Mass Spectra of Copolymers: article from Mass Spectrometry Reviews, 2002, 21, 108–144

MASS SPECTRA OF COPOLYMERS Maurizio S. Montaudo Instituto per la Chimica e la Tecnologia dei Materiali Polimerici, Consiglio Nazionale delle Ricerche, viale A. Doria 6, 95125 Catania, Italy Received 25 January 2002; revised 14 May 2002; accepted 28 May 2002

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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II. Structural Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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III. Copolymer Composition . . . . . . . . . . . . . . . . . . . . . . . . . . A. Composition by the Direct Method . . . . . . . . . . . . . . . B. Composition by the Method Based on Statistics . . . . . . C. Copolymer Composition by the Hard-Ionization Method.

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IV. Copolymer Sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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V. Molar Mass Determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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VI. MS as a Detector for Liquid Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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VII. Composition Drift and the Compositional Distribution Histogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII.

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Quantitation and Methods to Correct Mass Spectral Intensities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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IX. Copolymer Mixtures and Blends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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X. Copolymers Obtained by Specific Synthetic Routes . A. Copolymers Produced by Enzymes or Occurring B. Block Copolymers . . . . . . . . . . . . . . . . . . . . . C. Random Addition Copolymers . . . . . . . . . . . . D. Random Condensation Copolymers . . . . . . . . .

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XI. Summary and Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Recent and older literature (covering the last 12–13 years) in the field of mass spectra of random and block copolymers is reviewed. A detailed description is given of the information on copolymer properties that can be recovered from the analysis of the low-mass region of the spectrum (the region below 500 Da) and the high-mass region. The features of mass spectra of copolymers obtained by different synthetic routes are discussed, such as free radical, condensation, ring-chain equilibration, microbial synthesis, ring-opening, simple anionic, cationic, Ziegler-Natta, and/or metallocene catalysis, along with some random and block copolymers that occur in Nature. The emphasis is on copolymer composition and average molar mass determination, and on the benefits of coupling mass spectrometry (MS) with separation techniques such as size-exclusion

———— Contract grant sponsor: National Council of Research (CNR, Rome). *Correspondence to: Maurizio S. Montaudo, Instituto per la Chimica e la Tecnologia dei Materiali Polimerici, Consiglio Nazionale delle Ricerche, viale A. Doria 6, 95125 Catania, Italy. E-mail: [email protected]

Mass Spectrometry Reviews, 2002, 21, 108– 144 # 2002 by Wiley Periodicals, Inc.

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chromatography (SEC) and high performance liquid chromatography (HPLC). # 2002 Wiley Periodicals, Inc., Mass Spec Rev 21:108–144, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mas.10021 Keywords: copolymer composition and sequence; copolymer molar mass; block copolymers

I. INTRODUCTION

Random and block copolymers are quite common materials, and thousands of tons are produced each year. Although most of them do not occur naturally, the equipment (i.e., the mass spectrometer) used to analyze them is identical to those used to analyze proteins and nucleic acids. The only difference consists in sample preparation, because biomolecules are often water-soluble, whereas random and block copolymers are often water-insoluble and one has to use organic solvents.

MASS SPECTRA OF COPOLYMERS

The characterization of random and block copolymers is sometimes complex, due to the large number of quantities to be determined; namely, the molar mass distribution (MMD) and MMD averages, the structure of the repeat units (here, A and B are the repeat units), the branch points, the estimation of the relative abundance of macromolecular chains terminated with one type of end group or another, the average molar fraction of A and B units in the copolymer, the average length of long AAAA and BBBB blocks, the weight of copolymer chains that possess a given composition (referred to as the compositional distribution histogram), the variation of copolymer composition as the molar mass of the macromolecular chain grows (usually called ‘‘composition drift’’), and in solid samples, the variation of copolymer properties when moving from the surface to the bulk. The great promise of mass spectrometry (MS) is to determine all these quantities at one time, by simply recording the copolymer’s mass spectrum and (carefully) decoding the information contained in it. In the present review, it will be shown that some success in this field has been achieved, and this success represents a great advance, because, prior to MS, the task of full copolymer characterization required the use of a combination of methods of analysis (Bark & Allen, 1982; Elias, 1984; Mitchell, 1987; Allen & Bevington, 1989; Pethrick & Dawkins, 1999). The average molar masses (MM) and the molar mass distribution (MMD) in copolymers is usually determined by light scattering (LS), or size-exclusion chromatography (SEC), and that topic is thoroughly discussed elsewhere (Elias, 1984; Mitchell, 1987; Provder, Barth, & Urban, 1995). Mass spectrometers equipped with electron impact (EI), chemical ionization (CI), or other hard-ionization ion sources cannot give any information on the average MM and the MMD, because the polymeric chain breaks apart and it is no longer intact. Therefore, soft-ionization techniques such as fast atom bombardment (FAB), electrospray ionization (ESI), and matrix-assisted laser desorption/ionization (MALDI) must be used. Desorption chemical ionization (DCI) and secondary ion mass spectrometry (SIMS) are soft-ionization techniques, too; however, it is often difficult to avoid that some polymeric chains break apart with those methods. To our best knowledge, this review is the first to focus on copolymers. Some fields, however, have been reviewed; namely, FAB applied to polymers (Montaudo, Scamporrino, & Vitalini, 1989a; Montaudo, 2001a), MALDI applied to polymers (Raeder & Schrepp, 1998; Nielen, 1999; Montaudo, Montaudo, & Samperi, 2002b), SIMS applied to polymers (Hercules, 2002), and the entire field of MS of polymers (Scamporrino & Vitalini, 1999; Scrivens & Jackson, 2000; Hanton, 2001). In the following, it will be demonstrated that mass spectrometry (MS) can be successfully used to analyze

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random and block copolymers produced in a wide variety of synthetic pathways.

II. STRUCTURAL ANALYSIS

When the chemical structure of the copolymer sample is unknown, one usually uses atomic absorption (AA) to reconstruct the ‘‘raw formula’’ of the chain, and one records the infrared (IR) or the nuclear magnetic resonance (NMR) spectrum (Koenig, 1999) to detect the chemical groups present in the repeat units. The method is identical for low MM and high MM compounds (Williams, 1973; Cooper, 1980; Shriner, Fuson, & Curtin, 1980; Silverstein, Bassler, & Morril, 1991). The structural analysis of copolymers by MS represents a valuable alternative to the above methods of identification, because the mass spectrum of a polymer possesses ‘‘characteristic’’ peaks that are peculiar to a polymer structure (Garozzo & Montaudo, 1985; Statheropoulos, Georgakopoulos, & Montaudo, 1991; Statheropoulos & Montaudo, 1991). The determination of polymer structure by MS can also be performed on insoluble samples. This aspect is a particularly attractive feature of MS, because it is more versatile than other techniques that strictly require sample dissolution. Often, one records the mass spectrum of the polymer with an instrument equipped with a hardionization source such as electron impact or SIMS source operated at high energy. Hard-ionization produces ion fragments and fragmentation patterns that are usually highly specific for a polymer type, and therefore, are useful for determining the polymer structure. Furthermore, mass spectra of two polymers that posses different repeat units will produce widely different mass spectra, because the spacing between peaks will be different. Thus, in poly(ethylene adipate) the spacing is 172.2 g/mol; in poly(butylene adipate) the spacing is 200.2 g/mol. This peculiar feature of polymer mass spectra can be used for polymer identification. In some cases, softionization techniques can be useful for structure elucidation and polymer recognition. PyGCMS is very a well-known technique for structural analysis in which MS is used as a detector at the end of a gas-chromatographic (GC) column to identify the pyrolysis products, which are volatile. For reasons of brevity, that discussion is omitted. The interested reader can find books and reviews on the subject (Allen & Bevington, 1989; Haken, 1998; Pethrick & Dawkins, 1999). Tandem MS, MS/MS/MS, and Post-Source Decay give useful structural information when applied to synthetic copolymers (Adamus et al., 2000; Koster et al., 2000; Chen, 2001; Keki et al., 2001; Przybilla et al., 2001) and to natural copolymers, such as copolysaccharides (Desaire, 109

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Sirich, & Leary, 2001; Ekeberg, Knutsen, & Sletmoen, 2001; Huang, Mechref, & Novotny, 2001; Ludwiczak et al., 2001; Si, Zhong, & Chen, 2001; Zaia, McClellan, & Costello, 2001). In the case of saccharides, an automated procedure for recovering structural information has been developed (Gaucher, Morrow, & Leary, 2000). This topic will be discussed below, in the section that deals with copolymer sequence. End-group identification and chain structure identification by MS are done at the same time, and they cannot be separated in two, different steps (as in the case of conventional techniques). In fact, the mass of the ion is the sum of three contributions; namely, the mass of the polymeric chain’s backbone, the masses of the end groups, and the masses of the cations (in the case of loss of a proton, one adds
1). From the first beginning, mass peak assignment in copolymers is done by hand (even when dozens of peaks are present; see Nuwaysir, Wilkins, & Simonsick, 1990; Plage & Schulten, 1991a). One produces a table with the masses of the compounds that are known to be present in the sample, and checks if the masses correspond. Some authors felt the necessity to discuss in-depth this timeconsuming but trivial step (which will be referred to as check-for-masses) (Yoshida, Yamamoto, & Takamatsu, 1998; Wilczek-Vera et al., 1999b). A method to speed-up the check-for-masses has been proposed (Danis & Huby, 1995); it has been improved by introducing autocorrelation (Wallace, Guttman, & Antonucci, 1999). In the case of oligosaccharides, peak assignment is very difficult (due to the large number of isobaric compounds), and computer programs are adopted for this purpose (Garozzo et al., 2000; Gaucher, Morrow, & Leary, 2000). Mass peak assignment is easier when the mass spectrum is recorded at high resolution and FT instruments equipped with high magnetic fields, operated at low trapping voltages; long ion-cooling periods are capable of

FIGURE 1. Expansion of the m/z region 900–1,000 of the ESI spectrum

of a triblock copolymer with units of ethylene oxide and propylene oxide. Reprinted from Van Rooji et al. (1998) with permission from American Chemical Society (copyright 1998).

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yielding superb mass resolution (Shi et al., 1998; Van Rooji et al., 1998; Aaserud, Prokai, & Simonsick, 1999). VanRooij et al. analyzed a triblock copolymer with units of ethylene oxide (EO) and propylene oxide (PO) (Van Rooji et al., 1998). Figure 1 reports an expansion of the ESI spectrum in the mass region 900–1,000 Da. The end-groups are H and OH, and the ions are sodiumcationized. The labels report the number of EO and of PO units in the macromolecular chain. The mass resolution is 15,000 and it can be seen that all peaks are well-resolved. The authors also recorded the ESI spectrum in the narrowband mode. They showed that mass resolutions higher than 100,000 can be obtained, and that it is possible to resolve the signal due to EO4PO13 from the signal due to PO16 with two carbon-13 atoms. Even higher resolutions are possible (Shi et al., 1998; Aaserud, Prokai, & Simonsick, 1999). However, FT instruments are quite expensive pieces of equipment, and it is necessary to make some considerations on more affordable apparata too. The signal-to-noise ratio (S/N) is another quantity that affects mass peak assignment, because the MS signals are sometimes ‘‘buried’’ in the noise. The following equation holds: ðS=NÞcopo ¼ d00 MIN½ðS=NÞA ; ðS=NÞB 

ð1Þ

where (S/N)A, (S/N)B, and (S/N)copo are the signal-to-noise ratios for the mass spectra of homopolymer A, of homopolymer B, and of the copolymer. The order of magnitude of the factor d00 is a matter of much debate, expecially in MALDI. Some authors believe that d00 is in the range 0.2– 0.25, with a fourfold-fivefold worsening with respect to the homopolymer (Beshah K, personal communication, 2001, evidences can also be found in Satoh 2000). Their argument is based on the fact that there are a huge number of different chemical species in a copolymer (much larger than for homopolymers) and the ion current is low. Other authors believe the worsening is small—that d00 is equal to 1 (at least when the structures of two repeat units are not completely different), and they report MALDI spectra of copolymers with an excellent S/N (Carroccio, Rizzarelli, & Puglisi, 2000; Chen et al., 2001). When the S/N ratio is low, mass peak assignment may become cumbersome. In fact, it may happen that the mass accuracy is low, the theoretical and the observed masses are sligthly different, and the check-for-masses may fail. Guttman et al. considered a series of low-intensity MS peaks with a low S/N and low mass accuracy. They showed that linear regression is a remedy for their specific case (Guttman, Blair, & Danis, 1997). Meyer et al. discussed the connection between check-for-masses and mass accuracy in great detail, and they developed a method for the detection of isobaric interferences (Meyer et al., 2001).

