MALDI TIME-OF-FLIGHT MASS SPECTROMETRY OF SYNTHETIC

Michel W.F. Nielen Akzo Nobel Chemicals Research, P.O. Box 9300, 6800 SB Arnhem, The Netherlands Received 26 April 1999; accepted 12 August 1999

I. Introduction ...... 309 II. Matrix Selection for Analysis ...... 310 A. Matrices for UV±MALDI ...... 310 B. Matrices for IR±MALDI ...... 317 III. Cationization in Polymer Analysis ...... 319 IV. Sample Preparation Techniques ...... 320 V. Discrimination Issues ...... 321 A. High and Low Mass Discrimination ...... 321 B. Oligomer Speci®c Discrimination ...... 324 VI. MS/MS in MALDI TOF MS of Synthetic Polymers ...... 325 VII. Chromatography/MALDI Coupling ...... 327 A. Thin Layer Chromatography/MALDI TOF MS ...... 327 B. Off-line Size-Exclusion Chromatography/MALDI TOF MS ...... 328 C. On-line and Direct Deposition Size-Exclusion Chromatography/MALDI TOF MS ...... 329 D. Other Liquid Chromatography Modes Coupled with MALDI TOF MS ...... 330 VIII. Polymer Applications ...... 331 A. Homopolymers ...... 331 B. Blends ...... 333 C. Copolymers and Resins ...... 334 D. Miscellaneous ...... 334 IX. Conclusion and Outlook ...... 334 References ...... 336

Mass spectrometry of intact synthetic polymers has been limited as size exclusion chromatography (SEC)/MALDI have been to ®eld desorption (FD) MS for many years. More recently, soft developed. Many different polymer applications appeared in ionization techniques such as electrospray (ESI) and matrix- recent literature, but most studies deal with homopolymers. assisted laser desorption/ionization (MALDI) and the revival of Many challenges remain, particularly in the ®elds of haloge- time-of-¯ight analyzers created new opportunities for the nated polymers, polyole®nes, copolymers, blends, and in the characterization of polymers. In this review emphasis is put sequencing of block-copolymers. # 1999 John Wiley & Sons, on MALDI time-of-¯ight mass spectrometry of polymers. The Inc., Mass Spec Rev 18: 309±344, 1999 selection of an appropriate MALDI matrix, cationization salt and sample preparation techniques are critical success factors for obtaining a reliable mass spectrum and to infer structural I. INTRODUCTION information such as monomer mass(es) and end-groups. However even under optimized conditions mass discrimination Among the analytical techniques currently used in the in the analysis of polydisperse polymers and speci®c oligomer characterization of synthetic polymers, mass spectro- discrimination might occur. Hence hyphenated techniques such metry is of increasing importance (Smith et al., 1997). In ÐÐÐÐ the past, mass spectrometry of synthetic polymers was hardly possible: as a rule, polymers had to be degraded Correspondence to: Michel W.F. Nielen; e-mail: michel.nielen@ akzonobel.com thermally or chemically prior to mass spectrometric

Mass Spectrometry Reviews, 1999, 18, 309± 344 # 1999 by John Wiley & Sons, Inc. CCC 0277-7037/99/050309-36 97 u&Oo,19;Ree crp,1998). Schrepp, & Raeder 1998; Odom, & the Wu Simonsick, & 1997; be until Jackson can 1996; topic (Montaudo, same literature elsewhere the found on the reviews short covers 1999; of beginning and polymers synthetic kDa. 35 to up mass) end-groups cation the the known (i.e., (i.e., residue plus mass increment the and mass mass) complexityÐthe repeating monomer polymer the of the of and range determination mass & the monomer allowingÐdepending Brown the in thereby on 1955; resolution (FWHM) McLaren, offer 10,000±20,000 & which (Wiley 1995) Lennon, source extrac- delayed with tion equipped molecular instruments re¯ectron state-of-the high the art and 1996); very been Li, has & MDa (Schriemer which 1 demonstrated beyond time-of- analyzed, in be the can polymers analyzer fragmentation; weight any (TOF) mole- hardly ¯ight quasi mass with single-charged the of mainly cular simplicity show the which of spectra because analysis polymer analyses. FTD limited and are all studies MWD and most of to far ESI characterization so the although samples, distributions, to ®ve contribute polymer can of MS MALDI complexity the Despite hntepoenstaindet h oxsec fseveral of coexistence the to distributions: due well, situation complex as protein more the is analysis than situation polymer polymer synthetic to the amenable although and are ESI that an MS recognized as have been MALDI such has still biopolymers It . and of and analysis had proteins the 1988) Hillen- on al., as impact & enormous et Tanaka such (Karas 1988; MALDI kamp, techniques matrix-assisted and desorption/ionization, 1990), ionization al., laser et (Prokai, (Fenn soft spectro- kDa ESI 10 electrospray, mass to Modern up the polymers for 1990). intact option of analysis only sector metric the on (FD) was desorption ®eld instruments years, many For analysis. & 310 NIELEN * * * * * hsrve srsrce oMLITFM of MS TOF MALDI to restricted is review This for suited ideally is MS time-of-¯ight MALDI c)oyesso nacietr distribution dendritic). branched, architecture cyclic, an (linear, show (co)polymers present, are sequence distribution additional block-length copolymers, and block dis- of composition case in chemical a (CCD), tribution addition in chains polymer the show copolymers, random of case func- in (FTD), a distribution creating type and end- tionality thereby initiation different processes, different to have termination due might chemistries chains group polymer synthesis, the polymer of result (MWD) to a distribution have as weight we molecular weight, a with molecular deal single a of instead eeto a eetatdfo oye n matrix and polymer 1998): from Owens, & extracted (Hanton matrix data for be solubility guidelines can general some selection applied. polymers, be could other peptides of same For the analysis because the simply in MS, used MALDI matrices of days early studied the in already polymer were polypropyleneglycol any and giving leneglycol spots those the for for even only signal. cases intensity not signal in search and polymers, resolution to but good has showing synthetic spots one sweet often of are and preparations MALDI sample obtained are inhomogeneous samples In rather homogeneous typically obtained. routines rule, a being acquisition in as well quite wherein, data perform analysis which 1997) within manu- al., automated et Instrument (Suckau distributions offer above. not outlined the is facturers as this of samples situation In polymer protein sample because . and and peptide matrix straightforward the salt and to sample homogeneous solvent, contrast is of matrix, experiment MALDI cocrystallization of any in goal preparation ®nal The ANALYSIS POLYMER FOR SELECTION MATRIX II. fet,eg,dtrnli ucsflyue ihsle salts silver with used background successfully and is dithranol incompatibilities e.g., the effects, of aware are mass be should solvents MALDI matrix the and in polymer molecular included. addition, high observed molecular In the actually The spectra. to 1. end refer Table weight therein documented in indicated with given weights is polymers matrices synthetic MALDI of overview An UV±MALDI for Matrices A. literature. in found some be point, and can starting UV±MALDI IR±MALDI a for for as hints process; matrix error on and analysis suggestions Nevertheless, trial many polymer a investigation. for often Polystyrene still optimization is under and Polydimethylsiloxane selection polymer matrix the polarity matrix glycol the with match Polytetramethylene to recommended is it Polymethylmethacrylate Generally, Polymers Diphenylbutadiene trans all Dithranol glycol acid Polypropylene Indoleacrylic acid Ferulic acid 2,5-Dihydroxybenzoic Matrices Caohdoyinmcai oyiy acetate Polyvinyl acid -Cyano-hydroxycinnamic ae-oul ytei oyessc spolyethy- as such polymers synthetic Water-soluble biul aymti pin r vial u one but available are options matrix many Obviously Rtni cdPolybutadiene acid -Retinoic Hydrophobic $ Hydrophilic MALDI TOF MS OF SYNTHETIC POLYMERS &

TABLE 1. Matrices for UV±MALDI mass spectrometry of speci®c synthetic polymers Polymer Matrix Matrix solvent Polymer solvent MW Reference

PEG SA 1500 Zenobi, 1994 PEG HABA THF 24,000 Montaudo et al., 1995a PEG DHB water acetonitrile±water 2400 Pasch & Rode, 1995 PEG DHB ethanol 24,000 Montaudo et al., 1995c PEG HABA dioxane methanol 4500 Whittal & Li, 1995 PEG ACA/MSA ethanol methanol 8000 Tang et al., 1995b PEG DHB dichloromethane 5000 Weidner et al., 1996b PEG glycerol/graphite methanol 8000 Dale et al., 1996 PEG NBA/graphite methanol 8000 Dale et al., 1996 PEG hydroxymethoxycinnamic methanol methanol 1000 Fei & Murray, 1996 acid PEG MBT 14,000 Xu et al., 1997 PEG AMBT EtOH/THF/water EtOH/THF/water 14,000 Xu et al., 1997

PEG K4[Fe(CN)6]/glycerol methanol/glycerol chloroform 8000 Zollner et al. 1997 PEG glassy azodyes 1500 Blair et al., 1998 PEG, pyrene- HABA dioxane methanol Whittal et al., 1996 PEG, derivatized- DHB ethanol/water DMSO 5000 Weidner & Kuhn, 1996 PEG, dimethyl- salicylamide ethanol/water water 1600 Krause et al., 1996 PEG, dimethyl- salicylanilide ethanol water 5200 Krause et al., 1996 PEG, carbonate- CHCA acetone water/acetone 7000 Hagelin et al., 1998 PTMEG DHB 1000 King et al., 1995 PTMEG DHB THF THF 9000 Jackson C et al., 1996 PPG ACA/MSA 6500 Tang et al., 1995a PPG DHB methanol±water (1:1) 2400 Barton et al., 1995 Polyether, CHCA acetonitrile/water methylene chloride 5000 Peiris et al., 1998 -azoxyaromatic Coal SA chloroform/methanol 70,000 Herod et al., 1994a Coal SA 270,000 Herod et al., 1994b Coal trihydroxyanthracene 200,000 Herod et al., 1995 Coal, -tar pitch SA, DHB 260,000 John et al., 1994 Coal, -tar various NMP NMP 100,000 Domin et al., 1997 Coal, -tar pitch none 10,000 Johnson et al., 1998 PMMA IAA acetone acetone 260,000 Danis & Karr, 1993 PMMA DHB acetone acetone 260,000 Danis & Karr, 1993 PMMA DHB water/acetonitrile acetone 9000 Danis et al., 1993 PMMA DHB water/ethanol THF 3000 Lehrle & Sarson, 1995 PMMA HABA THF 90,000 Montaudo et al., 1995a PMMA DHB THF 9500 Lloyd et al., 1995 PMMA ACA/MSA ethanol toluene 8000 Tang et al., 1995b PMMA DHB 50,000 Cottrell et al., 1995 PMMA IAA acetone acetone 50,000 Belu et al., 1996 PMMA IAA acetone acetone 7000 Larsen et al., 1996 PMMA DHB ethanol±water THF 4000 Lehrle & Sarson, 1996 PMMA DHB THF THF 15,000 Jackson C et al., 1996 PMMA DHB THF 3000 Weidner et al., 1996b PMMA DHB methanol methanol 6000 Dogruel et al., 1996 PMMA DHB ‡ CsI methanol methanol 6000 Dogruel et al., 1996 (Continued)

