Comprehensive Two-Dimensional Liquid Chromatography Analysis of

injection to the 2nd-D LC by use of a trap column, which allows an efficient interface between the two LC separa- tions. Over 200 different block copo...
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Anal. Chem. 2007, 79, 1067-1072

Comprehensive Two-Dimensional Liquid Chromatography Analysis of a Block Copolymer Kyuhyun Im, Hae-Woong Park, Youngtak Kim, Bonghoon Chung, Moonhor Ree, and Taihyun Chang*

Department of Chemistry and Polymer Research Institute, Pohang University of Science and Technology, Pohang, 790-784, Korea

A two-dimensional liquid chromatography (2D-LC) method, normal phase liquid chromatography (NPLC) for one dimension and reversed phase liquid chromatography (RPLC) for the other dimension, was employed to map the molecular weight distribution (MWD) of the individual blocks of a polystyrene-block-polyisoprene (PS-b-PI) diblock copolymer. The first-dimension (1st-D) NPLC separates PS-b-PI according to the PS block length while the second-dimension (2nd-D) RPLC separates PS-b-PI according to the PI block length. For the first-dimension NPLC separation, the column temperature was controlled to improve the resolution while the 2nd-D RPLC was run isothermally to reduce the separation time. The MWD information of individual blocks provides equivalent information to MWD and chemical composition distribution of a block copolymer. In this analysis, the effluent from the 1st-D LC separation is concentrated before the injection to the 2nd-D LC by use of a trap column, which allows an efficient interface between the two LC separations. Over 200 different block copolymer species could be identified from the 2D-LC chromatogram. Synthetic polymers are rarely homogeneous chemical species but have distributions in multiple molecular characteristics such as molecular weight, chain architecture, tacticity, and composition.1 A full characterization of such multivariate distribution is not a trivial task, if not impossible. Nonetheless, increasing interests in polymer materials with tailored properties demand more precisely controlled synthesis as well as high-precision characterization. Recent development in liquid chromatography (LC) analysis of complex polymers shows a clear trend to combine more than one LC separation mechanism together with multiple detections.2,3 It is a quite natural direction to characterize complex polymer systems with multivariate distributions in molecular characteristics. By combining different LC separation modes, it is possible to separate polymers selectively with respect to different molecular characteristics such as hydrodynamic volume, chemical composition, and functionality.4-9 * To whom correspondence should be addressed. Phone: +82-54-279-2109. Fax: +82-54-279-3399. E-mail: [email protected]. (1) Chang, T. Adv. Polym. Sci. 2003, 163, 1-60. (2) Berek, D. Prog. Polym. Sci. 2000, 25, 873-908. (3) Pasch, H. Adv. Polym. Sci. 2000, 150, 1-66. (4) Trathnigg, B. Prog. Polym. Sci. 1995, 20, 615-650. (5) Pasch, H. Macromol. Symp. 2001, 174, 403-412. 10.1021/ac061738n CCC: $37.00 Published on Web 01/06/2007

