Anal. Chem. 2004, 76, 335-344
Nonaqueous Capillary Electrophoresis-Mass Spectrometry of Synthetic Polymers Carolina Simo´,† Herve´ Cottet,‡ Willy Vayaboury,‡ Olivia Giani,‡ Matthias Pelzing,§ and Alejandro Cifuentes*,†
Department of Food Analysis, Institute of Industrial Fermentations (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain, Organisation Mole´ culaire, EÄ volution et Mate´ riaux Fluore´ s, UMR CNRS 5073, Universite´ de Montpellier 2, Case courrier 017, Place Euge` ne Bataillon, 34095 Montpellier, France, and Bruker Daltonik GmbH, Permoserstrasse 15, D-04318 Leipzig, Germany
In this work, the separation and characterization of ionizable organic polymers nonsoluble in water is carried out using nonaqueous capillary electrophoresis-ion trap mass spectrometry (NACE-MS). The polymers studied are poly(NE-trifluoroacetyl-L-lysine) (poly(TFA-Lys)) obtained by ring-opening polymerization of the corresponding N-carboxyanhydride. Different parameters (i.e., liquid sheath nature and flow rate, electrospray temperature, and separation buffer composition) are optimized in order to obtain both an adequate CE separation and a high MS signal of the samples under study. The optimum NACEMS separation conditions allow the molecular mass characterization of poly(TFA-Lys) up to a degree of polymerization of 38. NACE-MS provides interesting information on the chemical structure of (i) the polymer end groups and (ii) other final byproducts. The MS spectra obtained by using this CE-MS protocol confirm that the polymerization was initiated by the reaction of n-hexylamine (initiator) on the monomer. CE-MS-MS and CEMS-MS-MS results demonstrate that two different termination reactions occurred during the polymerization process leading to the transformation of the reactive amine end group into a carboxylic or a formyl groups. Byproducts such as 3-hydantoinacetic acid or diketopiperazine were also detected. To our knowledge, this is the first work in which the great possibilities of NACE-MS and NACEMSn for characterizing synthetic polymers are demonstrated. Among the different methodologies available, Capillary electrophoresis (CE) has been demonstrated to be a powerful analytical technique to characterize synthetic polymers. Thus, frontal analysis continuous capillary electrophoresis,1 capillary gel electrophoresis,2 and isotachophoresis3 have been described to * Corresponding author. E-mail:
[email protected]. Fax: 34-91-5644853. † Institute of Industrial Fermentations (CSIC). ‡ Universite´ de Montpellier 2. § Bruker Daltonik GmbH. (1) Staggemeier, B.; Huang, Q. R.; Dubin, P. L.; Morishima, Y.; Sato, T. Anal. Chem. 2000, 72, 255-258. (2) Grosche, O.; Bohrisch, J.; Wendler, U.; Jaeger, W.; Engelhardt, H. J. Chromatogr., A 2000, 894, 105-116. (3) Whitlock, L. R.; Wheeler, L. M. J. Chromatogr. 1986, 368, 125-134. 10.1021/ac034995q CCC: $27.50 Published on Web 12/05/2003
© 2004 American Chemical Society
characterize different types of macromolecules, usually bearing electrical charge. Recently, our group has shown that micellar electrokinetic chromatography (MEKC) can also be used to separate copolymers having a variable number of hydrophobic groups.4 By applying this procedure, the chemical composition of the poly(N-vinylpyrrolidone-co-2-hydroxethyl methacrylate) was determined. Also, we have demonstrated the potential of MEKC for the analysis of copolymerization reactions between ionic and nonionic comonomers.5 Electrically driven size exclusion chromatography or sizeexclusion electrochromatography (SEEC)6-8 has also been developed to separate macromolecules. This technique employs capillary columns (i.d. 30-100 µm) packed with bare silica particles (typically 3-10 µm), together with high dielectric constant solvents such as water, acetonitrile, or dimethylformamide (DMF). Under these conditions, after applying the high voltage, a strong electroosmotic flow is generated and with it the macromolecules move within the capillary. Polymers are separated according to their hydrodynamic volume due to the differential exclusion from the liquid fraction contained in the pores. Unfortunately, the durability of the packed capillaries is still a problem. Moreover, with SEEC, the retention window is smaller than under pressure conditions, and besides, it appears to strongly depend on the ionic strength of the mobile phase. Recently, the use of rigid polymer monolithic capillary columns for the separation of polystyrenes in capillary electrochromatography was shown.9 However, the reported chromatogram shows low selectivity and only polymers with a very large difference in molecular mass could be separated on these columns. An interesting strategy could be to extend the use of nonaqueous CE (NACE), which is now mainly used for the separation of drugs and other small molecules,10 to polymer analysis. Following (4) Gallardo, A.; Lemus, R.; San Roma´n, J.; Cifuentes, A.; Dı´ez-Masa, J. C. Macromolecules 1999, 32, 610-617. (5) Aguilar, M. R.; Gallardo, A.; San Roma´n, J.; Cifuentes A. Macromolecules 2002, 35, 8315-8322. (6) Venema, E.; Kraak, J. C.; Tijssen T.; Poppe, H. Chromatographia 1998, 48, 347-356. (7) Venema, E.; Kraak, J. C.; Tijssen, T.; Poppe, H. J. Chromatogr., A 1999, 837, 3-15. (8) Kok, W. Th.; Stol R.; Tijssen, R. Anal. Chem. 2000, 72, 468A-476A. (9) Peters, E. C.; Petro, M.; Svec, F.; Frechet, J. M. J. Anal. Chem. 1998, 70, 2296-2302.