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TABLE 1. (Continued) Polymer Matrix Matrix solvent Polymer solvent MW Reference

PMMA IAA THF THF 110,000 Schweer et al., 1996 PMMA DHB ‡ KtFAc THF THF 10,000 Spickermann et al., 1996 PMMA MBT Xu et al., 1997 PMMA dithranol ‡ surfactants 6500 Kassis et al., 1996 PMMA dithranol THF THF 20,000 Jackson AT et al., 1997a PMMA HABA THF acetone or THF 50,000 Rashidzadeh et al. 1998a PMMA, end-groups DHB acetone 4000 Maloney et al., 1995 PMMA, end-groups DHB THF 3500 Pasch and Gores, 1995 PMMA, end-groups IAA acetone 9200 Scrivens et al., 1995 PMMA, end-groups DHB acetone acetone 6000 Hunt et al., 1995 PMMA, end groups dithranol HFIP HFIP 13,000 Jackson, AT et al., 1997b PBA DHB acetone acetone Easterling et al., 1998 PBA IAA THF THF 200,000 Nielen & Malucha, 1997 PBMA IAA acetone acetone 100,000 Danis et al., 1995 Polyacrylate, ¯uorinated DHB THF trichlorotri¯uoroethane 25,000 Latourte et al., 1997b PS IAA THF Danis & Karr, 1993 PS HABA THF 50,000 Montaudo et al., 1994b PS none chloroform or THF 1400 Mowat & Donovan, 1995 PS HABA THF 46,000 Montaudo et al., 1995a PS dithranol ‡ AgtFAc 10,000 Scrivens et al., 1995 PS 9-Nitroanthracene THF 11,000 Lloyd et al., 1995 ‡ AgtFAc PS dithranol ‡ AgtFAc chloroform chloroform 40,000 Belu et al., 1996 PS dithranol ‡ AgtFAc THF THF 3500 Thomson et al., 1996 PS HABA THF THF 35,000 Liu & Schlunegger, 1996 PS 4-(Phenylazo)-resorcinol THF THF 35,000 Liu & Schlunegger, 1996 PS chromoionophor IV THF THF 35,000 Liu & Schlunegger, 1996 PS dithranol ‡ AgtFAc THF THF 7500 Chaudhary et al., 1996 PS IAA ‡ Agacac THF THF 125,000 Danis et al., 1996a PS POPOP ‡Agacac THF THF 22,000 Danis et al., 1996a PS all-trans-Retinoic THF (puri®ed) THF (puri®ed) 1:5 MDa Schriemer & Li,

acid ‡ AgNO3 1996 PS dithranol ‡ AgtFAc THF THF 35,000 Schweer et al., 1996 PS dithranol ‡ cations THF THF 3000 Deery et al., 1996 PS dithranol ‡ AgtFAc 210,000 Lee & Han, 1996 PS all-trans-Retinoic THF THF 500,000 Brown et al., 1997

acid ‡ AgNO3 PS dithranol ‡ AgtFAc THF THF 200,000 Nielen & Malucha, 1997 65 PS dithranol ‡ Cu(acac)2 THF THF 7000 Burgers & Terlouw, 1998 PS dithranol ‡ Ag/Cu/Pd 4000 Rashidzadeh et al. 1998b

312 MALDI TOF MS OF SYNTHETIC POLYMERS &

TABLE 1. (Continued) Polymer Matrix Matrix solvent Polymer solvent MW Reference

PS, nitroxide- dithranol ‡ AgtFAc 5000 Beyou et al., 1998 PS, dedtc- dithranol ‡ AgtFAc 5000 Beyou et al., 1998 PS, macrocyclic- dithranol ‡ AgtFAc 4400 Pasch et al., 1997 PS, -methyl 9-nitroanthracene THF THF 1600 Kukulj et al., 1998 ‡ AgtFAc PVC IAA THF Danis & Karr, 1993 PVC HABA THF Danis & Karr, 1993 PVAc DHB methanol Danis & Karr, 1993 PVAc HABA acetone acetone Cornett et al., 1998 PC HABA THF 17,000 Montaudo et al., 1995a PC IAA THF Danis & Karr, 1993 PC HABA THF Danis & Karr, 1993 PC DHB chloroform 5000 Weidner et al., 1996b PC HABA 20,000 Montaudo et al., 1994c PC IAA THF THF 100,000 Nielen & Malucha, 1997 PC dithranol THF 5000 Pasch et al., 1996 PB none 1300 Mowat & Donovan, 1995 PB POPOP ‡Agacac THF THF 11,000 Danis et al., 1996a

PB DHB ‡ AgNO3 THF THF 6000 Pastor & Wilkins, 1997 PB all-trans-Retinoic THF THF 300,000 Yalcin et al., 1997 acid ‡ Cu(II)nitrate PB, hydroxylated- dithranol ‡ AgtFAc THF THF 5000 Latourte et al., 1997a Polyisoprene all-trans-Retinoic acid THF THF 150,000 Yalcin et al., 1997 ‡ Cu(II)nitrate Polyisoprene dithranol ‡ AgtFAc THF THF Cornett et al., 1998 Squalane, Squalene NPOE ‡ AgtFAc toluene toluene 550 Weidner et al., 1997 Squalene, -epoxidized THAP ethanol/water 2000 Kuehn et al., 1997 Paraf®nes, Waxes DHB, NPOE ethanol/water toluene 3000 Kuehn et al., 1996 PDMS DHB THF 280,000 Montaudo et al., 1995b PMPS DHB THF THF 2500 Montaudo et al., 1996c PMPS IAA THF THF 2000 Montaudo et al., 1996c PMPS dithranol THF THF 2500 Montaudo et al., 1996c PAN 4-Hydroxy benzylidene acetone DMF 4500 Linnemayr et al., malononitrile 1998 Polycaprolactone HABA 14,000 Montaudo et al., 1994c Polycaprolactone HABA THF 10,000 Montaudo et al., 1995a Polycaprolactone DHB water LC mobile phase: THF 1400 Pasch & Rode, 1995 Polycaprolactone DHB methanol/water ethyl acetate 7000 Cordova et al., 1998 Polycaprolactone, DHB ‡ NaCl 5500 Miola et al., 1998 -benzyl/hydroxy PEF 2000 Hanton & Parees, 1995 PA6, -aminolized HABA 8000 Montaudo et al., 1994a (Continued)

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TABLE 1. (Continued) Polymer Matrix Matrix solvent Polymer solvent MW Reference

PA6, -diamino HABA tri¯uoroethanol 3000 Montaudo et al., 1995a PA6, -monoamino HABA tri¯uoroethanol 6200 Montaudo et al., 1995a PA6, -dicarboxyl HABA tri¯uoroethanol 6400 Montaudo et al., 1995a PA6 HABA tri¯uoroethanol 7000 Montaudo et al., 1996d polyamines, Triazine- DHB formic acid formic acid 5000 Braun et al., 1996 Poly(butylene adipate), HABA Montaudo et al., -glycolized 1994a Poly(butylene adipate) HABA THF 4000 Montaudo et al., 1995a Poly(butylene adipate) HABA, DHB THF 48,000 Montaudo et al., 1995d Poly(butylene adipate) IAA acetone acetone 3000 Williams et al., 1997 Poly(butylene adipate) dithranol ‡ AgtFAc THF THF 6500 Liu & Schlunegger, 1996 Poly(decamethylene DHB water THF/hexane (45:55) 3000 Pasch & Rode, 1995 adipate) Poly(ethylene DHB, THAP chloroform 3500 Weidner et al., 1995 terephtalate) Poly(ethylene DHB 5±10 mg/mL chloroform 3000 Weidner et al., terephtalate) 1996a Poly(ethylene DHB chloroform Weidner et al., terephtalate) 1996b Poly(ethylene 3000 Yates et al., 1996 terephtalate) Poly(ethylene dithranol THF HFIP 4000 Jackson A et al., terephtalate) 1997a Polyester, -aliphatic, DHB, IAA water±methanol, THF water, THF, THF/TFA, TFA 8000 Blais et al., 1995 -aromatic Poly(trimethylene IAA 4800 Williams et al., 1995 glutarate) Poly(trimethylene IAA acetone acetone 3000 Williams et al., 1997 glutarate) Poly(trimethylene IAA acetone acetone 3000 Williams et al., 1997 adipate) Poly(trimethylene IAA acetone acetone 3000 Williams et al., 1997 succinate) Polyester DHB THF, acetone THF, acetone 5000 Guittard et al., 1996b Poly(ethylene DHB THF 4000 Pasch et al., 1996 terephtalate, -isophtalate) Poly(dihydroxymethyl- HABA THF THF 120,000 Scampporrino et al., /phtalate) 1996 Poly(neopentyl IAA THF THF 40,000 Nielen & Malucha, terephtalate) 1997 Poly(ethylene adipate) IAA acetone acetone 3000 Williams et al., 1997 Poly(neopentyl sebacate) IAA acetone acetone 3000 Williams et al., 1997 Polyester, -methylated 5-nitrosalicylic acid 2500 Guittard et al., 1997 Polyester, -cyclic 5-CSA 8000 Guittard et al., 1996a Poly(diethyl- DHB water/acetonitrile acetone 2000 Feast et al., 1997 3-hydroxyglutarate) Poly(phenylglycidyl- DHB ethanol THF 6000 Leukel et al., 1996 phtalic anhydride)

314 MALDI TOF MS OF SYNTHETIC POLYMERS &

TABLE 1. (Continued) Polymer Matrix Matrix solvent Polymer solvent MW Reference

Poly(hydroxystearate) IAA THF Danis & Karr, 1993 Dendrimers, -aromatic DHB water/acetone water/acetone, chloroform, 5200 Sahota et al., 1994 polyester ‡ sodium chloride Dendrimers, -aromatic IAA THF THF 14,000 Leon & Frechet, polyether 1995 Dendrimers IAA acetone THF, chloroform Milberg & Garden, 1996 Dendrimers all-trans-Retinoic acid methylene chloride THF, chloroform >20,000 Milberg & Garden, 1996 Dendrimers, -carbosilane, DHB water Wu & Biemann, -t-amino 1997 Dendrimers, -carbosilane, DHB ‡ ammonium citrate water Wu & Biemann, -sulfonated (1:1) 1997 Dendrimers, IBU- dithranol THF THF 11,000 Srinivasan et al., 1998 Dendrimers, -aromatic- benzyloxycyano-cinnamic 5000 Gooden et al., 1998 polyether acid PU HABA 10,000 Tang et al., 1995a PU, -pyrolysate 5500 Lattimer, 1998 PU, dendritic wedges CHCA acetone acetone 6500 Puapaiboon & Taylor, 1999 PI dithranol ‡ AgtFAc chloroform chloroform 5000 Belu et al., 1996 Polysul®de, -linear 9-Nitroanthracene THF THF 2800 Mahon et al., 1998 ‡ AgtFAc PAA Danis et al., 1992 PAA, -¯uorinated end DHB THF THF 2000 Latourte et al., 1997b PSS SA 180,000 Danis et al., 1992 PSS DHB, SA, CSA water, water, THF 100,000 Raeder et al., 1995 PSS SA water 450,000 Danis & Karr, 1995 Polylactide DHB THF THF 6000 Montaudo et al., 1996b Polylactide HABA THF THF 10,000 Montaudo et al., 1996b Polydextrane DHB/HIC (3:1) water/acetonitrile water 2000 Bornsen et al., 1995

