 water/NaCl DHB/acetone catalytic chain transfer under emulsion condit. [51]up to 3 800 THF DHB/THF influence of cations [52]25 000 THF IAA/acetone coupling with GPC [53]260 000 acetone IAA/acetone [27]1 6 90/5 2 20-mix ture THF Dithranol/THF influence of mol. weight distribution [25]l1 000 THF DHB/acetone back biting [46]up to 100 000 THF IAA/AgTFA, THF rate coefficients [73]

PS 52 000 THF HABA/THF variation of polydispersity [43]

12 000 THF NA + AgTFA comparison to SEC [47]1150 (syndiotactic) CCl4 NA/acetone, water chain ends methyl and ethyl [54]up to 7 500 THF Dithranol/AgTFA variation of cation [52]mixtures THF Dithranol/AgTFA influence of cation: time-lag focusing [55]40 000 CHCl3 various comparison of different matrices [56]125 000 THF IAA/Ag(acac) [57]up to 100 000 THF Dithranol/AgTFA, THF rate coefficients [73]1 500 000 THF all-trans retinoic acid highest polymer distribution recorded up to now [9]

PS-sulfonic acid 43 000 SA comparison of acid, salt [58]3 700 91 600 THF, water SA, DHB/water polym. analog. sulfonation [42]

PEG 23 600 THF HABA/THF varying polydispersity [43]35 000 THF HABA/THF comparison of different matrices [59]5 000 THF DHB/THF comparison to GPC [60]11 000 CHCl3 Dithranol/CHCl3 [56]

mixture HFIP Dithranol/HFIP influence of cation; time-lag focusing [55]600, reacted withchlorine acetic acid

THF IAA/THF detection of 3 distributions [20]

alkoxy terminated THF DHB/water separation by SEC [61]fluorescence labeled methanol HABA/1,4-dioxane time-lag focusing [62]

Nylon 6 3 000 6 000 TFE HABA/THF varying polydispersity [43]3 000 6 000 TFE HABA/TFE end group determinatio n, determination of

cyclic structures[63]

Polycarbonate 17 000 THF HABA/THF varying polydispersity [43]Polylactides 1 000 THF DHB, THAP/THF [64]Polybutyleneadipate 4 000 THF HABA/THF varying polydispersity [43]

39 000 THF HABA, DHB/THF GPC fractions [43]Polycaprolactone 10 000 THF HABA/THF varying polydispersity [43]Poly-THF 2 000 THF THAP/THF [20]

Polybutylmethacrylate 10 000 acetone IAA/acetone comparison GPC, laser light scattering [65]Polyesteralcohol 2 500 THF DHB/THF [20]PDMS 21 000 THF DHB/THF GPC fractions [45]

6 000 HPLC separ. of mixtures, detection of cyclics [66]5 000 CHCl3 Dithranol/AgTFA,

CHCl3

[56]

Polybutadiene 10 000 THF POPOP/Ag(acac) [57]5 000 CHCl3 Dithranol comparison of different matrices [56]

Polyisoprene 10 000 THF POPOP/Ag(acac), THF [57]5 000 acetone IAA/Ag, acetone comparison of different matrices [56]

Polyesters several 1 000(PEA; PBA; PTS)

acetone IAA/acetone acidolysis [67]

(aromatic; aliph.) THF DHB cyclic structures, differences to SEC [60](PET) THF Dithranol/AgTFA, THF influence of cation, time-lag focusing [55]

9000(adipic acid ester)

THF Ditrhaonol/AgTFATHF

transesterification with 1,4-butandiol [68]

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analysis of complex polymeric structures (homopoly-mers; block or random copolymers; mixtures)

influence of preparation conditions of the MALDIsamples (pH; type of preparation; matrix; salts addedfor cationization)

use of MALDI-MS as a detector for GPC, HPLC, LC.Simplifying, there are two main trends observable at the

moment:1. A great deal of work is devoted to basic research in

order to clarify the capabilities of MALDI-MS by lookingat the influence of molecular, preparation, and instrumen-tal parameters.

2. MALDI-MS is applied more or less routinely some-

times in connection with other separation techniques forthe analysis of the chemical composition of polymers,copolymers or polymer mixtures by determining possibleend groups and thus chemical heterogeneity distributions.

Examples from the literature and our own work for themost important points will be described in more detail inthe following. Basic research has been mostly performedby using standard polymers like PMMA or PS. To illustratethe second point listed above we have mainly chosen che-mical systems with relevance to applications.

3.1. Molecular weight distributions

After the first success with relatively high mass poly-mers (see Table 1) it turned out that one of the features

offered by the MALDI technique the possibility to eluci-date molecular weight distributions over a wide massrange has to be considered with some caution. Possiblereasons and remedies are described in this section. Thequestion arising was: Does MALDI-MS provide the cor-rect molecular weight distribution if the polydispersity ishigher than, say, 1.5?

Montaudo et al. [43] compared samples of varying poly-dispersity of PMMA, PEG, and PS by MALDI and GPCmeasurements. Molecular weight distributions agreed onlyfor very narrow distributions. Especially in cases of poly-dispersity A2, only spectra decreasing in intensity and cor-responding to the low molecular weight branch of the dis-

tribution are obtained (an example for PDMS is shown inFig. 9 below). The same holds for nylon-6 and poly(carbo-nate) samples. For polymers with narrow molar mass dis-tributions (polydispersity a 1.1) (see e.g. Lloyd et al. [47];PMMA and PS) good agreement between MALDI-MSand SEC results is found.