Analytical Chemistry, Vol. 76, No. 2, January 15, 2004 335
Figure 1. Synthesis of poly(N-TFA-L-lysine) via ring-opening polymerization of NCA in the presence of n-hexylamine as initiator. R ) CH2CH2-CH2-CH2-NH-CO-CF3.
this idea, NACE has been applied to the separation of polyethers11 or N-phenylaniline oligomers (i.e., highly hydrophobic compounds) with polymerization degrees of 2, 4, 6, and 8.12 In a previous work, we showed the potential of NACE for the separation of synthetic polymers taking poly(N-trifluoroacetyl-Llysine) (poly(TFA-Lys)) as model compound.13 A general limitation of all CE methods described above is the necessity to account with standards in order to conveniently identify the polymers under study. The use of MS detection could help to overcome this drawback while it should provide interesting information about the chemical structure of the compounds under study and, as a result, the most probable polymerization reactions involved. However, despite these advantages, to our knowledge, the potential of neither CE-MS nor NACE-MS to characterize synthetic polymers has been addressed. The goal of this work is, therefore, to demonstrate for the first time the possibilities of NACE-MS to separate and characterize synthetic macromolecules, using as target poly(TFA-Lys), a nonwater-soluble polymer synthesized in our laboratory. These synthetic polymers are biomaterials of high interest due to their biocompatibility, their potential to mimic proteins, and their large range of applications such as drug delivery and tissue engineering.14 The development and application of the NACE-MS method is demonstrated to provide the following: (a) characterization of the main reaction products; (b) characterization of other minor compounds; and (c) knowledge about how both the polymerization and termination reactions take place. These results demonstrate the large possibilities of CE-MS within the polymer characterization area. (10) Riekkola, M. L.; Jussila, M.; Porras, S.; Valko, I. E. J. Chromatogr., A 2000, 892, 155-170. (11) Okada, T.; J. Chromatogr., A 1995, 695, 309-317. (12) Cottet, H.; Struijk, M. P.; Van Dongen, J. L. J.; Claessens, A.; Cramers, C. A. J. Chromatogr., A 2001, 915, 241-251. (13) Cottet, H.; Vayaboury, W.; Kirby, D.; Giani, O.; Taillades, J.; Schue´, F. Anal. Chem., in press. (14) Deming, T. J. Adv. Drug Delivery Rev. 2002, 54, 1145-1155.
336 Analytical Chemistry, Vol. 76, No. 2, January 15, 2004
EXPERIMENTAL SECTION Polymer Synthesis. Two poly(N-trifluoroacetyl-L-lysine) samples (polymer 1 and polymer 2) were synthesized by ringopening polymerization of N-trifluoroacetyl-L-lysine N-carboxyanhydride (Lys NCA) with n-hexylamine in DMF as described in more detail elsewhere (see Figure 1 for the propagation reaction).13 Briefly, the Lys NCA was prepared either by nitrosation of NR-carbamoyl-N-trifluoroacetyl-L-lysine (Lys NCAA) using the NO/O2 gas mixture15 (polymer 1) or by phosgenation of an R-amino acid16 (polymer 2). The molar ratio of monomer to initiator was 10 for both polymers. After 48 h of reaction at room temperature, the solvent was evaporated under vacuum at 100 °C until obtaining a solid. The polymer was used without further purification. Samples and Reagents. Polymer samples for CE-MS analysis were prepared as follows: 10 mg of the dry polymer was dissolved in 1 mL of pure MeOH. The sample solution was then diluted 2-fold by adding 1 mL of the more concentrated buffer separation. This sample solution at 5 g/L (in 50/50 v/v, MeOH/ electrolye) was filtered on a 0.45-µm PTFE membrane before injection. Methanol, acetonitrile, DMF, sodium dodecyl sulfate (SDS), acetic acid, formic acid, ammonium hydroxide, ammonium acetate, and sodium hydroxide were from Merck (Darmstad, Germany) and used without further purification in the various running buffers. Distilled water was deionized with a Milli Q system (Millipore, Bedford, MA). Separation Buffers. Two different separation buffers were tested. The first one (more concentrated) was prepared as follows: 6 g of acetic acid, 0.154 g of ammonium acetate, and 75 mL of a (87.5/12.5 v/v) MeOH/MeCN mixture were introduced into a 100-mL flask. After complete dissolution, the flask was filled to 100 mL with MeOH/MeCN (87.5/12.5 v/v). The second one was prepared using the same quantities of acetic acid and (15) Collet, H.; Bied, C.; Mion, L.; Taillades, J.; Commeyras, A. Tetrahedron 1996, 37, 9043-9046. (16) Cornille, F.; Copier, J. L.; Senet, J. P.; Robin, Y. U.S. Patent 6479665 B2, 2002.