MASS SPECTRA OF COPOLYMERS

III. COPOLYMER COMPOSITION

Copolymer composition is preferentially determined with 13 C-NMR (Bovey, 1972; Randall, 1977; Tonelli, 1989). The method is very powerful, especially when literature data are available that report the assignment of the peaks in the 13 C-NMR spectrum. However, literature data are rarely available for unusual monomers. Even when the monomers are the usual ones in anionic or cationic polymerization (styrene, methylstyrene, p-methoxystyrene, methylmetacrylate, butylacrylate, hexylacrylate, metacrylic acid, maleic anhydride, vinylpiridine, vinylpirrolidone, styrenesulfonic acid, acrylonitrile, glycidyl-methacrylate, glycolic acid, lactic acid, vinyl acetate, vinyl alcohol, ethylene oxide, propylene oxide, tetrahydrofuran, isobutylene oxide, ethylene sulfide ethylene, butadiene, isoprene, piperylene, propylene, phenylbutadiene, dimethyl-siloxane, hydromethyl-siloxane, propiolactone, d-valerolactone e-caprolactone), it sometimes happens that the 13C-NMR spectrum of the copolymer is not reported. This result is due to the fact that there are two repeat units (at least), and thus thousands of possible combinations. To cope with this difficulty, one can try to theoretically predict the assignment of the peaks in the 13C-NMR spectrum. However, the theory that predicts the chemical shift associated with a given sequence is incomplete (in the sense that the predicted chemical shifts sometimes differ from the actual ones). Therefore, one has to turn to the last recourse; namely, the standard method to find the assignment; that method implies the (time-consuming) synthesis of model compounds and performing two-dimensional NMR experiments (mainly heteronuclear correlation studies), which require some specialistic knowledge (at least in the interpretation stage). Mass spectra of copolymers do not suffer of the cited drawback, because the abscissa associated with a given sequence is merely the mass of the sequence, and it is related in a trivial manner (see above) to the mass of the repeat units. Three methods have been developed to extract compositional information from the MS spectrum; namely, the direct method, the method based on chain statistics, and the hard-ionization method. In the following, the usefulness and limitations of the three methods will be discussed. The quantity to be determined is always the same namely, the molar fraction of A units in the copolymer (referred to as cA ). A. Composition by the Direct Method

The direct method (Nuwaysir, Wilkins, & Simonsick, 1990; Montaudo, 1992b; Montaudo et al., 1992c; Simonsick & Prokai, 1995) has many variants. The ‘‘full’’ variant of the direct method estimates cA, using a combination of MS intensities:

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XX cA ¼ ð1=z1 Þ m IðAm Bn Þ ð2Þ PP where z1 is ðm þ nÞIðAm Bn Þ, the summations are over m and n, and I(Am Bn) is the mass spectral intensity of the oligomer Am Bn. The above equation is referred to as the ‘‘composition estimates’’ equation (Montaudo, et al., 1992b; Montaudo & Montaudo, 1992c). Figure 2 reports the composition estimate formula for a quite common case—namely, the mass spectrum of an AB copolymer that spans from dimers to nonamers. It can be seen that the formula is quite complex, and it is apparent that paper-and-pencil calculation takes too much time. The same consideration applies to the composition estimates formula for an ABCD copolymer (Montaudo & Montaudo, 1992b), which is much more complex. To cope with this difficulty, other variants exist. The simplest one (Simonsick & Ross, 1996) is to spot the tallest peak in the spectrum, and to assign it to ions due to chains Ap Bq. The molar fraction of A units in the copolymer is given by cA ¼ p/(p þ q). The above formula is straight and simple, but it is seldom used because it is inaccurate when the tallest peak is due to chains shorter than decamers. Vitalini and Scamporrino developed a very effective graphical variant, in which the composition is related to the slope of a line (Vitalini, & Scamporrino, 1992b). Because unwanted contributions to MS peak intensity such as those deriving from isotopes can affect the calculation, it is necessary to eliminate them. This process (referred to as deisotopization or deisotoping) can be done by hand (Affrossman et al., 1993; van Alebeek, Schols, & Voragen, 2001) or automatically (Perseptive Biosytems, proprietary software). B. Composition by the Method Based on Statistics

The method based on statistics consists in generating a theoretical mass spectrum, and comparing it with the experimental one. Sometimes a minimization is employed (Montaudo, Ballistreri & Montaudo, 1991b). In order to generate a theoretical mass spectrum, one needs a sequence distribution model (SEDIM). Interesting SEDIMs are the first-order Markoffian (Montaudo & Montaudo, 1992b; Zoller & Johnston, 1997), the secondorder Markoffian (Montaudo & Montaudo, 1993a), and the sequential (Montaudo & Montaudo, 1992b) models. The most popular SEDIM is the Bernoulli model, which predicts that the molar fraction, I(AmBn), of the oligomer Am Bn is given by: IðAm Bn Þ ¼ g2 ðcA Þm ðcB Þn

ð3Þ

where g2 ¼ [(m þ n)!]/[(m)! (n)!] and cB ¼ 1
cA. Some authors used a modified formula in which a multiplicative damping factor (referred to in the following 111

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FIGURE 2. Composition estimate formula for the mass spectrum of an AB copolymer, which spans from dimers to nonamers.

as a00) is inserted between (cA)m and (cB)n (Spool & Kasai, 1996; Servaty et al., 1998). The use of a damping factor (or similar) has two disadvantages—namely, it cannot deal with distributions of molar masses that are bimodal or trimodal (see also Satoh, Kamigaito, & Sawamoto, 2000) and furthermore, two parameters (namely a00 and cA) are needed to generate a theoretical mass spectrum, and this fact complicates the process of extraction of useful information from the spectrum. To circumvent these two problems, a hybrid method can be adopted (Montaudo & Montaudo, 1992b). The sum of dimers (Q2), trimers (Q3), tetramers (Q4), pentamers (Q5), etc. are obtained from the experimental mass spectrum. The theoretical intensities of dimers are multiplied by Q2, trimers are multiplied by Q3, tetramers are multiplied by Q4, etc. In this way, only one parameter (namely cA) is needed to generate a theoretical mass spectrum, and the minimization process proceeds quickly. Furthermore, distributions of molar masses that are bimodal or trimodal can be accounted for by using splines or, alternatively, by fitting Q2, Q3, Q4, Q5, etc., with a combination of MMDs. It is common practice to ‘‘squeeze’’ experimental intensities so that Q2 ¼ 1, Q3 ¼ 1, Q4 ¼ 1, Q5 ¼ 1, Q6 ¼ 1 (this ‘‘squeeze’’ is done by multipling I(AmBn) by 1/Q2, or 1/Q3, or 1/Q4, etc). This procedure met with a wide success because it is simple. On the other hand, when using minimization, there is a (remote) possibility that some low112

intensity peaks (with an unfavorable S/N) p erturb the minimization and alter the final result, making it useless (a weighted best-fit is transformed into an unweighted best-fit). The composition of ABC and ABCD copolymers can also be determined (Montaudo, Scamporrino, & Vitalini, 1989a). Plage and Shulten recorded the EI spectrum of a copolymer with units of styrene and acrylonitrile (Plage & Schulten, 1991a). Almost all of the peaks in the spectrum turned out to possess a double assignment. This result is (most probably) the first case in which such massive superposition occurred (more than seventy peaks; all with an uncertain assignment). Plage and Shulten introduced a method that will be referred to as ‘‘pruning,’’ which consists in selecting one of the assignments and neglecting the others. Since then, a number of cases have been reported in which most peaks in the spectrum possess a double or triple assignment. For instance, Wilczek-Vera et al. (1999b) recorded the MALDI–TOF mass spectrum of a copolymer with units of 4-vinyl pyridine and a-methylstyrene. They detected a large number of peaks with an uncertain assignment and they adopted pruning (Wilczek-Vera et al., 1999b). The method based on statistics allows one to avoid pruning. When two oligomers have the same mass, the theoretical intensities are summed together, and thus the

MASS SPECTRA OF COPOLYMERS

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of copolymers with units of ethylene oxide (EO) and propylene oxide (PO), which were described in detail by many authors (Schriemer, Whittal, & Li, 1997; Chen et al., 2000, 2001; Gallet et al., 2002). It must be noted that isobaric interferences disappear when ultra-high resolution instruments are used, as in the case of EO/PO (see Fig. 1, Van Rooji et al., 1998). C. Copolymer Composition by the Hard-Ionization Method

FIGURE 3. The method based on statistics. Scheme of the deconvolution process, when the mass spectrum possesses a massive number of peaks with uncertain assignment. The two contributions to peak intensity are depicted as empty and filled boxes.

theoretical spectrum is a convolution. Figure 3 reports an example of the convolution process. The two contributions to peak intensity are depicted as empty and filled boxes. When the theoretical spectrum matches exactly the experimental one, the experimental MS intensities are deconvoluted. This result is a fascinating feature, and it implies that the method based on statistics is ideally suited in those cases when most peaks in the spectrum possess a double or triple assignment. The direct method is useful and quick, but, when pruning is adopted, there is a possibility that the formula fails, giving incorrect compositional results. In those cases, the method based on statistics must be preferred. The MALDI spectrum of a random copolyester sample with units of butylene succinate, butylene adipate, and butylene sebacate was recorded, and it was shown that the method based on statistics gives the correct result and that the direct method fails (Montaudo et al., 1998a). Another example, which deals with a glycine-lactic copolymer analyzed by MALDI (Montaudo, 1999b), is reported in this review. It can be shown rigorously that, at masses above 10000 Da, most peaks have a double or triple assignment, independent of whether the difference between the masses of the repeat two units is small or large. The method based on statistics can be also used to enhance artificially the resolution. In fact, one can generate many theoretical spectra at a very high mass resolution (say one million), and find the best match with the experimental MS intensities. The best theoretical spectrum possesses a surperb resolution (artificially enhanced). It cannot be excluded that the method based on statistics can help to solve isobaric interferences in MALDI–TOF mass spectra

The hard-ionization method consists in identifying an MS peak that is due to monomer A, an MS peak that is due to monomer B, and calculating the ratio of their intensities. Some polymer samples do not possess enough monomeric species for this purpose, and other polymers are made of monomers that are highly volatile and they are deleted in the high vacuum of the mass spectrometer. However, suitable ions can be created by chain scission, and thus Electron Impact or any other hard-ionization ion source can be used. Zoller et al. used photoionization (vacuum-UV at 111 nanometers) to analyze copolymers with units of ethylene, propylene, and butene (Zoller et al., 1999). The majority of the studies have been carried out with TOF-SIMS. Only a brief account will be given, because the topic has been reviewed (Chen & Weng, 2000; see also the tabulation that appeared in Hanton, 2001). Leadley et al. applied it to biodegradable copolymers with units of b-malic acid and butyl malic acid (Leadley et al., 1997), and to copolyanhydrides with units of sebacic acid and ricinoleic acid maleate (Leadley et al., 1998). The composition of copolymers with units of St and MMA (Van den Eynde, Bertrand, & Penelle, 2000; Van den Eynde, Reiths, & Bertrand, 2001), ethyleneglycolmethacrylate and MMA (Briggs & Davies, 1997), siloxane (Zhuang, Gardella, & Hercules, 1997a), and other units (Li et al., 1998, 2001) were also determined. An interesting study on a crosslinked copolymer appeared (Pinto, Stika, & Lloyd, 1995). There is a claim that careful ‘‘ratioing’’ of MS intensities yields copolymer sequence (Galuska, 1996). Other systems studied include copoly (ether-ester)s (Bhatia & Burrell, 1991; Burrell, Bhatia, & Michael RS, 1994) glycolide/ trimethylene carbonate copolymers (Brinen et al., 1993; Brinen, Greenhouse, & Jarrett, 1991), ethylene/vinylacvetate (Galuska, 1994), nylon6/nylon66 (Briggs, 1987), and some copolymers containing ethyl methacrylate (Briggs & Ratner, 1988; Lub et al., 1989). Surface segregation and other effects at copolymer surfaces are often studied by X-ray photoelectron scattering (XPS). SIMS can be used to study surfaces of copolymer and blends. Dynamic SIMS can monitor the variation of copolymer properties when moving from the surface to the bulk. 113