The discrepancy between MALDI-MS, which in princi-ple is a particle counting technique, and SEC results forbroader distributions led to consideration of the possiblereasons. C. Jackson et al. [48] demonstrated that the mostprobable peak value (Mp) and the shape of the distributioncurve as determined by MALDI-MS and SEC are a func-

tion of how the data are displayed. Mp is the mass value forthe most abundant point on the SEC curve. In general SECresults are presented as weight fractions versus the loga-rithm of molecular weight. Plotting the mass spectral data

Table 1. Continued.

Substance Mol. weight Solvent Matrix/solvent Remarks Ref.

Copolymers

PS-block-p(a-methyl-S) THF IAA/THF information on bothconstituent parts [69]

tri-block H(EO)n(PO)m(EO)nOH after LC separation [61]

p-Butyleneadipate-co-butylen-succinate (PBAS)

THF DHB, HABA/THF fractions from GPC [70]

bisphenol-A copolyester 64 000 correction procedure for Mwdetermination

[71]

PMMA/PS THF DHB/acetone, water chain transfer reaction [72]S/MA THF Dithranol/AgTFA, THF rate coefficients [73]MMA/n-BMA 2 000 THF DHB/Na-salt, THF chain length distribution [74]altern. p[(o-cresyl glycidylether)-co-formaldehyde]

acetone 1,4-diphenyl-butad iene time-lag focusing [75]

glycidylend-capped p[(bisphenol-A)-co-epichlorhydrin]

1,4-dioxane HABA/dioxane time-lag focusing [75]

p[[propylene glycol)-S-(ethyleneglycol)-S-(propylene glycol)] bis-(2 aminopropylether)

1,4-dioxane HABA/dioxane end group analysis [75]

PNVP/PVAc 1 700 THF IAA/acetone high resolution reflectron spectrum [76]Some special compoundsLignin 2 600 (maximum) acetone/

waterDHB/acetone [77]

Technical waxes 3 000 toluene,xylene

2-NPOE/AgNO3,xylene

results good at intermediate masses [78]

Phenolic resin novolacs several 1 000 acetone DHB/acetone detailed chem. composition [79]Epoxy novolacs several 1 000 THF Dithranol/LiCl, THF detailed chem. composition [79]

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as number fraction on a linear (or square root) mass scalehas a considerable influence on the shape of the spectrum.The SEC diagram thus obtained is much more similar to

the corresponding MALDI-MS spectrum plotted in thesame way (see Fig. 8).

A reasonable way to circumvent problems with broaddistributions is the MALDI investigation of samples frac-tionated by GPC. An example from Montaudo et al. [45]shows that a PDMS sample ofMp 30 000 Da could be char-acterized by measuring different GPC fractions. Of specialinterest is a comparison of the MALDI-MS spectrum ofthe unfractionated sample showing only the usual decreasein signal intensity of a broadly distributed polymer and thespectrum of a low-molecular weight fraction exhibitingthe expected shape (see Fig. 9) and resolution which

allows the determination of the end groups. By this charac-terization of GPC fractions MALDI-MS can be used for anabsolute mass calibration of the GPC so that an accuratedeconvolution of the chromatogram can be achieved.

Other examples are given by Danis et al. [76] for PMMA,PVAc and PNVP/VAc.

Although it has been shown (see [76]) by investigation

of mixtures of low polydisperse PMMA standards that theresponse of MALDI-MS is fairly constant between 6000and 60 000 Da, the loss of high molecular weight informa-tion just described arises with samples that have a lowmolar quantity of higher mass molecules. Systematicexperiments on mixtures of different PMMA and PS stan-dards have been performed by K. Martin et al. [80]. Thehigher mass share of the mixtures was found to be defi-nitely underrepresented in the MALDI spectra. Theauthors pointed out that higher laser power would benecessary for the desorption/ionization process of thehigher molecular weight polymers. Whereas the detection

efficiency of conventional GPC detectors such as UV-absorption, refractive index or light scattering is very higheven for small quantities of high mass material, in massspectroscopy, which counts the number of molecules, the

Fig. 8. Comparison of MALDI-MS and SEC-spectra. Top: MALDI mass spectrum of PEG (Mp = 1400 Da.) Bottem left: SEC spectrum ofthe same PEG as above (Mp = 4000 Da). Plotted is the weight fraction over a logarithmic mass scale. Bottom right: Plotted is the numberfraction over the square root of the molecular mass. (Reprinted with permission from [48].)

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high mass signal is easily lost in the noise of the baseline.Detector saturation also seems to be of importance for thedecrease of signal intensity at high molecular weights (seeSect. 3.2).

Effects of instrumental settings on the spectra will bedescribed in the next section.

3.2. Influence of instrumental parameters

Since the beginnings of MALDI-MS applications forpolymers it turned out that ionization and detectionmechanisms have an influence on the mass range detect-able and the shape of the distribution curve so that para-meters such as preparation conditions (dried-drop, spincoating, spraying), matrices, addition of salt, type of analy-zer (linear or reflection), and acceleration voltage are ofprime importance. Some of these points will be elucidatedin this section.

