Polymer characterization by combining liquid chromatography with MALDI and ESI mass spectrometry

Transcription

1 Anal Bioanal Chem (2002) 373 : DOI /s REVIEW Renata Murgasova David M. Hercules Polymer characterization by combining liquid chromatography with MALDI and ESI mass spectrometry Received: 6 November 2001 / Revised: 12 April 2002 / Accepted: 20 April 2002 / Published online: 12 June 2002 Springer-Verlag 2002 Abstract High-performance polymers are complex mixtures of materials of different size and chemical composition and with different end groups and architecture. To determine the molecular heterogeneity of such systems, hyphenation of several techniques is required. The value of coupling mass spectrometry (MS) with separation techniques has already been recognized such methods have proved to be among the most powerful for molecular characterization of complex polymer systems. The review focuses on matrix-assisted laser desorption/ ionization (MALDI) and electrospray ionization (ESI) MS coupled with liquid chromatography (LC). Such hyphenation has been used for most polymer analysis by mass spectrometry coupled with separation techniques. The advantages and/or limitations of these techniques for polymer characterization are discussed. Future prospects are briefly outlined. Keywords Liquid chromatography Matrix-assisted laser desorption/ionization Electrospray ionization Polymers Hyphenation Introduction Polymers are intrinsically complex materials characterized by different distributions; that most commonly examined is the molar mass. The synthesis of functional polymers (block, random, grafted) and other polymer architectures generates a series of distributions owing to the presence in the sample of polymer chains of different chemical composition and with different end groups and architecture. In practice no single technique can be used to investigate all these distributions, and complete characterization R. Murgasova D.M. Hercules ( ) Department of Chemistry, Vanderbilt University, Nashville, Tennessee, 37235, USA hercules@ctrvax.vanderbilt.edu of complex polymer mixtures at the molecular level requires combination of at least two techniques that are, preferably, selective toward one type of heterogeneity. The polymer chains are usually first fractionated by means of a separation technique such as liquid chromatography, supercritical-fluid chromatography (SFC), temperaturerising elution fractionation (TREF), etc., and/or chemical modification. The second step can be performed by a spectral technique, either off-line or in combination with a separation technique. Because of its high sensitivity, broad dynamic range, specificity, and selectivity, mass spectrometry (MS) has become an indispensable tool for determination of the structure of organic and inorganic polymeric materials. Gas chromatography mass spectrometry (GC MS) is a very early example of a hyphenated technique. GC MS, which uses electron impact or chemical ionization, has frequently been applied to the analysis of the thermal degradation products of polymers. Although hyphenation of thermal techniques to mass spectrometry provides much information about the chemical structure of the polymer, its thermal stability, and impurities present in the material, etc. [1], the chain length and the chain length distribution cannot be determined from the spectra of fragmented polymers. Two significant developments in ionization techniques in the late 1980s matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) greatly affected the use of mass spectrometry for polymer characterization. These techniques enable ionization of large non-volatile molecules with little or no fragmentation, thus enabling determination of the molar mass of intact polymers by MS. The coupling of MALDI and ESI MS to a separation technique also enables deconvolution of individual molecular heterogeneity in complex polymer systems, so that more complete molecular characterization can be accomplished. The primary goal of this review is to demonstrate the utility of the combination of condensed-phase separation techniques with mass spectrometry for polymer characterization, and to outline the advantages and/or limitations of

2 482 these methodologies. We shall focus on MS techniques equipped with the MALDI and ESI methods of ionization, because these techniques have been used for most polymer analyses by mass spectrometry coupled with separation techniques. Mass spectrometry Matrix-assisted laser desorption/ionization MS Since the appearance in the literature [2] of the first MALDI mass spectrum of poly(ethylene glycol) the structural and compositional analysis of synthetic polymers by MALDI MS has developed rapidly. The MALDI technique involves embedding the analyte in a matrix which absorbs at the wavelength of the laser. The energy is then transferred from the matrix to the analyte, which is desorbed and subsequently ionized in the gas phase. The origin of ions in MALDI MS is currently a matter of active