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IV. COPOLYMER SEQUENCE

Important quantities in copolymer sequence determination are the number-average length of like monomers and the run-number (Koenig, 1999). A widely adopted technique consists in obtaining a low MM copolymer by subjecting the sample to partial degradation. An interesting account is available on this method (Harwood, 1965). The partial degradation is associated with a cleavage of the bonds in the macromolecular backbone. In some cases, repeat units A are preferentially attacked and repeat units B are left intact, whereas in other cases there is a slight propensity for A repeat units. The partial degradation process is indicated accordingly as totally selective or partially selective. It is difficult to find a truly non-selective process. Even during the attack of aliphatic copolyesters by a strong acid in presence of methanol (methanolysis), the two comonomers are not completely equivalent (Montaudo et al., 1998b). In the late seventies, a series of articles appeared in which PyGC is used to determine number-average lengths and run-numbers (Tsuge et al., 1975; Blaszo & Varhegyi, 1978; Varhegyi, 1978; Tsuge et al., 1979a, 1979b; Nagaya, Sugimura, & Tsuge, 1980). The PyGC experiments were conducted at relatively low temperatures (in order to avoid total degradation), and the cleavage is partially selective, because some chemical bonds are preferentially attacked. These experiments are important in this context because the authors reconstruct the sequence of the undegraded copolymer from the analysis of a partially degraded sample, and thus they begin to explore the connection between the sequences of partially degraded and undegraded samples. MS can be used to determine copolymer sequence. The analysis of high molar mass copolymers can indeed be performed (Montaudo, Scamporrino, & Vitalini, 1989a), but it is not very appealing for a series of reasons. Some MS instruments are limited in mass. The resolution in the highmass region is low. Furthermore, the number of isobaric structures increases with mass, complicating the spectrum. To cope with such limitations, the most popular technique consists in obtaining a low MM copolymer by subjecting the sample to partial degradation, in analyzing the low MM copolymer, in determining its sequence, and in reconstructing the sequence of the undegraded copolymer. Methods to shorten the length of copolymer chains include methanolysis, rupture of photolabile bonds (photolysis), attack of C = C bonds by ozone (ozonolysis), attack by amino compounds such as piperidine (aminolysis), attack by sodium methoxode (methoxidation), attack by strong bases (saponification), hydrolysis, and pyrolysis. Some information on the topic can be found in a review on FAB-MS (Montaudo, Scamporrino, & Vitalini, 1989a). Furthermore, in some ionization sources such as DCI, a partial degradation of the copolymer sample occurs, in 114

the sense that the length of copolymer chains detected is much shorter than the length prior to MS. This phenomenon is observed with a variety of copolymers, namely hydroxyalkanoates (Abate et al., 1992), alkyl isocyanates (Majumdar et al., 1991), and fluorinated isocyanates (Chen et al., 1997b). In other ionization sources such as SIMS, partial degradation of the copolymer sample also occurs (Bletsos et al., 1990a). There are two cases in which the process of reconstructing the sequence of the undegraded copolymer from the analysis of the partially degraded sample is particularly simple. The first case is when non-selective partial degradation takes place, because the sequence is invariant. The second case is when totally or partially selective cleavage occurs; the partially degraded sample contains high oligomers—namely, octamers, nonamers, and decamers; and sequence information is available on the latter oligomers. The exact connection between the sequence of higher oligomers in the partially degraded sample and the sequence of the undegraded sample was studied experimentally (by recording a series of spectra of copolymers at different degradation stages, see Montaudo, Scamporrino, & Vitalini, 1991a) and theoretically. Some theoretical studies use Monte Carlo simulation, which can simulate the partial degradation process and allows one to extrapolate at time zero, when the sample is undegraded (Montaudo, 1993c). However, Monte Carlo simulation is used by a restricted number of researchers, and thus, a method has been developed (Montaudo & Montaudo, 2002a) that gives the correct answer, and avoids a Monte Carlo simulation. Independent of the method used to obtain them, the results are the following. The relative abundances of dimers and trimers are strongly affected by partially or totally selective cleavage, whereas the relative abundances of pentamers and hexamers are weakly affected. Lastly, when the low MM copolymer (obtained subjecting the sample to partial degradation) is made mainly of nonamers, decamers, and undecamers; the relative intensities are essentially unaffected (Montaudo & Montaudo, 2002a). In the case of block copolymers, partial degradation should be avoided, because it affects the lengths of AAAA and BBBB blocks by reducing them. Tandem MS and other MS/MS techniques (e.g., PostSource Decay), when applied to copolymers, give a number of product (daughter) ions, NUDAI for brief, which is larger than the NUDAI in the case of homopolymers. It has been conjectured that the increase in NUDAI is small for a block copolymer (because many sequences are systematically absent in block copolymers) and large for a random copolymer. There have been some attempts to verify experimentally this conjecture (Adamus et al., 2000; Koster et al., 2000; Chen et al., 2001; Desaire, Sirich, & Leary,

MASS SPECTRA OF COPOLYMERS

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2001; Keki et al., 2001; Przybilla et al., 2001; Zaia, McClellan, & Costello, 2001; see also Ekeberg, Knutsen, & Sletmoen, 2001; Huang, Mechref, & Novotny, 2001; Ludwiczak et al., 2001; Si, Zhong, & Chen, 2001). It is easy to forecast that, in the near future, someone will derive a formula to connect the intensity of fragment peaks to the number-average length of like monomers; that connection is of the uppermost importance to achieve a better discrimination between random and block copolymers. However, at the moment, the lack of the cited formula isolates the Tandem MS and other MS/MS methods to the group of qualitative methods.

V. MOLAR MASS DETERMINATION

Copolymer molar mass determination by MS is performed by assuming that the abundances of the chains are reflected in the intensities of MS peaks. Let Ni indicates the number of chains with mass mi (Elias, 1984; Mitchell, 1987; Pethrick & Dawkins, 1999). In this case, the number n , is given by: average molar mass, is given by: M
X .
X  n ¼ M Ni ð4Þ mi Ni  w, In a similar manner, the weight-average molar mass, M is given by:
X .
X  w ¼ M mi Ni ð5Þ m2i Ni Wilczek-Vera, Danis, & Eisenberg (1996) recorded the MALDI–TOF spectrum of a triblock styrene/AMST copolymer (AMST ¼ a methylstyrene) obtained anionically with secondary butyl lithium as initiator. MALDI peak n intensities were inserted in the equations that define M  w , and the result was M  n ¼ 4,273, M  w ¼ 4,411, in fair and M agreement with the molar mass averages obtained by SEC  n ¼ 4,190, M  w ¼ 4,510). They also analyzed with suc(M cess a diblock copolymer with the same repeat units  n and M  w. (Wilczek-Vera et al., 1999a), and determined M Molar Mass determination by MS can be performed also on copolymer samples that possess molar masses higher than the previous ones. For instance, the analysis of the MALDI–TOF mass spectrum of a block copolymer with units of t-butyl acrylate and methyl acrylate yielded  n ¼ 30,217, M  w ¼ 35,958 (Ma & Wooley, 2000). Figure 4 M reports the MALDI–TOF mass spectrum of styrene/ methylmetacrylate copolymer obtained anionically with tert-butyl sodium as initiator. The peak due to singly charged ions is at 27,000 Da, whereas the peak due to dimeric n¼ ions is at 54,000 Da. The SEC analysis yielded M 1 26,000, and the composition (determined by H-NMR) turned out to be almost equimolar. This result implies that  n ¼ 13,000. the styrene and MMA blocks have both M

FIGURE 4. MALDI–TOF mass spectrum of styrene/methylmetacrylate copolymer obtained anionically.

MS yields directly the MM distribution (MMD). However, to compare the MMD of the copolymer obtained by MS with the MMD obtained by SEC, a transformation must be applied and the intensity of each point of the MS spectrum must be multiplied by the mass. Broberg et al. analyzed amylopectin, which is a naturally occurring branched polymer (Broberg et al., 2000). They recorded the MALDI–TOF spectrum of an Amylopectin sample derived from Potato (referred to as AMPP). They processed their spectra, and they obtained the MMD of their samples. They also recorded the SEC trace of the two samples. Figure 5 reports the MMD for

FIGURE 5. MMD for sample AMPP as resulting from MALDI–TOF (upper trace) and SEC (lower trace). Reproduced from Broberg et al. (2000) with permission from Elsevier (copyright 2000).

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sample AMPP from MALDI–TOF (upper trace) and SEC (lower trace). The MM distribution initially has a rapid growth, maximizes for chains with size 15–20, and falls slowly (it is asymmetrical with respect to the point of maximum), with a structure for chains with size 45–50. The agreement between the two methods is good. When the sample possesses a broad MM distribution to calculate the MM, it is necessary to consider the intensities of peaks that span over a wide range of mass numbers. This spanning constitutes a serious problem in MS because the ion yield is likely to change with mass. In other words, mass discrimination is likely to occur, and the number of ions does not reflect the number of molecules in the sample. Mass discrimination occurs in many ionization techniques—namely, in SIMS (Hercules, 2002), ESI (Hanton, 2001), in MALDI (Nielen, 1999), etc. Erra-Balsells et al. identified another source of error— namely, the fact that the ionization yields of the two monomers may differ. They corrected MS intensities for such an effect, and they were able to evaluate the MMD of their copolymer (Erra-Balsells et al., 2000).

Another limitation comes form the fact that when two detectors are used, it is difficult to estimate the volume between the detectors (see, for instance, Provder, Barth, & Urban, 1995). In order to circumvent these obstacles, the SECMALDI method (and more in general, the SEC-MS method) can be applied. The method has two steps. In the first step, the sample is injected in the SEC apparatus, the SEC fractions are collected, and MALDI–TOF spectra (or MS spectra) of selected fractions are recorded. In the second step, Mn , and Mw are computed, by applying a calibration that is correct because it is ‘‘internal’’ and, therefore, genuine (Montaudo et al., 1995b, 1997, 1998c; Montaudo & Montaudo, 1999c; Carroccio, Rizzarelli, & Puglisi, 2000; Esser et al., 2000; Montaudo, 2002d). Carroccio et al. applied the SEC-MALDI method to random copolyester samples (referred to as Bionolle 3020 and B5050) with units of butylene succinate and of butylene sebacate (Carroccio, Rizzarelli, & Puglisi, 2000). Figure 6 reports the SEC traces of Bionolle 3020 and

VI. MS AS A DETECTOR FOR LIQUID CHROMATOGRAPHY

The most-used method for molar mass determination in copolymers is based on size-exclusion chromatography (SEC). A very detailed account can be found elsewhere (Gores & Kilz, 1993; Rudin, 1995). One injects the sample in the apparatus, and the SEC data-acquisition program (e.g., CHROM2000TM, GPC-CaliberTM, PSS-winSECTM, Millennium-32TM) records the trace, corrects the trace for  n and M  w by applying detector response, and computes M a calibration curve (conversion from elution vomes to masses). SEC of copolymers suffers of a series of limitations, and in the following, an attempt will be made to summarize them. It is customary (Uglea, 1996) to apply a calibration curve of the type log (M) ¼ b00 þ Ve b01, where b00 and b01 are calibration constants, and Ve is the elution volume. Unfortunately, the law of propagation of errors (Allen & Bevington, 1989) predicts that a small inaccuracy in b00 and b01 has catastrofic effects, and yields a large error in  n and M  w . Indeed, calibration constants b00 and b01 M are inaccurate (in most cases) due to the lack of suitable calibrants. The standard method to cope with this limitation (Gores & Kilz, 1993; Rudin, 1995) is to use interpolation formulas to improve the accuracy of b00 and b01. However, a peculiar effect, referred to as the ‘‘hydrodynamic interaction factor’’ (Montaudo et al., 1998c), may arise and interpolation formulas may give highly biased results. 116

FIGURE 6. SEC trace of Bionolle 3020 (a) and of B5050 (b) along with the respective SEC calibration lines. Reproduced from Carroccio, Rizzarelli, & Puglisi (2000) with permission from John Wiley & Sons, Ltd. (copyright 2000).

MASS SPECTRA OF COPOLYMERS

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FIGURE 7. MALDI–TOF spectrum of sample YAL1344. Reproduced from Yeung & Marecak (1999) with permission from Elsevier (copyright 1999).

B5050 and the calibration lines. Although the two samples possess identical structures, the lines are different. For instance, the chains in Figure 6a for which log (M) ¼ 4.85 (these chains have mass 70,000 Da) elute at 31 mL, whereas the chains in Figure 6b for which log (M) ¼ 4.85 elute much later, namely at 33 mL. The computation (GPCCaliberTM, see above) of MM averages for the sample in  w ¼ 52,000 and M  n ¼ 28,000. Figure 6a yielded M A variant of the SEC-MALDI method employs gelfiltration chromatography (GFC). Yeung et al. analyzed by GFC-MALDI yaluronic acid, which is an exactly alternating copolymer with units of glucuronic acid and N-acetyl glucosamine (Yeung & Marecak, 1999). The GFC fraction that eluted at 13.44 mL will be referred to as YAL1344. Figure 7 reports the MALDI–TOF spectrum of YAL1344. It can be noted that the fraction has a narrow distribution, centered around 11,000 Da, with well-resolved peaks. They noted that standard methods for SEC calibration yield  n and M  w due to unavaila large error in the resulting M ability of suitable calibrants. Instead, the GFC-MALDI method yields the correct result. MM averages were computed (Millennium-32TM, see above) and the result was  w ¼ 38,600, M  n ¼ 22,700. M Another variant of the SEC-MALDI method is the SEC-ESI method, which employs electrospray ionization (ESI) and has the distinct advantage of being less time-

consuming. In fact, the SEC apparatus is coupled on-line with the ESI mass spectrometer, and thus fraction collection is avoided (Simonsick & Prokai, 1995; Shi et al., 1998; Aaserud, Prokai, & Simonsick, 1999). Deery et al. studied a permethylated arabinogalactan sample, where methylated pentose and methylated hexose residues are found along the macromolecular chain. In that case, double detection was performed, i.e., SEC-ESI and SEC-MALDI (Deery, Stimson, & Chappell, 2001). When samples possess strong compositional drifts and/or broad compositional distributions, another potential source of error appears in the SEC of copolymers, as pointed out by Gores & Kilz (1993), which derives from the fact that, for compositionally homogeneous copolymers, the correspondence between masses and elution volumes is univocal (similar to the case of homopolymers), whereas for compositionally heterogeneous copolymers, the cited correspondence is not univocal and chains at different masses co-elute in the same fraction. This result implies that the sequence distribution of the SEC fractions may differ from the sequence of the unfractionated copolymer, and thus, the MMD measured by the SEC method may be skewed or distorted. This effect will be referred to as the HET-broadening of SEC fractions. SEC-MS can be used to measure HET-broadening and to evaluate this potential source of error. 117

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FIGURE 8. MALDI spectrum of sample FR6. The inset shows an expansion in the region 2,500–3,200. Reproduced with permission from Montaudo (1998d).

