Simple and Comprehensive Two-Dimensional Reversed-Phase HPLC

Feb 3, 2004 - Department of Polymer Science and Engineering, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku,. Kyoto 606-8585, Japan, Kyoto Monot...
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Anal. Chem. 2004, 76, 1273-1281

Simple and Comprehensive Two-Dimensional Reversed-Phase HPLC Using Monolithic Silica Columns Nobuo Tanaka,*,† Hiroshi Kimura,† Daisuke Tokuda,† Ken Hosoya,† Tohru Ikegami,† Norio Ishizuka,‡ Hiroyoshi Minakuchi,‡ Kazuki Nakanishi,§ Yukihiro Shintani,| Masahiro Furuno,| and Karin Cabrera⊥

Department of Polymer Science and Engineering, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan, Kyoto Monotech Co., 376-5-206 Tsukiyama-cho, Kuze, Minami-ku, Kyoto 601-8203, Japan, Department of Material Chemistry, Graduate School of Engineering, Kyoto University, PRESTO, JST, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan, GL Sciences, Iruma, Saitama 358-0032, Japan, and Merck, Darmstadt, D-64293, Germany

Simple and comprehensive two-dimensional (2D)-HPLC was studied in a reversed-phase mode using monolithic silica columns for second-dimension (2nd-D) separation. Every fraction from the first column, 15 cm long (4.6mm i.d.), packed with fluoroalkylsilyl-bonded (FR) silica particles, was subjected to the separation in the 2nd-D using one or two octadecylsilylated (C18) monolithic silica columns (4.6-mm i.d., 3 cm). Monolithic silica columns in the 2nd-D were eluted at a flow rate of up to 10 mL/ min with separation time of 30 s that meets the fractionation every 15-30 s at the first dimension (1st-D) operated at a flow rate of 0.4-0.8 mL/min. Three cases were studied. (1) In the simplest scheme of 2D-HPLC, effluent of the 1st-D was directly loaded into an injector loop of 2nd-D HPLC for 28 s, and 2 s was allowed for injection. (2) Two six-port valves each having a sample loop were used to hold the effluent of the 1st-D alternately for 30 s for one 2nd-D column to effect comprehensive 2D-HPLC without the loss of 1st-D effluent. (3) Two monolithic silica columns were used for 2nd-D by using a switching valve and two sets of 2nd-D chromatographs separating each fraction of the 1st-D effluent with the two 2nd-D columns alternately. In this case, two columns of the same stationary phase (C18) or different phases, C18 and (pentabromobenzyloxy)propylsilyl-bonded (PBB), could be employed at the 2nd-D, although the latter needed two complementary runs. The systems produced peak capacity of ∼1000 in ∼60 min in cases 1 and 2 and in ∼30 min in case 3. The three stationary phases, FR, C18, and PBB, showed widely different selectivity from each other, making 2D separations possible. The simple and comprehensive 2D-HPLC utilizes the stability and high efficiency at high linear velocities of monolithic silica columns. While high-efficiency separation methods such as ultrahighpressure liquid chromatography (UHPLC)1,2 can produce peak * Corresponding author. E-mail: [email protected]. † Kyoto Institute of Technology. ‡ Kyoto Monotech Co. § Kyoto University. | GL Sciences. ⊥ Merck. 10.1021/ac034925j CCC: $27.50 Published on Web 02/03/2004