research. Chemical and physical pathways proposed for MALDI ion formation have been critically reviewed by Zenobi and Knochenmuss [3]. The ions produced can be analyzed in a TOF mass analyzer, a natural choice for pulsed-ionization techniques. TOF MS techniques have two major advantages. First, the mass range is limited only by the detector, ion transmission, and ionization processes; second, in contrast with quadrupole analyzers, because all the ions are registered simultaneously, without scanning, TOF analyzers can address the scan speed/elution time problem; this makes them applicable to separations combined with MS [4]. During the last decade improvement of MALDI TOF MS instrumentation, chiefly arising from the use of reflectron technology [5], time-lag focusing [6], and Fourier-transform (FT) technology [7], has dramatically increased mass resolution, sensitivity, and mass-measurement accuracy. MALDI TOF MS can now be used to detect synthetic polymer molecules with molar masses in excess of 10 6 Da. State-of-the-art MALDI MS instruments equipped with time-lag focusing and operations in the reflector mode enable qualitative determination of chemical composition, including end group identification, for molar masses up to 36 kda. A resolving power of 800,000 (FWHM) with human insulin can be achieved by use of MALDI FTMS [8]. The strength of MALDI MS lies in the simplicity of the mass spectra, which contain mainly singly-charged quasi-molecular ions, and the relatively high tolerance of contamination of the analyte. Nielen has produced an excellent overview on the MALDI MS of synthetic polymers, covering the literature to the beginning of 1999 [9]. Despite the successes of the technique, several areas of difficulty have been encountered and reported in polymer MALDI MS research. Molar mass determination by MALDI MS depends on how accurately the ion abundance over the mass range represents the composition of the polymer. To obtain precise mean molar mass values the efficiency of the ionization process in MALDI should be independent of chemical composition and mass. In addition, the ions produced must be transmitted and detected without mass discrimination so they correctly represent the abundance of detected ions. In other words, discrimination in ionization, transmission, and/or detection should be avoided so that the intensity of each peak is representative of the molar concentration of the particular molecular species. These requirements can be fulfilled only for narrow-distribution homopolymers (polydispersity index below ~1.2), for which molar masses and molar mass distributions can be determined reasonably accurately [10]. For polydisperse homopolymers, however, the solubility, probability of desorption, and detection efficiency decrease with increasing analyte mass. Although a procedure based on smoothing and offset subtraction can provide reliable estimates of molar mass and molar-mass distribution directly from the MALDI mass spectra of polydisperse polymers [11, 12], this approach has not found wide application, mainly because its reliability depends on the dynamic range of the mass spectrometer. It is possible that new types of detector for TOF MS, e.g. direct ion-to-photon detectors, will circumvent the limited dynamic range of the channel plate detectors currently used [13, 14]. For complex polymer systems with several distributions mass discrimination will be more pronounced, because of the different ionization efficiencies of chemically and/or architecturally different polymer chains. For such samples the use of separation techniques and/or chemical pretreatment steps in combination with MALDI MS is essential. Electrospray ionization MS Electrospray ionization (ESI) is used to denote the overall process by which an intense electric field disperses a sample liquid into a bath gas as a fine spray of highly charged droplets. Evaporation of these charged droplets produces gas-phase ions by mechanisms that remain the subject of much argument and debate [15]. Since the first quadrupole ESI MS [16] the technique has become one of the most widely used in analysis. Advances in ESI MS instrumentation have also involved the TOF mass analyzer. The difficulty in coupling a continuous ESI source to a TOF instrument was circumvented by use of orthogonal injection, which transforms a continuous ion beam into a pulsed mode [17]. Employment of TOF analyzers has enabled more efficient coupling of ESI MS to liquid chromatographs, primarily because of rapid data acquisition and improved dynamic range. Novel designs such as quadrupole and orthogonal TOF (Q-TOF) analyzers [18, 19] became the basis for commercial Q-TOFMS and MS MS instruments with an upper mass range of 20 kda and a resolution of 20,000 (FWHM) [20]. It should be noted, however, that polymer molar mass and structural analysis by ESI MS has been less fruitful than use of MALDI MS, and analysis of synthetic polymers by ESI MS continues to be a challenging analytical problem. Occasionally the insolubility of polymers in