ammonium acetate but dissolving up to 200 mL with MeOH/ MeCN (87.5/12.5 v/v). CE-MS Conditions. The analyses were carried out in a P/ACE 5500 (Beckman Instruments, Fullerton, CA) CE apparatus, equipped with an UV-visible detector working at 200 nm and coupled using an orthogonal electrospray interface (ESI, model G1607A from Agilent Technologies, Palo Alto, CA) to the MS detector (an Esquire 2000 ion trap mass spectrometer from Bruker Daltonik GmbH, Bremen, Germany). Bare fused-silica capillary with 50-µm i.d. was purchased from Composite Metal Services (Worcester, England). The detection length to the UV detector was 20 cm, and 87 cm was the total length (corresponding to the MS detection length). Injections were made at the anodic end using N2 pressure of 0.5 psi for 15 s (1 psi ) 6894.76 Pa). All the separations were done using 25 kV as running voltage. The CE instrument was controlled by a PC running the System GOLD software from Beckman. Electrical contact at the capillary outlet was established via a sheath liquid delivered by a 74900-00-05 Cole Palmer syringe pump (Vernon Hills, IL). The mass spectrometer was operated in the positive or in the negative ion mode as indicated in each experiment. The spectrometer was scanned over 400-2200 m/z range at 13 000 u/s. For the connection between the CE system and the electrospray ion source of the mass spectrometer, the outlet of the separation capillary was fitted into the electrospray needle of the ion source and a flow of conductive sheath liquid established electrical contact between capillary effluent and dissolution for electrospray needle. MS operating conditions were optimized by adjusting the needle-counter electrode distance and applying electrospray potentials while the polymer solution was injected by direct infusion using N2 pressure at 0.5 psi in the CEESI-MS system. The pneumatic-assisted ESI was performed at a nebulizer pressure of 4 psi (N2). Nitrogen was used as dry gas at a flow rate of 3 L/min at 200 °C. MS-MS and MS-MS-MS experiments were performed with an isolation width of 4 m/z and a fragmentation amplitude of 5.0 V. The instrument was controlled by a PC running the Esquire software 5.1 from Bruker Daltonics. As described by Cottet et al.,13 the synthetic polymers used in this work have a strong tendency to adsorb onto the capillary wall; as a result, an exhaustive washing strategy had to be used in order to obtain repeatable results from run to run. A denaturating solution was prepared by dissolving 300 mg of NaH2PO4, 30 g of urea, and 1.728 g of SDS in a 100-mL flask with ultrapure water; the pH of the washing solution was then adjusted up to 7.0 with pulverulent NaOH. The washing procedure consisted of flushing the capillary, prior to each run, with different solutions as follows (all flushes done using 20 psi of pressure): 1 min (flush) H2O, 2 min (flush) washing solution, 15 min (voltage) 15 kV (buffer inlet, washing solution); 2 min (flush) washing solution, 1 min (flush) H2O, 3 min (flush) buffer separation, 5 min (voltage) 15 kV electrolyte (in order to equilibrate the capillary), 1 min (flush) electrolyte (this last flush was found to give better results in the baseline stability during the run). Before first use, uncoated capillaries were conditioned using a 20-min rinse with 0.1 M NaOH followed by a water rinse for other 20 min. At the end of the day, the capillary was rinsed as follows: 1 min (flush) H2O, 2 min (flush) wash solution, 15 min
(voltage, 15 kV), 2 min (flush) wash solution, 1 min (flush) H2O, and 0.5 min (flush) air. RESULTS AND DISCUSSION The synthesis of poly(N-TFA-L-lysine) in DMF via ring-opening polymerization gives rise to non-water-soluble polymers. The reaction of propagation using a primary amine (n-hexylamine) as initiator is depicted in Figure 1.17 Ideally, this polymerization should be a living polymerization- however, different side reactions can interfere with reactions of Figure 1 leading to byproducts or polymers of different chemical structure. Among others, these side reactions could be reactions of termination, other initiation steps, or other propagation