Copolymers and resins PVP/PVAc IAA THF Danis & Karr, 1993 PVP/PVA 3500 Schaer, 1995 EO/PO DHB water acetonitrile±water 1400 Pasch & Rode, 1995 EO/PO Kalinoski et al., 1995 EO/PO dithranol HFIP HFIP Scrivens et al., 1998 PO/EO/PO, HABA dioxane 2000 Schriemer & Li, -diaminopropylether 1995 EO/PO/EO, PO/EO/PO CHCA ‡ potassium iodide 8600 Lacey et al., 1996 EO/pyrene HABA 1,4-dioxane methanol 10,000 Lee et al., 1996 Jeffamine ACA/MSA ethanol methanol 2000 Tang et al., 1995b Jeffamine HABA dioxane dioxane 3000 Schriemer et al., 1997 Jeffamine DHB methanol methanol Goldschmidt & Owens, 1997 PPE-b-PEO dithranol THF 3500 Francke et al., 1998 PS/PMeS IAA ‡ AgIAA THF THF 5000 Wilczek-Vera et al., 1996 PMMA/MeSTY, IAA acetone acetone 9000 Guttman et al., 1997 -block(SRM 1487) (Continued)

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TABLE 1. (Continued) Polymer Matrix Matrix solvent Polymer solvent MW Reference

PMMA/PBMA DHB acetone THF 2500 Suddaby et al., 1996 PMMA/PBMA DHB acetone 4500 Haddleton et al., 1997 PMMA/PMAA IAA THF THF 150,000 Nielen & Malucha, 1997 PDMS/PHMS NPOE THF THF 15,000 Servaty et al., 1998 Copoly(aryletherketone/ dithranol chloroform chloroform 4000 Wang et al., 1997 arylethersulfone), -cyclic Copoly(arylether/ether dithranol ‡ AgtFAc 6000 Wang & Hay, 1997 sul®des) Copolycarbonate/copper HABA THF THF 3000 Vitalini et al., 1996 complex Copolyether-bisphenolA/ HABA THF THF 4000 Vitalini et al., 1996 copper complex Copolybisphenol HABA 1,4-dioxane dioxane 10,000 Schriemer et al., A/epichlorohydrin 1997 Copolyester PBA/PBS HABA, DHB THF 80,000 Montaudo et al., 1995d Copolyester PBA/PBSe HABA THF/chloroform SEC fraction THF or 30,000 Montaudo et al., chloroform 1998a Copolyester PBSu/PBA HABA THF/chloroform SEC fraction THF or 30,000 Montaudo et al., chloroform 1998a Copolyester PBSu/PBSe HABA THF/chloroform SEC fraction THF or 30,000 Montaudo et al., chloroform 1998a Copolyester HABA THF/chloroform SEC fraction THF or 30,000 Montaudo et al., PBSu/PBA/PBSe chloroform 1998a Copolyester HABA THF/chloroform chloroform 15,000 Montaudo et al., 1998c Epoxy resin DHB acetone acetone 3500 Epoxy resin, DHB SEC fractions 6000 Lo & Huang, 1998 -thiodiphenol Poly(cresylglycidyl/ 1,3-diphenylbutadiene acetone 2800 Schriemer & Li,

formaldehyd) ‡ AgNO3 1995 Poly(cresylglycidyl/ 1,4-diphenylbutadiene acetone acetone 3500 Schriemer et al.,

formaldehyde) ‡ AgNO3 1997 PF resin, -epoxy DHB acetone 2000 Pasch et al., 1996 PF resin DHB acetone 4000 Pasch et al., 1996 PF resin, -alkyl dithranol chloroform chloroform 2000 Mandal & Hay, 1997b PF resin, -polycyclic 2700 Mandal & Hay, carbonate of- 1997a PF resin, t-Butyl-, cyclic dithranol ‡ AgtFAc chloroform ‡ THF chloroform 2500 Mandal & Hay, siloxanes 1998a PF resin, Phenyl-, dithranol ‡ AgtFAc chloroform ‡ THF chloroform 2500 Mandal & Hay, cyclic siloxanes 1998a PF resin, -cyclic dithranol chloroform chloroform 2700 Mandal & Hay, phosphonate 1998b

Miscellaneous Fullerene CHCA benzene benzene 800 Cordero et al., 1996 Fullerenes MSA 756 Rogner et al., 1996 Fullerenes CHCA acetone chloroform Linnemayr & Allmaier, 1997 Fullerenes sulfur carbon disul®de 992 Brune, 1999 Calixarenes HPA water/acetonitrile chloroform Linnemayr & Allmaier, 1997

316 MALDI TOF MS OF SYNTHETIC POLYMERS &

TABLE 1. (Continued) Polymer Matrix Matrix solvent Polymer solvent MW Reference

Calix[3]indoles SA dichloromethane 1200 Lidgard et al., 1996

Triton X305 K4[Fe(CN)6]/glycerol chloroform 2000 Zollner et al., 1996 Tween 85 HABA acetonitrile/water acetonitrile/water 2000 Giang & Chang, 1997 Tween 20 CHCA acetonitrile/water water or isopropanol 2000 Cumme et al., 1997 Sorbitols, methyl, FA Kim et al., 1998 benzylidene- Surfynol DHB methanol methanol 1600 Parees et al., 1998 Alkylphenolethoxylate, DHB methanol 1000 Barry et al., 1997 -derivatized Surfactants, -ethoxylated CHCA ethanol iso-propanol or water 1800 Bartsch et al., 1998 Azodyes, polysulfonated- HABA ‡ diammonium ethanol water 1400 Sullivan & Gaskell, citrate 1997

TABLE 2. Physical-chemical data of UV±MALDI matrices

1 ®lm (cm ) Matrix MW at 337 nm PA (kJ/mol) Reference 2,5-DHB 154 0.79 Â 105 ± Allwood et al., 1996 841 866 Jorgensen et al., 1998 854 Æ 14 Steenvoorden et al., 1997 854 Æ 16 Burton et al., 1997 -CHCA 189 2.18 Â 105 ± Allwood et al., 1996 841 Jorgensen et al., 1998 933 Æ 9 Steenvoorden et al., 1997 766 Æ 8 Burton et al., 1997 Sinapinic acid 224 1.10 Â 105 ± Allwood et al., 1996 887 Jorgensen et al., 1998 894 Æ 13 Steenvoorden et al., 1997 Dithranol 226 ± 874 Æ 8 Burton et al., 1997 IAA 187 ± 900 Æ 16 Burton et al., 1997 HABA 242 ± 943 Jorgensen et al., 1998 766 Æ 8 Burton et al., 1997

in MALDI of polystyrenes but the stability of this mixture protein studies; as a consequence, af®nity data are is limited to a few minutes only. Matrices such as IAA and investigated while cation af®nity data are actually all-trans-retinoic acid show self-polymerization upon required in the synthetic polymer situation. Nevertheless, standing and ®nally might obscure the mass spectrum up some idea about the energetics can be obtained and should to several thousand Daltons. Initially promising matrices be considered in practice. It has been shown that matrices such as HABA (Montaudo et al., 1994b) showed with low PAvalues induce more fragmentation of peptides signi®cant mass discrimination in later studies (Rashid- than matrices with high PAvalues (Jorgensen et al., 1998). zadeh & Guo, 1998a; Nielen & Malucha, 1997). Some of the matrices listed in Table 1 should be considered B. Matrices for IR±MALDI ``experimental'' and are less suitable for routine applica- tions: graphite (Dale et al., 1996) and liquid matrices such Only one signi®cant study in the ®eld of IR±MALDI of as 2-nitrophenyl octylether, NPOE, (Williams et al., 1996) synthetic polymers is known so far, see the data in Table might cause severe contamination of the ion source. 3. Infrared lasers such as the Nd±YAG laser (3.27 mm, Physical-chemical data are only known for a few equivalent to 3050 cm1) excite the C±H stretch vibra- generally used matrices and are summarized in Table 2. tions, so, many matrices for UV±MALDI can be applied Most of the data in Table 2 originate from peptide and in IR±MALDI as well. In addition, matrix-less, i.e., IR±

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TABLE 3. Matrices for IR±MALDI mass spectrometry of synthetic polymers (Weidner et al., 1998) Polymer Matrix Matrix solvent Polymer solvent MW PMMA CCA THF THF 3500 PMMA SA THF THF 3500 PMMA DHB THF THF 3500 PMMA THAP THF THF 3500 PEG CCA THF THF 14,000 PEG SA THF THF 14,000 PEG DHB THF THF 14,000 PEG THAP THF THF 14,000

FIGURE 1. FT±IR spectra of IR±MALDI matrices: grey bars cover the absorption region for an infrared laser at 3.27 mm. Reproduced from Weidner et al., 1998, with permission from John Wiley & Sons, Ltd.