The matrices used most frequently for MALDI-MS arementioned in Table 1. The influence of different cationsadded to the matrix solution on the spectra of PMMA,

PEMA, PBMA and PHMA is reported by Lloyd et al. [81].A typical result is shown in Fig. 10. Following the argu-mentation of Thomson et al. [82] based on the principle ofhard and soft acids and bases, it can be concluded at least

in partial agreement with experimental practice that softbases (accessible valence electrons like the aromatic p-electrons of PS) might be compatible with soft acids suchas silver atoms with their unshared electrons. On the otherhand polar molecules with hard basic sites (O, N) such asPEG, PMMA, PVP should complex more readily withhard acids like the sodium or potassium cation.

The influence of different matrices (see Fig. 11), laserpower and effects of different end groups on the ion yield

are studied in a comprehensive paper by Belu et al. [56]. Inaddition and as a confirmation of the remarks made above,the cationization potential of different cations for variouspolymer structures can be inferred from Table 2, taken

Fig. 9. Top: MALDI-TOF mass spectrum of unfractionated PDMS30 000. Bottom: MALDI mass spectrum of a low molecular weight

fraction of the PDMS 30 000; A denotes Li adducts, B Na adducts.(Reprinted with permission from [45].)

Fig. 10. Influence of alkali salts (Na+/Cs+) on the MALDI massspectrum of PMMA. (Reprinted with permission from [81].)

Fig. 11. Comparison of MALDI-TOF mass spectra obtained for PS5000 using different matrices: IAA (top), NPOE (middle), anddithranol (bottom). No oligomer signals were obtained for thematrices HABA, SA and carbostyryl. AgTFA was used as cationi-

zation agent in all sample preparations. (Reprinted with permissionfrom [56].)

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from [56]. According to our own experience a too highlaser power (see also [80]) shifts the maximum of the dis-

tribution to lower mass values, most probably due toincreased fragmentation processes; on the other handhigher molecular weight oligomers become detectable butmass resolution is reduced as demonstrated in Fig. 12.Furthermore, fragmentation can be observed in the case ofPS. For a series of endfunctionalized PSs([1Si(CH3)31C6H5], [1Si(CH3)2 (CH2)2C6F13] no cleartrend in the relative ion yields in mixtures with H-termi-nated PS were found [56].

Instrumental effects in the analysis of polymers withlarge polydispersity are tackled in a recent paper by Mc-Ewen et al. [37]. The authors use a PMMA sample with a

polydispersity of 1.8 for the recording of MALDI-MSspectra in the linear and reflection mode. Whereas thespectrum in the linear mode more or less shows a simpledecay, the spectrum recorded in the reflection mode

ressembles more closely the corresponding SEC result. Inboth MALDI-MS modes (linear and reflection) the maxi-mum detectable mass is around 20000 Da whereas withSEC polymers up to 40000 Da can be detected (seeFig. 13). By gating the signal such that only a narrow pulseof ions in a selected mass range reaches the detector in thereflectron mode, these authors demonstrated that ions

above 25000 Da can be detected directly from the originalsample without fractionation. From the results of thispaper it must be concluded that the MALDI process is cap-able of producing ions over the entire molar mass distribu-tion, but that the high-mass tail cannot be detected whenlower mass ions are allowed to reach the detector; thus onereason for high-mass discrimination in highly polydispersesamples is detector saturation.

Another point well-known in MALDI-MS is the factthat higher acceleration voltages expand the detectablemass range (see Tang et al. [83]). This is due to the detec-tion system (mostly channel-plates for the transformation

of ions into electrons), for which a minimum velocity ofthe incident particles is necessary to achieve a usable sig-nal, especially for high mass species. Another examplefrom our lab is shown in Fig. 14.

Table 2. Metal salts added to the matrix in order to cationize poly-mer chains.

Metals that serve to cationizepolymer chains when added

to the matrix

a

Polymer AgAlkali metals

(i. e., Li, Na, K)

(1CH2C0

a

H )1

PS

Yes No

(1CH2CH2CHCH2 )1PBD

Yes NA

CH30

(1

CH2C2

CHCH2 )1

PI Yes NA

(1CH2CH2 )1

PENo NA

(1CH2CH2O )1

PEONo Yesb

(1CC0

CH3

0C2O0

OCH3PMMA

H2 )1Yes Yes

(1SiO0

CH3

0CH3

PDMS

)1Yes Yesb

a NA= not attempted.b As determined from published reports.

Fig. 12. Influence of laser power on a MALDI mass spectrum ofPMMA 4500 (dithranol/THF, KTFA). The loss in resolution is evi-dent. Laser fluence is doubled (lower trace) from threshold (uppertrace) to higher values (lower trace).

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3.3. Chemical composition of polymers

After the description of some pitfalls of MALDI-MS inparticular with respect to the determination of molecularweight distributions in this section we will give an impres-sion of the benefits of this method.

