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widely used practice of collecting a few discrete fractions of eluate for subsequent analysis is not discussed, because it does not provide a continuous distribution of the measured parameter. MS detection ESI is the softest ionization method currently available in MS. Soft ionization combined with ESI’s inherent multiple charging mechanism have made MS an ideal tool for studying biopolymers and for accurately determining their molar masses. Beyond its application to perfectly monodisperse analytes, ESI-MS has also been used in a variety of other polymer applications (23). As an on-line detection method, ESI-MS has been used to obtain more accurate SEC calibrations than those obtained via traditional approaches, to obtain chemical composition distribution information on copolymers, and to study humic and fulvic acids. Because only ~1% of the SEC effluent is needed for ESI-MS, other detectors are usually used. Because of their low microliters-per-minute flow rates, micro-SEC columns can be utilized. Deery et al. used SEC/ESI-MS to study 5000-Da and 12,000-Da dextrans and enzymatically digested arabinoxylan polysaccharides (24). They also used SEC/ESI-MS n to determine glycosidic linkage of permethylated arabinogalactan oligomers. In contrast to ESI-MS, multiple charging is not usually observed in MALDI MS; therefore, interpretation of the MALDI information and its use in calculating M are more straightforward than in ESI-MS. Fei and Murray combined SEC on-line with aerosol MALDI for the complete mass spectra, across the entire elution profile, of 1000-Da poly(ethylene glycol) and poly(propylene glycol) (25). A more popular approach is continuous off-line MALDI. Using a homemade apparatus, Lou and van Dongen electrosprayed the SEC eluent and a coaxially added matrix material onto a MALDI plate (26). Others have used a commercially available interface in which the SEC eluent in sprayed directly through a heated capillary nozzle onto a moving MALDI target precoated with an appropriate matrix. For polydisperse macromolecules, a continuous track of sample is deposited onto the matrix surface of the target; spectra are obtained from different positions on the track. Esser et al. used this approach to study polystyrene 32500 and poly(methyl methacrylate) (PMMA) 10900 to obtain both M and copolymer composition of a diblock copolymer of n-butyl methacrylate and PMMA (27 ). Matrix selection is an important challenge, and currently only a few different types of matrix materials have been coated onto commercially available targets. Recently, Liu et al. used poly(dimethyl siloxane)s to compare continuous off-line SEC/MALDI TOFMS with on-line SEC/ESI TOFMS (28). They found that the electrospray technique is more effective at characterizing Some detectors appear to bridge the gap between classes; for example, IR and NMR can function as either chemical or physical detectors. low-M oligomers and the MALDI technique is more effective at characterizing high-M oligomers. Inductively coupled plasma MS (ICPMS) is a quite popular detection method for the SEC quantitation of metals across the elution profile of polymers. Sadi et al. listed several precautions that must be taken when coupling SEC to ICPMS (29). The use of organic solvents should be kept to a minimum because they affect the stability of the plasma. The ionic strength of the mobile phase buffers must be adjusted carefully because they can help limit non-SEC behavior, but they may also denature the organometallic complexes of interest. In addition, the salt content of the mobile phase should be kept to a minimum to avoid clogging the nebulizer and to reduce wear on the sampler and skimmer cones. Sample preparation for this technique is also tricky because the species of interest must be extracted from the sample matrix without altering the nature of these species. Gardner et al. used SEC/ICPMS along with a UV detector to study metals in natural waters (30). Hall et al. used SEC/ICPMS to fractionate lead-bound ligands in human amniotic fluid and to detect and quantitate lead (31). In the realm of glycopolymers, Szpunar’s group has used SEC/ ICPMS along with DRI. The concentration-sensitive detector characterized the elution profiles of water-soluble and enzymatically digested polysaccharides, and the MS detector provided the distribution patterns of select metals in high- and low-M fractions of apple and carrot samples (32). Other MS methods have also been coupled on-line to SEC, including chemical reaction interface MS to analyze nucleic acids, proteins, and other biopolymers (33), as well as atmospheric-pressure chemical ionization MS to determine pesticide binding by humic substances (34). Spectroscopic detectors The IR detector can act as either a chemical or a physical detector, and it can be used in on-line or continuous off-line modes. In off-line use, the same commercially available hardware used for SEC/MALDI can often be used for SEC/FTIR. The applications of FTIR as a physical detector have thus far been on-line and limited to characterizing the short-chain branching content of polyolefins. Markovich et al. used SEC/FTIR to measure the methyl group content per 1000 carbons, as a function of M, of ethylene-based polyolefin copolymers (35). Figure 6 shows a more recent example by DesLauriers, who used on-line SEC/ FTIR and chemometrics to quantitate the ethyl and/or butyl content of ethylene 1-olefin copolymers as a function of M and related the results to trends resulting from catalysis and process changes (36). Cotts and Ouano used IR as a chemical detector to study poly(vinyl butyral), which is actually a random terpolymer and is MARCH 1, 2005 / ANALYTICAL CHEMISTRY 111 A
