Anal. Chem. 1999, 71, 3477-3483
Separation of Carotenoid Isomers by Capillary Electrochromatography with C30 Stationary Phases Lane C. Sander,* Matthias Pursch, Bernd Ma 1 rker,† and Stephen A. Wise
National Institute of Standards and Technology, Gaithersburg, Maryland 20899
The use of a polymerically bonded C30 stationary phase is demonstrated in capillary electrochromatography (CEC) for the separation of carotenoid isomers. Column selectivity in CEC was found to be equivalent to that observed with liquid chromatography (LC); however, CEC column efficiency was significantly improved over LC efficiency. Gradient elution capability was achieved by the combined use of a liquid chromatographic pump and a CEC apparatus. The separation of carotenoid isomers from food and algal extracts is demonstrated. Interest in capillary electrochromatography continues to grow at a rapid pace as refinements in the technique are reported.1-5 The motivation for this interest is the promise of high separation efficiencies comparable to those obtained with gas chromatography or capillary electrophoresis. The limits in column efficiency predicted by Knox and co-workers6,7 have not yet been achieved, and the focus of much of the current research in CEC is with column technology. Columns utilizing 1.5 and 0.5 µm diameter substrates have been reported,8-10 and approaches utilizing continuous beds prepared in situ have been described.11-13 Column efficiencies in CEC commonly exceed LC column efficiencies by a factor of 2-3 (i.e., 200 000-300 000 plates/m), and reduced plate heights for well-packed CEC capillaries are typically less than 2.3,7 Less importance has been placed on the study of column selectivity in CEC. Most work to date has utilized octadecyl (C18) stationary phases; however, as with conventional † Current address: Department of Analytical and Environmental Chemistry, University of Ulm, D-89081 Ulm, Germany. (1) Robson, M. M.; Cintas Moreno, P.; Myers, P.; Melvin, R. E.; Bartle, K. D. J. Microcolumn Sep. 1997, 9, 357-372. (2) Colon, L. A.; Guo, Y.; Fermier, A. Anal. Chem. 1997, 69, 461A-467A. (3) Dittmann, M. M.; Wienand, K.; Bek, F.; Rozing, G. P. LC-GC 1995, 13, 800-814. (4) Dittmann, M. M.; Rozing, G. P. J. Chromatogr., A 1996, 744, 63-74. (5) Miyawa, J. H.; Alasandro, M. S. LC-GC 1998, 16, 36-41. (6) Knox, J. H.; McCormack, K. A. J. Liq. Chromatogr. 1989, 12, 2435-2470. (7) Knox, J. H.; Grant, I. H. Chromatographia 1991, 32, 317-328. (8) Seifar, R. M.; Kok, W. T.; Kraak, J. C.; Poppe, H. Chromatographia 1997, 46, 131-136. (9) Behnke, B.; Bayer, E. J. Chromatogr., A 1994, 680, 93-98. (10) Luedtke, S.; Adam, T.; Unger, K. K. J. Chromatogr., A 1997, 786, 229235. (11) Ericson, C.; Liao, J. L.; Nakazato, K.; Hjerten, S. J. Chromatogr., A 1997, 767, 33-41. (12) Peters, E. C.; Petro, M.; Svec, F.; Frechet, J. M. J. Anal. Chem. 1997, 69, 3646-3649. (13) Peters, E. C.; Petro, M.; Svec, F.; Frechet, J. M. J. Anal. Chem. 1998, 70, 2288-2295.
10.1021/ac990311w Not subject to U.S. Copyright. Publ. 1999 Am. Chem. Soc.
Published on Web 07/17/1999
LC, the use of different stationary-phase chemistries should provide a way of altering selectivity in CEC. Carotenoids constitute a class of isomeric compounds that are important as nutrients and as naturally occurring pigments. These compounds exhibit antioxidant properties and may provide benefits in the prevention of disease. The measurement of individual carotenoid species is challenging due to the complexity of natural sources and the similarity of isomers within these mixtures. Previously we described the development of an LC stationary phase based on C30 polymeric surface modification chemistry that exhibited enhanced selectivity for carotenoid isomers.14 Better separations of complex carotenoid mixtures were demonstrated with this column than could be achieved with C18 columns, even though efficiency was somewhat lower than for C18 columns. Solid-state NMR investigations of C30 phases revealed that the enhanced selectivity results from highly ordered alkyl chains, providing excellent molecular shape recognition for carotenoids and tocopherols.15-17 In this paper, we report the first use of polymeric C30 stationary phases in CEC for the separation of carotenoid isomers. The combination of high efficiency and high selectivity permits better separations of this class of compounds than has been previously achieved. EXPERIMENTAL SECTION18 Materials. Carotenoid standards were obtained from sources previously identified.19a Structures of the various trans carotenoid isomers with numbering are shown in Figure 1. Cis isomer mixtures were prepared by iodine isomerization of trans standards by the general method of Zechmeister.19b A β-carotene preparation derived from Dunaliella algae was obtained from Betatene Ltd. (Melbourne, Australia). A composite food sample similar to SRM 2383 “Baby Food Composite” was extracted and saponified using the procedure of Sharpless et al.20 The ingredients in this sample are as follows: creamed corn (30%), macaroni/tomato/beef (30%), (14) Sander, L. C.; Sharpless, K. E.; Craft, N. E.; Wise, S. A. Anal. Chem. 1994, 66, 1667-1674. (15) Pursch, M.; Strohschein, S.; Handel, H.; Albert, K. Anal. Chem. 1996, 68, 386-393. (16) Strohschein, S.; Pursch, M.; Lubda, D.; Albert, K. Anal. Chem. 1998, 70, 13-18. (17) Albert, K.; Lacker, T.; Raitza, M.; Pursch, M.; Egelhaaf, H. J.; Oelkrug, D. Angew. Chem., Int. Ed. Engl. 1998, 37, 777-780. (18) Certain commercial equipment, instruments, or materials are identified in this report to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose. (19) (a) Emenhiser, C.; Sander, L. C.; Schwartz, S. J. J. Chromatogr. 1995, 707, 205-216. (b) Zechmeister, L. Cis-Trans Isomeric Carotenoids, Vitamins A, and Arylpolyenes, 1st ed.; Academic Press: New York, 1962.