Often synthetic polymers are not only characterized bydifferences with respect to their molar mass distributions,they also exhibit chemical distributions (i.e. different func-tional groups as well as different sequences of monomers

and different sequence length) and structural heterogene-ities (i.e. linear, cyclic, grafted, branched parts). In a vari-ety of publications it is demonstrated that MALDI-MSalone and in combination with various separation techni-

ques such as SEC, HPLC and LC is a very useful tool forthe elucidation of these chemical structures.

As already mentioned in the introduction, mass resolu-tion obtainable in MALDI-MS allows the detection of sin-gle oligomers and the determination of end groups. Someexamples, mainly from technically relevant polymers,described in the following will illustrate the potential inpolymer research. New developments for MALDI-MSsuch as time-lag focusing (see section 3.6) will furtherenhance the capabilities of MALDI-MS in this area.

Montaudo et al. [63] describe the analysis of differentlyterminated nylon-6 samples (diamino-, monoamino-,dicarboxyl- and amino-carboxyl terminated). Cyclic oligo-mers characterized by a mass difference of 18 Da to the

Fig. 13. Wide polydisperse (D = 1.8) PMMA 17 000. Reflectron mass spectrum (top) MALDImass spectrum (bottom) of the wide polydisperse PMMA 17 000 standard with the parent ionselector set to transmission of ions of mass 28 000 with a 4000 Da window. (Reprinted from [37].)

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main oligomer series could be detected and a completestructural characterization of the molecular species presentcould be achieved. An example from our laboratory onpolysulfones for the structural elucidation of by-productsis shown in Fig. 15.

A series of aliphatic polyesters has been studied by Wil-liams et al. [67], revealing asymmetric oligomer distribu-tions, hetero-terminated linear chains and cyclic oligo-mers. Comparison to ESI-MS, NMR and end group titra-

tion results confirm the MALDI-MS interpretation of thechemical composition. The feasibility of acidolysis for thestructural characterization of higher molecular weight spe-cies or insoluble portions of the starting material wasexplored by following the progress of the reaction in theMALDI spectra.

The mode of termination for a thermally initiated freeradical polymerization of styrene and MMA with AIBN asinitiator has been evaluated by Zammit et al. [84]. For lowmolecular weight molecules excellent agreement betweenMALDI-MS and SEC data was achieved. As widelyaccepted, termination can occur via either disproportiona-

tion or combination, resulting in chains with one or twoinitiator fragments, respectively. The ratios of the termina-tion modes (disproportionation to combination)determinedfor MMA and styrene were 4.37 l 1.1 and 0.057 l 0.032,

respectively, at 908C, in excellent agreement with literaturedata (see [84]). In the case of styrene even subtle propertiesof the reaction could be revealed: i.e. a Diels-Alder rearran-

gement product due to thermal initiation as well as a chainscission product dueto theMALDI conditions.Spickermann et al. [12] clearly demonstrate that quanti-

fication of oligomers with different end groups represents

Fig. 14. Polymer standard PMMA 12 300 (dithranol/THF, KTFA)measured by MALDI-MS at 30 kV (top) 20 kV (bottom) accelerat-ing voltage, resulting in lower intensity of the spectrum and a slightalteration of the molecular weight distribution.

Fig. 15. Top: MALDI mass spectrum of polysulfones (dithranol/THF, AgTFA). The insert shows details of the spectrum. Bottom: Aconsistent assignment of the different signals.

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a challenge. The authors describe the characterization ofoligostyrene macromonomers by isocratic and gradientHPLC and MALDI-TOF MS. Though the molecularweight distributions coincide between the different techni-ques, mixing experiments revealed that the MALDI peakareas do not represent the mixing-ratio of the different oli-gomer types. Thus conclusions on concentration ratiosdirectly from the MALDI spectra have to be consideredwith caution as ionization probabilities for different spe-

cies are not known a priori. In the case of the macromono-mers the MALDI results lead to an identification of newcoupling products formed as impurities during the courseof the anionic polymerization.

Further examples describing the combination ofMALDI-MS with other separation techniques will bedescribed below.

The use of MALDI-MS as a powerful (off-line) alterna-tive to conventional refractive index or ultraviolet detec-tion in the liquid chromatography of polymers is describedby Pasch and Rode [61]. Figure 16 shows an example fortechnical polyethylene oxides, widely used as surfactant

additives, under chromatographic conditions, in whichchain length as well as the end groups direct the separation in contrast to the normal SEC mode giving separation bythe hydrodynamic radius of the polymer species. Mainly

three fractions result, all showing a peak-to-peak massincrement of 44 Da, demonstrating that all fractions con-sist of ethylene oxide-based oligomer chains. The endgroups calculated from the spectra for the three fractionsare 18 Da (+44n) for fraction 1, 200 Da (+44n) for fraction

2 and 228 Da (+44n) for fraction 3, n denoting the numberof repetition units. Under the assumption that the materialinvestigated is a pure fatty alcohol ethoxylate the endgroups can be assigned to PEG (a,x-dihydroxy endgroups), C13-terminated PEO (a-tridecyl x-hydroxy endgroups) and C15-terminated PEO (a-pentadecyl-x-hydroxy end groups), respectively. The materialsdescribed are good examples of a technically importantclass of surfactants that might be favorably characterizedby MALDI-MS.