1.2 SEC 30 dW/d(log M ) 0.8 0.4 0 3 4 5 log M FIGURE 6. Short-chain branching in polyolefins by SEC/FTIR. 6 7 Distribution of the number of short-chain branches (SCB) with one methyl chain-end (1 Me CE) per 1000 total carbons (TC) across the MMD of an ethylene 1-olefin copolymer. Each type of symbol represents one run through the column. (Adapted from Ref. 36; figure provided courtesy of Paul DesLauriers, Chevron Phillips.) 20 10 0 SCB / 1000 TC (1 Me CE) more accurately referred to as poly(vinyl butyral-co- vinyl alcohol-co-vinyl acetate) (12, 37 ). By monitoring the OH stretch of the hydroxyl group and the butyral ring vibration, they found the vinyl alcohol content of the polymer to be independent of M. Most applications of FTIR as a chemical detector are continuous and off-line. The SEC eluent is directed to a heated nozzle for evaporation of the solvent and deposition of the analyte onto a rotating germanium disk; spectra are obtained from any location on the disk (38, 39). To date, all SEC/NMR experiments have been performed in continuous (not stop-flow) mode, and all reports have dealt with SEC/ 1 H-NMR (40). The use of NMR as a physical detector appears to be thus far restricted to measurement of the tacticity distribution in mixtures of isotactic and syndiotactic PMMA (41). As a chemical detector for SEC, on-line coupling of NMR has been used to determine the MMD of uniformly isotactic PMMA (42) and to study the chemical heterogeneity of synthetic copolymers (i.e., the change in chemical composition as a function of molar mass; 43). Neiss and Cheng reported recently on the SEC/NMR analysis of alginates, in which the microstructure of these plant polysaccharides was determined in combination with Markov statistical models, off-line 13 C-NMR, and enzymology (44). In addition to its widespread use as a concentration-sensitive detector, UV–vis has also been used to determine the chemical heterogeneity of copolymers, for example, in the study of poly(styrene-co-methyl methacrylate)s by SEC/DRI/UV/MALS (45). The DRI served as a concentration-sensitive detector, and the UV detector monitored = 262 nm, where styrene presents a strong absorption band but methyl methacrylate does not absorb. The weight fraction of styrene was quantitated across the MMD of the copolymers. Fluorescence spectroscopy is growing in popularity as a detection method for SEC. Fauser and colleagues separated airborne organic molecules from bitumen by SEC, identifying these particles by on-line fluorescence detection (46). In studying dissolved organic matter from Baltic Sea water, Lepane isolated the hydrophobic and hydrophilic fractions by adsorption chromatography and then characterized them by SEC/fluorescence (47). Moon and co-workers monitored the coupling of fluorescence-labeled anhydridefunctional polystyrene and PMMA in dilute polymer blends with SEC/ fluorescence (48). Maliakal et al. used the same method to measure polymer–polymer chain-end reaction rate constants (49). Edwards et al. attempted to use SEC/FT-Raman MS with a low-volume flow cell to characterize the microstructural variation in polybutadiene polymers as a function of elution volume (50). As an on-line detector, the Raman spectrometer lacked the necessary sensitivity to provide quantitative information for the particular analytes being examined because of its low sensitivity at the different excitation wavelengths. This setup is still promising, however, provided the proper experimental conditions can be found for the analyte solutions so that they exhibit a high scattering intensity relative to the solvent or mobile phase. The use of conductivity detection in SEC has been rather sparse, despite its great potential for the analysis of polyelectrolytes. Rinaudo et al. used SEC/DRI/VISC/conductivity to measure the charge distribution, which was superimposed upon the MMD, for a series of sodium carboxymethylcelluloses (51). Conclusions For the purposes of determining both fundamental and end-use properties of macromolecules, multidetector SEC has come of age (1). The current focus is thus on obtaining the proper combination of detectors to characterize the physicochemical properties of interest. Physically, SEC/DRI/MALS/ VISC has been shown to be extremely powerful and can now be augmented by on-line QELS. The combination of chemical detectors has also become impressive with the recent report of characterizing polymer additives using SEC/UV/ 1 H-NMR/ESI-MS/FTIR—the first three detectors were on-line, and the FTIR operated in continuous off-line mode (52). The constant conversion of analytical techniques into SEC detection methods provides a growing choice of parameters that can be measured. André M. Striegel is an assistant professor at Florida State University. His research interests include the study of natural and synthetic polymers— in particular, applying multidetector SEC and related techniques to determine structure–property relationships of macromolecules and elucidating fundamental aspects of separation science. Address correspondence about this article to Striegel at Department of Chemistry & Biochemistry, Florida State University, Tallahassee, FL 32306- 4390 (striegel@chem.fsu.edu). 112 A ANALYTICAL CHEMISTRY / MARCH 1, 2005
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