© 2007 American Chemical Society

Two-dimensional liquid chromatography (2D-LC) is a combination of adequately selected two different LC separation methods. The 2D-LC has become increasingly popular particularly in protein analysis.10-12 The best configuration of a 2D-LC separation is to employ two LC methods exclusively sensitive to one of the molecular characteristics while suppressing the effect of the others so that each separation becomes orthogonal to each other.13-15 However, such an ideal situation is rarely achieved and a compromise needs to be made in reality. For most 2D-LC separations of synthetic polymers reported so far, the 1st-D separation was done by interaction chromatography (IC) separating the polymers by chemical heterogeneity while the 2nd-D separation was done by size exclusion chromatography (SEC) separating the polymer chains with respect to the molecular size. The reason why IC × SEC 2D-LC configuration has been most popular is that SEC is the most widely used separation method for polymer characterization and SEC analysis can be done fast.7,16 However, SEC separation is not sensitive to chemical composition and suffers from a low resolution due to the band broadening.17-19 Therefore, the use of IC for the 2nd-D separation is often desirable since IC is sensitive to the chemical composition and the band broadening problem is far less severe than SEC under a proper IC separation condition.8 For an example of IC × IC 2D-LC analysis, Murphy et al.20 and Jandera et al.21 separated individual blocks of alcohol ethoxylate and ethylene oxide-propylene oxide (6) Berek, D. Macromol. Symp. 2001, 174, 413-434. (7) van der Horst, A.; Schoenmakers, P. J. J. Chromatogr. A 2003, 1000, 693709. (8) Chang, T. J. Polym. Sci., Polym. Phys. 2005, 43, 1591-1607. (9) Shalliker, R. A.; Gray, M. J. Adv. Chromatogr. 2006, 44, 177-236. (10) Bushey, M. M.; Jorgenson, J. W. Anal. Chem. 1990, 62, 161-167. (11) Opiteck, G. J.; Jorgenson, J. W.; Anderegg, R. J. Anal. Chem. 1997, 69, 2283-2291. (12) Wagner, K.; Racaityte, K.; Unger, K. K.; Miliotis, T.; Edholm, L. E.; Bischoff, R.; Marko-Varga, G. J. Chromatogr. A 2000, 893, 293-305. (13) Venkatramani, C. J.; Xu, J.; Phillips, J. B. Anal. Chem. 1996, 68, 14861492. (14) Slonecker, P. J.; Li, X.; Ridgway, T. H.; Dorsey, J. G. Anal. Chem. 1996, 68, 682-689. (15) Im, K.; Kim, Y.; Chang, T.; Lee, K.; Choi, N. J. Chromatogr. A 2006, 1103, 235-242. (16) Pasch, H.; Adler, M.; Rittig, F.; Becker, S. Macromol. Rapid Commun. 2005, 26, 438-444. (17) Lee, W.; Lee, H.; Cha, J.; Chang, T.; Hanley, K. J.; Lodge, T. P. Macromolecules 2000, 33, 5111-5115. (18) Popovici, S. T.; Kok, W. T.; Schoenmakers, P. J. J. Chromatogr. A 2004, 1060, 237-252. (19) Baumgarten, J. L.; Busnel, J. P.; Meira, G. R. J. Liq. Chromatogr. Relat. Technol. 2002, 25, 1967-2001.

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co-oligomers, respectively, by using comprehensive normal phase liquid chromatography (NPLC) × reversed-phase liquid chromatography (RPLC) 2D-LC methods. Such IC × IC 2D-LC separations often encounter difficulty arising from the mobile phase incompatibility. In IC separations the eluent is usually not a strong solvent. If the eluent of the 1st-D separation is a stronger solvent than the eluent for the 2nd-D separation, the polymer samples in the 1st-D effluent are often not separated in the 2nd-D LC separation but elute together with the injection solvent as a solvent plug.22 One method to overcome the breakthrough problem associated with the transfer of a solute in a stronger solvent environment from the 1st-D to the 2nd-D is to add a poor solvent to the 1st-D effluent prior to the injection to the 2nd-D column.23-26 The other method to overcome the problem of solvent incompatibility is the use of a trap column to replace the solvent.9,24,27 The use of a trap column provides an additional advantage in 2D-LC separations. In the direct interface of two LC, the amount of the analyte transferred to the 2nd-D LC is small, which in turn requires use of highly sensitive detectors.7,16 The trapping of the 1st-D LC effluent to a trap column can effectively concentrate the sample before the injection to the 2nd-D column.6,28 The precise characterization of copolymers having both molecular weight distribution (MWD) and chemical composition distribution (CCD) is much more complicated than the analysis of homopolymers. The ultimate goal in the characterization of copolymers with such a bivariate distribution is to construct a twodimensional map, one in MWD and the other in CCD. An attractive method for the purpose is 2D-LC in which two chromatographic separation methods, each exclusively sensitive to one molecular characteristic, are used in sequence. We have previously shown that the offline NPLC × RPLC separation can fractionate the individual blocks of polystyrene-block-polyisoprene diblock copolymers (PS-b-PI).29 The MWD information of the individual blocks provides equivalent information to MWD and CCD of a block copolymer. This offline 2D-LC characterization required a reconcentration procedure before the 2nd-D LC analysis. In this study, we report on the comprehensive online 2D-LC analysis of the same PS-b-PI. A combination of RPLC and NPLC effectively separated the PS-b-PI according to the individual block lengths and allowed mapping of the bivariate distribution of the two blocks. In so doing, the solvent exchange and re-concentration of the 1st-D LC effluents were accomplished before the injection into the 2nd-D LC by online full sorption/ desorption method. This interface of the two LC separations was efficient to make a high-resolution separation of the individual blocks possible. (20) Murphy, R. E.; Schure, M. R.; Foley, J. P. Anal. Chem. 1998, 70, 43534360. (21) Jandera, P.; Fischer, J.; Lahovska, H.; Novotna, K.; Cesla, P.; Kolarova, L. J. Chromatogr. A 2006, 1119, 3-10. (22) Jiang, X.; van der Horst, A.; Schoenmakers, P. J. J. Chromatogr. A 2002, 982, 55-68. (23) Moore, A. W., Jr.; Jorgenson, J. W. Anal. Chem. 1995, 67, 3456-3463. (24) Trathnigg, B.; Rappel, C.; Raml, R.; Gorbunov, A. J. Chromatogr. A 2002, 953, 89-99. (25) Haefliger, O. P. Anal. Chem. 2003, 75, 371-378. (26) Murahashi, T. Analyst 2003, 128, 611-615. (27) Sweeney, A. P.; Shalliker, R. A. J. Chromatogr. A 2002, 968, 41-52. (28) Nguyen, S. H.; Berek, D.; Chiantore, O. Polymer 1998, 39, 5127-5132.