© 2004 American Chemical Society

capacity (PC)3,4 as much as 300, increasing demand for the separation of very complex mixtures prompted the study of multidimensional separation techniques,5 including two-dimensional (2D)-GC,6 2D-CE,7 and 2D-HPLC.8-19 Some of comprehensive 2D-HPLC studies are aimed at proteomics studies.8-11,15-17,19 2D-HPLC can potentially give a variety of combinations of separation modes as well as large PC by utilizing a wide range of stationary phases and mobile phases. It is attractive, especially with the recent development of LC/MS instrumentation, to achieve the analysis of hundreds or thousands of compounds at a time. Various plumbing schemes have been developed to alleviate the problem of the slow elution process of HPLC compared to GC or CE.8-17 2D-HPLC can be carried out in various ways. In a popular peptide separation scheme where ion-exchange-mode separation or size-exclusion-mode separation was followed by reversed-phase separation, relatively long separation time was allowed for both first-dimension (1st-D) and second-dimension (2nd-D) separa(1) MacNair, J. E.; Patel, K. D.; Jorgenson, J. W. Anal. Chem. 1999, 71, 700708. (2) Lan, K.; Jorgenson, J. W. Anal. Chem. 1999, 71, 709-714. (3) Giddings, J. C. Anal. Chem. 1967, 39, 1027-1028. (4) Grushka, E. Anal. Chem. 1970, 42, 1142-1147. (5) Mondello, L.; Lewis, A. C.; Bartle, K. D. Multidimensional Chromatography; John Wiley & Sons Ltd.: Chichester, England, 2002. (6) Bertsch, W. J. High Resolut. Chromatogr. 2000, 23, 167-181. (7) Rocklin, R. D.; Ramsey, R. S.; Ramsey, J. M. Anal. Chem. 2000, 72, 52445249. (8) Bushey, M. M.; Jorgenson, J. W. Anal. Chem. 1990, 62, 161-167. (9) Holland, L. A.; Jorgenson, J. W. Anal. Chem. 1995, 67, 3275-3283. (10) Opiteck, G. J.; Lewis, K. C.; Jorgenson, J. W.; Anderegg, R. Anal. Chem. 1997, 69, 1518-1524. (11) Opiteck, G. J.; Jorgenson, J. W.; Anderegg, R. Anal. Chem. 1997, 69, 22832291. (12) Wehr, T. LC-GC 2002, 20, 954-962. (13) Kohne, A. P.; Dornberger, U.; Welsch, T. Chromatographia 1998, 48, 9-16. (14) Kohne, A. P.; Welsch, T. J. Chromatogr., A 1999, 845, 463-469. (15) Wagner, K.; Racaityte, K.; Unger, K. K.; Miliotis, T.; Edholm, L. E.; Bischoff, R.; Marko-Varga, G. J. Chromatogr., A 2000, 893, 293-305. (16) Unger, K. K.; Racaityte, K.; Wagner, K.; Miliotis, T.; Edholm, L. E.; Bischoff, R.; Marco-Varga, G. J. High Resolut. Chromatogr. 2000, 23, 259-265. (17) Wagner, K.; Miliotis, T.; Marko-Varga, G.; Bischoff, R.; Unger, K. K. Anal. Chem. 2002, 74, 809-820. (18) Haefliger, O. P. Anal. Chem. 2003, 75, 371-378. (19) Davis, M. T.; Beierle, J.; Bures, E. T.; McGinley, M. D.; Mort, J.; Robinson, J. H.; Spahr, C. S.; Yu, W.; Luethy, R.; Patterson, S. D. J. Chromatogr., B 2001, 752, 281-291.