LDI, has potential for the characterization of rather apolar However, one should also note some disadvantages when samples such as waxes (Weidner et al., 1998). FT±IR considering IR±MALDI. First, one IR±laser shot will spectra of the matrices in Table 3 are given in Fig. 1: in desorb more material than a corresponding UV±laser shot all cases, excitation of the matrix by a 3.27 mm IR-laser is due to its greater penetration depth, thus only a few mass feasible. For proteins and peptides, a lower degree of spectra can be acquired from a speci®c spot and more prompt and metastable fragmentation has been observed frequent spot change is required. Second, the pulse length as compared to corresponding UV±MALDI measure- of the IR±laser is generally longer, for example 10±20 ns ments (Berkenkamp et al., 1997; Zhang et al., 1998). for a YAG pumped OPO laser vs. 3±5 ns for a

318 MALDI TOF MS OF SYNTHETIC POLYMERS & conventional 337 nm nitrogen laser. These disadvantages observation, some papers deal with conformations and explain, at least partly, the much lower (7±20 times) energetics and compare theoretical ®ndings with experi- resolution obtained in IR±MALDI of polymers. The peak mentally observed MALDI data. The experimentally width due to the initial velocity distribution might be observed absence of sodium cationized poly(ethylene effectively reduced by delayed-extraction techniques glycol) oligomers below n ˆ 5 has been attributed to too (Weidner et al., 1998; Zhang et al., 1998), similar to low binding energies and the requirement of multiple UV±MALDI experiments. Nevertheless, it was con- interactions, i.e., folded oligomer conformations (Helden cluded that in general UV±MALDI is to be preferred et al., 1995; Reinhold et al., 1998). Also, for polyester for the analysis of synthetic polymers (Weidner et al., trimers, folded conformations are energetically more 1998), but there might be potential for some interesting favorable, but the difference was smaller for sodium vs. niche applications too, such as the characterization of lithium cations (Gidden et al., 1997). Actually, different halogenated polymers, which often show extensive cations such as lithium, sodium, and cesium ef®ciently prompt fragmentation in UV±MALDI (McEwen et al., wrap the polymer around them but the detailed structure 1998), and in the analysis of polymers absorbing at of the inner coordination sphere of polyether oxygen 337 nm by themselves. atoms around the cation was found to be cation dependent, i.e., the larger cesium cations prefer higher (11-fold) coordination (Wyttenbach et al., 1997). Varying the cation III. CATIONIZATION IN POLYMER ANALYSIS type and size in the analysis of polymethacrylates showed molecular weight distribution (MWD) shifts as much as Contrary to MALDI of biopolymers, the ®nal ionization 20±35%, and higher mass averages with the larger of synthetic polymers is usually by cationization rather cations, these effects being more pronounced for poly- than protonation. Consequently, matrix optimization disperse samples (Dogruel et al., 1996; Scrivener et al., alone is not suf®cient and should be considered together 1996). It was concluded that larger cations should be able with cationization issues. For relatively polar polymers to form a more stable conformation with a larger oligomer sodium and/or potassium adduct ions might be observed as more oxygens should be available for coordination with in the MALDI mass spectrum, even when they were not the cation. Similar MWD shifts were observed when intentionally added (Danis & Karr, 1993). These cations poly(ethylene terephtalate) was measured in the presence are present as impurities in glassware, solvents, reagents, of lithium, sodium, potassium, rubidium and, cesium etc., and polymers having relatively high cation af®nities (Jackson et al., 1997a). do not necessarily require high cation concentrations in Polyesters are less ¯exible than polyethers and the MALDI sample. Most of the synthetic polymers polyacrylates, and consequently the most favorable having heteroatoms will show cationization after addition interactions with the cation are expected to occur at of sodium or potassium salts, e.g., polyethers (King et al., higher molecular weight oligomers. Cationization of 1995), polyacrylates, polyesters, polyamides. Use of the polystyrene was investigated using silver, zinc, copper, delayed ion extraction technique allows more time for cobalt, aluminum, palladium, and platinum salts. It cation attachment, and a substantial increase in signal was found that silver, copper, and palladium yielded intensity of cationized polymers might be obtained ef®cient cationization of polystyrene oligomers and it was (Mowat et al., 1997). Apolar synthetic polymers without argued that cationization occured by gas phase ion± heteroatoms such as polystyrene, polybutadiene, and reactions rather than pre-formed ions from the polyisoprene can be successfully ionized after the condensed phase (Deery et al., 1996; Hoberg et al., 1997; addition of silver or copper salts, which interact with Rashidzadeh & Guo, 1998b). The disadvantage of the the double-bonds of these polymers (Mowat & Donovan, silver and copper cation option are their two signi®cant 1995). Polymers without hetero-atoms and without any isotopes which complicate the isotope patterns of the double bonds such as polyethylene and polypropylene are cationized polystyrene oligomers. Therefore, the use of a still not amenable to MALDI analysis because of the monoisotopic cation, 65Cu(II)acetylacetonate, was advo- extremely low binding energy of the cation-oligomer cated (Burgers & Terlouw, 1998). complexes (Reinhold et al., 1998); in these cases ®eld Only one study dealt with effects of the counter ions desorption MS or medium-energy electron ionization MS in MALDI. In all cases the cation was sodium and the might be an alternative to some extent (Prokai, 1990; polymer was poly(methyl methacrylate) but the counter Ludanyi et al., 1999). ions were iodide, bromide, or chloride. Decreasing ion Cationization has been studied by several groups but yields were observed in the order I > Br > Cl. It was argued systematic approaches and cation af®nity data are that the counter ion in¯uences the amount of gas phase generally lacking and remain interesting research oppor- cations available for cationization in the expanding plume tunities for the near future. As exceptions to this general (Hoberg et al., 1998).

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IV. SAMPLE PREPARATION TECHNIQUES fast crystallization method was introduced (Weinberger et al., 1993; Nicola et al., 1995) in which the target is put Before using a particular sample preparation technique, in a vacuum chamber in order to promote rapid crystal- solvent(s) must be selected for the matrix, sample, and lization, typically within a few seconds. As a result, cationization salt solutions; suggestions are included in smaller crystals are obtained with less segregation, giving Table 1. Ideally only one solvent is present in the ®nal enhanced reproducibility, signal intensity and resolution. mixture which reduces the risk of segregation during Accelerated drying using a stream of high-purity nitrogen crystallization on the MALDI target. However, salts are gas is an alternative means for reaching similar goals hardly soluble in organic solvents used for nonpolar (Castoro et al., 1992; Gusev et al., 1995a). Improved synthetic polymers. Typically, a stock solution of the salt homogeneity and better sensitivity were also obtained by in an intermediate solvent such as propanol can be spin-coating sample preparation techniques (Perera et al., prepared and subsequently diluted with the matrix and 1995). In the thin-layer method, ®rst a matrix layer is polymer solvent. However, even relatively low concen- prepared and allowed to crystallize and next the sample is trations of a polymer nonsolvent in the ®nal mixture can added and dried. Particularly in the analysis of peptides affect ®nal signal reproducibility and cause errors in the and proteins (Vorm et al., 1994) but also in the analysis of MWD data obtained. The effect of the water and an aromatic polyester (Guittard et al., 1995) and organic methanol content in THF used for the MALDI analysis dendrimers (Milberg & Garden, 1996) improved results of PS 7000 was studied and the data showed very poor were reported. reproducibility and 13% lower Mn values when 5% water One of the most promising techniques for sample was present in the ®nal mixture (Yalcin et al., 1998). preparation of synthetic polymers is electrospray deposi- Similar phenomena were observed for PMMA (Chen & tion (Axelsson et al., 1997). A typical experimental set-up Guo, 1997; Yalcin et al., 1998). Note that water is a is shown in Fig. 2. Using an applied potential of 8 kV, a nonsolvent for PS and methanol for PMMA. If actually a distance from needle to sample slide (target) of 2±4 cm mixture of solvents is used in the sample preparation then and a ¯ow rate of 10±100 mL/min, a 1 cm diameter the solvent composition will change during the solvent circular spot on the target was obtained. A comparison evaporation process because of differences in volatility. was made for PS and other polymers, using both the dried As a consequence, the solubility of the polymer changes droplet (i.e., one-layer) and the thin-layer (i.e., two-layer) as well, thus when some less volatile non-solvent is technique, with and without electrospray deposition. The present in the preparation, the polymer might precipitate dried-droplet technique without electrospray deposition before matrix crystal formation. In these cases, even an showed large variations in shot-to-shot reproducibility and initial clear sample preparation might change into a signal intensities ranging between zero and the maximum problematic situation during the drying process. The best observed. The thin-layer method without electrospraying way is to select a solvent system that will allow matrix yielded a large number of spectra without any polymer crystallization to take place simultaneously or prior to distribution. Electrospray deposition on the other hand polymer precipitation. with either the one-layer or the two-layer approach Of course, the molar ratio of polymer sample/matrix/ yielded much higher signal intensities and much better salt in the sample preparation determines whether a mass shot-to-shot and spot-to-spot reproducibility, slightly spectrum will be obtained or not. However, in the high favoring the one-layer electrospraying approach. The mass range, multimer formation might occur which is a improved results of the electrospray deposition method source of errors in the MWD calculations. Increasing the can be explained by the small and evenly-sized cocry- matrix to polymer ratio might improve such an unintended stals formed (Axelsson et al., 1997; Sadeghi & Vertes, situation (Schriemer & Li, 1997a). 1998). Following the selection of MALDI matrix, cationiza- Recently, microscopy and surface analysis techniques tion salt and solvent(s) for sample, matrix and salt, several such as scanning electron microscopy (SEM), confocal options are available for transferring the mixture onto the ¯uorescence microscopic imaging, X-ray photoelectron MALDI target. The oldest procedure is the so-called spectroscopy (XPS) and time-of-¯ight secondary ion mass dried-droplet method (Karas & Hillenkamp, 1988). In this spectrometry (TOF±SIMS) have been successfully method the three solutions are mixed by volume and applied to study the homogeneity of the ®nal MALDI approximately 0.5±1 mL of the mixture is applied to the sample preparations. The microscopy techniques showed target and air-dried at room temperature. The crystal- better homogeneity of the sample preparation for fast lization is relatively slow, thereby increasing the risk of crystallization methods (Westman & Barofsky, 1995; Dai segregation between sample and matrix and cationization et al., 1996). Evidence for segregation of cationization salt salt, or segregation within one of the distributions of the from matrix crystals was obtained by TOF±SIMS images synthetic polymer (cf. INTRODUCTION). Later on, the both for slow drying preparations such as for a PEG 1500