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Figure 1. Experimental setup for the coupling of NP-TGIC and RPLC with two 10-port switching valves for comprehensive 2D-LC.

EXPERIMENTAL SECTION Materials. PS-b-PI diblock copolymer was prepared by sequential anionic polymerization. Details of the apparatus and the polymerization procedure were reported previously.30 The block copolymer was characterized by SEC coupled with a light scattering detection as Mw ) 2.4 kg/mol and Mw/Mn ) 1.06.29 NP-TGIC and RPLC Analysis. For the NP temperature gradient interaction chromatography (TGIC) separation,31 a diolbonded silica column (Nucleosil, 7 µm, 100 Å, 250 × 7.8 mm i.d.) was used. The mobile phase was an iso-octane/THF mixture (97/3 v/v, J.T. Baker/Samchun, HPLC grade). The temperature of the separation columns was controlled by circulating fluid from a programmable bath/circulator (ThermoHaake, C25P) through a column jacket. For the RPLC separation, a C18-bonded silica column (Kromasil, 5 µm, 100 Å, 150 × 4.6 mm i.d.) and the mobile phase, a mixture of CH2Cl2/CH3CN (53/47 v/v, Samchun, HPLC grade), were used as stationary and mobile phase, respectively. Chromatograms were recorded with a UV absorption detector (TSP, UV 100) operating at a wavelength of 260 nm. 2D-LC. For the NP-TGIC × RPLC 2D-LC analysis, the same conditions as in the 1D-LC analysis were used except for the flow rate. The flow rate of the 1st-D NP-TGIC was set low at 0.05 mL/ min to synchronize with the 2nd-D RPLC separation operated at a flow rate of 1.5 mL/min. The schematic description of the comprehensive 2D-LC apparatus is shown in Figure 1. Three HPLC pumps (two Bischoff compact pumps and a Spectra series P100) and two UV detectors (TSP, UV100), a six-port injector valve (7125, Rheodyne), and two 10-port valves (Alltech, SelectPro) were used. To trap the polymers in the 1st-D LC effluent, two C18bonded silica trap columns (Alltech C18, 3 µm, 100 Å, 33 × 7.0 mm i.d.) were used. The key component interfacing the two LC systems is the two switching valves that enable continuous and alternate sampling of the 1st-D effluent and injection to the 2nd-D (29) Park, S.; Cho, D.; Ryu, J.; Kwon, K.; Lee, W.; Chang, T. Macromolecules 2002, 35, 5974-5979. (30) Kwon, K.; Lee, W.; Cho, D.; Chang, T. Korea Polym. J. 1999, 7, 321-324. (31) Chang, T.; Lee, H. C.; Lee, W.; Park, S.; Ko, C. Macromol. Chem. Phys. 1999, 200, 2188-2204.