mechanisms different from those described in Figure 1. Different side reactions in reaction conditions similar to ours were proposed in the past. However, there was generally no direct confirmation of the structures. Knowledge of the chemical structure of the polymers, especially the nature of the end groups, as well as the chemical structure of small molecules (byproducts) present in the polymer sample is fundamental for a good understanding of the polymerization process. The development of a NACE-MS method able to separate and characterize such compounds could provide such crucial information. CE-ESI-MS Optimization. To develop the NACE-MS method to characterize the polymers under study, the liquid sheath nature, the liquid sheath flow rate, the electrospray temperature, and the separation buffer composition were optimized. These parameters were adjusted for simultaneously obtaining a high MS signal/noise ratio and a good peak resolution during the NACE-MS analysis. The results obtained during optimization of the liquid sheath nature are shown in Figure 2. As can be deduced from electropherograms A-C, neither the addition of buffer (5%) nor the addition of acids (0.01% formic acid or 0.01% acetic acid) to the sheath liquid improved the signal/noise ratio. Interestingly, the use of the sheath liquid without any additive (i.e., methanol/ acetonitrile, 87.5:12.5, v/v) provided the best results, as can be seen by comparing Figure 2D with Figure 2A-C. This is probably due to the fact that the ionization of the polymers is already favored by the acidic buffer coming from the separation capillary. Therefore, the use of a higher acidic content in the sheath liquid only brings an increase of chemical noise in the MS, as can be deduced from Figure 2A-C. Hence, the conditions of Figure 2D were then chosen for the next experiments. Moreover, the complexity of the sample under study can be also deduced from the multiple peaks shown in the electropherogram of Figure 2D. The identification of the different peaks in Figure 2D will be discussed further. Once the liquid sheath nature was optimized, the liquid sheath flow rate and the electrospray temperature were studied. The signal/noise ratio for the highest peak (namely, peak 5; see below for identification) detected in the total ion electropherogram (TIE) and in the extracted ion electropherogram (EIE) was followed in this study. The results of liquid sheath flow rate optimization are given in Figure 3A, and the results on the optimization of ESI temperature are shown in Figure 3B. As can be seen by comparing Figure 3A and B, the effect of the flow rate on the signal/noise (17) Kricheldorf, H. R. R-Amino acid-N-Carboxy-Anhydrides and Related Heterocycles; Springer-Verlag: New York, 1987.
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Figure 2. Effect of the sheath liquid composition on the CE-ESI-MS separation. Sheath liquid composition: (A) 5% concentrated buffer in methanol/acetonitrile, 87.5:12.5 (v/v); (B) 0.01% acetic acid in methanol/acetonitrile, 87.5:12.5 (v/v); (C) 0.01% formic acid in methanol/acetonitrile, 87.5:12.5 (v/v); (D) methanol/acetonitrile, 87.5:12.5 (v/v). CE separation buffer: 1 M acetic acid, 20 mM ammonium acetate in methanol/acetonitrile, 87.5:12.5 (v/v). Run voltage: +25 kV. Injection: 15 s at 0.5 psi. Bare silica capillary: 87 cm of total (and detection) length with 50)µm i.d. ESI-MS conditions: nebulizer at 3 psi; dry gas at 4 L/min; dry temperature: 100 °C. Sheath liquid flow: 0.24 mL/h (4 µL/min).
Figure 3. Effect of the sheath liquid flow (A) and electrospray temperature (B) on the MS S/N ratio measured for both the TIE (triangles) and the EIE (squares) modes. Sheath liquid composition: methanol/acetonitrile, 87.5:12.5 (v/v). CE separation buffer: 1 M acetic acid, 20 mM ammonium acetate in methanol/acetonitrile, 87.5:12.5 (v/v). Run voltage: +25 kV. Injection: 15 s at 0.5 psi. Bare silica capillary: 87 cm of total (and detection) length with 50-µm i.d.. ESIMS conditions: nebulizer at 3 psi; dry gas at 4 L/min; dry temperature: 100 °C in Figure A. Sheath liquid flow: 0.24 mL/h (4 µL/min) in Figure B.