320 MALDI TOF MS OF SYNTHETIC POLYMERS &

FIGURE 2. Electrospray sample deposition set-up employed for preparation of MALDI sample slides. Reproduced from Axelsson et al., 1997, with permission from John Wiley & Sons, Ltd. sample with DHB and sodium cations dissolved in chromatography (SEC) forced several groups to search methanol/water, as well as for a fast drying system of for explanations of mass discrimination in MALDI MS. PMMA 2900 with DHB and sodium dissolved in acetone But before going into more detail of these mass (Hanton et al., 1999). Upon changing from the dried discrimination phenomena, one has to consider the droplet to the electrospray deposition method homoge- fundamental differences between SEC and MS (Jackson neous cocrystallization occurred and very homogeneous et al., 1996): A narrow MWD as typically obtained by chemical images were obtained. Similarly, XPS analysis anionic ``living'' polymerizations can be theoretically showed improved sample homogeneity in MALDI of described by a Poisson distribution. The calculated proteins for the so-called crushed crystal method vs. the number fraction distribution, as to be expected in MS dried droplet method (Smith et al., 1997). where each oligomer chain acquires ideally one charge Negative ion MALDI analysis of polymeric acids independent of its molecular weight, is shown in Fig. 3a. such as sulphonated polystyrene and poly(acrylic acid) In SEC, on the other hand, a weight fraction distribution requires desalting and conversion into the hydrogen form, vs. the logarithm of the molecular weight will be obtained thereby putting an additional demand on sample prepara- as shown in Fig. 3b. In this case the peak molecular tion. The addition of ion exchange beads to the sample/ weight values, Mp, of both distributions are theoretically matrix mixture might be adequate for the purpose of very similar and only two monomer units higher in SEC desalting but one should be aware of different solubilities vs. MS. In fact, excellent comparisons between MALDI of the polymer in its neutralized form vs. the original salt MS and SEC data for narrow-distributed (polydispersity form. Precipitation of the neutralized polymer onto the ion <1.1) polymers have been experimentally observed exchange beads might occur thereby obstructing the ®nal indeed in several studies where sample preparation and/ analysis. More sophisticated desalting options such as or instrumental discrimination factors could be success- membrane desalting (Bornsen et al., 1995; Worrall et al., fully ruled out (e.g., Cottrell et al., 1995). For wide 1998) and on-probe desalting using self-assembled MWDs as obtained in many polymer syntheses, the monolayers (Brockman et al., 1997) are currently situation can be described by a Schulz or Flory investigated for MALDI of biomolecules and might yield distribution. The comparison of the theoretical results some valuable spin-off for the analysis of acidic synthetic for a condensation polymer is given in Fig. 4. A decaying polymers in the negative ion mode. number fraction distribution is to be expected in MS and Mp will be the monomer mass, in sharp contrast to the SEC situation where Mp will be equal to the weight- V. DISCRIMINATION ISSUES average molecular weight, Mw. So the huge discrepancy between MS and SEC data in Fig. 4 originates simply from how the data are being displayed. In theory, A. High and Low Mass Discrimination examination of the decaying mass spectrum up to very Discrepancies between MWD data of polymers as high m/z values, smoothing, and background subtraction calculated from MALDI TOF MS and size exclusion would be suf®cient to obtain reliable MWD data directly

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FIGURE 4. Simulated distribution for a wide polymer distribution FIGURE 3. Simulated narrow distribution of oligomers for 20.5 from, e.g., a polycondensation reaction: (a, top) plotted on a linear and monomers reacted per initiator: (a, top) plotted on a linear and (b, (b, bottom) plotted on a logarithmic scale. Reproduced from Jackson C bottom) plotted on a logarithmic scale. Reproduced from Jackson C et al., 1996, with permission from the American Chemical Society. et al., 1996, with permission from the American Chemical Society. from the mass spectrum, even in the case of polydisperse over, the high molecular weight end in the mass spectrum synthetic polymers (Montaudo et al., 1996a). will disappear much earlier into the baseline noise than the In practice, however, baseline subtraction on a very high molecular weight side of the SEC distribution noisy and decaying signal is not trivial, and signi®cant (compare Figs. 4a and b): a few high molecular weight errors and poor reproducibility of MWD data are molecules represent only a few ions in the MS but still obtained. Moreover, such a data treatment assumes that show a signi®cant bulk property as detected by the no mass discrimination occurs in sample preparation, refractive index detector in a SEC apparatus. As a result, desorption/ionization, transmission, and detection. Upon too low MWD data are to be expected in such a situation. realizing that a polymer with a polydispersity >2 will On the other hand, experimentally obtained MALDI mass have masses over an m/z range of a few hundred Da up to a spectra showing a decaying pattern as in Fig. 4a few hundred thousand Da, it becomes obvious that it is immediately show that the polymer sample under unrealistic to expect discrimination-free experiments in investigation is polydisperse (>1.1) and that direct MALDI MS of polydisperse synthetic polymers. More- MWD calculation from the mass spectrum will be biased

322 MALDI TOF MS OF SYNTHETIC POLYMERS & to (far) too low values and pre-fractionation will be required (cf. Section VII). Basically, two different origins for mass discrimina- tion in MALDI TOF MS can be distinguished: sample preparation on the one hand and instrumental factors on the other. Both contributors can be minimized, yielding reliable and reproducible mass spectra for narrow distributed synthetic polymers having average masses up to several hundred thousands Da. Sample preparation issues were covered in the previous sections: one should select the right MALDI matrix, the right salt for cationization, preferably use one type of solvent, optimize mixing ratios, and apply the best preparation technique, e.g., electrospray deposition. Each of these sample preparation factors might ruin the ®nal goal of homo- geneous cocrystallization of polymer, matrix and salt. With regards to the instrumental factors, desorption/ ionization, transmission, and detection might be respon- sible for mass discrimination. Channel plate detectors have a limited dynamic range and can get saturated easily by low molecular weight components such as matrix ions and/or oligomers; particularly in the case of polydisperse polymers where the majority of the ions (cf. Fig. 4) will be low molecular weight (McEwen et al., 1996; Schriemer and Li, 1997b; Zhu et al., 1998). When the more abundant lower mass ions in polydisperse polymers are de¯ected from reaching the detector, then the high-mass ions can be observed indeed which proves that at least in part saturation of the detector is responsible for high mass discrimination in MALDI TOF MS (McEwen et al., 1997). But the sensitivity at the high molecular weight end of the MWD will be insuf®cient anyway due to the lower impact velocity of a high MW ion vs. a low MW ion which generally causes a lower number of secondary electrons (Axelsson et al., 1996; Larsen et al., 1996). Instrument manufacturers coped with the detector problem in different ways. For example, using a conversion dynode plus a microchannel plate and a cesium iodide scintillator mounted on the entrance window of a photomultiplier. FIGURE 5. The effect of laser power on the MALDI±MS of Unfortunately, most of the solutions compromise the monodisperse PMMA (an equimolar mixture of n ˆ 20, 35, and 50). resolving power of the detection system. Alternatively, a Reproduced from Sakurada et al., 1998, with permission from John procedure might be considered in order to correct for the Wiley & Sons, Ltd. decreasing detector response with increasing mass (Scamporrino et al., 1998). Fig. 5 (Sakurada et al., 1998). An increase in laser power Ideally the laser power should be so chosen that the results in an increase of ion intensities of the high ionization probability is the same for all ions, but the laser molecular weight species, accompanied by an increase in power is not so high as to cause fragmentation. In practice, the matrix peak intensity. Higher laser powers can also however, higher laser powers are required in order to reach induce dimerization by clustering in the gas phase, the threshold ¯uence for the observation of higher yielding an MWD shift towards higher masses (Axelsson molecular weight oligomer ions (Lloyd et al., 1995). et al., 1996). Moreover, higher laser power can initiate Many authors observed mass discrimination effects unintended scissions in the polymer structure, as shown related to the applied laser power: Equimolar amounts for example in the characterization of hyperbranched of monodisperse PMMA's were mixed and the effect of polyesters (Feast et al., 1997) and in the degradation of laser power was studied. A typical example is shown in PMMA (Lehrle and Sarson, 1995; 1996).

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The ion transmission in MALDI TOF MS has been reported as a source of mass discrimination as well, particularly when a polydisperse polymer is analyzed by an instrument having a long ¯ight tube and a detector of a small detection area (Guo et al., 1997b). Mixtures of narrow disperse PS were used to investigate similar discrimination effects. It was shown that the lensing action provided by the source electrodes in TOF MS is responsible for a mass-dependent distribution of the ions in the plane of the detector (Schriemer & Li, 1997b). In delayed-extraction instruments the lensing properties can be altered by adjusting the amplitude of the pulse voltage. However, depending on the pulse voltage and time delay applied, only a narrow mass range will be focussed, i.e., only a part of the molecular weight distribution will have a better resolution. Depending on the calculation method, peak areas of the unfocused peaks are overestimated compared to the well-focused ion peaks and, as a FIGURE 6. Comparison of relative ion yields of an equimolar mixture consequence, different molecular weight averages are of per¯uoroalkylsilyl- and H-terminated polystyrene oligomers by calculated (Zhu & Li, 1998). MALDI TOF MS. Reproduced from Belu et al., 1996, with permission Also, on the low molecular weight end of the polymer from the American Society for Mass Spectrometry. distribution, mass discrimination might occur, yielding too high MWD values for MALDI mass spectrometry vs. alternative methods (Barry et al., 1997). This phenomenon was investigated. A typical result for the per¯uoroalk- can be at least partly explained by the lower binding ylsilyl-PS is shown in Fig. 6. The impact of the end group energy of cations for shorter oligomers (cf. Section III) on the cationization ef®ciency will be more pronounced and losses of low molecular weight components by for the lower oligomers. Obviously, the end group evaporation. Derivatization has been successfully applied modi®cation allows these oligomers to ionize more to reduce the low-mass discrimination in ethoxylate easily. The improved ionization at higher oligomer polymers (Barry et al., 1997). numbers as shown in Fig. 6 has probably resulted from In summary, it has to be concluded that MALDI±TOF improved interaction of the functionalized PS with the mass spectrometry cannot be considered as an absolute matrix (Belu et al., 1996). In a second example, oligomer method in the measurement of polymers with high speci®c mass discrimination both from end group polydispersity, whereas it can be really an absolute functionality and from different oligomer architecture is method in the analysis of narrowly distributed polymer demonstrated. The MALDI mass spectrum of an aromatic samples (Martin et al., 1996). polyester based on terephthalic acid (TPA) and neopen- tylglycol (NPG) is given in Fig. 7. The spectrum shows ions of cyclic(TPA/NPG) oligomers, linear (TPA/NPG) B. Oligomer Speci®c Discrimination n n oligomers and linear (TPA/NPG)nTPA oligomers. Direct Apart from high- and low mass discrimination in the calculation from the mass spectrum yields the following molecular weight distribution (MWD), speci®c discrimi- composition: 14 Mol% cyclic (TPA/NPG)n oligomers, 20 nation might occur in the functionality type distribution Mol% linear (TPA/NPG)n oligomers, and 66 Mol% linear (FTD). Oligomers having chemically different end group (TPA/NPG)nTPA oligomers. However, according to structures might in¯uence their respective cocrystalliza- established reference methods, only 6 Mol% cyclic tion behavior during sample preparation and/or might (TPA/NPG)n oligomers are present in this sample (Nielen show different cationization ef®ciencies as such. Surpris- & Buijtenhuijs, 1999) and only 3% linear (TPA/NPG)n ingly, very limited data have been published concerning oligomers (based on titration and on NMR). Thus this topic. The relative ion yields of two differently oligomer speci®c mass discrimination in MALDI of funtionalized polystyrenes, terminated with a dimethyl- functionalized and/or differently shaped oligomers is phenylsilyl end group and with a per¯uoroalkylsilyl end responsible for signi®cant errors in composition calcula- group, were compared relative to the standard proton- tions and conclusions based thereon. Much more research terminated PS. By ratioing the signals of the functiona- effort is required in this ®eld in order to establish the lized to unfunctionalized oligomers at each degree of impact of end group on cationization yield and polymerization, the effect of the end group on ion yield to derive suitable response correction functions which