Other examples of successful combinations withMALDI-MS are given by Kruger et al. [20, 64]. Linear andcyclic fractions of polylactides can be separated. The

authors use the liquid adsorption chromatography at criti-cal conditions (LCACC) for the preseparation. Even incombination with conventional GPC (see above) MALDI-MS can reveal a variety of information. On-line couplingof these two methods using MALDI-MS as an on-lineGPC detector is possible as shown by Fei et al. [85]. Theinvestigation of HPLC fractions by MALDI-MS isdescribed by Just et al. [66]. The authors were able to sepa-rate cyclic siloxanes from linear silanoles and to character-ize their chemical heterogeneity. MALDI-MS is also suita-ble for investigating thin-layer chromatography platesdirectly. A quantitative analysis can be achieved for

cocaine hydrochloride as demonstrated by Nicola et al.[86].The examples presented in this section clearly indicate

that MALDI-MS as a detector for other separation techni-ques represents an interesting means for the detailed char-acterization of the composition of polymers not only withrespect to molar mass distribution but also for their chemi-cal composition. Furthermore, it can be very helpful forGPC calibration in cases where no standards are available(see [63]).

3.4. Characterization of particular chemical reactions

That MALDI-MS due to its mass resolution and sensi-tivity can be used for the characterization of the productsof a chemical reaction in general has been describedalready in sect. 3.3. Some examples with emphasis on spe-cial types of reactions will be given in the following.

MALDI-MS has been used for molecular weight deter-minations for the emulsion polymerization of MMA andMMA-styrene. Suddaby et al. [10] found that catalyticchain transfer polymerization is an extremely effectivemethod to control the molecular weight even under emul-sion conditions.

Pasch et al. [46] reveal that in group-transfer polymeri-zation of MMA, in addition to the expected linear oligo-mers, those with cyclic end groups (back-biting) areformed. It is shown that for technical products also cyclic

Fig. 16. Separation of a technical polyethyleneoxide by liquidchromatography at the critical point of adsorption and analysis offractions by MALDI-MS. Peak assignment indicates degree ofpolymerization. Column Nucleosil 100 RP-18 (125 N 4 mm I.D.);

eluent acetonitrile/water (70:30 v/v). (Reprinted with permissionfrom [61].)

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end groups can be formed in significant amounts, whichmay influence the application performance of the material.

The mechanism of the anionic copolymerization ofanhydride-cured epoxides was investigated by Leukel etal. [87]. They investigated the strictly alternating copoly-

merization of phenyl glycidylether and phthalic anhydride,initiated by different imidazoles. Especially the questionof whether the initiator remains chemically bound duringthe whole course of the reaction was not solved unambigu-ously. That this in fact is the case was proved by an endgroup analysis by means of the MALDI-MS spectra.

A newly frequently used powerful tool to investigatefree radical propagation rate coefficients is pulsed laserpolymerization followed by a determination of the mole-cular weight distribution. Schweer et al. [73] demonstratethat, for the analysis of the free radical polymerization ofstyrene and MMA, MALDI-MS can be used successfully.Propagation rate constants could be obtained in good

agreement with SEC results. Another example for thedetermination of propagation rate constants for MMA isgiven by Zammit et al. [88].

The polymerization of MMA using zirconocene initia-tors was investigated by Li et al. [89]. MALDI mass spec-tra of low-molecular weight species reveal that this processdoes not represent a living polymerization and that back-biting cyclization is involved.

Thomson et al. [90] deal with the surfactant free emul-sion polymerization of MMA with ammonium persulfateinitiator and especially the remaining water soluble MMA-based oligomers after polymerization. Background is the

general knowledge that latex stability is provided by thenegatively charged sulfate groups remaining at polymerchain ends and by the in-situ surfactant formation during

the aqueous phase initiation of the reaction. MALDI couldgive valuable hints on these so-called in-situ surfactants.So the mean number of MMA repeat units was found byMALDI-MS to be 8 to 9. This appears to be the first directmeasurement of surfactants produced in-situ from mono-

mer and persulfate initiator.

3.5. Some selected examples of MALDI-MS application to

non-standard polymers

As can be inferred from the literature and Table 1, agreat deal of work has been performed for polymer stan-dard materials and mostly for homopolymers. Work oncopolymers is only rarely reported. As the finding of anefficient matrix for a special polymer is a crucial point it isevident that the finding of a suitable matrix for two ormore types of monomers might become a limiting factorfor the MALDI-MS analysis. The examples presented inthis chapter will elucidate that nevertheless valuable infor-mation can be extracted in these cases from the spectra.

Wilczek-Vera et al. [69] investigated block copolymersof PS-block-(p-a-methylstyrene). For polymers in themass range up to 10000 Da interesting details could beobtained from the data. So the individual block-length dis-tribution derived from the spectra is in good agreementwith predictions from a Schulz-Zimm model. Furthermore,the authors confirm that block copolymers with narrowmolecular weight distributions may have complex andeven bimodal compositional distributions. It was found

that the polydispersity factors observed for the individualparts could be higher than for the whole polymer. Theblock copolymer was prepared by sequential anionic poly-

Fig. 17. Bivariate distribution in composition and chain length obtained from the deconvolution of the corre-sponding MALDI mass spectrum. Here composition of the random copolymer of MMA and BMA is expressedas the number of BMA units. The degree of polymerization is given as DP, the vertical scale gives the relativeintensity. (Reprinted with permission from [74].)