ratio is more pronounced than the effect of the ESI temperature in the range studied for these compounds. The minimum variation observed for the ESI temperature effect seems to indicate that the transfer of analytes from liquid phase into the gas phase already takes place adequately at the minimum temperature tested (i.e., 100 °C), which is also favored by the nonaqueous nature of the CE medium used. On the other hand, the sheath liquid flow rate has a marked effect on the ionization of these compounds, as can be deduced from Figure 3A, where a maximum for both the TIE and the EIE signal is obtained at 0.3 mL/h (i.e., 5 µL/ min). Different effects taking place simultaneously can explain this maximum value obtained. Thus, it is necessary to achieve a 338
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minimum flow rate in order to keep the spray stable; this minimum probably is not achieved at flow rates around 0.1-0.2 mL/h, which can explain the low values obtained below 0.2 mL/h. On the other hand, at flow rates higher than 0.35 mL/h, a decrease in the signal/noise ratio is also observed. This decrease is due to both the ionization efficiency in the ESI drop decreases at higher flow rates and the dilution of analytes induced by the higher sheath flow. Therefore, the best results for both the EIE and TIE signal/ noise ratio were obtained using a flow rate of 0.3 mL/h (Figure 3A) and a ESI temperature of 300 °C (Figure 3B), and these values were used for the next experiments. It is also noteworthy from Figure 3, that the better sensitivity (or signal/noise values) can be achieved working with the EIE compared with the TIE. Thus, in some cases, signal/noise ratios up to 30 times better could be obtained using EIE instead of TIE for these polymers. On the basis of the results obtained in Figure 2, in which the best signal/noise ratio was obtained for the minimum ionic strength into the sheath liquid, two different nonaqueous separation buffers with different ionic strengths were next tested, namely: (a) 1 M acetic acid, 20 mM ammonium acetate in methanol/acetonitrile, 87.5:12.5 (v/v) and (b) 0.5 M acetic acid, 10 mM ammonium acetate in methanol/acetonitrile, 87.5:12.5 (v/ v). Although a very slight improvement was observed in the signal/noise ratio by using the more diluted buffer, the separation resolution with this buffer was much worse due to the increase in electroosmotic flow as already mentioned in our previous work.13 Therefore, the more concentrated buffer was selected (i.e., 1 M acetic acid, 20 mM ammonium acetate in methanol/ acetonitrile, 87.5:12.5 (v/v)) for all the experiments. Poly(N-TFA-L-lysine) Separation of CE-MS: Optimum Conditions. Once the liquid sheath nature, the electrospray temperature, the liquid sheath flow rate, and the separation buffer composition were optimized, the next optimum conditions were achieved: running buffer made of 1 M acetic acid, 20 mM ammonium acetate in methanol/acetonitrile, 87.5:12.5 (v/v); ESI temperature of 300 °C, together with a sheath liquid composed of methanol/acetonitrile, 87.5:12.5 (v/v) flowing at 0.3 mL/h (5 µL/min). Using these conditions CE-MS separations such as the one shown in Figure 4 were typically obtained. In our previous work,13 the tendency of poly(TFA-Lys) macromolecules to adsorb onto the silica wall was described. To overcome this limitation, a
Figure 4. CE-MS TIEs under optimum CE-MS conditions; MS and MS-MS spectra of some compounds. Sheath liquid composition: methanol/ acetonitrile, 87.5:12.5 (v/v). CE separation buffer: 1 M acetic acid, 20 mM ammonium acetate in methanol/acetonitrile, 87.5:12.5 (v/v). Run voltage: +25 kV. Injection: 15 s at 0.5 psi. Bare silica capillary: 87 cm of total (and detection) length with 50-µm i.d. ESI-MS conditions: nebulizer at 3 psi; dry gas at 4 L/min; dry temperature: 300 °C. Sheath liquid flow: 0.3 mL/h (5 µL/min).
specific washing procedure based on electrophoretic desorption between each injection had to be developed, providing high efficiency and good repeatability. Concerning the CE-MS experiments, a similar washing routine had to be developed. The routine developed is described at the end of the section CE-MS Conditions, and it allows the obtaining of adequate reproducibility for migration times with RSD values lower than 3% for the same day (n ) 5) and efficiency values up to 30 000 plates/m for the different polymers detected (see CE-MS TIE in Figure 4). Characterization of the Polymerization Products by CEMS and CE-MSn. Once optimum analytical conditions were achieved, an exhaustive study of the reaction products was carried out by CE-MS. First, the peaks that migrate before the electroosmotic flow (EOF) marker were studied. These compounds,
carrying a global positive charge and migrating between 18 and 36 min are numbered in the CE-MS TIE of Figure 4 according to their degree of polymerization. The peak assignment was confirmed by the MS spectra as exemplified by some typical MS and MS-MS spectra given in Figure 4. As can be seen, good and informative MS spectra could be obtained even inside the broad peak migrating at 35 min (peak marked as 13-38 in the CE-MS TIE). For example, from the MS spectra of peak 3 and peak 5 (see Figure 4), two main ions were obtained and they are assigned as [M + H+] and [M + Na+], since their mass difference is 22.2 Da. To corroborate this, a small quantity (0.01%, w/v) of lithium acetate was added to the sheath liquid and the [M + 6.9]+ was then detected, which confirms the assignment of the MS spectra. These mass values together with the MS-MS fragmentation Analytical Chemistry, Vol. 76, No. 2, January 15, 2004
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Figure 5. Proposed structure and fragmentation pattern for living polymer.