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tion.8-11,15-17,19 The 1st-D separation, ion-exchange mode, in many cases, is known to be relatively slow and inefficient. Some studies reported recently included the use of two or four columns at the 2nd-D.11,15-17 A kind of 2D separation can be carried out with one column packed successively with ion-exchange-type particles followed by reversed-phase packings utilizing step gradient elution of an ion-exchange column.20 In comprehensive 2D-HPLC separations, the 2nd-D column should ideally be eluted at very high speed to meet the rate of fractionation at the 1st-D separation, as practiced in 2D-GC.5,6 Because a particle-packed column cannot be operated at adequately high flow rate, various approaches were taken in the past; (i) small columns were employed at 1st-D compared to 2nd-D,8,14,18 (ii) the first column was eluted slowly or intermittently,9-11,13,14 or (iii) two or more sets of chromatographs were used at the 2ndD.15-18 Limited PC, long separation time, and high pressure drop have been encountered in comprehensive 2D-HPLC. High permeability, high efficiency, and high stability of a packed bed are simultaneously required for 2nd-D columns. This seems to be the greatest difficulty seen with comprehensive 2D-HPLC reported using particle-packed columns in 2nd-D. Monolithic silica columns seem to be well suited for this purpose because of the high permeability and high efficiency at high linear velocity.21-24 Highspeed separation at the 2nd-D is expected to enable comprehensive 2D-HPLC in a simpler scheme. In addition to high efficiency and high permeability, 1st-D and 2nd-D columns must possess adequate difference in selectivity to effect 2D separations. Ideally the 1st-D and 2nd-D should have orthogonal selectivity or different separation mechanisms. Ionexchange mode and reversed-phase mode, or size-exclusion mode and reversed-phase mode have often been combined to effect 2D separations for peptide mixtures in proteomics.8-11,15-17,19,20 We previously reported that three stationary phases, fluoroalkyl (FR), pentabromobenzyl (PBB), and octadecylsilyl (C18) bonded silicas, showed widely different selectivity in common mobilephase, methanol-water mixtures, in reversed-phase LC.25 Here we report an attempt of comprehensive 2D-HPLC in simple reversed-phase mode using monolithic C18 or PBB-bonded phases, or both, at the 2nd-D and a particle-packed FR column at the 1stD. EXPERIMENTAL SECTION Materials. (a) Columns. Fluoroalkyl bonded (FR; 5,5,6,6,7,7,7heptafluoro-4,4-bis(trifluoromethyl)heptyldimethylsilyl bonded silica column, sold as Fluofix, 4.6-mm i.d., 15 cm) was obtained from NEOS. Short monolithic silica columns (4.6-mm i.d., 3 cm) clad with PEEK resin were similar to those previously reported26 and (20) Wolters, D. A.; Washburn, M. P.; Yates, J. R., III. Anal. Chem. 2001, 73, 5683-5690. (21) Minakuchi, H.; Nakanishi, K.; Soga, N.; Ishizuka, N.; Tanaka, N. Anal. Chem. 1996, 68, 3498-3501. (22) Minakuchi, H.; Nakanishi, K.; Soga, N.; Ishizuka, N.; Tanaka, N. J. Chromatogr., A 1998, 797, 121-131. (23) Tanaka, N.; Kobayashi, H.; Nakanishi, K.; Minakuchi, H.; Ishizuka, N. Anal. Chem. 2001, 73, 420A-429A. (24) Tanaka, N.; Kobayashi, H.; Ishizuka, N.; Minakuchi, H.; Nakanishi, K.; Hosoya, K.; Ikegami, T. J. Chromatogr., A 2002, 965, 35-49. (25) Turowski, M.; Morimoto, T.; Kimata, K.; Monde, H.; Ikegami, T.; Hosoya, K.; Tanaka, N. J. Chromatogr., A 2001, 911, 177-190. (26) Cabrera, K.; Lubda, D.; Eggenweiler, H.-M.; Minakuchi, H.; Nakanishi, K. J. High Resolut. Chromatogr. 2000, 23, 93-99.

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were used as 2nd-D columns after a chemical bonding reaction on column with demethyloctadecylsilyl (C18) or dimethyl-2,3,4,5,6pentabromobenzyloxypropylsilyl (PBB) groups. The preparation of bonded phase on monolithic columns was carried out as previously reported for silica-packed capillary columns.27 A toluene solution of N,N-diethylaminodimethyloctadecylsilane (20% v/v) was pumped through a monolithic silica column at a flow rate of 0.03 mL/min for 6 h, while the column was kept at 60 °C. Prior to the reaction, the column was washed with 2-propanol and then with toluene. After the reaction, column was washed with toluene and then with 2-propanol at 1 mL/min for 30 min each at room temperature. Sample compounds and solvents were obtained from commercial sources, polynuclear aromatic hydrocarbons (PAHs) from AccuStandard, and the others from Nacalai-tesque or Tokyo Chemical Ind. The samples were numbered as follows except for Figure 2. Alkylbenzenes: (1) benzene, (2) toluene, (3) ethylbenzene, (4) propylbenzene, (5) butylbenzene, (6) amylbenzene, (7) hexylbenzene, and (8) heptylbenzene. PAHs: (9) naphthalene, (10) fluorene, (11) anthracene, (12) pyrene, (13) triphenylene, (14) benz[a]pyrene, and (15) benzo[ghi]perylene. Alkyl phenyl ketones: (16) acetophenone, (17) propiophenone, (18) butyrophenone, (19) valerophenone, (20) hexanophenone, (21) heptanophenone, (22) octanophenone, (23) nonanophenone, and (24) decanophenone. Phthalate esters: (25) diethyl phthalate and (26) benzyl butyl phthalate. Bulky aromatic hydrocarbons (Ar-Ar): (27) diphenylmethane, (28) triphenylmethane, (29) o-terphenyl, and (30) triptycene. Benzene derivatives: (31) anisole, (32) thioanisole, (33) fluorobenzene, (34) chlorobenzene, (35) bromobenzene, (36) iodobenzene, (37) 1,3,5-trichlorobenzene, (38) nitrobenzene, (39) 2,4dinitrochlorobenzene, (40) R,R,R-trifluorotoluene, (41) 1,4-bis(trifluoromethyl)benzene, (42) o-difluorobenzene, (43) m-difluorobenzene, (44) p-difluorobenzene, and (45) 1,3-bis(trifluoromethyl)benzene. (b) Instrument. The following components were assembled to compose the 2D chromatographs described below: three sets of pumps, two PU-611 (GL Sciences) and one LC-10ADvp (Shimadzu), three detectors, two SPD-10AVvp (Shimadzu) and one UV-702 (GL Sciences), an injector valve (8125, Rheodyne), two six-port valves (C2-0346D, VICI), and a data processing system (EZChrom Elite-2.61, GL Sciences). The detector for the 1st-D has a pressure-resistant cell (8 µL) that can withstand pressure of up to 7 MPa. All solenoid valve operations were programmed and operated by a remote controlling unit, RT730 (GL Sciences). The 2D-HPLC systems were assembled as shown in Figure 1 so that the sample loops of the 2nd-D injector were back-flushed to minimize band broadening. Case 1: The outlet tubing of 1st-D detector was connected to a 500-µL loop of the 2nd-D injector. In this case, two sets of HPLC instruments were simply connected in series,9 as shown in Figure 1a. The 500-µL loop allowed variation of 1st-D fraction volume subjected to separation at 2nd-D. Case 2: Two six-port valves each equipped with a 500-µL loop were connected as shown in Figure 1b and used as an injector of 2nd-D. This scheme uses two HPLC instruments with an added loop on the 2nd-D injector valve for sample storage.8,10 A 10-port (27) Tanaka, N.; Kinoshita, H.; Araki, M.; Tsuda, T. J. Chromatogr. 1985, 332, 57-69.