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meriziation with secondary butyllithium as initiator. Underthe reaction conditions chosen the butyl segment is fol-

lowed by a short a-methyl-styrene segment, then a styrenesegment followed again by another short a-methylstyrenesegment, so that in fact a triblock copolymer is formed.

A random copolymer of MMA and n-butyl MA synthe-sized by free radical copolymerization was investigated bySuddaby et al. [74]. By deconvolution of the mass spectraboth the composition and chain-length distribution ofMMA/BMA copolymers could be obtained simulta-neously. The distribution in composition and chain-lengthobtained is shown in Fig. 17. The authors developed amethodology which allows the determination of two reac-tivity ratios, two chain-transfer constants, and a measure ofthe reinitiation reaction by using these reaction conditions.

Condensation polymers such as phenolic novolacs orresols were investigated by Pasch et al. [79]. They demon-strated that for polycarbonates and polyesters different endgroups and cyclic oligomers can be found depending onthe reactant ratio (see Fig. 18). Reaction products charac-terizing the product spectrum are shown in the scheme ofFig. 18.

Although polar materials can be investigated favorablywith the MALDI technique, reports on nonpolar speciesare scarce. The following two cases deal with hydrocarbonmolecules. As these examples are to our knowledge theonly ones described up to now, we give the preparation

conditions in somewhat more detail, which, in our experi-ence, have to be followed strictly. Kuhn et al. [78] describethe characterization of technical waxes in the mass rangeup to 3000 Da. Their sample preparation was as follows: 2-

Fig. 18. Top: MALDI mass spectrum of ethylene glycol-terephtha-lic/isophthalic acid polyester. The inset shows an enlarged part of

the spectrum indicating different molecular ions and homologousseries; matrix DHB, solvent THF. Bottom: a scheme for the assign-ment of the different signals of the spectrum above. (Reprinted withpermission from [79].)

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nitrophenyl octyl ether (1% solution in xylene) wasdropped onto the sample holder. After drying, silver nitrate(5% solution in water) was added and the matrix driedagain. The samples (15 mg/ml in toluene or xylene) aredropped onto the matrix/salt layer. A co-crystallization of

matrix and sample is assumed because the aromatic sol-vents used for the samples should redissolve the matrix.

The investigation of polyisoprene (Mp 11000 Da) andpolybutadiene (Mp 10000 Da) is reported by Danis et al.[57]. Here sample preparation is the following: by ultraso-nication for 30 min saturated solutions of POPOP (1,4-di-(2-(5-phenyloxazolyl)) benzene) and silver(I) acetonyl-acetate in THF were prepared. The solutions were centri-fuged and the supernatant solution used for the prepara-tions. A 10:1:1 v/v/v mixture of the POPOP, silver saltand polymer solution was prepared and applied to the sam-ple holder.

That it is possible to characterize the molecular structure

of the hard segments of the polyurethane foams widelyused in the automotive industry with respect to chainlength distribution and end groups has been demonstratedin the authors lab [91]. It was found that the length of thethe hard segments decreases much more slowly for poly-ester foams than for polyether foams. This fact was attribu-ted to a lower solubility of the hard segments in polyethers,leading to an earlier onset of phase separation.

3.6. Recent developments

Key instrumental parameters of MALDI-MS are massresolution and mass accuracy, which allow the separationof single oligomers and end group determination. For con-ventional instruments, resolution of single oligomersceases depending on the mass of the repetition unit somewhere between some thousand and perhaps20000 Da. Consequently there have been attempts toenhance mass resolution by coupling the MALDI principlewith mass spectrometers of higher mass resolution, e.g.Fourier transform (FT) ion cyclotron resonance (ICR)mass spetrometers as described by van Rooij et al. [92].They achieve a mass resolution of 6000 at mass 4000 and amass accuracy of 15 ppm. Unit mass resolution for a PEG

4000 could be achieved. It is interesting to note that evenbefore the application of MALDI-MS to synthetic poly-

mers there have been experimental efforts to apply laserdesorption FT-MS without using a matrix. Brown et al.[93] achieved well-resolved spectra for PS, PEI, poly(ethy-lene glycol methyl ether) (PEG-ME) and PEG up to Mpvalues of about 6000 Da by using a CO2 laser for ionization

and the addition of alkali chlorides (Na; K) to the samples.Recent developments in MALDI-MS make use of the

long-known fact that by the so-called delayed extraction ortime-lag focusing (see sect. 2.3), first applied in 1955 byWiley and McLaren [38], mass resolution and signal-to-noise ratio is enhanced. This technique has been appliedwith great success to the analysis of synthetic polymers[75].

Jackson et al. [55] describe the application of time-lagfocusing to polymer standards such as PS, PMMA, PEGand PET samples. An example from our own work isshown in Fig. 19. In a recent publication oligomeric reso-lution for PS is achieved up to 50 000 Da [94].