3 ESI-compatible solvents, and their electroneutrality, prohibits ion formation by the ESI mechanism. As an alternative such samples can be derivatized. Use of ESI MS to characterize synthetic polymers has, nevertheless, produced promising results [21, 22, 23]. The unique ability of ESI to produce multiply-charged ions extends this extremely soft technique to a higher mass range, even for analyzers with a limited mass range. Unfortunately, as a result of multiple charging, even polymers with narrow dispersity and high mass give very complex mass spectra. The problem can be overcome depending on polymer molar mass and complexity by using high-resolution ESI FTMS to determine the charge states from the spacing between the isotope peaks, and subsequently transforming multiply-charged spectra into the singly charged representation. A mass resolving power of ESI FTMS of 430,000 (FWHM) for a sample of bovine ubiquitine (MW=8564) has been reported [24]. Another approach is to reduce the number of components entering the ESI source by coupling ESI MS with a separation technique, as discussed below. It is worth mentioning that a recently reported ionization technique impact desolvation of electrosprayed microdroplets (IDEM) [25] has shown promise for the analysis of mixtures. In IDEM analytes are electrosprayed in a vacuum, producing highly charged microdroplets that are accelerated to velocities of several km s 1 and collide with a target surface. The energetic impacts vaporize the droplets and release gas-phase ions largely free from adducts. Compared with atmospheric-pressure ESI the technique has the advantages of significantly less multiple charging and lower susceptibility to solution impurities. Liquid chromatography 483 Fig. 1 Molecular weight (M) retention volume relationships for exclusion- and interaction-based separation mechanisms. In the exclusion mode the retention volume decreases with increasing M (curve a) whereas in the interactive LC mode the retention volume increases with increasing M (curve b). At the transition point the retention volume is independent of M (curve c) Liquid chromatography (LC) is currently the most widespread technique for polymer separation. In LC synthetic macromolecules can be separated on the basis of exclusion, adsorption, partition, or phase separation, the main mechanism depending on interactions among the analyte, mobile phase, and column packing. On the basis of the main mechanism used for discrimination of polymers two major groups of separation methods can be distinguished (an in-depth review is available elsewhere [26]). Size-exclusion chromatography (SEC) is an entropybased separation method in which macromolecules are separated according to their size (hydrodynamic volume) via selective permeation through, and/or exclusion from, one part of column. SEC is always performed in a mobile phase of constant eluent strength (isocratic mode), typically a pure solvent. Unless a molar-mass-sensitive detector is used SEC requires calibration, ideally by use of samples of the same polymer with narrow or, at least, known molar-mass distributions. Few polymers are available commercially as sets of such standardized samples. The molar mass (M) retention volume relationship (calibration curve) for exclusion-based separation mechanisms, which, in combination with the SEC chromatogram, provides molar mass and distribution data, is illustrated in Fig. 1a. Interaction-based LC involves several enthalpy-based separation techniques which separate according to chemical composition and/or molar mass. Thus chemical composition and molar mass distribution data for polymers can both be determined by use of interactive LC. On the basis of the main separation mechanism interactive LC can be classified into: liquid adsorption chromatography (LAC) (also called normal-phase LC), in which separation is governed by attractive interactions between the stationary phase and the macromolecules; liquid partition chromatography (LPC) (also called reversed-phase LC), in which separation is governed by differences between the thermodynamic stability of the macromolecules in the mobile and stationary phases; and precipitation liquid chromatography (PLC), in which separation is governed by precipitation and (re)dissolution of macromolecules. Interactive LC of oligomers can be performed in the isocratic mode. Higher molar-mass polymers usually require controlled mobile-phase composition (eluent gradient) or temperature (temperature gradient) conditions to circumvent an exponential increase of retention with molar mass (curve b in Fig. 2). The resulting techniques are sometimes denoted gradient polymer elution chromatography (GPEC) and temperature gradient interaction chromatography (TGIC). In practice, the above-mentioned main separation mechanisms are usually accompanied by secondary separation processes (e.g. adsorption in SEC, exclusion in LAC, etc.) which further complicate quantitative data processing.