Figure 1. (a) Tubing connection at 2nd-D injector of simple 2D-HPLC in case 1. (b) Tubing connection of two six-port valves used as 2nd-D injector in case 2. (c) Scheme of 2D-HPLC using two 2nd-D columns in case 3.

valve holding two sample loops can substitute for the two 6-port valves when properly connected. An eight-port valve can also substitute the two six-port valves for holding two loops, but accompanied by different flow direction in a loop.8,10 Case 3: 2D-HPLC using two 2nd-D columns consists of three sets of chromatographs, as shown in Figure 1c. The outlet of the 1st-D detector was connected to a switching valve directing the flow to either of the two injector loops (500 µL) of the 2nd-D instruments. (c) Operation of 2D-HPLC. Case 1: The loop at the 2nd-D HPLC was loaded with the 1st-D HPLC at 0.4 mL/min for 28 s, and then the injection valve was turned to inject the fraction for 2 s into the 2nd-D HPLC operated at 10 mL/min, and turned back for loading for the next 28 s, resulting in fractionation at the 1st-D every 30 s with loss of 1st-D effluent for 2 s in each cycle. The time allowed for injection, 2 s, was adequate to inject a 200-µL fraction by back-flushing the 2nd-D injector loop. Case 2: The two loops in Figure 1b were loaded alternately for 30 s at 0.4 mL/min, and the loaded effluent from the 1st-D was injected at 10 mL/min into the 2nd-D every 30 s. Back-flushing completely swept the loaded sample at the 2nd-D injector. This scheme allows comprehensive 2D-HPLC without loss of sample. Separation time in 2nd-D in case 1 and case 2 was set to be 30 s

to elute all the analytes in this study. Sampling frequency should be varied depending on the elution time at 2nd-D. Case 3: Two 2nd-D columns, two C18 columns or C18 and PBB columns, were used. In the latter case, two runs were needed to complete 2D chromatography. This scheme was convenient to show the unique selectivity of a PBB column, which was not easily produced with high reproducibility by on-column derivatization. The operation of the switching valve, every 15 s, allowed loading of a fraction from the 1st-D to the injector loop of the 2nd-D alternately, and the 2nd-D separation was allowed to proceed within 30 s. Fifteen seconds after each injection, the injection valve was turned back for the next loading. The two 2nd-D chromatographs were operated at up to 10 mL/min, while the 1st-D chromatograph was operated at 0.4-0.8 mL/min. In each case, a contour plot was constructed by using software, Deltagraph 5.0.

