Anal. Chem. 2003, 75, 5539-5543
Development of Water-Based Liquid Chromatography at the Critical Condition S. L. Phillips, L. Ding, M. Stegemiller, and S. V. Olesik*
Department of Chemistry, The Ohio State University, Columbus, Ohio 43210
Liquid chromatography at the critical condition (LCCC) is a chromatographic technique that allows for the isolation of one area of the polymer matrix so that other areas of the polymer may be probed with size-exclusion or adsorptive chromatographic modes. This technique has been successfully applied to the analysis of functionality distributions in functionalized oligomers and to polymer distributions within copolymers. Herein, the critical conditions of two polar polymers, poly(acrylic acid) and polystyrene sulfonate, are determined. These conditions were identified by varying buffer concentration, organic modifier within the mobile phase, or both. At the critical condition of poly(acrylic acid), the retention characteristics of a copolymer of acrylic acid and vinyl pyrrolidinone were determined. This extension to water-based mobile phase conditions will substantially broaden the possible applications of LCCC. Water-soluble poly(alkenoic acid)s, such as poly(acrylic acid), have been incorporated into polymer systems used in applications ranging from dental and pharmaceutical to food because of their hydrophilic and nontoxic nature.1 The mechanical properties of these polymer systems have been well-documented, but information about reaction byproducts and molecular weight are not completely established.2 Variations in polymer structure can include several species incorporated into the backbone, side-chain, and end-groups of the polymer system. The three-dimensional arrangement of these structures further adds to the heterogeneity available to a polymer system. Characterization of polymer systems has been proven to be a nontrivial task because of the range of concentrations of some species within the polymer system. In addition, the low solubility of high molecular weight species can hinder polymer analysis, as well. Traditional modes of analysis used for the characterization of small molecules, such as IR and NMR, are not always amenable to macromolecular systems. Although functional groups are sometimes identifiable using these techniques, the distribution of these groups within the polymer matrix often cannot be ascertained. Liquid chromatography at the critical condition (LCCC) is a technique that can readily determine trace component analysis in polymer systems, functionality distributions in telchelic poly* To whom correspondence should be addressed. E-mail:
[email protected]. (1) Culbertson, B. M. Prog. Polym. Sci. 2001, 26, 577-604. (2) Huang, Y.; Schricker, S. R.; Culbertson, B. M.; Olesik, S. V. J. Macromol. Sci., Pure Appl. Chem. 2002, A39, 27-38. 10.1021/ac034456l CCC: $25.00 Published on Web 09/13/2003
© 2003 American Chemical Society
mers, and polymer molecular weight distributions in copolymers. In LCCC, the critical condition (CC) of a portion of a polymer is determined. The critical condition corresponds to the chromatographic conditions under which the Gibbs free energy of transfer for that portion of the polymer is zero (i.e., the enthalpy term and the entropy term in the Gibbs equation exactly compensate each other). For example, at the critical condition of polystyrene, oligomers of varying molecular weights of polystyrene elute at the same retention time. However, if a functionalized polystyrene polymer is injected at the CC for polystyrene, a separation due entirely to functionality distribution results (i.e., the polystyrene backbone is “chromatographically invisible”). This research group previously demonstrated that enhanced-fluidity liquid mobile phases could be used to improve the approach to, identification of, and maintenance of the critical condition for a number of polymers.3 These attributes are due to the precise control of solvent strength that is possible in enhanced-fluidity liquids through pressure and temperature variation.4 Others have been able to also identify the critical conditions for polyisoprene,5,6 methacryloyl-terminated polyoxyethylene,7 poly(methyl methacrylate), poly(decyl methacrylate),8 poly(ethylene glycol),9 and polyoxyethylene,10 which has led to the characterization of a multitude of polymeric architectures. To our knowledge, LCCC using primarily water-soluble polymers has not been attempted to date. The typical method development involved in the analysis of a polymer system at the critical point of adsorption involves the initial establishment of an appropriate size-exclusion chromatographic system. Column surface and mobile phase conditions are chosen so that the polymer undergoes a separation based on the hydrodynamic volume of the polymer with essentially no interaction with the surface of the separation media. The critical condition is then established by adding an appropriate amount of “nonsolvent” to the chromatographic system so that the polymer no longer experiences a separation based on hydrodynamic volume and begins to interact with the stationary phase surface. The critical condition elution (3) Souvignet, I.; Olesik, S. V. Anal. Chem. 1997, 69, 66-71. (4) Souvignet, I.; Olesik, S. V. J. Phys. Chem. 1995, 99, 16800-16803. (5) Lee, H.; Chang, T.; Lee, D.; Shim, M. S.; Ji H.; Ninidez, W. K.; Mays, J. W. Anal. Chem. 2001, 73, 1726-1732. (6) Czichocki, G.; Heger, R.; Goedel, W. A.; Much, H. J. Chromatogr., A 1997, 791, 350-356. (7) Murgasova, R.; Capek, I.; Lathova, E.; Berek, D.; Florian, S. Eur. Polym. J. 1998, 34, 659-663. (8) Pasch, H.; Augenstein, M. Makromol. Chem. 1993, 194, 2533-2541. (9) Brun, Y.; J. Liq. Chromatogr. Relat. Technol. 1999, 22, 3027-3065. (10) Trathnigg, B.; Kollroser, M.; Gorbunov, A.; Skvortsov, A. J. Chromatogr., A 1997, 761, 21-34.