Figure 2. (A) NP-TGIC chromatogram of PS-b-PI. Temperature program is shown in the plot. Column: Nucleosil diol-bonded silica, 250 × 7.8 mm i.d., 7 µm, 100 Å. Eluent: iso-octane/THF (97/3 v/v) at a flow rate of 0.8 mL/min. (B) RPLC chromatogram of PS-b-PI. Column: Kromasil C18, 150 × 4.6 mm i.d., 5 µm, 100 Å. Eluent: CH2Cl2/CH3CN (53/47 v/v) at a flow rate of 0.5 mL/min.

column through two identical trap columns. The first 10-port valve was connected to two 100 µL loops that store the 1st-D effluent alternately for 2 min each at a flow rate of the 1st-D separation (0.05 mL/min). While one loop (Loop2 in Figure 1) is being loaded with the 1st-D effluent, the effluent stored in the other loop (Loop1) is swept by CH3CN (delivered by Pump 2) and delivered to the trap-column1 connected to the second 10-port valve. The flow rate of Pump 2 was 1.5 mL/min, which is fast enough to sweep the void volume of the trap-column (∼0.6 mL) in 2 min. At the same time, the polymers trapped in trap-column2 in the previous cycle are separated by the 2nd-D RPLC. The trap-columns are also the C18-bonded silica column and the trapped polymers are desorbed by the 2nd-D RPLC eluent driven by Pump 3 and separated by both the trap column and the 2nd-D column. The trapping process can be repeated many times before the injection to the 2nd-LC column as far as the full sorption of the polymer sample is maintained and the resolution of the 2nd-LC separation is not degraded due to the overloading.32 The multiple trapping increases the amount of the sample injected into the 2nd-D separation, which enhances flexibility in employing less sensitive but useful detectors for polymer analysis such as refractive index, UV absorption, and light scattering detectors. The number of multiple trappings can be adjusted depending on the analysis time of the 2nd-LC separation. In this study, the sample trapping was repeated six times because 12 min was required for the 2nd-LC separation. It is equivalent to a singletrap process with a 600 µL storage loop but the multiple trap method allows software control in varying the collection amount of the 1st-D effluent without changing the loop. RESULTS AND DISCUSSION For a good IC separation, a proper separation condition needs to be selected first. To separate the PS-b-PI according to the PS block length, a diol-bonded silica and an iso-octane/THF mixture

(97/3 v/v) were used as stationary and mobile phase, respectively.29 The eluent condition is close to the chromatographic critical condition for PS while it is a strong solvent for PI chains and homo-PI elutes in the SEC regime (before the injection solvent peak).33 Therefore, PI block hardly interacts with the polar stationary phase while PS block has a sufficient interaction for IC separation. Figure 2A displays the NP-TGIC chromatograms of the PS-b-PI recorded by a UV detector. To enhance the resolution, the temperature of the column was changed in three steps as shown in the plot.34 The injection solvent peak appears at tR ≈ 10 min. The PS-b-PI is resolved very well according to the degree of polymerization of the PS block. Each peak has a homogeneous PS block length but variable PI block length. On the other hand, the separation according to the PI block distribution can be accomplished by RPLC using a C18-bonded silica column and CH2Cl2/CH3CN (53/47 v/v) mobile phases, as shown in Figure 2B. The RPLC resolution according to the degree of polymerization of PI block appears not as good as the NPLC resolution according to the PS block length. The poor resolution of RPLC is likely due to two reasons. First, the separation condition is set in such a way that the interaction strength of PI block with the stationary phase is not strong, as can be seen from the shorter analysis time of RPLC than that of NPLC separation. We could have made the interaction stronger and improved the resolution by choosing a weaker eluent composition (higher CH3CN content), but it would increase the analysis time that has to be reconciled with the resolution. Second, the RPLC retention is affected mainly by the PI block length but also by the PS block length to some extent. Once the block copolymer is fractionated by NPLC according to the PS block length, the resolution of the (32) Park, S.; Park, I.; Chang, T.; Ryu, C. Y. J. Am. Chem. Soc. 2004, 126, 89068907. (33) Macko, T.; Hunkeler, D. Adv. Polym. Sci. 2003, 163, 61-136. (34) Ryu, J.; Chang, T. Anal. Chem. 2005, 77, 6347-6352.