RESULTS AND DISCUSSION Performance of Monolithic Silica Columns. A 3-cm C18modified monolithic silica column was able to generate ∼1500 theoretical plates with t0 of 3.5 s in 80% methanol at linear velocity of ∼10 mm/s with the shortest time constant setting of the detector (0.05 s). The monolithic silica column was able to Analytical Chemistry, Vol. 76, No. 5, March 1, 2004

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Figure 2. Chromatograms obtained in 80% methanol for various hydrocarbons on the three stationary phases: (a) FR, 4.6-mm i.d., 15 cm; (b) monolithic C18, 4.6-mm i.d., 5 cm; and (c) monolithic PBB, 4.6-mm i.d., 5 cm. Solutes, alkylbenzenes (1-7 C6H5CnH2n+1, n ) 0-6); 8-11 bulky polyaromatic hydrocarbons (Ar-Ar) (8 diphenylmethane, 9 triphenylmethane, 10 o-terphenyl, 11 triptycene); and PAHs (12 naphthalene, 13 fluorene, 14 anthracene, 15 pyrene, 16 triphenylene). Flow rate, 1 mL/min.

withstand a linear velocity of greater than 10 mm/s. The pressure drop with the monolithic column was lower than 10 MPa at 10 mL/min in 80% methanol-20% water at room temperature. Properties of a 10-cm monolithic silica columns were reported previously.26 Difference in Selectivity of the Three Stationary Phases. 2D chromatography usually uses two columns of different modes that can provide orthogonal, or independent, selectivity for a sample mixture. Here we show that 2D separation can be carried out in a simple reversed-phase system by using FR, C18, and PBB stationary phases in methanol-water mobile phase. A similar approach was reported with C18 and another stationary phase of aromatic functionality with intermittent injections into 2nd-D.13,14 Figure 2 shows the chromatograms obtained for various hydrocarbons in methanol/water ) 80/20 on the three stationary phases. The elution orders for these hydrocarbons were considerably different. The three groups of compounds, alkylbenzenes (17), aromatic compounds having bulky, nonplanar structures (ArAr, 8-11), and PAHs (12-16) were eluted in a relatively similar range from the C18 column in Figure 2. While the PBB phase showed preferential retention for PAHs compared to alkylbenzenes, the FR phase showed the exactly opposite tendency, and the C18 phase was between the other two phases. The widely different selectivity seen among the three stationary phases can be explained based on the difference in the contribution of London 1276 Analytical Chemistry, Vol. 76, No. 5, March 1, 2004

(dispersion) interactions between the bonded organic moieties and the solutes.25,28 Case 1: Simple 2D Separation with One Column at the 2nd-D. Direct connection of the outlet of the 1st-D to the injector loop of a 2nd-D chromatograph, as shown in Figure 1a, is the simplest approach to comprehensive 2D-HPLC. Loading the injector loop with effluent of the 1st-D allows the flow rate of 2nd-D LC to be independent from that of the 1st-D all the time.8-10 The 2nd-D injector loop is large enough to hold a fraction from 1st-D for up to 75 s at 0.4 mL/min. Because of the high flow rate in the 2nd-D separation, 10 mL/min, the injection of 100-500 µL needed only a few seconds, while the injector loop of the 2nd-D is filled with the 1st-D effluent for the rest of the time. In the present case, an injection time of 2 s completely carried away the loaded fraction of less than 200 µL by back-flushing. The 2nd-D injector valve was at the “load” position (the solid line in Figure 1a) for 28 s and at the “inject” position (the dashed line in Figure 1a) for 2 s. Thus, the 2D separation with a single 2nd-D column is accompanied by the loss of ∼7% of the effluent from the 1st-D. This can be avoided by peak parking.13,14 Injection of a large-volume sample can cause significant band broadening. The use of a smaller 1st-D column with a larger 2nd-D column is a possible approach to avoid this problem but is (28) Turowski, M.; Yamakawa, N.; Meller, J.; Kimata, K.; Ikegami, T.; Hosoya, K.; Tanaka, N.; Thornton, E. R. J. Am. Chem. Soc. 2003, 125, 13836-13849.