324 MALDI TOF MS OF SYNTHETIC POLYMERS &

FIGURE 7. Delayed extraction re¯ectron MALDI TOF mass spectrum of a terephthalic acid/ neopentylglycol polyester sample. Matrix, 2,5-dihydroxybenzoic acid. Reproduced from Nielen & Malucha, 1997, with permission from John Wiley & Sons, Ltd. would ideally allow composition calculations directly precursor ion selector in the ®eld-free region of the from the mass spectrum. linear ¯ight path and a scanning re¯ectron voltage, a PSD MS/MS daughter spectrum can be obtained. Despite the low internal energy and low fragmentation ef®ciency VI. MS/MS IN MALDI TOF MS OF SYNTHETIC obtained, MALDI PSD is very useful for controlled POLYMERS fragmentation such as required in, e.g., peptide sequen- cing. In addition, a pulsed collision gas cell can be Being a soft ionization method, fragmentation is mounted in the ®eld-free region of MALDI TOF systems generally not very pronounced in MALDI mass spectro- for high energy (keV) collisions thus yielding additional metry. However, under speci®c instrumental and experi- fragments in peptide analysis (PSD/CID). In contrast to mental conditions, different types of fragments can be the large number of references on MALDI PSD (and observed in MALDI TOF MS. At high laser ¯uences, PSD/CID) of peptides, only a few MS/MS data of fragmentation might occur in the ion-source region, often synthetic polymers can be found in MALDI TOF referred to as ``prompt fragmentation''. In addition, in literature. A good example of a PPG mass spectrum delayed extraction ion sources, fast metastable fragmen- including ISD fragment ions is shown in Fig. 8 (Mowat tation might occur during the delay time before et al., 1997). ISD fragmentation was also observed in the extraction, i.e., on the 100±1200 ns time scale, referred MALDI analysis of hyperbranched polyesteramides to as in-source decay (ISD). The increase in ISD which showed rapid dissociation of higher oligomers fragmentation with laser energy at a ®xed delay time is into lower oligomers minus a water molecule (Kwak- caused by a greater number of collisions in the expanding kenbos et al., 1999). Next, PSD/CID of mass-selected plume leading to a greater amount of collisional ions was applied in order to discriminate between these activation in the source. Prompt and ISD fragmentation ISD fragment ions and the isobaric cyclic oligomers. yield fragment ions in the mass spectra from both the From these results and reference data obtained by linear and the re¯ector detector. In contrast, metastable techniques as NMR, titration, and FD MS, it was obvious fragmentation in the ®eld-free region, referred to as post- that MALDI TOF MS yielded wrong conclusions about source decay (PSD), occurs on the 10 ms time scale and the chemical composition distribution and functionality yields fragment ions in the mass spectrum from the type distribution for these hyperbranched samples. re¯ector detector only. With the aid of a suitable Structural information of dendritic polyurethanes was

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FIGURE 8. MALDI-ISD TOF MS/MS spectrum of PPG 2000 under delayed extraction and high laser intensity conditions. (4 kV extraction pulse, 1.21 ms delay time, laser power 120 mJ). The symbols

represent the following series: *58n1, ‡ 58n ‡ 10,  58n ‡ 23, 58n ‡ 39, 458n ‡ 41. Reproduced from Mowat et al., 1997, with permission from John Wiley & Sons, Ltd.

FIGURE 9. Proposed mechanism for the fragments observed in the MALDI±PSD TOF MS/MS spectrum of a third generation polyurethane dendritic wedge. Reproduced from Puapaiboon et al., 1999, with permission from John Wiley & Sons, Ltd. obtained by PSD experiments (Puapaiboon et al., 1999). fragments from direct cleavage (labeled A and B) is given Most of the fragment ions were sodium adducts arising in Fig. 11. The formation of fragment ions was compared from the cleavage of the or amide bond, see Fig. 9. with high energy MALDI±CID experiments in a A MALDI/PSD spectrum of the 10-mer of PMMA is MALDI/hybrid sector oaTOF instrument (Scrivens et al., shown in Fig. 10, and a proposal for the origin of 1997). Despite the different time-scales for dissociation,

326 MALDI TOF MS OF SYNTHETIC POLYMERS &

FIGURE 10. MALDI±PSD TOF MS/MS spectrum of the 10-mer of PMMA. Reproduced from Scrivens et al., 1997, with permission from Elsevier Science B.V. the main difference between Fig. 10 and the mass VII. CHROMATOGRAPHY/MALDI COUPLING spectrum obtained in the hybrid instrument when no collision gas was applied, is the ratio of the fragment ion A. Thin Layer Chromatography/MALDI TOF MS peaks arising from direct cleavages (A and B series) and rearrangement processes (C,D,E, and F series). The same The coupling of thin layer chromatography (TLC) with comparison for the 15-mer of PEG yielded similar MALDI mass spectrometry has been realized via an conclusions: both MS/MS spectra may be readily extraction method (Gusev et al., 1995b): following TLC interpreted in terms of polymer structure but enhanced separation a drop of extraction solvent was added and the resolution and signal-to-noise ratio was observed in the (peptide) analyte was extracted from the TLC spot into MALDI/hybrid sector oaTOF instrument (Jackson et al., the solvent. Next, the MALDI matrix was added, mixed 1996). Actually, these authors applied the MALDI/hybrid with the extracted analyte and allowed to cocrystallize on sector oaTOF to a variety of synthetic polymers and the TLC plate. Finally, MALDI measurements were studied high-energy MALDI±CID spectra of polyacry- carried out directly onto the plate using a modi®ed lates, polyethylene glycols, polystyrenes, and polyesters. LAMMA 1000 laser microprobe instrument. The main PSD MALDI TOF MS was successfully used in order to advantage of such a direct measurement is the direct TLC differentiate between linear and branched PEG (Kowalski imaging capability. Later on, the authors modi®ed the et al., 1998). Using a precursor ion selector having a matrix deposition method in order to reduce the analyte resolution of 150, the MS/MS spectra of mass selected spreading, by using a method in which pre-dried matrix linear PEG 2000 oligomers showed fragmentation by was pressed onto the TLC gel, and an ultimate spatial cleavage on both sides of the oxygen atoms, while the resolution of 50 mm for TLC imaging was obtained MS/MS spectra of mass selected branched PEG 2000 (Gusev et al., 1995c). A further improvement was re- showed a dominant single cleavage of intact ethylene ported recently by electrospraying the matrix material oxide monomer. PSD fragmentation patterns were also onto the TLC plate (Mowthorpe et al., 1999). The authors reported for mass selected oligomer ions of polyisobu- used a modi®ed commercial MALDI±TOF instrument tylene (Varney et al., 1997). for direct measurements on the cocrystallized spots and A very important and challenging application of applied their method on rapid impurity testing of MALDI TOF MS/MS in polymer analysis would be the pharmaceuticals. As a simple alternative to these direct sequencing of block copolymers and the mass spectro- methods, off-line coupling of TLC and MALDI TOF MS metric determination of the block length distribution. was reported for the analysis of pitch fractions (Herod Obviously, much more effort is required in order to reach et al., 1996). The silica spots containing the fractionated this goal. analytes were scraped from the plates and extracted using

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FIGURE 11. Proposed mechanism for the formation of the direct cleavage ion series A and B in Fig. 10. Reproduced from Scrivens et al., 1997, with permission from Elsevier Science B.V.

1-methyl-2-pyrrolidinone in an ultrasonic bath. Finally, coupled with MALDI TOF MS is size-exclusion the extracted material was deposited on the MALDI target chromatography (SEC) or gel permeation chromatogra- and measured without the addition of a matrix, i.e., the phy (GPC). As outlined in Section V, the characterization sample acted as its own matrix. A similar TLC/MALDI of polydisperse synthetic polymers is obstructed by mass approach was used for the analysis of organic reactions discrimination issues. A practical solution to this problem such as the base-catalyzed condensation between benzal- is the pre-fractionation of a polydisperse polymer sample dehyde and cyclohexanone (Hilaire et al., 1998). into several fractions each having a narrow MWD (polydispersity <1.2) by SEC. The fractions on their turn can be very well characterized by MALDI TOF MS B. Off-Line Size-Exclusion Chromatography/MALDI provided an optimized sample preparation protocol is TOF MS used. Finally, the mass-measured fractions can be used as The coupling of matrix-assisted laser desorption/ioniza- absolute calibration points (Log M vs. elution volume or tion to liquid separations such as capillary electrophoresis time) for the SEC chromatogram and the absolute MWD (CE) and liquid chromatography (LC) has been reviewed averages calculated from the calibrated chromatogram very recently (Murray, 1997). A good description of with the aid of a classical SEC software package. methods and their applications in the biochemical Actually, this is an ideal marriage overcoming the analysis ®eld was given but polymer data were rather shortcomings of both components: SEC is obstructed by scarce. The most relevant separation technique to be the very limited availability of well-characterized cali-

328 MALDI TOF MS OF SYNTHETIC POLYMERS &

FIGURE 12. Set-up for mSEC/MALDI TOF MS using a robotic interface. Inset (right): Close-up of the needle tip. Reproduced from Nielen, 1998, with permission from the American Chemical Society. bration standards while MS of polydisperse samples is MALDI TOF MS in the delayed-extraction re¯ectron quantitatively limited by discrimination issues; now mode (Nielen & Malucha, 1997). polydisperse synthetic polymers can be characterized using an absolute calibration method based on the C. On-Line and Direct Deposition Size-Exclusion investigated polymer sample itself. Moreover, the mass Chromatography/MALDI TOF MS spectra of the low molecular weight fractions can be used to infer structural information from the polymer such as On-line coupling of SEC and MALDI MS has been the monomer(s), from the repeating mass increment(s), presented, both in a continuous ¯ow set-up (Li et al., and, following extrapolation to zero monomer(s) and 1993) and in an aerosol MALDI set-up (Fei & Murray, subtraction of the cation mass, the combined end-group 1996). The former approach utilizes liquid MALDI mass. In a series of papers, this approach was applied to matrices and shows similarities with continuous ¯ow the analysis of polydisperse synthetic polymers such as FAB but might suffer from typical ion source contamina- poly(dimethylsiloxane)s (Montaudo et al., 1995b); poly- tion problems. The latter, aerosol approach, utilizes polar (butylene adipate) and poly(butylene adipate-co-butylene solvents such as methanol, instead of the more general succinate), (Montaudo et al., 1995d); PMMA, poly(vinyl SEC solvent tetrahydrofuran (THF). acetate) and poly(N-vinylpyrrolidone-co-vinyl acetate), It can be argued that off-line coupled SEC/MALDI (Danis et al., 1996b); PS, polybutylacrylate, polycarbo- MS is more attractive than on-line combinations since it nate, terephthalic acid/neopentylglycol polyester and allows MALDI optimization (e.g., detector settings) for poly(methylmethacrylate-co-methacrylic acid), (Nielen individual narrow fractions of the polymer distribution, & Malucha, 1997); PMMA, poly(dimethylsiloxane) and particularly the more demanding high molecular weight several copolyesters, (Montaudo et al., 1998b; Montaudo parts. However, off-line SEC/MALDI MS, as outlined et al., 1998a). A comparison of MWD data calculated above, involves fraction collection, evaporation, pipetting, from off-line SEC/MALDI TOF MS with reference data etc. and is de®nitely laborious and time-consuming. So, from manufacturers, from SEC with conventional cali- direct deposition methods in which SEC fractions and bration using narrow standards, and from SEC with on- MALDI matrix are directly and automatically deposited line intrinsic viscosity (IVD)- and light scattering onto the MALDI target are to be preferred. The feasibility detection (RALLS), is given for a vareity of polydisperse of such a direct deposition was demonstrated by spray- polymers in Table 4. Generally, a very good agreement deposition of approximately 15% of a SEC ef¯uent onto a with reference data is obtained. It is also obvious that continuous rotating matrix-coated substrate using a samples such as polybutylacrylate cannot be analyzed by modi®ed LC-TransformTM Series 100 IR interface (Kassis conventional SEC with calibration based on PMMA et al., 1997). The resulting polymer ``trail'' could be narrow standards, thereby underlining that SEC columns analyzed directly by MALDI MS but the mass spectra should be calibrated by the investigated polymer itself, as obtained were summed in order to calculate MWD obtained by off-line SEC/MALDI coupling. In addition, averages instead of using the MS data as calibration structural data of monomers and end groups could be points in the SEC chromatogram. As a consequence, the inferred from the isotopically-resolved oligomer spectra method still suffered from discrimination issues as of the low molecular weight fractions, as recorded by discussed in Section V.