Figure 6. CE-MS TIE and EIE of the living polymers (i.e., poly(N-TFA-L-lysine), from n ) 2 to n ) 38 monomers) under optimum CE-MS conditions. All the conditions as in Figure 4.
allowed us to identify the main reaction product as poly(N-TFAL-lysine) (see Figure 5). Thus, as can be seen from the MS and MS-MS spectra of Figure 4 and the fragmentation scheme of Figure 5, fragmentation of the molecule has released the hexylamine group and different monomer units with 224.2 Da of mass. Also, in all the MS-MS spectra of these compounds, a peak at m/z ) 449.4 is observed, corresponding to the dimer with an amine terminal group indicated in Figure 5. These results are in good agreement with the reaction scheme indicated above in Figure 1. Moreover, poly(N-TFA-L-lysine) polymers with monomer units up to 38 could be detected using our CE-MS procedure. This is demonstrated in Figure 6, where the EIE of the polymers from n ) 2 to n ) 38 are given, corresponding to a molecular mass ranging from 549.5 to 8621.6 Da for monocharged to tetracharged ions. Table 1 shows the comparison between the experimental masses obtained for these polymers and the theoretically expected. Table 1 also provides the charge of the detected ion according to its mass. As can be seen, good agreement is obtained between both types of values, corroborating the possibility of CE-MS to analyze and characterize synthetic polymers. 340 Analytical Chemistry, Vol. 76, No. 2, January 15, 2004
From peak 2 to peak 38 in Figure 4, we were not able to detect any other masses that could correspond to similar polymers initiated by molecules other than n-hexylamine. Moreover, the number-average molecular mass (2300 g/mol) calculated by 1H NMR, assuming that each polymer chain was initiated by nhexylamine, is in agreement with the ratio monomer to initiator of 10. For these two reasons, a possible second reaction path involving the initiation with TFA-L-lysine instead of n-hexylamine was discarded. It can be also deduced from the CE-MS TIE of Figure 4 that these separation conditions can discriminate the living polymers (peak 2 to peak 38) from dead polymers migrating after the EOF. The reaction of termination given in Figure 7 was proposed in the literature in experimental conditions close to ours.17-19 However, direct confirmation of the dead polymer structure has never been reported. NACE-MSn offers the possibility of obtaining some more information about the termination of this polymerization. It can be done through the characterization of the dead (18) Sela, M.; Berger, A. J. Am. Chem. Soc. 1955, 77, 1893-1899. (19) Lundberg, R. D.; Doty, P. J. Am. Chem. Soc. 1957, 79, 3961-3972.
Figure 7. Classical ending mechanism during the synthesis of poly(N-TFA-L-lysine) via ring-opening polymerization of NCA; R ) CH2CH2-CH2-CH2-NH-CO-CF3.
Table 1. CE-MS Experimental Mass Values vs Theoretical Values for the Living Poly(NE-trifluoroacetyl-L-lysine) Macromolecules Composed of Different Monomer Units (n Value) n
theoretical mass (Da)
experimetal mass (Da)
2
549.6
3 4 5 6 7 8 9
773.7 997.9 1222.1 1446.3 1670.5 1894.7 2118.8
10 11 12 13 14 15 16 17 18
2343.0 2567.2 2791.4 3015.6 3239.8 3463.9 3688.1 3912.3 4136.5
19 20 21 22 23 24 25 26 27 28 29
4360.7 4584.9 4809.0 5033.2 5257.4 5481.6 5705.8 5930.0 6154.1 6378.3 6602.5
30 31 32 33 34 35 36 37 38
6826.7 7050.9 7275.1 7499.2 7723.4 7947.6 8171.8 8396.0 8620.2
549.4 [from here ions detected as (M + H)+] 773.5 997.7 1221.9 1446.0 1670.2 1894.2 2118.2 [from here ions detected as (M + 2H)2+] 2343.4 2567.4 2791.6 3015.8 3240.0 3464.2 3688.6 3912.4 4136.7 [from here ions detected as (M + 3H)3+] 4361.4 4585.8 4809.6 5033.4 5258.4 5482.2 5706.9 5931.0 6155.1 6378.9 6603.6 [from here ions detected as (M + 4H)4+] 6827.6 7052.4 7276.4 7501.2 7724.8 7948.8 8173.6 8396.0 8620.8
polymers migrating after the EOF (peaks A1 and A2 in Figure 8). According to the mechanism given in Figure 7, the amine end group of the living polymer is transformed into a carboxylic acid leading to a dead polymer unable to further react with the monomer. Considering the structure of this dead polymer, it is expected to obtain macromolecules with molecular weights of 593.6 (n ) 1); 817.7 (n ) 2); 1041.9 (n ) 3); ... (369.5 + (n × 224.15)). These values were used in the EIE to seek masses