Figure 3. Two-dimensional separation of a mixture of hydrocarbons and benzene derivatives in simple 2D-HPLC, case 1. (a) Chromatogram obtained in the 1st-D on FR column in 60% methanol-water. (b) Chromatograms obtained in the 2nd-D on C18 column in 80% methanol-water. The insets a and b are expanded views of panels a and b, respectively. (c) A contour plot obtained for 2D-HPLC. Fractionation every 30 s at the 1st-D. Flow rate: 0.4 mL/min for 1st-D and 10 mL/min for 2nd-D.

associated with considerable dilution of solutes.8,14,18 When a mobile phase of lower elution strength is used for the 1st-D separation than for the 2nd-D, band broadening can be minimized.13 This is the reason an FR column with the lower retentive property was used in 60/40 methanol-water in the 1st-D and the C18 phase was used in the 2nd-D with 80/20 methanol-water mobile phase. The loss of column efficiency at the 2nd-D was avoided even with the injection of 200-µL fractions. Figure 3 shows chromatograms obtained for a mixture of hydrocarbons and benzene derivatives with the 2D-HPLC system using a particle-packed FR column at the 1st-D and a C18 monolithic column at the 2nd-D. A flow rate of 10 mL/min allowed the elution of solutes having retention factors (k values) of up to 8 at 2nd-D within 30 s with t0 of 3.5 s. Figure 3a presents the chromatogram obtained at the 1st-D showing many overlapping peaks. PAHs were eluted as mixtures from the FR column between 9 and 13 min as shown in the Figure 3a inset, and some

are separated at the 2nd-D, as indicated in the Figure 3b inset. Some peaks, including peaks 12 and 13, appeared in more than one 2nd-D chromatogram. Tailing of such a huge peak can also be observed in a contour plot in Figure 3c. From the 2nd-D chromatograms in Figure 3b, a contour plot was obtained, as shown in Figure 3c. The 2D plots indicate that several types of hydrocarbons and benzene derivatives were clearly distinguished from each other. A group of compounds showed similar behavior characterized by their relative affinity to the two stationary phases; thus, 2D reversed-phase HPLC can afford structural information of the solutes, especially when the separation mechanism on each stationary phase is known and different. The FR column gave ∼11 000 theoretical plates, while the C18 column gave ∼1100 plates in the 2D system that is lower due to long connection tubing than that measured independently. The peak width at the 1st-D was 0.31-2.34 min for elution time of 5.4-63.7 min. Fractionation every 30 s at 1st-D separation may Analytical Chemistry, Vol. 76, No. 5, March 1, 2004

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Figure 4. (a) Set of 2nd-D chromatograms of simple and comprehensive 2D-HPLC separation of a mixture of hydrocarbons and benzene derivatives using FR column in the 1st-D and a C18 column as the 2nd-D column, case 2. Two six-port valves were used as 2nd-D injector as shown in Figure 1b. Other conditions were similar to those for Figure 3. (b) 2nd-D chromatograms expanded for case 2. (c) 2nd-D chromatograms expanded for case 1, similar to Figure 3b inset.

mix separated peaks before the injection into the 2nd-D. Thus, the PC is limited either by the fractionation time, 30 s, until an elution time of 13.6 min, or by the peak width at the 1st-D thereafter. PC of the 1st-D was estimated to be ∼61 for the separation time of 65 min by taking into account the fractionation interval and the peak width. When multiplied with the PC of ∼17 at the 2nd-D, total PC of ∼1040 for the 2D separation was obtained, although this can be an overly optimistic value in practice. Unexpectedly great scattering in Figure 3c is caused by a large difference in interactions between PAHs and the two stationary phases. The extra space seen in Figure 3c may not necessarily be used for other types of compounds. A short t0 at a high flow rate is highly desirable to increase PC per unit time, especially in isocratic conditions. This is one of the advantages of a monolithic silica column with high stability against high flow rate. In reported examples, short t0 values similar to the present study were recorded with gradient elution on a short column.15,17 Much longer t0 was recorded under isocratic conditions using a particle-packed column at the 2nd-D. About 6.7% of the 1st-D effluent in volume was wasted at the 2nd-D in a simple 2D system. The maximum possible loss during 2-s injection time is estimated to be ∼17% for the first peak eluting at 5.4 min with the peak volume (width) of 0.12 mL (18 s) at a flow rate of 0.4 mL/min in the present system. Maximum loss will occur when the middle of the peak is wasted during injection at the 2nd-D. It may be possible to adjust the timing of injection of the 2nd-D to the valley of peaks on the 1st-D separation as long as the fraction volume does not exceed 500 µL. The high reproducibility of isocratic HPLC runs may allow this kind of programmed fractionation to minimize sample loss. Although this simplest 2D-HPLC scheme may not be used for quantitation of 1278 Analytical Chemistry, Vol. 76, No. 5, March 1, 2004