A random copolyester sample with units of butylene adipate (A) and of butylene sebacate (B) was analyzed by SEC-MALDI (Montaudo et al., 1998d). The sixth SEC fraction is referred to as FR6. Figure 8 reports the MALDI spectrum of FR6, and the inset shows an expansion in the region 2500–3200 with decamers, undecamers, and dodecamers. The detailed assignment for MS peaks can be found elsewhere (Montaudo et al., 1998d). Because oligomer of different sizes (m þ n) are present, the molar fraction of butylene adipate units, cA was determined for all sizes. A model has been developed (Montaudo et al., 1998d) to describe how the skewing depends on the parameters that characterize the column, and on the parameters that characterize the copolymer sample. The model predicts that, in this case, HET-broadening is small. Figure 9 reports cA versus (m þ n) for sample FR6. It can be seen that the agreement between theory and experiment is good, and that the slope is small, which implies that the HET-broadening is small (Montaudo et al., 1998d). SEC-ESI can be performed on-line to offer the unique possibility of recording single ion currents and thus to 118

FIGURE 9. Compositional drift for sample FR6. Molar fraction of Adipate versus (m þ n) for sample FR6. The variation of the molar fraction of Adipate is reported as a function of chain size (m þ n). Reproduced from Montaudo (2002d) with permission from Elsevier (copyright 2002).

MASS SPECTRA OF COPOLYMERS

FIGURE 10. SEC calibration curve for MMA/BA determined by SEC-

ESI. Reproduced from Simonsick & Prokai (1995) with permission from American Chemical Society (copyright 1995).

measure the volume at which each single oligomer is eluted (see, for instance, Simonsick, Ross, & Prokai, 1996b for the application to an aliphatic/aromatic copolyester). Simonsick et al. applied SEC-ESI to a random copolymer with units of methylmetacrylate (MMA) and butylacrylate (BA) (Simonsick & Prokai, 1995). Figure 10 reports the SEC calibration curve for MMA/BA determined by SECESI. From the inspection of the figure, it can be seen that oligomers tend to group themselves along a line, and that none of the oligomers elutes distant from the line. This result implies that the HET-broadening is small. High performance liquid chromatography (HPLC) is often used when large amounts of samples must be separated (preparative LC). HPLC is widely adopted for separating chains with different A/B ratios. Mori injected a random copolymer sample with units of styrene and methyl-methacrylate in an HPLC apparatus, and showed that the abundance of styrene in the copolymer grows as the elution volume increases; styrene-rich chains are eluted first whereas styrene-poor chains are eluted last (Mori, 1988). Kilz et al. separated a mixture of 16 star-shaped copolymer samples with units of styrene and butadiene by HPLC, and demonstrated that styrene-rich chains are eluted first, whereas styrene-poor chains are eluted last (Kilz et al., 1995). HPLC may also separate by size, but the efficiency of the separation mechanism in HPLC is lower than in SEC, with the result that long chains are eluted simultaneously (Uglea, 1996), and thus HPLC is confined to low MM copolymers. The HPLC apparatus is equipped with an UVdetector and, when the two repeat units in the copolymer

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are widely different (for instance, when an aromatic group is present, as in styrene/methyl-methacrylate or in styrene/ butadiene), the detector may be ‘‘blind’’ to some chains. One can replace the UV detector with an evaporative lightscattering detector, but the response of the latter still has some non-linearities. Moreover, the relationship between elution times and masses is expected to be extremely complex. Chains with a given A/B ratio should be eluted at a well-defined volume, say Ve. Instead, short chains are eluted before Ve and long chains are eluted after Ve. This effect is referred to as strong coupling between size and sequence separations (see Protschka & Dubin, 1993; Kuchanov, 2001). In some cases, strong coupling can be useful (Jandera, Holcapek, & Theodoridis, 1998; Jandera, Holcapek, & Kolarova, 2000). For instance, Jandera et al. analyzed a copolymer sample with units of ethylene oxide (A) and ethylene (B), using an HPLC apparatus connected with an APCI mass spectrometer, and they were found that it is possible to find chromatographic conditions in which almost all Am Bn chains are eluted at different times (Jandera, Holcapek, & Theodoridis, 1998). When the structure of the repeat units is similar (e.g., methylmetacrylate and butylacrylate), the HPLC apparatus separates co-oligomers exclusively on the basis of their size, independent of their composition, and thus the relationship between elution times and masses becomes extremely simple. In this case, one can use HPLC-MS, which consists of two steps—namely, fractionating a copolymer, and analyzing selected fractions. Optionally, one can recover the average molar masses, using the calibrated chromatographic trace. One of the first copolymer analyses by HPLC-MS dates to 1989. A copolymer sample with units of hydroxybutyrate (HB) and of hydroxyvalerate (HV) was subjected to partial methanolysis, and the resulting low MM copolymer (referred to as metHB/HV) was injected into an HPLC column (Ballistreri et al., 1989a). Selected fractions were collected and analyzed by FAB-MS. The mass spectra possessed superb quality, and their analysis indicated that the fractions that elute after 20 mL possess all the same composition, namely, cA ¼ 0.86. The composition of the partially methanolyzed sample was be identical to the composition of the undegraded sample (non-selective cleavage). Figure 11 reports the HPLC chromatogram of metHB/HV and the calibration line (Ballistreri et al., 1989a), which can be used for elution volume-to-masses conversion. The trace can be used to compute the average molar masses of the partial methanolyzed sample, which  n ¼ 800, M  w ¼ 1,300. are M MS can be used as a detector for liquid chromatography at the critical conditions (LCCC), and it will be discussed in the section, which deals with block copolymers because it is used mainly to analyze the cited kind of copolymers. 119

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FIGURE 11. HPLC chromatogram of sample ‘‘metHB/HV’’ along with

the calibration line, which can be used for elution volume-to-masses conversion. Reproduced from Ballistreri et al. (1989a) with permission from American Chemical Society (copyright 1989).

VII. COMPOSITION DRIFT AND THE COMPOSITIONAL DISTRIBUTION HISTOGRAM

A discussion of the importance of the Composition Drift, the Bivariate Distribution of Chain Length and Composition, and the COmpositional DIstribution HIstogram (CODIHI) can be found in standard textbooks of polymer science (Flory, 1971; Elias, 1984). The COmpositional DIstribution HIstogram (CODIHI) reports the weight of copolymer chains that possess a given composition and it is a curve, whereas the Bivariate Distribution is a surface. The theory predicts that, for addition copolymers reacted at low conversion, the CODIHI is flat almost everywhere (with the lone exception of a small region centered around the average composition), and that the shape of the Bivariate Distribution is very simple (Flory, 1971; Elias, 1984). Conventional methods exist for measuring the cited quantities. The composition drift is often estimated with a SEC apparatus equipped with two detectors (Allen & Bevington, 1989; Gores & Kilz, 1993), whereas the shape of the CODIHI is measured by HPLC fractionation (Arai et al., 1999; Yoshie & Inoue, 1999) or NMR (Watanabe et al., 2001). Furthermore, the method traditionally used for measuring the bivariate distribution is the ‘‘chromatographic cross-fractionation’’ (also referred to as ‘‘two-dimensional chromatography’’ or ‘‘orthogonal chromatography’’). Macromolecules with different composition are separated in a silica column, and a SEC column is used to elute chains with different sizes (Allen & Bevington, 1989). As an alternative to conventional methods, MS can be used. 120

MS was used to monitor the compositional drift in AB copolymers, and the changes of the molar fraction of A units was followed in the ranges 1,000–11,000 Da (Guttman, Blair, & Danis, 1997) and 2,000–11,000 Da (Moore et al., 2002). MS was used to estimate the weight of copolymer chains that possess a given composition, and to draw the CODIHI in the case of copolymers with units of methylmethacrylate, butylacrylate, styrene, and maleic anhydride (Montaudo, 2002d). The bivariate distribution of chain lengths and compositions for some copolymers was reconstructed from MS (DeVries & Hunziker, 1996; Suddaby, Hunt, & Haddleton, 1996; Wilczek-Vera, Danis, & Eisenberg, 1996; Van Rooji et al., 1998; Wilczek-Vera et al., 1999a, 1999b; Falkenhagen et al., 2000). In the case of copolymers of high molar mass, some problems arise due to the loss of resolution at mass above 12 kDa in the mass spectra. Nevertheless, a variant of the SEC-MALDI method can be used to overcome the problem. The variant consists in fractionating the whole copolymer by SEC, collecting the fractions, and recording the MALDI and NMR spectra (Montaudo, 2002d). In fact, due to the high sensitivity of the MS and NMR methods, the sample amount contained in a narrow fraction provided by an analytical SEC device is sufficient to run both types of spectra. Figure 12 reports the bivariate distribution of a commercial ST/MMA sample (Montaudo, 2001a). The sample was reacted at high conversion, and it possesses an asymmetrical bivariate distribution, showing the composition drift expected for such kind of sample.

FIGURE 12. Bivariate distribution of a commercial ST/MMA sample.

MASS SPECTRA OF COPOLYMERS

VIII. QUANTITATION AND METHODS TO CORRECT MASS SPECTRAL INTENSITIES

Some authors reported that the intensities of the peaks in the MS due to different co-oligomers do not reflect the relative abundances of oligomers. Vitalini and Scamporrino recorded the FAB spectra of a series of random copolyethers with porphyrin and Bisphenol-A units at different compositions (Vitalini & Scamporrino, 1992b). The FAB spectra was not quantitative. In particular, the analysis of the FAB spectrum of one of the samples (indicated as copolymer V) suggested that the molar fraction of porphyrin units in the macromolecular chain was ca. 30%, whereas less than 1% porphyrin units were present (more specifically, the molar ratio of the comonomers was 5/995). In a similar manner, Vitalini et al. recorded the MALDI spectra of a series of copolyethers at different compositions in which a Shiff-base/copper complex (referred to as A) and Bisphenol-A (referred to as B) units are found randomly along the chain (Vitalini et al., 1996). The analysis of the MALDI spectrum of the sample indicated as copolyether IV yields a molar fraction of A units cA ¼ 0.12, whereas the correct value is known to be between cA ¼ 0.44 and cA ¼ 0.48. This result implies that the intensities of the MALDI peaks reflect relative abundances in a distorted manner. When the intensities of the peaks in the MS due to different oligomers do not reflect the relative abundances of oligomers, they must be corrected. Ramjit and Sedwig reported a method to correct MS intensities (Ramjit & Sedgwick, 1976). They considered a copolymer with units of ethylene adipate (A) and trimethylene adipate (B). They determined two correction factors (1.55 and 1.27, respectively), and corrected MS intensities accordingly. Unfortunately, the cited method applies exclusively to MS peaks due to dimers and nobody has ever reported how to extend it to trimers or higher oligomers. Polce et al. reported the MALDI spectra of three copolymers with units of quinoxaline (QX) and ethersulfone (ESU) obtained from different feeds—namely, 75/25, 50/50, and 25/75. They noted that the MS intensities are not quantitative, because peaks due to ESU-rich chains are underestimated. They performed some molecular mechanics calculations that pointed out that the gas-phase basicity in QX/ESU oligomers decreases when the ESU content grows, causing a lowering of the ionization efficiency (Polce et al., 2001). It cannot be excluded that molecular mechanics calculations can also help to correct MS-peak intensities in other cases. The MS analysis of EO/PO copolymers is a matter of much debate because some authors believe that, in this case, MS is quantitative and others believe that it is not quantitative.

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Chen et al. analyzed a block copolymer with units of ethylene oxide (A) and propylene oxide (B) obtained with a mixture of two initiators (Chen et al., 2000). They recorded the MALDI–TOF spectrum of the sample, and noted that the peaks are due to ions of the type H-(CH2)16O-AmBnH,,,Naþ and H-(CH2)18O-AmBn-H,,,Naþ. They recorded the spectra, using three different matrices (retinoic acid, HABA, and ditranol) and noted that the relative peaks intensities change as the matrix changes. They also prepared the sample from three different solvents (acetone, methanol, THF); they recorded the spectra, and noted that the peak intensities depend on the solvent. They prepared the sample with three different analyte-to-matrix ratios, and once again, differences were easy to detect. Figure 13 reports a section of the MALDI–TOF spectrum when the analyte-to-matrix ratio is 1:500, 1:50, and 1:10 (Chen et al., 2000). There are strong differences indeed. On the other hand, when the sample preparation protocol is fixed (matrix, solvent, analyte-to-matrix ratio) one can use MS to analyze EO/PO samples with small differences and detect them (Chen et al., 2001). The group of scientific papers in which the authors assume implicitly that the MS response reflects (at least approximatiely) the abundances of the macromolecular species is vast. The group contains the overwhelming majority of the reports in which the method based on chain statistics and the direct method for composition determination is adopted. The group also contains other reports. For instance, Meyer et al. analyzed Cremophor EL (a polyethoxylate), and they were able to isolate the watersoluble fraction, Faqua, using cation exchange chromatography. Unfortunately, there are some MS peaks in the spectrum of Faqua, that possess an uncertain assignment. There are two ions, referred to as RIC-X-H,,,Kþ and RICX-RIC,,,Naþ, which possess approximately the same mass. The authors assumed that no selective supression occurs (i.e., that MALDI is approximatively quantitative) and they

FIGURE 13. EO/PO block copolymer. Section of the MALDI–TOF

spectrum, when the analyte-to-matrix ratio is 1:500 (A), 1:50 (B), and 1:10 (C). Reproduced from Chen et al. (2000) with permission from John Wiley & Sons, Ltd. (copyright 2000).