4 484 Fig. 2 Characterization of polyurethanes by use of chemical degradation and SEC MALDI MS. (A) MALDI spectrum for PUR hydrolysate; the oligomer ion peaks in the spectrum correspond to PUR hard blocks of different chain length. (B) MALDI spectrum for PUR degraded with ethanolamine; the oligomer ion peaks in the spectrum correspond to PUR soft blocks of different chain length. (C) SEC chromatogram of ethanolamine-degraded PUR (D) MALDI spectra of selected SEC fractions from ethanolaminedegraded PUR. The locations of the fractions are shown in (C). (E) MALDI SEC calibration curve for PUR degradants (squares). The points of the SEC calibration curve based on PS standards are indicated by triangles The situation becomes more difficult for complex polymers one LC separation method might not be sufficient for simultaneous discrimination among several types of molecular heterogeneity. To increase LC selectivity toward one molecular characteristic, two or more separation mechanisms are combined within one LC system (coupled LC methods) [26]. So far several approaches have been proposed; most apply a combination of exclusion and adsorption at the transition point. At the transition point the effects of both separation mechanisms mutually compensate so that molarmass-independent separation occurs (vertical curve c in Fig. 1). Of the coupled separation methods proposed, liquid chromatography at the critical adsorption point (often called LC under critical conditions; LCCC) [27] has already been successfully combined with MS. LCCC is performed at a special temperature and mobile phase composition (typically a multicomponent liquid) at which all chains with the same repeating unit elute at the same elution volume (irrespective of their length) (curve c in Fig. 1). This means that the polymer chain (or one block) will become chromatographically invisible. Under these conditions separation according to a structural unit other than the repeat unit, e.g. functionality, or the length of the block, can be achieved in either SEC or LAC mode. LCCC can be used to discriminate among oligomers according to their functionality (end group distribution), among binary polymer blends, and among block and graft copolymers. Occasionally separation according to architecture can also be achieved. It should be noted, however, that LCCC is generally feasible for molecules up to ~10 5 Da only. For characterization of complex polymer systems LC methods can be combined on-line or off-line in several in-

5 dependent LC systems to achieve multidimensional separations (multidimensional chromatography). For example, in the first dimension macromolecules are separated according to their chemical composition by interactive LC; this is followed by chromatography in the second dimension in which SEC separates polymer chains according to molecular weight. Direct transfer within several dimensions faces two major problems related to the detection step when multicomponent mobile phases are used to control retention preferential solvation can complicate quantitative detection of macromolecules by non-specific detectors; because of substantial dilution of the sample in the course of multiple separations, an analyte must be reconcentrated before detection with common LC detectors. These problems can be overcome by use of a sensitive MS detector. LC MS coupling LC combined with MALDI MS SEC MALDI MS Hydrodynamic volume is a measure of relative molar mass and depends on both chemical composition and architecture. To obtain absolute molar mass values by SEC, therefore, either the exact relationship between hydrodynamic volume and molar mass for a given polymer must be known or molar-mass-sensitive detectors must be employed. Because the mass axis in MALDI MS is independent of the nature of the material being analyzed the technique can, similarly to light scattering and viscometry, can be used as an SEC detector for absolute molar mass measurement. To overcome MALDI MS polydispersity discrimination, a high-polydispersity sample can be fractionated by analytical or preparative SEC, yielding fractions with very narrow distributions which can subsequently be analyzed by MALDI MS. The coupling of MALDI MS with SEC has advantages for both techniques. SEC provides MALDI MS with