all the solutes with one run, another run with a slightly shifted timing for fractionation at 1st-D may allow the quantitation of all the peaks in either chromatogram. If preparative separations are intended, or if quantification of all the solutes is not intended, the simple 2D-HPLC using one column at the 2nd-D is a practical means for providing large peak capacities not obtainable even with ultrahigh efficiency separation methods such as UHPLC or CE. Case 2: Simple and Comprehensive 2D Separation with One Column at the 2nd-D. As shown in Figure 1b, two six-port valves each equipped with a loop of 500-µL capacity were used to hold the fraction from the 1st-D alternately. Because the effluent from the 1st-D was loaded in either of the two loops, all the fractions of the 1st-D separation were introduced into the 2nd-D column to effect comprehensive 2D-HPLC. The fractions collected in the loop for 30 s were injected into the 2nd-D column alternately and subjected to the separation for 30 s. The 2nd-D chromatograms are shown in Figure 4a, which is very similar to Figure 3b, indicating the minor effect of partial loss of each 1st-D fraction in case 1. Small differences in peak height between the two 2nd-D chromatograms (Figure 4b and c) could also be caused by the slight variation in valve motion. Although a part of a solute band can be carried over to the next chromatogram with slight difference in fractionation, the comprehensive 2D-HPLC scheme can still afford a similar contour plot (not shown). The results from the comparison of case 1 versus case 2 indicate that one can readily modify a standard HPLC to comprehensive 2D-HPLC by adding a 6-port valve (or by simply using a 10-port valve with two sample loops as an injector, as mentioned in the Experimental Section) at the 2nd-D. Very fast elution at 2nd-D, possible with a short monolithic silica column, allows this approach to achieve high PC.

Figure 5. Chromatograms obtained at the 2nd-D in 2D separation using two C18 monolithic columns at the 2nd-D, Case 3. Fractionation every 15 s at the 1st-D. Other conditions were similar to those for Figure 3.

Case 3: Comprehensive 2D Separation Using Two Columns at the 2nd-D. The 1st-D detector outlet was connected to a switching valve that directed the flow to either one of the two injector loops of the two 2nd-D HPLC systems, as shown in Figure 1c. The 2nd-D loops alternately accept all the effluent from the 1st-D for 15-30 s. The alternate injection into the two 2nd-D columns allowed the separation time to be twice as long as the fractionation interval at the 1st-D. In a 2D-HPLC system using four columns at the 2nd-D, so much longer time could be used for the 2nd-D separation using so many chromatographs.17 When two equivalent columns are used at the 2nd-D, comprehensive 2D-HPLC can be completed in a single run. Figure 5 shows the 2nd-D chromatograms for a mixture of hydrocarbons and benzene derivatives using two C18 monolithic silica columns at the 2nd-D at 10 mL/min. Fractionation at the 1st-D operated at 0.8 mL/min was carried out at every 15 s to produce 200-µL fractions. Injections were made at 5.75, 6.25, and 6.75 min for the C18-I column (Figure 5a inset), and at 6.00, 6.50, and 7.00 min for C18-II (Figure 5b inset). A 2D chromatogram (not shown) obtained by combining the two sets of chromatograms in Figure 5 was similar to Figure 3c, which was obtained in case 1 with loss of several percent of 1st-D effluent by using one C18 column at the 2nd-D. PC of 1050 was obtained in ∼30 min, indicating the reduction of total separation time to produce similar PC. When the 1st-D was operated at 0.4 mL/min with 15-s fractionation time and 2nd-D at 10 mL/min, PC of ∼1190 was obtained in 65 min. The slightly greater PC in case 3 than in case 1 or case 2 was provided based on the more frequent fractionation at 1st-D. Figure 6 shows another example of 2D-RPLC separation using C18 and PBB monolithic columns at 2nd-D. When two different columns are used in 2nd-D, the system can provide combinations of two 2D separations, but requires two runs to obtain complete chromatograms that cover all 1st-D fractions with each of the two