Analytical Chemistry, Vol. 75, No. 20, October 15, 2003 5539
volume lies at the retention volume corresponding to the total volume of the column where essentially no separation is expected to occur. The practice of water-based SEC of polar analytes is often complicated by interactions of the polymer with the separation surface. These interactions include ion exclusion and hydrophobic interactions with the stationary phase surface, thereby perturbing the intended separation based on the hydrodynamic volume.11 To reduce these non-size-exclusion effects, the mobile phase and stationary phase must be carefully selected. Hydrophobic interactions are often alleviated in a water-based SEC system with the addition of a hydrophobic solvent, such as methanol.12,13 Ionic exclusion effects are eliminated with the addition of buffers or electrolytes.12,13 On the basis of the theory of LCCC, it seemed likely that a critical chromatographic condition could be established for a polar analyte through careful manipulation of these variables. The addition of the appropriate type and amount of buffer and nonsolvent is expected to allow for the careful balance of the enthalpy and entropy of the polymer interaction with the chromatographic system. Herein, the establishment of the critical chromatographic condition for two anionic polymers, polystyrene sulfonate and poly(acrylic acid), is illustrated. The effects of both buffer type and concentration and nonsolvent concentration as well as column length are illustrated for each polymer sample. Finally, the LCCC conditions for the poly(acrylic acid) were used to characterize a statistical copolymer containing poly(acrylic acid). EXPERIMENTAL CONDITIONS Materials. Sodium salts of polystyrene sulfonate samples (Mw ) 1640, Mw/Mn ) 1.12; Mw) 2400, Mw/Mn ) 17 mM, adsorptive interactions controlled the chromatography and also caused the polymers to become insoluble, with the lowest molecular weight standard showing adsorption and having an elution volume beyond the total volume of the column. Figure 6 shows the calibration curve for the PSS standards at the critical condition of the PAA standards. At the CC for the PAA, the PSS standards are being separated primarily by a size-exclusion mechanism. The critical chromato5542 Analytical Chemistry, Vol. 75, No. 20, October 15, 2003
Figure 6. Calibration curves at LCCC conditions with 53% water (17 mM phosphate buffer)/47% acetonitrile, (b) polystyrene sulfonate, and (9) poly(acrylic acid) using the 250 × 2.0-mm column.
Figure 7. Calibration curve of PAA with a 150 × 2.0-mm dimension column with 53% water (17 mM phosphate buffer)/47% acetonitirile. (Dashed line indicates the critical elution volume.)