simplified, narrow-dispersity samples that improve the capacity of MALDI MS to obtain significant compositional information about the analyte. The molar masses of SEC fractions measured by MALDI MS can be used for effective calibration of the SEC system to give absolute values for the average molar masses of highly disperse (unfractionated) polymer samples, something that is rarely achieved by SEC alone. The mass retention volume relationship can also be used to study branching, copolymer block structure, SEC column performance, and broadening deconvolution [28]. An off-line variant of this approach was first described by Montaudo s group [29] for analysis of polydisperse polydimethylsiloxanes. Several applications demonstrating the use of this approach, which involves collecting fractions or depositing a sample on a prepared target before MALDI MS analysis [9], soon appeared in the literature. The SEC MALDI MS combination seems to be particularly useful for analysis of rigid-rod synthetic polymers such as polyalkylthiophenes [30]. Efforts have been made to optimize and simplify offline SEC MALDI MS, which turned out to be an efficient means of obtaining reliable molar masses and molar mass distributions, although labor-intensive and complex. A direct deposition method was developed in which the SEC effluent is spray-deposited on to a rotating matrix-precoated substrate and the resulting track subsequently analysed by MALDI MS, thus simplifying sample preparation [31]. The number-average molar masses determined for relatively narrow polydispersity (<1.2) poly(methyl methacrylate) (PMMA) samples using the direct deposition method were in fairly good agreement with the manufacturer s values, although the molar mass distribution values were lower. The direct spray deposition method was also used to analyze a broad poly(methyl methacrylate) (PMMA) material, which could not be analyzed by MALDI MS alone. The results were compared with those from a blend of five narrow-dispersity standards that mimicked the broadly dispersed material [32]. The technique was also applied to the analysis of a diblock copolymer of n-butyl methacrylate and methyl methacrylate, and provided information about both molar mass and chemical composition [33]. Two different approaches continuous flow and aerosol have been proposed for direct coupling of MALDI MS to liquid separation methods. Continuous-flow (CF)-MALDI [34] uses a flow probe similar to a continuous-flow fast atom bombardment probe. The maximum CF-MALDI flow rate is less than 5 µl min 1 ; thus a conventional LC column cannot be used. Another restriction is that the matrix itself must be a liquid. In the aerosol method [35] for MALDI liquid introduction the matrix and the analyte are dissolved in a solution that is sprayed directly into the mass spectrometer. Ions can be formed by irradiating the aerosol particles with pulsed 355-nm radiation from a frequency-tripled Nd:YAG laser. Flow rates in excess of 0.5 ml min 1 have been used. Poly(ethylene glycol) (PEG 1000) and poly(propylene glycol) (PPG 1000) have been analyzed by use of this method [36]. The major advantages of off-line SEC MALDI MS over on-line coupling are that it is relatively easy to implement and does not require instrument modification. It also enables independent system optimization, resulting in higher sensitivity and mass accuracy than the on-line approach. On-line coupling, on the other hand, enables rapid analysis and, after further refinements, might become a powerful analytical technique. Three recent reviews discuss in detail the strengths and limitations of on-line and off-line coupling of MALDI MS to different separation techniques, and the further technical improvement of both [4, 37, 38]. Two-dimensional LC MALDI MS 485 For complex polymer systems comprising molecules of different chemical composition and different size combination of SEC with MS cannot usually provide adequate