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FIGURE 14. MALDI spectrum of the water-soluble fraction, Faqua, of Cremophor EL, mixed with three

internal standards. Peaks due to the three internal standards are indicated by symbols (diamonds and circles). The other peaks are due to the sample. Reproduced from Meyer et al. (2001) with permission from Elsevier (copyright 2001).

were able to solve this ambiguity as follows. Faqua was mixed with three internal standards, and the MALDI spectrum was recorded, which turned out to possess an extraordinary good mass accuracy—namely, few milliDaltons (Meyer et al., 2001). Figure 14 reports the MALDI spectrum of Faqua. Peaks due to the three internal standards are indicated by symbols (diamonds and circles). The other peaks are due to the sample.

IX. COPOLYMER MIXTURES AND BLENDS

Mass spectra of genuine copolymers and of mixture of two homopolymers are quite different. The differences grow very quickly as the mass increases, and at the nonamerdecamer level, a quick glance to the spectrum is enough to differentiate between a copolymer and a mixture of two homopolymers (Montaudo & Montaudo, 2002a). The theory predicts that, by ‘‘ratioing’’ MS intensities, the composition of the mixture is derived. Nevertheless, it is common knowledge that, when the sample is a mixture of two homopolymers, MS can give large errors, and cases have been reported where peaks due to homo A or homo B are systematically absent or they are strongly suppressed. This result happened during the MS analysis of a mixture of PEG and PMMA (Montaudo, Scamporrino, & Vitalini, 122

1989a), and of a mixture of PS and PMMA (Nielen, 1999). Another example of selective supression was reported by Storey, Brister, & Sherman (2001). They recorded the MALDI spectrum a triblock copolymer with units isobutylene (middle block) and of e-caplactone. The spectrum is puzzling for two reasons-namely, all MS peaks are due to e-caplactone homopolymer (peaks due to the copolymer are systematically absent), and the MMD distribution is centered much too low in mass (Storey, Brister, & Sherman, 2001). Even when the sample is a mixture of two or three copolymers that differ exlusively in compositions, MS gives errors, although usually small ones. Falkenhagen et al. analyzed a sample, referred to as MIX3, which is a mixture of three polymers with units of ethylene oxide (A) obtained, using three different alcohols (Falkenhagen et al., 2000). The alcohols possess aliphatic chains with 10, 12, and 14 carbons. The abundance of the three components in the mixture is 33/33/33 (w/w/w). Figure 15 reports the MALDI–TOF spectrum of the mixture. The MS peaks are due to ions of the type H-(CH2)10-O-Am-H,,,Naþ, H-(CH2)12-O-Am-H,,,Naþ and H-(CH2)14-O-Am-H,,,Naþ. One can apply a transformation, and the intensity of each point of the MS spectrum can be multiplied by the mass. The weight fraction is thus obtained (see for instance, Broberg et al., 2000), which turns out to be 38/38/24, quite far from the correct value (33/33/33).

MASS SPECTRA OF COPOLYMERS

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dom condensation copolymers. In the following, the properties of copolymers that belong to each category and details about their mass spectra MS will be discussed. A. Copolymers Produced by Enzymes or Occurring in Nature

FIGURE 15. MALDI–TOF spectrum of MIX3, which is a mixture of

three block copolymers. Reproduced from Falkenhagen et al. (2000) with permission from Taylor & Francis Ltd, http://www.fandf.co.uk/journals (ß 2000).

In the case of a mixture of copoly-hydroxyalkanoates, MS analysis is quantitative, and therefore, it is possible to compare experiments with theory. Lets consider a mixture of two random copolymers with the same chemical structure. The quantities of interest are dA and eA, which represent the molar fraction of A units of the first and of the second copolymer, and X the molar fraction of the first copolymer in the mixture. The overall composition of the copolymer sample (cA) will be intermediate between the compositions of the two components: cA ¼ [X]dA þ [1
X] eA. A Mixture of copolymers can be analyzed by the statistical method. The SEDIM in Equation 3 is replaced with a different SEDIM, which predicts that I(Am Bn) is given by (Ballistreri et al., 1991): IðAm Bn Þ ¼ g2 g3

ð6Þ

where g2 is defined above, and g3 is given by X(dA)m (1
dA)n þ [1
X] (eA)m(1
eA)n. MS analysis is quantitative also in the case of a mixture of alkyl isocyanate copolymers (Hoke et al., 1995). Kowalczuk et al. compared theoretical predictions with experimental MALDI data, concerning a mixture of copolymers with units of pivalolactone and units of hydroxybutyrate (Kowalczuk, Adamus, & Montaudo, 2002).

X. COPOLYMERS OBTAINED BY SPECIFIC SYNTHETIC ROUTES

Mass spectra of copolymers have appeared in the literature, and the analyzed samples belong to four categories namely, copolymers produced by enzymes or occurring in Nature, block copolymers, random addition copolymers, and ran-

Polysaccharides and glycoconjugates fall in two broad classes—namely, those that are important in molecular biology (see Varki et al. (1999) for an overview of these compounds), and other naturally occurring materials (yaluronic acid, chitin, chitosan, cellulose, pectin, amylopectin, amylose, etc.; see Crescenzi, Dea, & Stivala, 1985; Collins, 1987). Some examples of saccharides that belong to the first class are chondroitin sulfate, some N-linked saccharides, and the mixture of glycans (referred to as glycan pool, see Rudd et al., 1997) obtained when glycoproteins are suitably treated. Contrary to proteins, which possess a fixed sequence, most polysaccharide samples possess heterogeneity, because they are a mixture of chains with different sizes and compositions. Among glycobiologists, it is a common belief that the heterogeneity in composition for glycans is smaller than for synthetic copolymers. In practice, the width of the COmpositional DIstribution HIstogram (see Section IV) for glycans is expected to be smaller than the width for synthetic copolymers. A number of different techiniques are used to assess polysaccharide composition (Hounsell, 1998). One of the protocols subjects the chains to total cleavage (down to monomers or to dimers) and measuring the abundances. Colorimetric methods are often used. Another standard tool for polysaccharide sequencing is NMR. Mass spectra of Polysaccharides have appeared. The comprehensive review on MALDI of carbohydrates by Harvey covers literature up to the end of 1998 (Harvey, 1999), and it contains references to research with MALDI along with some work done using Plasma Desorption, FAB, and DCI. Two approaches have been developed for polysaccharide sequencing by MS. In the first approach, the macromolecular species present in the sample are separated and carefully purified, and the mass spectrum of each single well-defined species is recorded. Usually, tandem MS is used (Hisamatsu et al., 1991; Gaucher, Morrow, & Leary, 2000; Spina et al., 2000; Desaire, Sirich, & Leary, 2001; Ludwiczak et al., 2001; Zaia, McClellan, & Costello, 2001), and a brief account on polysaccharide sequencing by MS is available (Garozzo, 1997). The purification step is time-consuming, and therefore, a second approach was developed, in which the step is omitted. In this method, a mixture of chains with different compositions and sequences is analyzed by MS (Rudd et al., 1997). 123

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Broberg et al. analyzed amylopectin from potato and wheat (see Fig. 5, Broberg et al., 2000), whereas Yeung et al. analyzed yaluronic acid (see Fig. 7, Yeung & Marecak, 1999). Garozzo et al. recorded the MALDI spectrum of a polysaccharide sample, and detected peaks up to 12000 Da, which are due to chains with 67 saccharidic units (Garozzo et al., 2000). Huang et al. analyzed a sample that is a mixture of biantennary and triantennary glycans (Huang, Mechref, & Novotny, 2001). Kabel et al analyzed a mixture of xylo-oligosaccharides (Kabel, Schols, & Voragen, 2001), whereas Si et al. analyzed a mixture of amino-oligosaccharides (Si, Zhong, & Chen, 2001). Ackloo et al. analyzed kappa-carrageenans short chains (Ackloo et al., 2001), whereas Ekeberg et al. were able to detect MS peaks due to kappa-carrageenans chains with twelve repeat units (Ekeberg, Knutsen, & Sletmoen, 2001). Chai et al. used a specific enzyme, namely heparinase I, to reduce the size of heparin, HPLC to separate oligomers with different sizes and then recorded the ESI spectrum of HPLC fractions (Chai et al., 1998). Rudd et al. obtained a glycan pool from human erythrocyte CD59 and the the MS analysis of the pool showed that it possesses a huge number of different components (Rudd et al., 1997), thus hinting that the saccharidic part of glycoproteins is disordered (similar to a random copolymer). Karlsson et al. studied a series of highly heterogeneous polyglycosylceramides. They carefully tabulated and interpreted hundreds of mass spectral peaks due to chains up to 41 repeat units (Karlsson et al., 1999). The structure of Lipopolysaccharides (LIPSAS) is extremely complex (Varki et al., 1999). Therisod et al. were able to record the mass spectrum and to identify the three mass regions due to lipid fragments, core fragments (simple copolysaccharides) and LIPSAS pseudomolecular ions, respectively (Therisod, Labas, & Caroff, 2001). Derivatization is often adopted in the analysis of polysaccharide samples (Hounsell, 1998). Methylation and acetylation are the most common derivatization routes (see for instance Yang & Montgomery, 2000). When the reactants are stoichiometrically unbalanced (Limberg et al., 2000a,b), random copolysaccharides are produced (see Sturiale, Naggi, & Torri, 2001 for a mixture of heparin-derived chains which spans from hexamers and octamers). Many authors believe that mass spectra of permethylated samples possess a better spectral quality (in terms of resolution and S/N ratio) with respect to native samples (Garozzo et al., 2000; Deery, Stimson, & Chappell, 2001). Deery et al. analyzed by MS a permethylated arabinogalactan sample (Deery, Stimson, & Chappell, 2001). Bacteria such as Pseudomonas Olevarans, Pseudomonas Putida, Alcaligenes Euthrophus, Rodospirillum Rubrum, and Ralstonia Eutropha are able to produce copolymers, mainly poly-hydroxyalkanoates. A comprehen124

sive review of their properties and structure can be found elesewhere (Sudesh, Abe, & Doi, 2000). It is practically impossible to construct a model for the polymerization reaction, because a huge number of enzymes form an extraordinarily complicated system in a living bacterium. This complexity implies that mixtures of copolymers with the same structure, but different composition, can be produced, and that the shape of the COmpositional DIstribution HIstogram (see Section VII) can deviate from the shape of a bell (Arai et al., 1999; Watanabe et al., 2001; Yoshie & Inoue, 2001). When a copolymer of this kind is left in the soil, it is attacked by the above bacteria (and also by other very common bacteria, such as Alcaligenes fecalis) and it depolymerizes. In a word, these copolymers are biodegradable, a very unusual (and attractive) feature. MS has been successfully employed to analyze polymers from bacterial synthesis. The most common and the most studied one is certainly the copolymer with units of b-hydroxybutyrate and units of b-hydroxyvalerate (Ballistreri et al., 1989a; Nedea, Morin, & Marchessault, 1991; Abate et al., 1993; Lang et al., 1998; Saeed et al., 1999; Adamus et al., 2000). Copolymers of bacterial synthesis in which three, four, up to seven (Ballistreri et al., 1990) different repeat units are found along the chain as well as those with unsaturated carbon bonds (Ballistreri et al., 1992) were analyzed by MS. Mixtures of two copolymers can be analyzed as well (Ballistreri et al., 1991). Abate et al. recorded the MALDI–TOF spectrum of a microbial copolymer with units of b-hydroxybutyrate (HB) and b-hydroxyvalerate (HV) (Abate et al., 1992). Prior to the MS analysis, the copolymer was reacted with an HCl/ methanol mixture, and a reduction of the length of the chains occurred (this process is referred to as partial methanolysis). They used MS intensites to estimate the molar fractions of HB and HV. They applied the method based on chain statistics, and found that HB/HV ¼ 87/13, with a good agreement between the experimental and theoretical data (Abate et al., 1993). The DCI spectrum (reagent gas: ammonia) of a microbial copolymer with four different repeat units [bhydroxyvalerate (HV), b-hydroxycaproate (HC), b-hydroxynonaoate (HN), and b-hydroxyundecanoate (HH)] was recorded (Abate et al., 1992). The authors used MS intensites to estimate the molar fractions of the four repeat units in the copolymer. They applied the method based on chain statistics, and found that HV/HC/HN/HH ¼ 2/31/65/2. This result is by no means trivial, because it is well known that, in this case, 1H-NMR and 13C-NMR analysis gives unsatisfactory results because all comonomeric units resonate at the same chemical shift. Figure 16 reports the FAB spectrum of a microbial copolyester that contains units of b-hydroxy caproate (HC), b-hydroxyoctanoate (HO), b-hydroxydecaoate

MASS SPECTRA OF COPOLYMERS

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1238.3 is due to the ion HO-B7A3-H,,,Naþ, whereas the peak at 1,540.6 is due to the ion H-O-B8A4-H,,,Kþ. It is interesting to note that the composition of the copolymer differs from the composition of the monomeric feed. The ultimate model for copolymerization cannot account for this experimental observation, whereas the terminal model can. This result implies that the concept of reactivity ratios (see Montaudo & Montaudo, 1992b; Suddaby, Hunt, & Haddleton, 1996) holds also for copolymers obtained using lipase (and more in general for enzymatic synthesis). FIGURE 16. FAB spectrum of a microbial copolyester. Reproduced

from Ballistreri et al. (2001) with permission from Elsevier (copyright 2001).