corresponding to such polymers into the electropherogram. Very small peaks could be detected after the EOF time (peak A2 ∼40 min) corresponding to n from 3 to 8, which seems to confirm that these compounds are formed and that the ending-mechanism of Figure 7 takes place. This dead polymer is partially deprotonated (slightly anionic) in the electrolyte and thus detected after the EOF. However, under our analytical conditions, some other different compounds were also detected, what seems to indicate that other side reactions are also playing a role. Thus, the compounds migrating inside the broad peak A1 just after the electroosmotic flow (i.e., between 36 and 38 min, Figure 8) were studied. The MS spectra from peak A1 gave rise to the EIEs shown in Figure 8. As can be seen, [M + Na]+ compounds with mass values ranging from 824.5 to 2170 Da were detected. As above, this mass assignment was ratified by using lithium acetate in the sheath liquid. It is also necessary to mention here that the maximum mass values detected for peak A1 (and also for peak A2) are probably limited as well by the maximum scan range that the ion trap can work with (i.e., up to 2200 Da for monocharged molecules). Despite this drawback, the important advantages provided by CE-MSn in obtaining both the exact mass of the detectable macromolecules and information about their structure has to be kept in mind. Once the molecular mass values for the different dead polymers detected in peak A1 were obtained (namely, from 801.5 to 2147.0 Da), MS-MS analyses from the protonated ions (i.e., [M + H+]) were generated, giving rise to the MS-MS spectra seen in Figure 8. As can be easily deduced by comparing these MS-MS spectra with the MS-MS spectrum in Figure 4, these compounds seem to be also polymers composed of monomer units with 224.2 Da of mass. Moreover, the hexylamine group is also in the structure of these dead polymers as can be deduced from the observed 101 mass lost. However, the rest of the structure cannot be clearly deduced either from the MS-MS spectra obtained or from the reaction scheme of Figure 7 (i.e., giving rise to an acidic group). Therefore, a deeper analysis of the fragmentation pattern was carried out by MS-MS-MS. From the MSMS spectra of these dead polymers it could be observed that after fragmentation a small fragment ion of m/z ) 477 always come out (see, for example, the two MS-MS spectra in Figure 8); therefore, this ion was selected for carrying out the MS-MSMS experiments. The MS-MS-MS spectra obtained from the ion at m/z ) 477 is given in Figure 8, in which fragments at m/z Analytical Chemistry, Vol. 76, No. 2, January 15, 2004
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Figure 8. CE-MS TIE, MS, MS-MS, and MS-MS-MS spectra of some compounds; and EIE of the detected dead polymers (from n ) 2 to n ) 8, peak A1) under optimum CE-MS conditions. All the conditions as in Figure 4.
Figure 9. Proposed structure and fragmentation pattern for dead polymer detected in peak A1.
equal to 449.2, 448.2, 253.1, 225.0, and 224.1 coul be observed. Putting all the above information together, the structure given in Figure 9 is proposed. According to this structure, the “dead polymer” detected in peak A1 is, like the living polymer, composed of the same monomer units and hexylamine. The main difference is the neutral N-formyl end group, which can also explain that these compounds migrate practically at the same analysis time as the electroosmotic flow (i.e., with an electrophoretic mobility nearly zero). The separation of this dead polymer from the EOF peak was probably due to an interaction of the polymer with the capillary wall, which induces a slight retardation in the migration time of the polymer. The N-formyl end group is believed to come 342 Analytical Chemistry, Vol. 76, No. 2, January 15, 2004
from the reaction scheme given in Figure 10, where the NCA should act as catalyst in the reaction. To our knowledge, this reaction between the DMF and the living polymer has never been reported. Nevertheless, the activation of the DMF by an electrophilic molecule inducing a reaction with an amine was already reported.20 The N-formylation of aliphatic primary amines with DMF promoted by 2,3-dihydro-1,4-phthalazinedione was reported by Iwata and Kuzuhara.21 This latter reaction gives some credit (20) Stierandova, A.; Safar, P.; Proceedings of the 23th European Peptide Symposium, September 4-10, 1994, Braga, Portugal, Maia, H. L. S., Ed.; ESCOM Science Publisher: Leiden, 1995; pp 183-184. (21) Iwata, M.; Kuzuhara, H. Chem. Lett. 1989, 2029-2030.
Figure 10. Proposed ending mechanism explaining the synthesis of a dead polymer with N-formyl end group. NCA; R ) CH2-CH2-CH2CH2-NH-CO-CF3.