329 & NIELEN

small quantities of polymer in each 10 sec spot, and the absence of optimization of the polymer/matrix ratio, MALDI TOF MS data were successfully used as calibration points for the mSEC/UV chromatogram and allowed the calculation of absolute molecular weight averages by using regular SEC software. The MWD averages thus determined for polydisperse poly(bisphenol A carbonate) and an aromatic copolyester resin, were in good agreement with reference data from manufacturer and off-line SEC fractionation/MALDI analysis. More- over, the method still allowed the characterization of end- groups and the chemical composition distribution in the copolyester using (isotopically-)resolved mass spectra of oligomers. Recently, an automated SEC/MALDI method was presented using a SEC system consisting of a 4.6 mm i.d. SEC column, a THF ¯ow of 0.3 mL/min, an analog refractive index (RI) or UV detector, and a post-column split 10:1 to a microfraction collector (Danis et al., 1998). Every 4 sec a SEC fraction was collected and MALDI data were acquired in the linear mode using an automated data acquisition and data analysis protocol. The mass spectra were smoothed to a high degree, the derivative of the smoothed data was taken, and the Mp of the spectra at zero intensity was used vs. retention time (from fraction number) in the data output table. Thus a SEC calibration curve could be constructed and used for the calculation of absolute MWD data of a polydisperse poly(bisphenol A carbonate) sample. As an alternative, a less sophisticated low-cost direct deposition interface might be constructed from a modi®ed pen plotter, programmed to apply 1.5 mL FIGURE 13. Mass spectra of three poly(styrene) samples with spots from a microLC system onto the MALDI target nominal molecular weights of (A) 330,000 (B) 600,000 and (C) (Stevenson & Loo, 1998). 900,000. Reproduced from Schriemer & Li, 1996, with permission from the American Chemical Society. D. Other Liquid Chromatography Modes Coupled with MALDI TOF MS In principle, all LC modes in polymer analysis can be Following some early developments (Dwyer & interfaced with MS. Recently, size-exclusion chromato- Botten, 1996), a direct deposition interface has become graphy (SEC), gradient polymer elution chromatography commercially available. In this system, the eluent from a (GPEC) and liquid chromatography at the critical point of conventional-sized SEC system is sprayed through a adsorption (LCCC) were on-line coupled with ESI TOF heated nozzle and spotted or track-deposited on MALDI MS, in a single experimental set-up (Nielen & Buijten- targets equipped with foils containing precoated MALDI huijs, 1999). Of course, coupling of GPEC and LCCC matrix. The disadvantages of this system are the high with MALDI TOF MS, either off-line or on-line, can be sheath gas consumption (300±900 L/h) and the signi®cant realized simply by using a gradient LC system and one of volumes of hazardous solvents which evaporate. To the SEC/MALDI couplings discussed above. In an early overcome these shortcomings, a miniaturized method example of off-line LCCC/MALDI TOF MS, a fatty was developed featuring the use of a mSEC system with a ethoxylate sample was separated into three main THF ¯ow of only 5 mL/min, UVabsorbance detection, and groups and fractionated for MALDI analysis (Pasch & a robotic interface in which the MALDI matrix is Rode, 1995). According to the mass spectra, the LCCC coaxially added to the SEC ef¯uent and directly spotted separation was proven to be controlled mainly by end- onto the MALDI target without the use of heat and sheath group interactions showing the elution order PEG, C13- gas, see Fig. 12 (Nielen, 1998). Despite the extremely terminated PEG and C15-terminated PEG.

330 MALDI TOF MS OF SYNTHETIC POLYMERS &

TABLE 4. Comparison of molecular weight distribution data of polymers (Nielen & Malucha, 1997)

Polymer and data source Mw Mn Mz Mp PD Note Polystyrene 48 kDa Manufacturer 48,000 22,500 78,000 2.13 SEC (PS) 47,700 22,300 83,500 45,100 2.14 a SEC/MALDI 47,000 23,600 78,600 45,100 1.99 SEC/IVD 47,500 18,000 81,100 43,400 2.64 Polybutylacrylate 62 kDa Manufacturer 59,200 19,900 2.98 SEC (PMMA) 78,200 17,400 187,000 44,800 4.49 b SEC/MALDI 62,200 16,500 138,400 38,000 3.76 SEC/IVD 63,600 19,000 131,900 3.35 SEC/RALLS 57,200 25,000 98,900 2.29 Aromatic polyester 8 kDa SEC/MALDI 7300 3900 11,400 6800 1.88 SEC/IVD 8100 4400 10,700 9000 1.83 Polycarbonate 29 kDa Manufacturer 28,800 17,300 1.66 SEC/MALDI 28,500 15,800 43,200 30,400 1.81 SEC/IVD 28,800 16,700 39,800 31,900 1.72 SEC/RALLS 30,500 19,200 40,500 32,300 1.59 Poly(methylmethacrylate-co-methacrylic acid) 34 kDa Manufacturer 34,000 15,000 2.27 SEC/MALDI 36,300 18,500 56,600 35,300 1.97 c SEC/MALDI 34,300 19,300 50,700 34,100 1.78 d aConventional calibration of the SEC column using narrowly-distributed PS standards. bConventional calibration of the SEC column using narrowly-distributed PMMA standards. cData based on MALDI measurements in the positive ion mode using IAA with sodium formate as matrix. dData based on MALDI measurements in the negative ion mode using DHB as matrix.

VIII. POLYMER APPLICATIONS MS to the determination of absolute molecular weights of polydisperse polymers has been discussed already in A. Homopolymers Section VII and typical results of MWD calculations were summarized in Table 4. In Table 1 several polymer applications of MALDI TOF The determination of end-groups in homopolymers MS are summarized. Homopolymers, in particular PEG, requires resolution of the oligomers and suf®cient mass PMMA, and PS, are being studied frequently for the accuracy. Time-lag focusing (in this review also referred purpose of matrix- and instrument optimization. Further- to as ``delayed extraction'') provides both improved more, absolute molecular weights have been determined resolution and signal-to-noise ratio, as well as enhanced for narrow-distributed (homo)polymers. An example of mass accuracy in MALDI TOF of polymers, thus enabling the determination of absolute molecular weights by the differentiation of end groups (Whittal et al., 1997; MALDI TOF MS is given in Fig. 13. The three mass Jackson AT et al., 1997b). In a linear TOF mass spectro- spectra of poly(styrene) were obtained in a linear TOF meter equipped with a time-lag focusing ion source, instrument after careful sample preparation with all- oligomer resolution was obtained for PEG of masses up to trans-retinoic acid as the matrix and silver nitrate for 25 kDa and for polystyrene (PS) even up to 55 kDa cationization. The PS oligomers remained unresolved at (Whittal et al., 1997). MALDI TOF with time-lag focus- these high masses and the distributions appeared as broad ing in the re¯ector mode yielded a resolution of almost peaks. At higher masses, multiple charged distributions of 9000, as can be seen in the mass spectrum of PMMA in [M ‡ nAg]n ‡ ions dominated the MALDI spectra. This Fig. 14 (Jackson AT et al., 1997b). At lower molecular feature was successfully applied in the MALDI analysis weights, isotopically-resolved mass spectra were obtained of PS having a nominal molecular mass of 1.5 million Da in the delayed-extraction re¯ector mode and used for end- (Schriemer & Li, 1996). The application of MALDI TOF group analysis of a variety of polymers (Nielen &

331 & NIELEN

FIGURE 14. MALDI time-lag focusing spectrum of a speci®c PMMA obtained in the re¯ectron mode of operation (the inset shows the expansion of the molecular adduct ion [M ‡ Na] ‡ peak region for the 36- mer of this PMMA and the theoretical isotope pattern). Reproduced from Jackson AT et al., 1997b, with permission from John Wiley & Sons, Ltd.

FIGURE 15. Detail of the delayed-extraction re¯ectron MALDI mass spectrum of a mSEC fraction of polycarbonate. Reproduced from Nielen, 1998, with permission from the American Chemical Society.

332 MALDI TOF MS OF SYNTHETIC POLYMERS &

Malucha, 1997). In Fig. 15, a detail of the isotopically- crimination effects. As an example, two narrow stan- resolved MALDI mass spectrum of a polycarbonate is dards, PS 10200 and PMMA 9200, were dissolved in shown, featuring [M ‡ Na] ‡ and [M ‡ K] ‡ ions of oligo- THF and mixed by volume in several ratios. Next, mers (Nielen, 1998). The repeat unit mass of the poly- samples were prepared using either indoleacrylic acid carbonate, 254.1 Da, was con®rmed and after subtraction (IAA) matrix with sodium formate, or dithranol matrix of the cation and n-times the repeat unit mass, the end with silver tri¯uoracetate. In a ®rst set of experiments the groups were inferred from the mass residues (214, 0, and volumetric ratio of PS vs. PMMA was increased from 0:1 94 Da) yielding the structure proposals phenylcarbonate up to 199:1, and the matrix was IAA/Na in these cases, (bisphenol A carbonate)nphenyl, cyclic poly(bisphenol A i.e., optimized for PMMA. The striking result is shown in carbonate)n and (bisphenol A carbonate)n phenyl. So far, Fig. 16: even when PMMA is present only as a 0.5% end group analysis by MALDI TOF has been successfully impurity in a blend with PS, the mass spectrum is still used in a variety of reactivity studies in polymer dominated by the [M ‡ Na] ‡ ions of the PMMA chemistry, and these data are usually found in polymer-, distribution. In a second set of experiments, the rather than mass spectrometry journals. volumetric ratio of PS vs. PMMA was decreased from 1:0 down to 1:9, and the matrix was dithranol/Ag in all cases, i.e., optimized for PS. In any mixture tested, the B. Blends PMMA distribution is dominant and when PS is present Blends of the same narrow-distributed polymers, but of as a 10% impurity relative to PMMA, the mass spectrum different molecular weights, were used in several studies will not show PS ions any longer but only the PMMA towards discrimination phenomena in MALDI TOF MS distribution. Of course, this general discrimination in (Spickermann et al., 1996; Goldschmidt & Owens, 1997). favor of PMMA might be exploited in an application for MALDI applications concerning blends of different types qualitative trace analysis of acrylates in PS samples, but of polymers are scarce, probably because of signi®cant from these results it is obvious that quantitative analysis differences in cationization ef®ciencies and other dis- of blends with MALDI is far from reality.