(HD), b-hydroxy dodecanoate (HE), andb-hydroxy tetradecanoate (HT) (Ballistreri et al., 2001). It can be seen that dimers and trimers are present. Mass spectral intensities were used to compute the composition (chain statistics). The best agreement was obtained for HC/HO/HD/HE/ HT ¼ 10/37/38/10/5. Enzymatic synthesis of copolymers is a promising field (see Koskinen & Klibanov (1996) for a review on enzymatic synthesis). Horseradish peroxidase was used to to produce a copolymer with units of para-functionalized phenols (Reihmann & Ritter, 2001). Lipase was used to produce a copolymer with units of hydroxydecanoic acid (A) and b-butyrolactone (B) (Jedlinski et al., 1999). Its MALDI spectrum (see Fig. 17) displays peaks due to ions cationized with sodium or potassium. Thus, the peak at

FIGURE 17. MALDI spectrum of a copolymer with units of hydro-

xydecanoic acid (A) and b-butyrolactone (B) obtained by enzymatic synthesis (lipase). Reproduced from Jedlinski et al. (1999) with permission from Elsevier (copyright 1999).

B. Block Copolymers

Books are available with a description of the various synthetic routes used to obtain block copolymers (Allport & Janes, 1973; Goodman, 1982). A very common route (referred to in the following as double-anionic) consists in adding a first monomer, performing anionic polymerization up to complete monomer consumption, and adding the second monomer. The MMD of the copolymer is usually narrow, even narrower than the polymeric precursor produced before the addition of the second monomer. EO/PO block copolymers terminated with NH2 (an amino end-group) are available commercially under the trade name Jeffamine. EO/PO/EO triblock copolymers are available commercially under the trade name Pluronics and Poloxamer. Mass spectra of copolymers obtained by double-anionic synthesis show negligibly low mass discrimination. Thus, one can record the mass spectrum, derive  n, M  w and cA (see formulas above), and use the formulas M that yield the number-average lengths of like monomers  n /k1, hnBi ¼ cB M  n /k1, hnAi and hnBi; namely, hnAi ¼ cA M where k1 is the average between the masses of the two repeat units. Polyurethane block copolymers are obtained from a polymeric diol (a polymer terminated with OH on both sides, such as polyethyleneglycol or polytetrahydrofuran) and di-isocyanates (the most common are methyl diisocyanate and 4-40 - diphenyl methane diisocyanate). In some cases, polymeric diols are used that contain ester linkages (such as OH-terminated polyethyleneadipate or polybutyleneadipate), and thus polyester-urethane block copolymers are formed. Bletsos et al. recorded the TOF-SIMS spectrum of a polyester-urethane sample obtained by reacting poly(hexylene azelate) with toluene diisocyanate (Bletsos et al., 1990a). Cohen et al. recorded the TOFSIMS spectrum of a polyester-urethane sample obtained by reacting poly(butylene adipate) with 4-40 -diphenylmethane diisocynate, and partially degrading the sample by attacking it with trifluoroacetic acid (Cohen et al., 1995). They found that the acid selectively cleaves the bonds between polyester repeat units. Mehl et al. used 125

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ethanolamine to attack polyurethanes and phenyl-isocyanate to attack polyester-urethane block copolymers. The partial degradation process turned out to be totally selective (Mehl et al., 2000). A quite uncommon method to obtain a block copolymer is to use a monomer and a bifunctional initiator namely, the dimer of a-methylstyrene (sodium salt). The initiator has a strong tendency to form aggregates, mainly dimeric and tetrameric, and thus, a block copolymer is formed. Guttman et al. recorded the MALDI–TOF spectrum of a copolymer that contained units of a-methylstyrene (A) and methylmethacrylate (B). The sample is a reference material named SRM1487. The MS intensities were used by the authors to determine the average number of A units in the chain, l(s), using the direct approach. The authors displayed the result of the calculation of l(s) along with the average number of A unit in the chain, determined by a double detector SEC device, and found an excellent agreement (Guttman, Blair, & Danis, 1997). Liquid chromatography at the critical conditions (LCCC) is a relatively new LC techique, and it is ideally suited for block copolymers due to a most peculiar feature—namely, the elution time depends on the length of the first block, and is independent on the length of the second block (Pasch, 2000). The LCCC-MALDI technique for block copolymers consists in fractionating a polymer,  n, M  w , and the analyzing selected fractions, and deriving M number-average lengths of the blocks. The MALDI–TOF spectra of the LCCC fractions display a smaller number of peaks with respect to the MALDI–TOF spectrum of the whole sample. In the case of diblock copolymers, the de-

crease (and the associated simplification) is very strong (Lee et al., 2000), whereas in the case of triblock copolymers, the decrease is less pronounced (Lee et al., 2001). Lee et al. analyzed a triblock LA/EO/LA copolymer (LA ¼ lactic acid, EO ¼ ethylene oxide), and Figure 18 reports the MALDI spectrum of an LCCC fraction (Lee et al., 2001). The spectrum is simple, because a small number of peaks are present. A variant of the LCCC-MALDI technique uses near Critical Conditions, where the separation is slightly (weakly) chain-length dependent (Falkenhagen et al., 2000). C. Random Addition Copolymers

Random addition copolymers can be obtained from a mixture of two monomers and a free-radical initiator. The composition of the copolymer usually differs from the composition of the monomeric feed. Although extremely accurate models have been developed to predict the composition of the copolymer (Allen & Bevington, 1989), the terminal model is the most used, because it is simple. The terminal model employs the ratios between the propagation constant (the reactivity ratios) to describe polymerization kinetics. MS can be used for reactivityratio determination (Montaudo, 1992b; Suddaby, Hunt, & Haddleton, 1996). When the monomer conversion is high, the resulting copolymer may be compositionally heterogeneous. Haddleton et al. synthetized a random copolymer with units of methylmetacrylate (A) and metacrylic acid

FIGURE 18. MALDI spectrum of an LCCC fraction of a triblock LA/EO/LA copolymer. Reproduced from

Lee et al. (2001) with permission from American Chemical Society (copyright 2001).

126

MASS SPECTRA OF COPOLYMERS

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FIGURE 19. ESI spectrum of a copolymer obtained, using catalytic chain transfer along with the structure

of the ions that may be present. Reproduced from Haddleton et al. (1996) with permission from The Royal Society of Chemistry (copyright 1996).

(B), using Cobalt(II) catalysis and the extremely common azo initiator AIBN (N,N0 -azo-bisisobutyronitrile) (Haddleton et al., 1996). Figure 19 reports the ESI spectrum and the structure of the ions that may be present (referred to as G and H). An inspection of the spectrum reveals that ions G are systematically absent, and that the ‘‘tallest’’ peak in the region 1950–2050 Da is the peak at mass 2013, which corresponds to A18B2. The fact that Arich chains are the most abundant is in perfect agreement with the fact that the overall composition of the copolymer is known (Haddleton et al., 1996) to be cA ¼ 0.923. Random addition copolymers can be obtained by copolymeric precursor. Li et al. synthetized a copolymer with units of styrene and of 4-methoxystyrene (Li et al., 1998). They reacted it with trimethylsilyliodide, and the 4-methoxystyrene units transformed themselves to 4-vinylphenol units (a demethylation occurred). The composition was determined by the hard-ionization method (Li et al., 1998). A homopolymeric precursor can also be used. Sometimes, a hydrolytic attack is

sufficient, and Dawkins recorded the MALDI spectrum of a copolymer with units of vinylacetate and vinylalcohol (Dawkins, 2001). Raeder et al. recorded the MALDI spectrum of a copolymer with units of styrenesulphonicacid and styrene obtained by reacting polystyrene with sulphuric acid (the reaction time was tuned in such a way to achieve almost complete conversion), and they determined the degree of sulfonation, which coincides with the composition of the copolymer (the direct method was applied, the result was 96%) (Raeder, Spickermann, & Mllen, 1995). When poly(N-vinylcarbazole) is reacted with acetyl sulfate, a random copolymer is formed, and MS allows one to determine the copolymer composition (also referred to as the degree of sulfonation, Weng, 2000a; Wang et al., 2000a). Montaudo recorded the MALDI spectrum of a copolymer with three different units—namely, styrene (ST), maleic anhydride (MAH), and maleic acid (MAC) (Montaudo, 2002e)—and determined the composition of 127

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D. Random Condensation Copolymers

FIGURE 20. Portion of the MALDI–TOF spectrum of a copolymer

obtained by ring-opening copolymerization of dilactide and 6-methyl2,5-morpholinedione (the cyclic glycine-lactic acid dimer) in the region 550–800. Reproduced from Montaudo (1999b) with permission from John Wiley & Sons Ltd. (copyright 1999).

the copolymer (method based on statistics) as ST/MAH/ MAC ¼ 50/44/6. When the two monomers in the feed are cyclic, one can use ring-opening copolymerization, and the polymerization rates may be high. The MALDI–TOF mass spectrum of a copolymer that contained units of lactic acid residue (L) and glycine (G) obtained by ring-opening copolymerization of dilactide and 6-methyl-2,5-morpholinedione (the cyclic glycine-lactic acid dimer) was recorded (Montaudo, 1999b). The synthetic scheme yields a quasi-random copolymer with a peculiar sequence distribution, because it deviates from the standard one, in which the two repeat units (L and G) are found at random along the copolymer chain. The deviation is due to chains that have sequences that contain two consecutive G units, which are not produced. This deviation is small, and is therefore difficult to detect. Figure 20 reports a portion of the MALDI–TOF spectrum of the copolymer in the region m/z 550–800. The peak at 588 is the most intense, and it is the sum of two contributions—namely, H-L5G3-OH Kþ and H-L5(L’)G2OH Naþ, where species L’ possesses a 13C atom. Analogously, the peak at 603 is the sum of H-L6G2-OH Kþ and H-L6(L’) G1-OH Naþ. Peaks at 617, 645, 660, 675, 690, 702, 717, 732, 474, and 762 also have a double assignment. The copolymer composition was derived from the MS. The direct method gives cL ¼ 0.70, whereas the method based on statistics gives cL ¼ 0.77. The latter is the correct result (Montaudo, 1999b), and thus, this example is another case in which the direct method fails. 128

Condensation Copolymers usually possess a quite broad MMD. As a consequence, low MM oligomers are very abundant. The theory predicts that the MMD follows the  w /M  n equals 2), and thus most probable MMD (the ratio M that the peaks associated with dimers, trimers, and tetramers dominate the spectrum (unless they are elimated during the workup). Partial degradation is often adopted in sequence determination of copolycondensates. Garozzo et al. recorded the EI spectrum of a series of random copolyester samples obtained by the reaction in the melt of p-acetobenzoic acid and polyethylene terephthalate (Garozzo et al., 1987). Partial degradation was achieved by pyrolysis, and the process is totally selective, because only the CH2 –CH2 bond is cleaved (see Fig. 21a). A series of strong peaks was observed due to hydroxyl-ended chains accompanied by another series due to vinyl-ended chains, and a mechanism was proposed in which chains that belong to both series have the same origin—namely, a cyclic oligomer (Garozzo et al., 1987). A series of four ether–sulfone/ether–ketone (PES/PEK) samples that possessed different composition and/or sequence were studied by FAB-MS (Montaudo et al., 1995a). Methoxidation was used to reduce the molar mass of the samples, and the partial degradation process is not selective because it attacks the ether bond (connecting the two phenyl rings) that is present in both repeat units (see Fig. 21b). Mass spectral peak intensities were used for sequence determination (chain statistics was applied), and the results compared very well with those from the 13 C-NMR analysis (Montaudo et al., 1995a). The chainstatistics method has been applied to the case of a copolyamide (Montaudo & Montaudo, 1992b) with units of adipoyl piperazine (referred to as A) and of truxilloyl piperazine (referred to as B). Photolysis was used for the copolymer degradation. This example is a case of totally selective cleavage, because the cyclobutane ring is split quantitatively into two parts by photolysis (see Fig. 21c). Remarkably, the FAB mass spectra have relatively few peaks, and the composition of the copolymer varies as the mass grows (compositional drift). These facts accurately correspond to the predictions of the totally-selective process theory (Montaudo & Montaudo, 1992b). The direct method and the chain-statistical method were both used to obtain copolymer composition. The direct method failed, or more specifically, it yields undesirable results. On the other hand, using the chain statistical method, the copolymer composition for the 80/20 sample was cA ¼ 0.80, with a very good agreement factor (AF ¼ 6%) (Montaudo & Montaudo, 1992b). Branched copolymers obtained by polycondensation can be analyzed by MS. A commercial polyester produced