6 486 information about both molecular weight and composition distribution. One way of addressing this problem is to add a second separation technique, e.g. LCCC. In the first step of such a procedure the mixture is separated according to composition or functionality (LCCC) yielding fractions which are chemically homogeneous. These fractions are transferred to a size-selective separation method (SEC) and analyzed in respect of molar mass. As a result of this two-dimensional separation information is obtained on both types of molecular heterogeneity [39]. LCCC and SEC combined off-line with MALDI MS detection have been applied to the characterization of poly(propylene glycol) [40] poly(l-lactide) block poly (ethylene oxide) block poly(l-lactide) triblock copoolymers [41] polyamides-6 [42], and poly(propylene oxide)s [43]. Chemical separation SEC MALDI MS Occasionally separation can be aided by chemical modification. It has been reported that MALDI MS hyphenated to SEC and combined with selective degradation can provide information about the soft-block oligomer distribution in polyurethanes (PUR) [44]. Phenylisocyanolysis and ethanolaminolysis can liberate soft blocks from polyester- and polyether-based PUR. On liberation the soft blocks are amenable to SEC MALDI MS analysis. Partial acid-catalyzed hydrolysis can also be used to determine the hard-block chain-length distribution for polyesterbased PUR [45]. The complete characterization of PUR by SEC MALDI MS combined with selective degradation is shown schematically in Fig. 2. LC combined with ESI MS A distinguishing feature of ESI MS is that samples are introduced in solution at atmospheric pressure. This results in the natural compatibility of ESI MS with many types of separation technique. The separation conditions (the mobile phase and its flow rate) must, however, be suitable for MS operation. For LC systems operating at flow rates of ml min 1 a post-column splitter is usually used to reduce the flow rate to the µl min 1 scale typical of ESI operation. Post-column addition can be used to provide a source of, e.g., alkali metal ions to promote cationization. Although ESI MS is easy to interface with LC there have been few reports of direct coupling of these techniques for the analysis of synthetic polymers, and applications have been limited to oligomer analysis. SEC ESI MS Prokai and Simonsick were the first to report the use of on-line SEC ESI MS to combine the quantitative power of SEC with the identification power of MS, for analysis of a complex polymer mixture of octylphenoxypoly(ethoxy)ethanol. Selected-ion chromatograms of sodiated species enabled monitoring of elution profiles of individual oligomers, and calibration of the SEC [46]. Further, by using the SEC with ESI quadrupole-ms detection they calibrated the SEC for ethylene-oxide-based non-ionic surfactants and acrylic macromonomers without the use of external calibrants. The chemical composition distribution of acrylic macromonomers was also profiled across the molarmass distribution [47]. The on-line SEC ESI MS approach was also used to study polytetrahydrofuran, poly(methyl methacrylate) (PMMA), polyesters, and polystyrene in terms of repeat units and end groups and was used to compute accurate calibration curves for the SEC column. Reconstructed ion currents and the number of oligomers observed in the spectra were found to be highly dependent both on the amount of the cationization salt and the conevoltage setting [48]. The on-line coupling of SEC to ESI- FTMS was evaluated with narrow-dispersity PMMA. Molar masses and distribution evaluated by use of the MS-generated calibration curves were in excellent agreement with the values specified by the supplier. The report demonstrates the advantage of coupling high-resolution MS and SEC for the analysis of complex polymer mixtures, e.g. a broad-dispersity glycidyl methacrylate butyl methacrylate (GMA BMA) copolymer. Except for absolute molar masses, determination of the end groups and cyclic reaction products for (GMA BMA) was possible [49]. Microcolumn (250 mm 0.5 mm i.d.) SEC coupled with ESI MS (µsec ESI MS), specifically a quadrupole ion-trap and FT ion cyclotron resonance analyzers, has been used for analysis of octylphenoxypoly(ethoxy)ethanol. The µsec ESI MS hyphenated technique obviates the need for effluent splitting and results in improved chromatographic resolution and reduced solvent and sample consumption. µsec can also be interfaced with other chromatographic techniques [50]. SEC ESI FTMS has been applied to the characterization of polyesters and polyoxyalkylenes [51]. Two-dimensional LC ESI MS An interesting