The value of time-lag focusing becomes evident in theanalysis of more complex systems. Schriemer et al. [75]apply the method to the analysis of copolymers. For thealternating copolymer of poly[(o-cresyl glycidyl ether)-co-formaldehyde] and glycidyl end-capped [(bisphenolA)-co-epichlorhydrin], apart from molecular weight distribu-tion, detailed information on structure and compositionwas obtained directly from the spectra. Analysis of blockcopolymers and random copolymers is found to be moredifficult because, due to the higher chemical complexity,the demand for mass resolution of single oligomers isincreased. Prior knowledge of end groups and repetition

units nevertheless could allow the determination of the dif-ferent molecular sequences composing a given oligomer.The assignment for a typical repeat pattern as shown inFig. 20 is given in Table 3 [75]. The authors also deal withthe question of whether there is a constant oligomer detec-tion efficiency over the whole molecular weight distribu-tion. They developed a model which removes the need forthe quantification of the absolute oligomer intensity whenthe purpose of the investigation is to compare the com-pound to a standard product.

For time-lag focusing in general a square wave pulse isused, which is mass-dependent [38]. In a recent paperWhittal et al. [95] suggested a special pulse form called

functional wave time-lag focusing which should providea high mass accuracy over a broad mass range. For record-

Table 3. Comparison of experimental mass data and calculated values for several peaks shown in Fig. 20.

Measd. mass [Da] Proposed EO/POa Calcd. mass Mass accuracy [%] Proposed EO/POa Calcd. mass Mass accuracy [%]

1798.03 36/2 1798.10 0.004 7/24 1798.26 0.0131842.12 37/2 1842.13 0.001 8/24 1842.29 0.0051886.12 38/2 1886.15 0.002 9/24 1886.31 0.0101930.18 39/2 1930.18 0.000 10/24 1930.34 0.0081974.16 40/2 1974.21 0.002 11/24 1974.37 0.0102018.11 41/2 2018.23 0.006 12/24 2018.39 0.014

2062.11 42/2 2062.26 0.008 13/24 2062.42 0.0152106.10 43.2 2106.29 0.009 14/24 2106.45 0.016

a The ratio between the number of ethylene oxide repeat units and the number of propylene oxide repeat units.

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Fig. 19. Top: MALDI mass spectrum of PMMA 34 000 (dithranol/THF, AgTFA) recorded in the delayed extraction mode. Bottom: Theextension of the spectrum clearly demonstrates that single oligomers can be resolved in the linear and in the reflectron mode. The spectrumwas recorded in collaboration with Dr. Mayer-Posner, Bruker-Franzen Analytik GmbH, Bremen, Germany.

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ing the mass spectra of a mixture of proteins between 5700Da and 29000 Da they use a pulse that decreases in ampli-tude with time. This principle should be applicable to theanalysis of synthetic polymers also.

As already pointed out in Sect. 2, matrices and samplepreparation are crucial points for the applicability ofMALDI-MS. There have been several attempts to find newmatrices and special preparation conditions to enhance thedesorption and ionization process. Nevertheless samplepreparation for MALDI-MSis still a more-or-less empiricalprocess. So there should be remaining potential from this

side for improvementsof mass range andsensitivity.Probably all MALDI-MS spectroscopists have encoun-

tered the effect that when the laser beam hits the sample, agreat variance in quality of the spectra for different areasof the sample can be observed because of the heterogeneityof the matrix/analyte distribution. Therefore variousattempts have been made towards more homogeneoussample preparations. In a recent patent application, Bie-mann et al. [96] claim a method for producing continuoushomogeneous layers of MALDI matrix materials that aresubstantially free of voids and large crystals. They describea spray nebulizer principally based on a device used for thedeposition of HPLC fractions. The important point of the

invention is the use of a non-reaction sheath gas that con-fines and entrains the spray, so that substantial solvent eva-poration occurs before the matrix material hits the target.

Perera et al. [97] report improvements of MALDI spec-tra by spin-coating solutions of matrix and analyte onto atarget that rotates at 300500 rpm. A special technique formatrices like DHB, SA, ferulic acid and so on has beenpublished by Allwood et al. [98]. They deposit a saturated

matrix or matrix/analyte solution on a soda lime glass andcrush the films of a thickness between 45 and 60 lm withpolished aluminum plates in order to break up larger crys-tals. Such crushed samples exhibit a lower threshold flu-ence for matrix ion detection than standard dried-dropletsamples. Drying the samples at refrigerated temperatures(l2.58C) results in samples of much better macroscopicuniformity than samples dried at room temperature. Thesefew points should indicate that really subtle effects caninfluence the ion yield in the MALDI process.

Although the goal of this review is primarily an over-view of synthetic polymers, we would like to mention atleast some interesting results reported recently from some

other areas of current chemistry.The design of a MALDI probe in transmission geometry

as reported by Lennon and Glish [99] may further broadenthe applicability of MALDI-MS. The key point is a quartzsample holder fitted to the end of a glass fiber, whichguides the laser light necessary for desorption of the sam-ple. This geometry allows use even in spatially constrainedion source regions such as in quadrupole mass spectro-meters for example. An advantage of the approachdescribed is the fact that with just one fiber optic, a laserand the probe described, MALDI can be performed onmultiple instruments in the lab. The authors have been able

to obtain a spectrum ofa

50 fmol of total loading of bom-besin, MS/MS has been performed on 5 pmol of des-Arg 9-bradikinin.