graphic mobile phase system established for the poly(acrylic acid) standards with the 250-mm-long column was then applied to the 150-mm-long column (the other dimensions of the chromatographic system were identical to those utilized with the longer column). Figure 7 shows the resulting calibration curves. The critical mobile phase composition of 53 vol % H2O/47 vol % acetonitrile with a phosphate buffer concentration of 17 mM provided primarily a size-exclusion mechanism on the shorter column. The shift of the CC with column configuration and operating conditions was also noted for the critical chromatographic system established for the sodium polystyrene sulfonate standards with an acetate buffer system. Previous studies have also documented similar variation in the critical condition with operating conditions. For example, enhanced-fluidity liquids enabled the routine establishment of the critical chromatographic mode with column lengths varying from 500 mm to 2 m long, utilizing a bare-silica stationary phase.14,15 These studies described the use of both temperature and pressure tuning of the solvent system. At a temperature of 343 K with a column length of 500 mm, the critical chromatographic mode for polystyrene standards was achieved at an inlet pressure of 280 atm with a mobile phase of 54 mol % CO2/46 mol % THF.15 Using the same temperature and mobile phase composition, a 250-µm-i.d. packed capillary column with a length of 1.8 m required an inlet pressure of 260 atm to establish the critical condition for polystyrene. In addition, when varying (14) Yun, H.; Olesik, S. V.; Marti, E. H. Anal. Chem. 1998, 70, 3298-3303. (15) Phillips, S. L.; Olesik, S. V. Anal. Chem. 2002, 74, 799-808.
Figure 8. (A) Chromatogram of acrylic acid/vinyl pyrrolidinone copolymer under SEC conditions (as described in Figure 5) or (B) at the critical condition of poly(acrylic acid) (as described in Figure 6) using the 250 × 2-mm column.
the column length of a Novapak-C18 stationary phase from 150 to 300 mm, Philipsen et al. noted that a temperature change of 10 K was needed to maintain the critical chromatographic mode for polystyrene standards with a THF/H2O mobile phase.16 In addition, while maintaining the same stationary phase and column length of a Nucleosil-C18 system but changing the stationary phase pore diameter from 100 to 4000 Å, a temperature change of 3 K was necessary in order to maintain the critical condition for polystyrenes with a dichloromethane/acetonitrile mobile phase.16 At this point, it does not appear likely that there is a buffer gradient established along the column. To fully clarify these observations, further studies utilizing different buffer/nonsolvent combinations, column materials, and temperature tuning should be attempted. Analysis of Block Polymers of Poly(acrylic acid). The LCCC conditions established for the poly(acrylic acid) standards were applied to the analysis of copolymers of acrylic acid and vinyl pyrrolidinone. The analysis of the 30/70 wt/wt acrylic acid and vinyl pyrrolidinone copolymer is illustrated in Figure 8. The weight-average molecular weight of this copolymer was determined to be 7000 using size-exclusion chromatography. Figure 5 shows the SEC calibration curve established utilizing a mobile phase of 99 vol % H2O/1 vol % acetonitrile with 5 mM phosphate (16) Philipsen, H. J. A.; Klumperman, B.; van Herk, A. M.; German, A. L. J. Chromatogr., A 1996, 727, 13-25.
buffer. When the copolymer was injected into the chromatographic system at the critical condition for poly(acrylic acid), the center of mass of the observed chromatographic band was at a retention time less than that of the critical condition for PAA and greater than that observed for the retention of the copolymer under SEC conditions. In other words, under the CC for PAA, the molecular weight variation for PAA will not contribute to the observed chromatogram, which leaves only the microscopic environment of the PVP to selectively interact with the chromatographic system. The measured retention time of the copolymer then shows an apparent weight loss for the copolymer, which is associated with removing the PAA from the retention mechanism. The chromatogram of the copolymer at the CC for PAA could be used to verify the composition of the copolymer (i.e., relative weight percentages of the PAA and PVP). However, to properly calculate the molecular weight of the PVP in the copolymer from the retention time of the copolymer at the CC of the PAA, the retention times of PVP standards would be needed to generate an appropriate SEC calibration curve. Those standards were not available for this effort. CONCLUSION This study successfully demonstrates the novel development of LCCC for primarily water soluble polymers. The variation in buffer concentration and the proportion of organic modifier in the mobile phase was used to approach the critical condition for the two polymer systems, polystyrene sulfonate and poly(acrylic acid). The critical condition of two polar polymer analytes utilizing two solvent systems was established. The critical condition of poly(acrylic acid) was then used to study the retention characteristics of a copolymer containing both acrylic acid and n-vinyl pyrrolidinone. Future work will entail further investigation into alternative column and buffer system parameters.
Received for review May 1, 2003. Accepted August 3, 2003. AC034456L
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