approach has been demonstrated by Nielen and Buijtenhuijs [52]. In one experimental arrangement three different LC methods were interfaced with ESI-orthogonal acceleration-tof MS. In SEC ESI MS, both absolute mass calibration of the SEC column and determination of monomers and end groups from mass spectra were achieved for a sample of polydisperse PMMA. By use of gradient polymer elution chromatography (GPEC) ESI MS, the chemical composition distribution within the oligomers of diproxylated bisphenol A adipic acid polyester resin was quantified. In addition, LCCC ESI MS of alkylated poly(ethylene glycol) and terephthalic acid neopentyl glycol polyester resin, with elution governed by end group functionality, enabled quantitative end-group determination. It should be pointed out that column-switching devices in which fractions from an LC column are directed to a

7 487 Fig. 3 Schematic diagram of two-dimensional LCCC SEC MS second column can be used to perform two-dimensional chromatography with on-line mass detection [53]. A simplified schematic representation of a possible arrangement for two-dimensional LCCC SEC with mass detection [54] is shown in Fig.3. The use of a MS detector in multidimensional chromatography has several advantages over common LC detectors: 1. because of the higher sensitivity of MS detectors, a diluted sample does not have to be re-concentrated before detection; 2. because solvent is removed from the effluent, the MS signal is not affected by the eluent; and 3. highly resolved mass spectra enable detailed analysis of the analyte. LC coupled with NMR and MS Fig. 4 Schematic diagram of LC NMR MS instrumentation Because of technological advances in LC, NMR, and MS, it has become possible to acquire both NMR and MS data simultaneously from a single chromatographic analysis. Several groups have begun to investigate the technique of LC NMR MS for the analysis of mixtures, especially those of pharmaceutical drug metabolites. There are principally two ways of linking NMR and MS with an LC system either in parallel (splitting before the NMR) or in series (splitting after the NMR). In most experiments the flow from the LC is split before the NMR and directed simultaneously to the NMR and to ESI MS, typically in the stop-flow mode, in the ratio 95:5 (Fig. 4), because the sensitivity of NMR is substantially lower than that of ESI MS. Practical aspects of the LC NMR MS technique have been discussed by Taylor et al. [55]. Experimental arrangements for coupling of a chromatographic separation system with an NMR spectrometer have already been discussed in detail [56]. On-line SEC NMR MS provides unique opportunities for analyzing complex polymer systems. By use of online SEC NMR MS SEC fractions can be simultaneously analyzed by NMR, to determine both polymer abundance and composition, and by MS, to determine the molar mass of each fraction. This leads to a direct measure of the bivariate distribution with regard to molar mass and copolymer composition. An off-line SEC NMR MALDI MS approach has been used for the analysis of high-conversion copolymers of PMMA and poly(butyl acrylate) [57] and of styrene maleic anhydride [58]. In summary, Table 1, which is not intended to be all-inclusive, lists some of the most important contributions to the LC MS of synthetic polymers. Conclusions and future directions Over the past decade both MALDI and ESI MS have become undisputedly valuable tools for the analysis of synthetic polymers. Polydispersity complications have been reduced by coupling MALDI and ESI MS with separation techniques, in particular with liquid chromatography. Coupled SEC MALDI MS and SEC ESI MS enable reliable determination of average molar masses and distributions for polydisperse synthetic homopolymers without the use of external calibrants. In contrast with SEC viscometry and SEC light scattering, structural information about repeat units, end groups, and the presence of minor species, are also provided by SEC MALDI MS and SEC ESI MS. Interfacing two different LC separation mechanisms (e.g. LCCC and SEC) with MALDI and ESI MS enables simultaneous quantitative determination of chemical composition or group distributions with molar mass distribution. Ultimately, a comprehensive combination of two-dimensional chromatography with NMR MS might provide information about different aspects of molecular heterogeneity for complex polymer mixtures. Although beyond the scope of this review, other separation technologies, e.g. supercritical-fluid chromatogra-