FIGURE 16. MALDI TOF of PMMA and blends of PMMA and PS, applying a matrix recipe optimized for PMMA, i.e., indoleacrylic acid and sodium formate for cationization. For conditions, see Section VIII. B.

333 & NIELEN

C. Copolymers and Resins long blocks of one monomer preferably grow from short blocks of the other monomer or is there no correlation In Table 1 several copolymer and resin applications of between block sizes. An example is the MALDI analysis MALDI TOF MS are included: from ethylene oxide/ of block copolymers of -methylstyrene and vinylpyr- propylene oxide (EO/PO) copolymers, copolystyrenes idine (Danis et al., 1997). The mass spectra of the block and copolyacrylates, to several copolyesters, and epoxy- copolymers prepared with varying molar ratios of and phenol-formaldehyde resins. In copolymer spectra monomers are shown in Fig. 18. With increasing relative the number of masses observed increases dramatically, amounts of the 4-vinylpyridine monomer, the average hence oligomer resolution is usually limited to relatively mass increased as well as the spectra became more low molecular weights. Provided that this resolution is complex. Nevertheless, the resolution and mass accuracy achieved, that the masses of the different monomers used of MALDI TOF in the re¯ector mode with additional use are suf®ciently different from each other, and that no of a delayed extraction ion source, allowed unambiguous oligomer-speci®c discrimination occurs, the chemical assignment of the composition of the [M ‡ Ag] ‡ ions composition distributions (CCD) can be calculated observed. Finally, the distributions of the -methylstyrene directly from the MALDI mass spectra. Thus, evidence units were plotted for different numbers of vinylpyridine might be obtained for the presence (or absence) of a units and from the similarity of the distributions it could chemical composition drift as a function of oligomer be concluded that there was no correlation between the mass. An example of the complexity of a copolymer mass size of the ®rst block and the size of the second block; i.e., spectrum, obtained after separation by micro size- the random coupling hypothesis was found to be valid. exclusion chromatography (mSEC), is given in Fig. 17 Some mass spectrometric studies about copolymers claim (Nielen, 1998).The aromatic copolyester investigated was block-length distribution data; however, one should note based on the building blocks, dipropoxylated bisphenol A that MALDI TOF allows conclusions about the chemical (D), adipic acid (A) and isophthalic acid (I); proposals for composition of the copolymer chains only, i.e., differ- the chemical composition of the [M ‡ Na] ‡ ions in Fig. entiation between random-, alternating-, and block- 17 are given in Table 5. Note that without the use of the copolymers is not obtained! Such information about the mSEC preseparation one would encounter an even more blockiness of copolymers would require a kind of mass complex situation caused by additional overlap due to the spectrometric sequencing, similar as in peptide analysis. presence of [M ‡ Na] ‡ ions of cyclic oligomers; in Tandem MS/MS approaches have to be developed for the mSEC/MALDI MS cyclic oligomers show up later in full characterization of synthetic copolymers. different spots/fractions. MALDI TOF MS can also be successfully applied to block-copolymers. Knowledge of the distribution of D. Miscellaneous blocks in a copolymer is very important for the ®nal Among the miscellaneous applications of MALDI TOF properties and to conclude whether the random coupling MS in the ®eld of synthetic (macro)molecules, data can hypothesis is valid for the system being studied, i.e., do be found in literature dealing with, a.o., fullerenes, calixarenes, and alkylethoxylate surfactants, see Table 1.

IX. CONCLUSION AND OUTLOOK In a period of six years MALDI TOF MS has gained wide acceptance as a tool for polymer characterization. This speci®c application ®eld can be considered as a spin-off of MALDI activities in the traditional ®eld of peptides and proteins. However, signi®cant differences have been observed: sample preparation is less straightforward due to the coexistence of several distributions, and homo- geneous cocrystallization between matrix and synthetic polymer is not so easily achieved. These seriously hinder automated spectra acquisition of large numbers of FIGURE 17. Detail of the delayed-extraction re¯ectron mass spec- polymer samples. Nevertheless, impressive results in trum of fraction nr. 15 of dipropoxylated bisphenol A/adipic acid/ isophthalic acid copolyester resin after mSEC/MALDI MS. Repro- structure elucidation of polymers are documented in duced from Nielen, 1998, with permission from the American literature: identi®cation of monomers, end groups, and Chemical Society. chemical composition distributions of copolymers in

334 MALDI TOF MS OF SYNTHETIC POLYMERS &

TABLE 5. Chemical composition of oligomers in the mass spectrum of Fig. 17 (Nielen, 1998) [M ‡ Na] ‡ ion at m/z Functionality Structure proposal

1858, 1878, 1898, 1918, 1938 HO...... COOH (DA)4,(DA)3DI, (DA)2(DI)2, DA(DI)3, (DI)4 1880, 1900, 1920, 1940 HO...... COONa (DA)4,(DA)3DI, (DA)2(DI)2, DA(DI)3 2006, 2026, 2046, 2066, 2086 HOOC...... COOH I(DA)4, I(DA)3DI, I(DA)2(DI)2, IDA(DI)3, I(DI)4 2028, 2048, 2068, 2088 HOOC...... COONa I(DA)4, I(DA)3DI, I(DA)2(DI)2, IDA(DI)3 2184, 2204, 2224, 2244, 2264 HO...... OH (DA)4D, (DA)3DID, (DA)2(DI)2D, DA(DI)3D, (DI)4D 2332, 2352, 2372, 2392, 2412 HO...... COOH (DA)4DI, (DA)3(DI)2,(DA)2(DI)3, DA(DI)4, (DI)5 2354, 2374, 2394, 2414 HO...... COONa (DA)4DI, (DA)3(DI)2,(DA)2(DI)3, DA(DI)4

FIGURE 18. Details of the MALDI±TOF mass spectra of block poly( -methylstyrene-co-vinylpyr- idine) samples prepared from different molar ratios of monomers. Reproduced from Danis et al., 1997, with permission from the American Society for Mass Spectrometry. chemically complex mixtures. Some polymers showed to fragmentation in MALDI (in contrast to electrospray be less amenable to MALDI: polyole®nes, which lack ionization). Possibly, the use of IR±MALDI yields better unsaturation and heteroatoms for stable cationization; results for these polymers. chlorinated and brominated polymers, which often show For some years it was thought that MALDI TOF MS photochemical fragmentation in the ion source; and would be a simple and fast method for the determination polyamine dendrimers, which show signi®cant prompt of the molecular weight distribution and the molecular

335 & NIELEN

weight averages Mw,Mn, and Mz. Nowadays, it has been Axelsson J, Hoberg A-M, Waterson C, Myatt P, Shield GL, admitted that MALDI TOF MS is quantitatively much less Varney J, Haddleton DM, Derrick PJ. 1997. Improved reliable than chromatography and that, apart from reproducibility and increased signal intensity in matrix- different manners of data presentation, mass discrimina- assisted laser desorption/ionization as a result of electro- tion occurs during ionization, transmission, and detection spray sample preparation. Rapid Commun Mass Spectrom 11:209. of wide polymer distributions. As a way out, samples having a polydispersity >1.2 are characterized by off-line, Barry JP, Carton WJ, Pesci KM, Anselmo RT, Radtke DR, Evans JV. 1997. Derivatization of low molecular or semi on-line, coupling of SEC and MALDI TOF MS weight polymers for characterization by matrix- (SEC/MALDI). Thus the mass spectrometer is used as a assisted laser desorption/ionization time-of-¯ight mass detector and provides both absolute calibration mass spectrometry. Rapid Commun Mass Spectrom 11: points for the SEC column based on the polymer itself, 437. and the structural data about monomers and end groups. Barton Z, Kemp TJ, Buzy A, Jennings KR. 1995. Mass spectral Many MALDI studies assume uniform cationization characterization of the thermal degradation of poly(pro- ef®ciencies for polymers and copolymers, having differ- pylene oxide) by electrospray and matrix-assisted laser ent end group chemistries and chain lenghts. Most likely, desorption ionization. Polymer 36:4927. this is far from reality and more research efforts are Bartsch H, Strassner M, Hintze U. 1998. Characterization and required in this ®eld. The feasibility of MALDI/CID/PSD identi®cation of ethoxylated surfactants by matrix-assisted of polymers has been demonstrated. Controlled MS/MS laser desorption/ionization mass spectrometry. Tensile Surf fragmentation of copolymers (sequencing) is extremely Det 35:94. important in order to establish the type of copolymer Belu AM, DeSimone JM, Linton RW, Lange GW, Friedman (block-, random-, alternating-); the position of the blocks, RM. 1996. Evaluation of matrix-assisted laser desorption and the block length distribution. Obviously, a lot of work ionization mass spectrometry for polymer characterization. still has to be done since hardly any data are found in J Am Soc Mass Spectrom 7:11. literature. Berkenkamp S, Menzel C, Karas M, Hillenkamp F. 1997. Although outside the scope of this review, alternative Performance of infrared matrix-assisted laser desorption/ mass spectrometric techniques have shown promising ionization mass spectrometry with lasers emitting in the 3 mm wavelength range. Rapid Commun Mass Spectrom results for the characterization of polymers. Among them 11:1399. are MALDI MS/MS studies using a hybrid sector Beyou E, Chaumont P, Chauvin F, Devaux C, Zydowicz N. instrument with an oaTOF as MS2; MALDI FTICR; and 1998. Study of the reaction between nitroxide-terminated SIMS. Of course, the mass range of these alternative polymers and thiuram disul®des. Toward a method of techniques is limiting their application to low molecular functionalization of polymers prepared by nitroxide medi- weight polymers. Recently, ESI TOF MS has become ated free ``living'' radical polymerization. commercially available, featuring an m/z range of 13,000 31:6828 and the inherent ability of multiple-charging. Besides, on- Blair WR, Guttman CM, Wallace WE. 1998. Investigation of line LC/ESI TOF MS is performed very easily (Nielen & chemical and mechanical modi®cations of MALDI sample Buijtenhuijs, 1999); consequently, some overlap with preparation techniques for synthetic polymers. Proc 46th MALDI TOF MS of polymers is foreseen. 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