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Figure 3. RPLC chromatogram of the NP-TGIC fraction boxed in Figure 2A when the sample was injected directly via a 100 µL sample loop. Column: Kromasil C18 150 × 4.6 mm i.d., 5 µm, 100 Å. Eluent: CH2Cl2/CH3CN (53/47 v/v) at a flow rate of 0.5 mL/min.

fractions according to the PI block length would be improved since the fraction has a homogeneous PS block length. Having established the separation conditions for each block, we combined the NPLC (fractionating the PS block) and the RPLC (fractionating the PI block) separation to map the individual block length distribution of the PS-b-PI. We chose the NP-TGIC system as the first dimension since it exhibits higher resolution than RPLC and the TGIC separation cannot be repeated rapidly. The fast repetition of the 2nd-D analysis is a key for the comprehensive 2D-LC analysis. While the column has to be cooled down before the next run in TGIC, 2nd-D RPLC is an isothermal elution that allows rapid runs repeatedly. The next thing to be considered is the compatibility of the effluent from the 1st-D separation with the 2nd-D separation. The effluent of the 1st-D is a mixture of iso-octane/THF (97/3 v/v), which is a strong solvent for the 2nd-D RPLC elution. If this effluent is directly injected into the RPLC system, the polymers are not separated but eluted together with the injection solvent. Figure 3 shows the RPLC chromatogram of a NP-TGIC fraction shown with a dotted box in Figure 2A. The fraction in iso-octane/THF mixed solvent was directly injected into the RPLC system (C18 column and CH2Cl2/CH3CN (53/47 v/v) mobile phase). The PS-b-PI fraction was not further resolved but eluted as a narrow peak near the system peak position. It is a typical “breakthrough” or “solvent-plug” phenomenon that polymers elute together with injection solvent.22 The breakthrough problem can be prevented if the injection solvent is identical to the eluent (ideal case) or its solvent strength is weaker than that of the eluent. In popular IC × SEC type 2DLC configuration, the breakthrough problem is not serious since the eluent for SEC separation is a strong solvent. In the IC × IC 2D-LC configuration, the breakthrough problem does not always exist. However, for more routine applications of the 2D-LC method, it is desirable to exchange the solvent in the 1st-D effluent prior to the injection into the 2nd-D column. In this 2D-LC study, the solvent of the 1st-D effluent was exchanged with a weaker solvent. The solvent exchange was accomplished by use of a small C18 trap-column and the eluent promoting the sorption of polymer 1070 Analytical Chemistry, Vol. 79, No. 3, February 1, 2007

Figure 4. RPLC chromatogram of the NP-TGIC fraction boxed in Figure 2A when the sample is injected via a trap-column. The singleinjection chromatogram (dashed line) was shifted to match the two chromatograms for easy comparison. The dotted line is the intensity of the single-injection chromatogram multiplied by six to compare with the multiple-injection chromatogram. The separation condition is the same as that in Figure 3.

to the trap-column. The trap-column has the same type of stationary phase as the 2nd-D column so that the complete desorption takes place by the eluent of the 2nd-D LC and the trapcolumn also serves for the 2nd-D separation together with the 2nd-D analytical column. The two switching valves in Figure 1 operate in an alternating manner. While Loop2 is being filled with the effluent from the 1st-D NP-TGIC separation and the sample trapped in trap-column2 is being separated by 2nd-D RPLC, Pump 2 delivers CH3CN to transfer the effluent stored in Loop1 to the trap-column1. The CH3CN is a nonsolvent for the polymer and induces the full sorption of the polymers in the 1st-D effluent to the trap-column. The success of this trapping method depends on how effectively the polymers are adsorbed in the trapping process and desorbed in the 2nd-D separation. To examine the efficiency of sorption and desorption, we carried out a similar experiment to the one shown in Figure 3 using a trap-column. Instead of injecting the fractionated sample (boxed peak in Figure 2A) to the 2nd-D RPLC via a 100 µL loop directly as in Figure 3, the sample was injected via a 100 µL loop and a trap-column where the eluent is replaced by CH3CN. Figure 4 shows the RPLC chromatograms of the sample injected via a trap-column: single injection (dashed line) and multiple injections (solid line). In the multiple injection experiments, 100 µL samples were transferred six times consecutively to the trap-column as shown with arrows in the chromatogram. The single-injection chromatogram was shifted to match the two chromatograms for easy comparison. Unlike the direct injection of the 1st-D effluent (Figure 3), the injection via a trap-column yields a well-resolved chromatogram according to the PI block length. It demonstrates that trapping of the polymer using a weak solvent prevents the breakthrough of the polymer sample. The chromatograms of a fraction with a monodisperse PS block are