MASS SPECTRA OF COPOLYMERS

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FIGURE 21. Some partial degradation reactions used to reduce the chain length: partial pyrolysis (A), partial methoxidation of an polyether (B), and partial photolysis (C).

from a dicarboxylic acid (1-4-ciclohexane dicarboxylic acid, referred to as A), a diol (neopentylglycol, referred to as B), and a triol (trimethylolpropane, referred to as C) was analyzed by MALDI and ESI (Hunt, Sheil, & Derrick, 1998). The resulting product is a branched copolyester in which AB and AC units are found at random along the chain. The molar fraction of AC units coincides with the fraction of branching points, and it is, therefore, connected to the degree of branching The MALDI mass spectra of condensation copolymers produced by adventious hydro-

lysis of hyperbranched polyesters were also recorded (Moore et al., 2002). Poly-silsesquioxanes have recently received much attention due to the fact that they make useful precursors to fully crosslinked, solid monoliths (Wallace, Guttman, & Antonucci, 1999; Tecklenburg, Wallace, & Chen, 2001; Williams et al., 2001). It has been known that they possess a large number of closed loops, NCL, but up to 1999, the exact amount of NCL and how NCL scales with the macromolecular size was unknown. Wallace et al. recorded the 129

TABLE 1. Reports on Mass Spectra of Copolymers Produced by Enzymes or Occurring in Nature

Copolymer samplea HB/HV (MET þ HPLC) HB/HV (TGRES) ALK7 (MET þ HPLC) Xyloglucans HB/HV (MIXT, MET þ HPLC) HB/HV (MET þ HPLC) HB/alkenyl-H HV/HC/HN/HU (MET þ HPLC) HB/HV HB/HP/HV HB/HV Heparin HB/HV/HC HB/HV (TRAN) Yaluronic acid Biantennary saccharides Bi/triantennary saccharides 12HAD/BBL (LIPASE) Polyglycosylceramides Amylopectin HC/HO/HD/HE/HT HB/HV (SAPON) Exopolysaccharide Oligosaccharides Pectin Pectin Lipopolysaccharides sulfated mannans Amylopectin Dextrin Oligogalacturonides Oligogalacturonides (Amidat) HC/HO/HD Permethylated arabinogalactan Cellulose acetate CremophorEL kappa-Carragenam kappa-Carragenam Chondroitin Sulfate Xylo-oligosaccharides (HPLC) Amino-oligosaccharides (HPLC) Lipoarabinomannans Lipopolysaccharides Heparin NFUR/BBOL Cyclomaltooctaose Glycosaminoglycan (chondroitin sulfate) triantennary saccharides Polyflavonoid tannins Hemicellulose

Instrb FAB FAB FAB FAB FAB FAB FAB FAB MALDI FAB SIMS ESI FAB MALDI MALDI MALDI MALDI MALDI MALDI MALDI FAB ESI MALDI MALDI MALDI MALDI ESI MALDI FAB FAB MALDI MALDI FAB MALDI MALDI MALDI ESI ESI Quadrupole MALDI ESI MALDI MALDI MALDI MALDI FAB ESI MALDI MALDI MALDI

Compc ST ST ST ST ST ST ST ST HI ST

ST ST

PLOT PLOT

DI

DEISO ST

DI

Author Ballistreri et al., 1989a Ballistreri et al., 1989b Ballistreri et al., 1990 Hisamatsu et al., 1991 Ballistreri et al., 1991 Nedea, Morin, & Marchessault, 1991 Ballistreri et al., 1992 Abate et al., 1992 Abate et al., 1993 Ballistreri et al., 1995 Lang et al., 1998 Chai et al., 1998 Ballistreri et al., 1999 Saeed et al., 1999 Yeung & Marecak, 1999 Geyer et al., 1999 Colangelo & Orlando, 1999 Jedlinski et al., 1999 Karlsson et al., 1999 Broberg et al., 2000 Impallomeni et al., 2000 Adamus et al., 2000 Garozzo et al., 2000 Spina et al., 2000 Limberg et al., 2000a Limberg et al., 2000b Gaucher, Morrow, & Leary, 2000 Erra-Balsells et al., 2000 van der Burgt et al., 2000a van der Burgt et al., 2000b van Alebeek et al., 2000 van Alebeek, Schols, & Voragen, 2001 Ballistreri et al., 2001 Deery, Stimson, & Chappell, 2001 Bashir, Critchley, & Derrick, 2001 Meyer et al., 2001 Ackloo et al., 2001 Ekeberg, Knutsen, & Sletmoen, 2001 Desaire, Sirich, & Leary, 2001 Kabel, Schols, & Voragen, 2001 Si, Zhong, & Chen, 2001 Ludwiczak et al., 2001 Therisod, Labas, & Caroff, 2001 Sturiale, Naggi, & Torri, 2001 Reihmann & Ritter, 2001 Shizuma et al., 2001 Zaia, McClellan, & Costello, 2001 Huang, Mechref, & Novotny, 2001 Pasch, Pizzi, & Rode, 2001 Jacobs et al., 2002

a HB, b-hydroxy butyrate; HV, b-hydroxy valerate; HP, repeat unit from pentenoic acid; HC, b-hydroxy caproate; HH, b-hydroxy heptanaoate; HO, bhydroxyoctanoate; HN, b-hydroxy nonaoate; HD, b-hydroxynonaoate; HU, b-hydroxyundecanoate; HE, repeat unit from eicosanoic acid; HT, repeat unit from tetradecenoic acid; ALK7, HV/HC/HH/HO/HN/HD/HU; TGRES, thermogravimetric residue; MET, prior to the MS analysis, the copolymer was reacted with an HCl/methanol mixture, and a reduction of the length of the chains occurred (this process is referred to as partial methanolysis); Amidat, prior to the MS analysis, the sample was attacked with ammonia; MIXT, two copolymers having different compositions were mixed together; Alkenyl-H, repeat unit with unsaturation; SAPON, prior to the MS analysis, the copolymer was reacted with a KOH/water/COAG mixture (COAG is the complexing agent, namely 18-crown-6) and a decrease in the average length of the macromolecular chains occurred (this process is referred to as partial saponification); TRAN, prior to the MS analysis, the copolymer was reacted with a sodium methoxide/methanol mixture; LIPASE, enzymatic catalysis (lipase); 12HDA, hydroxydecanoic acid; BBL, b-butyrolactone; NFUR, N-(hydroxyphenyl)-2-furamide; BBOL, benzoyl-oxybiphenyl-4-ol. b Ion source of the mass spetrometer used. c Method used to extract compositional information from MS intensities. DI, direct method (see Section 3.1); HI, hard ionization method (see Section 3.2); ST, statistics (see Section 3.3). PLOT, abundances were plotted and the overall composition can be easily deduced; DEISO, direct method with isotopic correction (deisotopization).

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TABLE 2. Reports on Mass Spectra of Block Copolymers

Copolymer samplea EO/PO (Jeffamine) EA/RMDI HAZ/TID ST/BU/ST (OZONO) DPMI/BUA EO/TFE DFMEO/TFEO ST/AMST triblock EO/PO (Jeffamine) EO/PO (triblock, TWAPET) SIL/ISDI (thick film) PHEE/EO EO/PO triblock (Pluronic L31) EO/ethylene MMA/BA EO/LL (LCCC) ST/AMST 4VP/AMST (triblock) EO/ethylene EO/PO DHX-VPO/EO DPMI/THF tert-BA/MA ST/IP PHEE/EO EO/PO LA/EO/LA CPL/IBU/CPL (triblock) CPL/SIL/CPL ST/TBST EO/PO (poloxamer) HB/PIV

Instrb

Compc

LD SIMS SIMS FAB SIMS LD REMPI MALDI MALDI MALDI SIMS MALDI FT APCI PB-EI MALDI MALDI MALDI MALDI MALDI MALDI SIMS MALDI SIMS MALDI MALDI MALDI MALDI MALDI MALDI MALDI MALDI

DI HI HI HI DI ST DI

HI DI DI DI DI DI

HI HI

ST

Author Nuwaysir, Wilkins, & Simonsick, 1990 Bletsos et al., 1990a Bletsos et al., 1990a Montaudo, Scamporrino, & Vitalini, 1991a Cohen et al., 1995 Simonsick & Ross, 1996 DeVries & Hunziker, 1996 Wilczek-Vera, Danis, & Eisenberg, 1996 Van der Hage et al., 1997 Schriemer, Whittal, & Li, 1997 Zhuang, Gardella, & Hercules, 1997a Francke et al., 1998 Van Rooji et al., 1998 Jandera, Holcapek, & Theodoridis, 1998 Murphy, Schure, & Foley, 1998 Lee et al., 1999 Wilczek-Vera et al., 1999a) Wilczek-Vera et al., 1999b Falkenhagen et al., 2000 Chen et al., 2000 Wang et al., 2000b) Mehl et al., 2000 Ma & Wooley, 2000 Mehl & Hercules, 2001 Przybilla et al., 2001 Chen et al., 2001 Lee et al., 2001 Storey, Brister, & Sherman, 2001 Kricheldorf & Langanke, 2001 Yang, Nonidez, & Mays, 2001 Gallet et al., 2002 Kowalczuk, Adamus, & Montaudo, 2002

a

MMA, methyl-methacrylate; BA, butylacrylate; MA, methylacrylate; EO, ethyleneoxide; PO, propyleneoxide; TFE, tetrafluoro ethylene; EA, ethylene adipate; DFMEO, difluoro methylene oxide; TFEO, tetrafluoro ethylene oxide; RMDI, dicyclohexylmethane-diisocyanate; HAZ, hexylene azelate; TID, toluene diisocyanate; DPMI, diphenyl methane isocyanates; THF, tetrahydrofuran; BUA, buty lene adipate; TWAPET, terminated with aminopropyl ether; TBST, tert butyl styrene; SIL, siloxane repeat unit; DHX-VPO, repeat unit with two aromatic rings and two hexamethylene sidegroups; ISDI, isophorone diisocyanate; ST, styrene; BU, butadiene; OZONOL, prior to MS, the sample was reacted with ozone, which attacks esclusively the butadiene unit; 4VP, 4-vinylpyridine; PHEE, phenylene-ethynlene; IBU, isobutylene; CPL, e-caprolactone; LA, lactic acid; IP, isoprene; HB, hydroxybutyrate; PIV, pivalolactone. b Ion source of the mass spetrometer used. PB, particle beam. c Method used to extract compositional information from MS intensities. DI, direct method (see Section 3.1), HI, hard ionization method (see Section 3.2), ST, statistics (see Section 3.3).

MALDI spectrum of a Poly-silsesquioxane sample, and measured the changes of NCL with the macromolecular size. They were able to elucidate the sample’s structure (Wallace, Guttman, & Antonucci, 1999). Other samples were analyzed by MS (Tecklenburg, Wallace, & Chen, 2001; Williams et al., 2001). Some condesation copolymers can undergo sequential reordering due to exchange reactions such as ester– ester and carbonate–carbonate exchange. For instance, exactly alternating AB copolymers can undergo thermal reorganization, and chains with two consecutive A or B

units can appear. The occurrence of such scrambling is difficult to detect with 1H-NMR and 13C-NMR because the spectra are usually limited at the dyads level. This limit is due to the fact that, in condesation copolymers, the distance between comonomeric units is large and the interaction is weak. On the other hand, MS can explore higher sequences, and sequential reordering is easily detected (Ramjit & Sedgwick, 1976; Giuffrida et al., 1986; Montaudo, Scamporrino, & Vitalini, 1989b; Montaudo et al., 1995a). Scrambling reactions also occur in polysiloxanes (Servaty et al., 1998). 131

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TABLE 3. Reports on Mass Spectra of Random Addition Copolymers

Copolymer samplea ST/MMA MMA/BA MMA/BMA/DEAEMA DFMEO/TFEO (Fomblin Z) HEA/LIPMO ACR/BUT ACR/ST HEA/LOARES NVMAZL/ST St/Bu (ozonolysis) DFMEO/TFEO (Fomblin Z) ETVPYR/12VPYR CAMO/ETIPROP SIL4DAAP ETVPYR/12VPYR ST/TODEST ST/TODEST MMA/BA VP/VA ST/SST ST/PADEST MMA/EGMA ST/BUT DHI/PADEDHI NVP/VA LA/GA MMA-MAAC DFMEO/TFEO (Fomblin Z) Ethylene/propylene ST/MAAC DGEBA/DBOXAND (addition) MMA/BA LA/EO MEEPI/HXI ACR/BUT MMA/EGMA (graft) MMA-AMST MLA/BMLA MMA-MAAC (SEC fractions) ETHYL/PROP SIL/SILF3 SIL/SILF9 GMA/BMA ST/4VP (from precursor) ST/BMA (CORA-TEMPO) HSIL/SIL (EQUILIB) GMA/BMA (SEC fractions) ITFN/HXI Ethylene/butene or hexane Ethylene/VA GLLA/LALA ACR/BUT (OZONOL) HYST/MOST ST/MMA Poly(vinilydene fluoride)