different columns. At the 1st-D, the FR column was operated at 0.4 mL/min in 60% methanol, as in case 1. Fractions were alternately injected every 30 s into PBB and C18 columns operated at 7 mL/min with 60-s separation time in 80% methanol. One run starting with the 2nd-D injection into PBB at 0 min and another run starting with the 2nd-D injection into PBB at 0.5 min were required to complete the 2D separation. From the two separate complete runs obtained with two different 2nd-D columns, two sets of 2nd-D chromatograms were obtained. Such chromatograms obtained for two runs on the C18 phase were similar to those shown in Figure 5. Figure 6a shows the 2D separation of the mixture with the FR phase in combination with the C18 phase similar to Figure 3c, while Figure 6b shows a 2D chromatogram with FR and PBB columns. The two contour plots were each constructed from the two separate runs. The reproducibility of two separate runs with two different columns at 2nd-D was better than the case with two PBB columns at 2nd-D because of the poorer reproducibility of PBB columns. Actually panels a and b of Figure 6 are chromatographic expressions of panels c and d of Figure 6, the plots of k values of the hydrocarbons and benzene derivatives on the three stationary phases with each other measured independently. The location of spots in Figure 6a and b were similar to plots in Figure 6c and d, but the prediction of k values in the 2D system from Figure 6c and d was not very accurate. This is because of the injection of nearly 200 µL of much weaker mobile phase into a small-sized column (∼460 µL void volume) at 2nd-D. In Figure 6b and Figure 6d, PAHs were located close to the vertical axis, indicating very small retention and separation on the FR column and the effective retention and separation on the PBB phase. The separation was not possible with the FR column that provided very small dispersion interaction with aromatic compounds rich in π-electrons. Panels b and d of Figure 6 also showed that fluorineAnalytical Chemistry, Vol. 76, No. 5, March 1, 2004

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Figure 6. (a, b) Contour plots obtained for FR-C18 system (a) and for FR-PBB system (b) for 2D-HPLC separations of a mixture of hydrocarbons and benzene derivatives, using FR in 1st-D and C18 or PBB as a 2nd-D column in case 3. Fractionation every 30 s at the 1st-D. (c, d) Plots of k values measured independently on C18 phase (c) and on PBB phase (d) against k values obtained on FR phase. Temperature, 30 °C; mobile phase, 60% methanol for FR phase and 80% methanol for C18 and PBB phase. See Experimental Section for solute identification.

substituted benzenes were preferentially retained on the FR column, while they are not well retained or separated on the PBB column. Aliphatic compounds showed relatively short retention on the PBB phase due to the small dispersion interactions, which made the retention on the FR phase look significant compared to aromatic compounds. Retention on the FR column can be provided by the mobile-phase phenomena, so-called hydrophobic interactions,28 but counteracted by the lack of contribution of dispersion interaction. As previously reported, the retention of solutes on PBB and FR stationary phases showed an opposite tendency when plotted against the magnitude of a refractive index of a solute, which is a measure of potential dispersion interactions. Compounds containing heavy atoms were retained preferentially by the PBB phase. With the larger halogen atom on a phenyl ring (halogenated benzene), an increase in retention was observed on the C18 and PBB phase, while a slight decrease with little separation was observed on the FR phase.25 While the results in Figure 6a and b are predictable from Figure 6c and d, respectively, the present results have proved the 1280 Analytical Chemistry, Vol. 76, No. 5, March 1, 2004

possibility of comprehensive 2D-RPLC using these stationary phases of widely different selectivity. PC of ∼850 was obtained for C18-FR system and ∼750 for FR-PBB system. The lower flow rate at 2nd-D resulted in a decrease in PC despite the increase in column efficiency. FR and PBB columns showed nearly orthogonal difference in selectivity that allowed grouping of separated peaks. The results shown here indicate that 2D-RPLC using the combination of C18, PBB, and FR stationary phases, which represent stationary phases of the greater and smaller contribution of dispersion interactions to solute retention, will be effective for separation and characterization of organic compounds. Greater PC can be expected by increasing PC of each dimension by using a column of higher efficiency or by employing higher flow rate (smaller t0). More than 10 times higher flow rate was employed at 2nd-D also in previous studies,8,18 but the flow rate at 1st-D was much lower than optimum for the column employed. In the present system, the 1st-D column was operated at a nearly optimum flow rate, and the 2nd-D column was about 12-25 times faster. The flow rate applied for the present column

of 4.6-mm i.d., however, led to consumption of a large amount of mobile-phase solvents. Thus, it is desirable to carry out 2D separations with a miniaturized system.

and Industry, through Kansai Bureau of Economy, Trade and Industry, and Osaka Science and Technology Center.

ACKNOWLEDGMENT This work was supported in part by a Grant-in-Aid for Scientific Research funded by the Ministry of Education, Sports, Culture, Science and Technology, and by the Ministry of Economy, Trade

Received for review August 8, 2003. Accepted December 9, 2003. AC034925J

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