Figure 5. NP-TGIC × RPLC 2D-LC chromatogram of PS-b-PI. 1st-D NP-TGIC: diol-bonded silica column (250 × 7.8 mm i.d., 7 µm, 100 Å), iso-octane/THF (97/3 v/v) at a flow rate of 0.05 mL/min. 2nd-D RPLC: C18-bonded silica column (150 × 4.6 mm i.d., 5 µm, 100 Å). CH2Cl2/ CH3CN (53/47 v/v) at a flow rate of 1.5 mL/min. Trap-column: C18-bonded silica column (3 µm, 100 Å, 33 × 7.0 mm i.d.). The temperature program for 1st-D NP-TGIC was practically the same as that in Figure 1 except for the prolonged elution: 2 °C during the first 120 min, linear increase to 25 °C for 480 min, linear increase to 67 °C for the next 400 min, and kept at 67 °C for the next 300 min.

better resolved than the unfractionated PS-b-PI shown in Figure 2B, indicating that the PS block affects the RPLC retention of PS-b-PI. The multiple-injection chromatogram shows a good resolution comparable to the single-injection chromatogram, demonstrating that the full sorption/desorption and the effective concentration of the injected sample occurs. Furthermore, the intensity of the multiple-injection chromatogram is almost exactly 6 times as high as that of the single-injection chromatogram, confirming a good sample recovery. The trap-columns used in this study are not very small. The column void volume is about 0.6 mL. Therefore, the CH3CN filling the void volume of the trap-column give rise to a huge solvent peak in the chromatograms shown in Figure 4, which can be alleviated by using a smaller column. The high-resolution chromatogram obtained despite the use of a large trap-column indicates that the polymers are trapped at a small volume near the entrance of the trap-column and desorbed effectively by the 2nd-D eluent.34 Otherwise, a significant loss in 2nd-D resolution should have been observed, in particular for the multiple-trapping case. Figure 5 shows a comprehensive 2D-LC chromatogram of the PS-b-PI sample. The flow rate of 1.5 mL/min through the RPLC column completes the 2nd-D separation in 12 min. During the 2nd-D separation, the 1st-D NP-TGIC separation elutes 600 µL at the flow rate of 0.05 mL/min. The loading of the 100 µL loop and

the transfer to the trap-column are executed six times to accumulate the polymers eluting for 12 min from the 1st-D NPLC column. In this way, all the effluent from the first column can be analyzed by the 2nd-D LC in a comprehensive manner. Each peak represents a PS-b-PI species with different degrees of polymerization of the two blocks. NP-TGIC separates the PS-b-PI according to the degree of polymerization of the PS block while RPLC separates the PS-b-PI according to the degree of polymerization of the PI block. The degree of polymerization of PS block (x) and PI block (y) were identified by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy using dithranol as a matrix and silver trifluoroacetate as a cationizing agent.29 The two LC separations are reasonably orthogonal to each other to fractionate each block nearly independently. The largest deviation from the orthogonal separation occurs for the PS-b-PI with short PI blocks and long PS blocks (left upper corner of the 2D-LC chromatogram). The behavior is consistent with the 1D NP-TGIC separation shown in Figure 2 in which the elution peak becomes broader as the PS block length increases. In summary, we carried out a comprehensive 2D-LC separation of low MW PS-b-PI. The separations according to PS and PI block length were carried out at NPLC and RPLC, respectively. The two LC separations are interfaced via online solid-phase extraction and the multiple trapping method, which enables one to concentrate Analytical Chemistry, Vol. 79, No. 3, February 1, 2007

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the 1st-D effluent and to use a common concentration detector for HPLC. The comprehensive 2D-LC separation successfully mapped the bivariate distribution of the block copolymer. Over 100 RPLC chromatograms were collected to construct the 2D-LC chromatogram and over 200 different block copolymer species were identified. The total analysis time in this study may be too long to be implemented as a routine analysis tool. However, for most synthetic polymers such a detailed mapping of the multivariate distribution would not be necessary and a reasonable

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compromise between the resolution and the analysis speed of this type of analysis would be very useful. ACKNOWLEDGMENT This work was supported by a research grant from KOSEF (Center for Integrated Molecular Systems) and the BK21 program. Received for review September 15, 2006. Accepted November 17, 2006. AC061738N