Figure 11. CE-MS TIE, MS spectrum of peak B and chemical structure of 3-hydantoinacetic acid (obtained from the MS-MS spectrum of the 493.2 m/z ion). All the conditions as in Figure 4.
to our interpretation since it is similar to that proposed in Figure 10 except for the nature of the catalyst. From the reaction depicted in Figure 10, a dimethylamine molecule is released in the reaction flask. This secondary amine should be able to initiate the polymerization, giving rise to another living polymer with a dimethylamine group for one end and an amine group for the other. However, under our separation conditions, this compound could not be detected, probably because its quantity is very low compared to the main one (i.e., poly(N-TFA-L-lysine) initiated with n-hexylamine) or because the sensitivity of our system was not
high enough due to supression of the ionization induced by the comigrating substances. As can be seen in Figure 11, the CE-MS TIE also showed a last peak (called peak B). According to its electrophoretic migration, the compound corresponding to peak B must bear a global negative electrical charge. The two main ions detected in the MS spectrum for peak B corresponds to compounds with mass values equal to 492.2 and 514.3 Da; this result was corroborated by two independent experiments, namely, using ESI in the negative mode and adding lithium acetate to the sheath liquid. Analytical Chemistry, Vol. 76, No. 2, January 15, 2004
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Figure 12. Reactions explaining the formation of 3-hydantoinacetic acid and diketopiperazin.
As can be seen in the MS spectrum of Figure 11, the compound with a molecular mass of 514.3 Da gives rise to different complexes with sodium (i.e., [M + Na]+, [2M +Na]+, and [3M + Na]+) that can be formed via ionic interactions under electrospray conditions in positive mode.22,23 From the MS-MS experiments, it was clear that this compound bears an acidic group; however, a complete molecular structure for this compound could not be clearly identified. On the other hand, MS-MS experiments carried out with the 493.2 m/z ion gave rise to mass fragments of 475, 447, 268, 250, 243, and 180 m/z. In this case, it was possible to arrive at a molecular structure for this compound. Thus, the compound that better matches all the results (i.e., electrophoretic behavior, mass value, and fragmentation pattern) is given in Figure 11, and it seems to correspond to 3-hydantoinacetic acid. The presence of 3-hydantoinacetic acid in the sample can be explained by reaction a in Figure 12 and proves that a certain quantity of deprotonated monomer is present in the reactor. The deprotonated monomer, also called activated monomer, is usually present when the initiator is a strong base (secondary or tertiary amine, for example) able to capture the proton on the NCA. In the presence of such a deprotonated monomer, a polymerization according to the mechanism of the activated monomer can occur forming a polymer bearing both NH2 and COOH end groups (i.e., without incorporating any initiator).17 However, this polymer could not be detected, probably because, as above, the quantity is too low or due to the supressing effects of the comigrating substances. Interestingly, the extracted ion electropherogram at m/z 448 shows a very small peak corresponding to diketopiperazin (a neutral compound with Mw ) 448.4); this compound was also detected in the EOF peak as both the [M + Na]+ and [M + Li]+ complex, which further corroborates the existence of 3-hydantoinacetic acid since from a chemical point of view these two compounds are coming from the same molecules (see routes a and b of Figure 12).
The two polymers (polymer 1 and polymer 2) both synthesized in the same conditions but with two different modes of preparation of the monomer, as described in the Experimental Section, were then characterized using the aforementioned NACE-MS optimal conditions. Their respective CE-MS TIEs were similar (data not shown). The respective mass spectra obtained for each sample indicate that there are not appreciable differences in the polymerization products coming from the use of NCA obtained by phosgenation of R-amino acid or by nitrosation of carbamoylamino acid.
(22) Van Stipdonk, M. J.; Ince, M. P.; Perera, B. A.; Martin, J. A. Rapid Commun. Mass Spectrom. 2002, 16, 355-363. (23) Koch, K. J.; Aggerholm, T.; Nanita, S. C.; Cooks, R. G. J. Mass Spectrom. 2002, 37, 676-686.
Received for review August 26, 2003. Accepted November 4, 2003.
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CONCLUSIONS In this work, the great potential of CE-MSn to characterize synthetic polymers has been demonstrated for the first time. From our work, the molecular structure of different polymers made up to 38 monomers of (N-trifluoroacetyl-L-lysine) could be confirmed. Moreover, in this CE-MSn work, some new and unexpected compounds were also determined for the first time such as polymers with a N-formyl end group coming from the reaction of the living polymer with the solvent (DMF). The detection of other byproducts (3-hydantoinacetic acid and diketopiperazin) and the determination of their structure were also very informative on the polymerization mechanism. This work clearly shows that coupling the high resolution power of NACE with a MS detector led to a powerful analytical tool very useful for studying complex mixtures of synthetic macromolecules. ACKNOWLEDGMENT The authors thank Dr. Gerard Bruin and Novartis Pharma AG (Basel, Switzerland) for the gift of the P/ACE 5500 instrument used in this work. C.S. thanks the Consejerı´a de Educacio´n y Cultura (Comunidad de Madrid) for a fellowship. The authors also thank CICYT (AGL2002-046210-C02-02) for financial support.
AC034995Q