Instrb LD LD SIMS SIMS Field ioniz. Field Ioniz. EI Field ioniz. SIMS FAB DCI DCI DCI DCI DCI SIMS SIMS SIMS Field des MALDI SIMS SIMS SIMS DCI MALDI SIMS ESI SIMS SIMS SIMS Plasma des. MALDI SIMS DCI Photoioniz. SIMS MALDI SIMS MALDI Photoioniz. MALDI MALDI ESI SIMS MALDI MALDI ESI DCI Photoioniz. Photoioniz. MALDI MALDI MALDI SIMS SIMS

Compc DI DI ST ST

HI ST ST ST ST DEISO

DI HI HI ST ST ST HI HI

ST HI ST ST HI DI HI HI

HI ST ST ST ST ST ST ST HI HI

Author Nuwaysir, Wilkins, & Simonsick, 1990 Nuwaysir, Wilkins, & Simonsick, 1990 Wilding et al., 1990 Bletsos et al., 1990b Plage et al., 1990 Plage & Schulten, 1991a Plage & Schulten, 1991a Plage & Schulten, 1991b Lee & Gardella, 1992 Vitalini & Scamporrino, 1992a Guarini et al., 1993 Xin et al., 1993 Vincenti & Sommazzi, 1993 Ranasinghe et al., 1993 Majumdar et al., 1993 Affrossman et al., 1993 Hittle, Proctor, & Hercules, 1994 Davies et al., 1994 Danis & Huby, 1995 Raeder, Spickermann, & Mu¨llen, 1995 Hittle, Proctor, & Hercules, 1995 Shard et al., 1995a Weng et al., 1995 Hoke et al., 1995 Schaer, 1995 Shard et al., 1996 Haddleton (1996) Spool & Kasai, 1996 Galuska, 1996 Davies et al., 1996 Klee, Haegele, & Przybylski, 1996 Suddaby, Hunt, & Haddleton, 1996 Shard et al., 1997 Chen et al., 1997a Zoller & Johnston, 1997 Briggs & Davies, 1997 Guttman, Blair, & Danis, 1997 Leadley et al., 1997 Nielen & Malucha, 1997 Zoller & Johnston, 1997 Yoshida, Yamamoto, & Takamatsu, 1998 Yoshida, Yamamoto, & Takamatsu, 1998 Shi et al., 1998 Li et al., 1998 Burguiere et al., 1999 Servaty et al., 1998 Aaserud, Prokai, & Simonsick, 1999 Chen et al., 1997b Zoller et al., 1999 Zoller et al., 1999 Montaudo (1999) Zoller & Johnston, 2000a Satoh, Kamigaito, & Sawamoto, 2000 Van den Eynde, Reiths, & Bertrand, 2000 Feng, Chan, & Weng, 2000 (Continued )

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MASS SPECTRA OF COPOLYMERS

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TABLE 3. (Continued )

Copolymer samplea

Instrb

Compc

NVCBZ/SUVCBZ NVCBZ/SUVCBZ MMA/BA PADE/PADB ST/HFHDIPAST ST/MMA LA/PO (SEC fractions)

SIMS SIMS MALDI Photoioniz. SIMS SIMS ESI

HI HI

MMA/PALLA (CHTSOM) ST/MAH/MAC

MALDI MALDI

HI HI

ST

Author Wang et al., 2000a Weng et al., 2000a Esser et al., 2000 Zoller et al., 2000b Weng et al., 2000b Van den Eynde, Reiths, & Bertrand, 2001 Chisholm, Navarro-Llobet, & Simonsick, 2001 Morrison, Eadie, & Davis, 2001 Montaudo, 2002e

a ACR, acrylonitrile; BUT, butadiene; ST, styrene; MMA, methyl-methacrylate; BA, butylacrylate; DFMEO, difluoro methylene oxide; TFEO, tetrafluoro ethylene oxide; HEA, 2-hydroxyethyl acrlylate; DEAEMA, (2-dimethylaminoethyl) methacrylate; LIPMO, lipid monomer (a methacrylate derivative with a C18H37 side chain); LOARES, acrylic repeat unit with a long aromatic-ester side chain; VP, vinyl pyrrolidone; VA, vinyl acetate; 4VP, 4-vinylpyridine; ETVPYR, 1-ethyl 4-vinylpyridinium monomer; 12VPYR, 1-alkyl 4-vinylpyridinium monomer with a long aliphatic chain; NVMAZL, N-vinylimidazole; SIL4DAAP, siloxane with dialkylaminopyridine substituents; MAAC, methacrylic acid; MEEPI, methoxy ethoxy propyl isocynate; HXI, hexylisocynate; ITFN, isocyanato-tri decafluoro-nonane; MLA, malic acid; BMLA, butyl malic acid; LA, lactic acid; GA, glycolic acid; EO, ethyleneoxide; PO, propyleneoxide; EGMA, ethyleneglycolmethacrylate; PHEE, phenylene-ethynlene; LL, dimer of lactic acid; AMST, a-methylstyrene; CAMO, carbon monoxide; DHI, dimethyl heptyl isocyanates; PADEDHI, partially deuterated dimethyl heptyl isocyanates; ETIPROP, ethylene/propylene; 4VP, 4-vinyl piridine; HYST, hydroxystyrene; NVCBZ, N-vinylcarbazole; SUVCBZ, sulfonated N-vinylcarbazole; MOST, methoxystyrene; GLLA, glicine-lactic dimmer; LALA, lactic dimer (lactide); CORA-TEMPO, controlled radical using TEMPO initiator; EQUILIB, obtained by ring-chain equilibration; HSIL, hydromethylsiloxane; BMA, butyl methacrylate; GMA, glycidil methacrylate; PADE, patially deuterated ethylene; DGEBA, bis [(epoxy propoxy)phenyl] propane; DBOXAND, NN0 dibenzyl-5oxanonane diammine; PADB, patially deuterated butene; PALLA, 2-phenyl-allyl-alcohol; MAH, maleic anhydride; MAC, maleic acid; SST, styrenesulfonic acid; NVP, N-vinylpyrrolidone; HFHDIPAST, hexafluorohydroxyisopropyl-alfa-styrene; SILF3, trifluoro substituted siloxane; SILF9, substituted siloxane with 9 fluorine atoms; CHTSOM, chain transfer isomerization; TODEST, totally deuterated (per deuterated) styurene; PADEST, partially deuterated styurene. b Ion source of the mass spetrometer used. c Method used to extract compositional information from MS intensities. DI, direct method (see Section 3.1); HI, hard ionization method (see Section 3.2); ST, statistics (see Section 3.3); DEISO, direct method with isotopic correction (deisotopization).

Considerable interest has arisen in the reactive blending of polyamides, polycarbonates, and polyesters (and also combinations of the above) and in the exchange reactions that may occur during the melt-mixing processes. The exchange reactions that lead to the formation of copolymer molecules from two homopolymers can be seen as the result of two consecutive processes. The formation of the copolymer from homopolymers by intermolecular exchange, and rearrangement of the copolymer (by intramolecular exchange) to a different sequence of A and B units along the copolymer chain: -A-A-A-A þ -B-B-B-B- ! -A-A-A-A-B-B-B-B-A-A-A-A-B-B-B-B-B- ! -A-B-B-A-A-B-B-A-BA copolymer sample produced by reactive blending of two homopolymers is made of three components: the two homopolymers and the copolymer formed. As the reaction proceeds, the two homopolymer sequences disappear and the copolymer sequences become abundant. The model for

this exchange process predicts that hnAi and hnBi decrease as the process proceeds. In the later stages, the sequence distribution exhibits a change-namely, it goes from Markoffian to Bernoullian. The experimental verification of the model’s prediction by 1H-NMR and 13C-NMR is very difficult for the reasons cited above. Instead, MS and the method based on chain statistics gives excellent results for these samples (Montaudo et al., 1992c; Montaudo, 1993c).

XI. SUMMARY AND OUTLOOK

Tables 1–4 summarize work on the mass spectra of copolymers. They report the type of repeat units that are found in the macromolecular backbone, the type of mass spectrometer (or the ionization source), and the method used to determine the composition. Some comments can be made. There has been an inversion during the years, and composition determination based on the analysis of the 133

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TABLE 4. Reports on Mass Spectra of Random Condensation copolymers

Copolymer samplea ET-OXB ET/ETX (partial deg) RSC/BPA-CAR (partial deg) TXPP-ADPP (PHOTOL) Copolycarbonates CDCA/NPTG/TRPG HISO/DMHISO DETOSUHID/DETOSUCDM MAIN-PORPH EA/ET melt mixing ET/ETX melt mixing ESU/EKE (PES-PEK) EMA/HEMA ESU/EKE (PES-PEK) SHIFF-ETH BISO/BADI Siloxane/amide SA/RAM BADI/BTE BSE/BSU/BADI/BTE SESQUI FUMA/SEBA NLO-PORPH BSU/BSE (SEC fractions) DADI/DISO SESQUI SESQUI QX/ESU LA/DMI Hyperbranched

Instrb

Compc

DCI FAB FAB FAB SIMS MALDI DEI SIMS FAB FAB FAB FAB SIMS FAB MALDI ESI SIMS SIMS MALDI FAB MALDI SIMS FAB MALDI ESI MALDI MALDI MALDI MALDI MALDI

ELS ELS ELS ST ST HI ST ST HI HI ST

DI DI ST

HI DI ELS

ST

Author Garozzo et al., 1987 Montaudo, Scamporrino, & Vitalini, 1989a Montaudo, Scamporrino, & Vitalini, 1989b Montaudo, Puglisi, & Samperi, 1989c Lub & Buning, 1990 Hunt, Sheil, & Derrick, 1998 Majumdar et al., 1991 Davies et al., 1991 Vitalini & Scamporrino, 1992b Montaudo et al., 1992c Montaudo, 1993c Montaudo et al., 1994 Pinto, Stika, & Lloyd, 1995 Montaudo et al., 1995a Vitalini et al., 1996 Simonsick, Ross, & Prokai, 1996b Senshu et al., 1997 Leadley et al., 1998 Montaudo et al., 1998a Montaudo et al., 1998b Wallace, Guttman, & Antonucci, 1999 Chen & Gardella, 1999 Vitalini, Mineo, & Scamporrino, 1999a Carroccio, Rizzarelli, & Puglisi, 2000 Koster et al., 2000 Tecklenburg, Wallace, & Chen, 2001 Williams et al., 2001 Polce et al., 2001 Keki et al., 2001 Moore et al., 2002

a

TMA, trimethylene adipate; EA, ethylene adipate; OXB, para-oxybenzoate; ET, ethylene terephthalate; ETX, ethylenetruxillate; RSC, resorcine; BPA-CAR, bisphenolAcarbonate; EMA, ethylmethacrylate; HEMA, 2-hydroxy-ethylmethacrylate; TXPP, truxilloil-piperazione; ADPP, adipoilpiperazione; PHOTOL, prior to the MS analysis; the copolymer was exposed to UV radiation at 333nm; FUMA, fumaric acid; SEBA, sebacic acid; ESU, ether sulphone; EKE, ether ketone; DETOSUHD, DETOSU-hexane diol; DETOSUCDM, DETOSU-trans cyclohexane dimethanol; DETOSU, spiro compound; MAIN-PORPH, copolyether with porphyrin units in the main chain; SESQUI, silsesquioxanes; BSU, butylene succinate; BADI, butylene adipate; BSE, butylene sebacate; BISO, butyleneisoterephthalate; BTE, butylene terephthalate; SA, sebacic acid; RAM, ricinoleic acid maleate; QX, quinoxaline; ESU, ethersulphone; HISO, hexyl isocyanates; DMHISO, dimethylheptyl isocyanates; DADI, diproproxylated BisphenolA adipate; DISO, diproproxylated BisphenolA isophthalate; SHIFF-ETH, copolyether with Shiff-base/copper complex; NLO-PORPH, copolyether with NLO porphyrin and a long aliphatic chain; CDCA, 1-4 cyclohexane dicarboxyil acid; NPTG, neopentylglycol; TRPGtrimethylolpropane; LA, actic acid; DMI, diphenylmethane diisocyanate. b Ion source of the mass spetrometer used. c Method used to extract compositional information from MS intensities, DI, direct method (see Section 3.1), HI, hard ionization method (see Section 3.2), ST, statistics (see Section 3.3), ELS, evaluated after the publication using statistics.

high-mass region of the spectrum (i.e., by chain statistics or by the direct method) has become more common than composition determination-based on the low-mass region (hard-ionization method). Furthermore, the number of reports increased dramatically in the last two-three years (for synthetic and naturally occurring copolymers), and this increase implies that MS is becoming a powerful tool for copolymer characterization (although it has some limitations, as discussed in the previous sections). There is 134

no reason to believe that the cited interest will decrease in the next years

ACKNOWLEDGMENTS

Partial financial support from the National Council of Research (CNR, Rome) is gratefully acknowledged.

MASS SPECTRA OF COPOLYMERS

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