Li et al. [100] present an approach for analyzing cellularproteins from one single erythrocyte with a total cellularvolume of 87 fl by MALDI-MS. A microspot sampledeposition system with fused silica capillaries is used. Twopeaks from apohemoglobins in the red blood cell could bedetected.

The direct analysis of non-peptide bead-bound combina-torial libraries is reported by Brummel et al. [101]. In com-parison to electrospray and time-of-flight secondary ionmass spectrometry it is shown that also MALDI-MS can

provide accurate molecular weight information on anangiotensin-II-antagonist target molecule synthesized on a40 lm PS bead. As there are first hints of the application ofcombinatorial methods in the area of functional polymers[102] future applications of MALDI analysis might occurin this very active branch of research.

Especially dendrimeric and fullerene compounds arevery suitable for investigation by MALDI-MS: in generalthe molecular weight distribution is narrow, the mass rangeis up to several 1000 Da, and as a soft ionization techniqueMALDI-MS allows the investigation of fragile structuressuch as are sometimes encountered in these two areas ofrecent research interest.

For example, Lorenz et al. [103] used MALDI-MS forthe characterization of carbosilane dendrimers withrespect to polydispersity. The molar mass characterization

Fig. 20. a) MALDI mass spectrum of poly[(propylene glycol)-b-(ethylene glycol)-b-(propylene glycol)]bis(2-aminopropylether). b)Expansion of the spectrum (reprinted with permission from [75])An assignment of the peak pattern is given in Table 3.

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of carbosiloxane dendrimers is described by Sheiko et al.[104]. Seebach et al. [105] use the method for the identifi-cation of dendrimers from (R)-3-hydroxybutanoic acid andtrimesic acid.

Ballenweg et al. [106] describe MALDI-TOF-MS as a

quick and facile method for the characterization of fuller-enes and fullerene derivatives as cyclopentane addends on[60]-fullerene. Isaacs et al. [107] describe the use MALDI-MS for the characterization of an C60 hexa-adduct (2 sub-stituted 1,3 butadiene). In many cases MALDI-MS gives adefinite and direct prove of the structure obtained.

4. Outlook

We believe that the material collected for this reviewwith a focus on synthetic polymers represents impressiveconfirmation that MALDI-MS has become a highly valu-

able tool for the molecular analysis of polymers in thespace of just a few years. Its possibilities range from deter-mination of molecular weight distributions to informationon chemical heterogeneities and the end products resultingfrom complex reaction mechanisms. In combination withother separation techniques, a great deal of detailed infor-mation on the molecular constitution, in many cases eventhe complete product spectrum, can be revealed.

Just recently the mass resolution has been increased con-siderably by pulsed extraction techniques, allowing, forexample, the separation of single oligomers of highermolecular weight distributions and a more exact determi-nation of the end-group chemistry due to enhanced mass

accuracy. Future developments will possibly concern on-line capability in combination with other separation tech-niques, further increase in sensitivity and stability, newinsights into the ionization mechanisms, new matrices, andpossibly procedures to circumvent detector saturation. Thequestion of the quantification of different species, espe-cially in broad molecular distributions, remains an impor-tant challenge for the future.

As a rather new technique in the polymer area thereseems to be enough potential for a prosperous develop-ment of MALDI-TOF-MS.

Acknowledgements

Continuous support of Prof. Dr. K. Mullen, Prof. Dr.W. Spiess and the Bundesministerium fur Bildung undForschung (BMBF project No. 03N6010A) as well as dili-gent assistance during measurement and interpretation byP. Boshans, H. Kullmann and G. Lupa is gratefullyacknowledged.

List of Abbreviations

AgTFA silver trifluoro acetateAIBN azo-bis-isobutyronitrile

BMA butylmethacrylateDa Daltondithranol 1,8,9-trihydroxy-anthraceneDHB 2,5-dihydroxy-benzoic acid

ESI-MS electrospray ionization mass spectro-metry

FWHM full width at half maximumFAB fast atom bombardmentFD field desorption

GPC gel permeati on chromatographyHABA 2-(4-hydroxy-phenylazo) benzoic acidHFIP hexafluorine-isopropanolHPLC high pressure liquid chromatographyIAA 3,b-indole acrylic acidKTFA potassium trifluorine acetateLC liquid chromatographyLD laser desorptionMALDI-TOF-MS matrix-assisted laser desorption ioni-

zation time-of-flight mass spectrome-try

Mn number average of the molecularweight distribution

Mp most probable peak of the molecularweight distribution

Mw weight average of the molecularweight distribution

PBMA poly (butyl methacrylate)PD 252Cf plasma desorptionPDMS poly(dimethyl siloxane)PEG poly(ethylene glycol)PEI poly(ethylene imine)PEMA poly(ethylene methacrylate)PET poly(ethylene terephthalate)PHMA poly(hexene methacrylate)

PMMA poly(methyl methacrylate)PNVP poly(N-vinyl pyrrolidone)PS poly(styrene)PVAc poly(vinyl alcohol)PVP poly(vinyl pyrrolidone)THAP 2,4,6-trihydroxy acetophenone

hydrateTHF tetrahydrofuranVAc vinyl alcohol

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Received December 12, 1997

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