8 488 Table 1 Representative LC MS references (Co)polymer LC MS Coupling Ref Polydimethylsiloxane SEC MALDI MS Off-line [29] Copolyesters SEC MALDI MS Off-line [10] PMMA, vinylpyrrolidone vinyl acetate copolymer SEC MALDI MS Off-line [28] Polystyrene, MMA methacrylic acid copolymer, SEC MALDI MS Off-line [59] polycarbonate Polyacylthiophenes SEC MALDI MS Off-line [30] PMMA SEC MALDI MS Semi on-line [32] Butyl methacrylate methyl methacrylate diblock SEC MALDI MS Semi on-line [33] copolymer Polyols SEC MALDI MS On-line [36] Poly(propylene glycol) LCCC SEC MALDI MS Off-line [40] L-lactide ethylene oxide lactic acid triblock copolymer LCCC SEC MALDI MS Off-line [41] Polyamides-6 LCCC SEC MALDI MS Off-line [42] Poly(propylene oxide) LCCC SEC MALDI MS Off-line [43] Polyurethanes Chemical separation SEC MALDI MS Off-line [44] Octylphenoxypoly(ethoxy)ethanol SEC ESI MS On-line [46] Acrylic macromonomers SEC ESI MS On-line [47] Poly(tetrahydrofuran), PMMA, polyether, polystyrene SEC ESI MS On-line [48] PMMA, glycidyl methacrylate methyl methacrylate SEC ESI FTMS On-line [49] copolymer Octylphenoxypoly(ethoxy)ethanol msec ESI MS and msec ESI MS FTMS On-line [50] Polyesters, polyoxyalkylenes SEC ESI FTMS On-line [51] PMMA, polyester resins SEC ESI MS, LCCC ESI MS, GPEC ESI MS On-line [52] PMMA methyl acrylate copolymers SEC NMR MALDI MS Off-line [57] Styrene maleic anhydride copolymers SEC NMR MALDI MS Off-line [58] phy and thermal field-flow fractionation, have produced promising results in the characterization of polymers by hyphenated techniques. Supercritical-fluid chromatography seems attractive, because it can be coupled directly with ESI NMR MS. The potential of high-resolution ESI ion mobility spectrometry (ESI IMS) as an analytical separation tool for analysis of polymer mixtures should also be emphasized. Mobility is related to the size and shape of the ion; this imparts a second dimension which enables IMS to separate isomeric species. Other soft-ionization MS techniques, e.g. fast atom bombardment and plasma desorption MS, can also be used in conjunction with a separation technique for low-mass polymer analysis. Future progress in the LC MS analysis of synthetic polymers will be governed by the development of new interfacing and separation technologies, and by instrumental improvement. Improvements in direct coupling of LC to MS should continue, because on-line LC MS techniques have the advantage of continuous sample detection and reaction monitoring. On-line MS monitoring of elution profiles of individual oligomer species can also facilitate better understanding of LC separation mechanisms. Novel multidimensional chromatography approaches utilizing micro or capillary columns should increase separation efficiency and selectivity leading to improvement in the detection step. The combination of LC MS with limited chemical reactions (e.g. solubilizing reaction, selective degradation) can enable total structural characterization of intractable complex polymer systems. New types of MS detector and ionization technique can overcome the limited mass range of current MS instrumentation and extend the application of LC MS to high molar-mass polymers. Finally, it should be emphasized that there is no universal solution to the analysis of complex polymer systems and that the analysis of each requires an individual creative approach. Acknowledgements This work was supported by the National Science Foundation, Grant CHE References 1. Reamaekers KGH, Bart JCJ (1997) Thermochim Acta 295: Tanaka K, Waki H, Ido Y, Akita S, Yoshida Y, Yoshida T (1988) Rapid Commun Mass Spectrom 2: Zenobi R, Knochenmuss R (1998) Mass Spectrom Rev 17: Tomer BK (2001) Chem Rev 101: Mamirin BA, Karataev VI, Shmikk DV, Zagulin VA (1973) Sov Phys JETP 37: Wiley WC, McLaren IH (1955) Rev Sci Instrum 26: Castro JA, Köster C, Wilkins C (1992) Rapid Commun Mass Spectrom 6: Nielen MWF (1999) Mass Spectrom Rev 18: Montaudo G, Garozzo D, Montaudo MS, Puglisi C, Samperi F (1995) Macromolecules 28:

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