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Figure 1. Structures of various carotenoid isomers, with numbering.
orange juice concentrate (20%), powdered infant formula (15%), papaya juice concentrate (3%), and creamed spinach (2%) (all percentages are mass fractions). HPLC grade solvents were used for preparation of buffers and mobile phases. Samples were prepared in solvent mixtures (acetone/buffer for CEC) with compositions similar to the mobile phase starting conditions. Column Preparation. Polymeric C30 stationary phases were prepared on Rainin 30 nm pore size, 3 µm particle size silica (Varian/Rainin HPLC, Walnut Creek, CA) or ProntoSIL 30 nm pore size, 3 µm particle size silica (Bischoff Chromatography, Leonberg, Germany) using procedures previously published.14 A
Figure 2. Block diagram of the gradient elution CEC apparatus. 3478 Analytical Chemistry, Vol. 71, No. 16, August 15, 1999
362 µm o.d. × 98 µm i.d. fused-silica tubing (Polymicro Technologies Inc., Phoenix, AZ) was packed using a liquid chromatographic pump or syringe pump as the pressure source. The bed length was 25 cm, and the detection window was placed adjacent to the outlet frit. Columns used with the HP 3D apparatus (see below) were 33 cm in total length. The slurry reservoir was built from a 10 mm i.d. semipreparative guard column cartridge (Upchurch Scientific, Oak Harbor, WA) by removal of the outlet frit. Connection was made between the fused-silica tubing and stainless steel fittings by use of 2 cm lengths of 381 µm (0.015 in.) i.d. × 1/ in. o.d. PEEK tubing (Upchurch Scientific). Microtight fittings 16 and tubing sleeves (Upchurch Scientific) were also used to make connections to fused-silica tubing. Packed capillary column frits were prepared by heating using an optical fiber splicer (Power Technology Inc., Little Rock, AR). Arc duration and current were adjusted to provide sufficient heat for frit consolidation. Suitable arc conditions were observed to vary with the solvent and pressure applied. Under an applied pressure of ∼28 MPa with an aqueous environment, good-quality frits were obtained using an arc current of 13 mA for 1.5 s. It was found that an aqueous environment resulted in the production of more consistent white homogeneous frits. Details of column production are described under Results and Discussion. Capillary Electrochromatography. Isocratic CEC separations were carried out using an HP 3D capillary electrophoresis instrument with vial pressurization modification (Hewlett-Packard, Wilmington, DE). Injections were made electrokinetically, typically for 3 s at 20 kV. Separations were carried out at 30 kV, with inlet and outlet vials pressurized to 1 MPa. Absorbance detection at 450 nm was used. All mobile phase solvents were degassed under vacuum prior to use. Mobile phase compositions are indicated in the figures. Buffer solutions were 1 mM borate buffer. Gradient elution CEC separations were carried out on an apparatus utilizing a liquid chromatographic pump to vary the mobile phase composition at the inlet of the CEC capillary. Details of the instrumental design are shown in Figure 2. A Varian 9012 liquid chromatograph (Varian Associates, Sugar Land, TX) was connected to a Valco 10 nL injector (Valco Instrument Co., Inc.,
Figure 3. Comparison of the separation of R- and β-carotene isomers by isocratic CEC and LC with absorbance detection at 450 nm: (a) CEC, R-carotene isomers; (b) LC, R-carotene isomers; (c) CEC, β-carotene isomers; (d) LC, β-carotene isomers.
Houston, TX) with 100 µm i.d. capillary tubing. The connection between the pump and PEEK cross was made with 150 µm i.d. × 150 cm length capillary tubing. The connection between the PEEK cross and injector was made with 75 µm i.d. × 6 cm length tubing. The inlet of the CEC column was placed directly in the outlet port of the injection valve to minimize dead volume. Injections were made either conventionally by valve switching or electrokinetically with the following procedure. With the LC pump stopped and without an applied potential, the injector is positioned in the load position and the rotor loop is filled. Next, the injector is positioned in the inject position and an electrical potential is applied for a short interval. Finally, the injector is repositioned in the load position, the pump gradient program is started, and the electric field is again applied. In this way, injections can be made with sample volumes less than the rotor volume. Connections were made using Microtight crosses (Upchurch Scientific) before the injector and after the detector, with platinum wire electrodes for
Figure 4. Isocratic CEC separations of the isomers of common carotenoid compounds with absorbance detection at 450 nm.
CEC operation. This design permitted column pressurization to 42 MPa, for pressure-assisted operation. A Linear model 205 UV/ vis detector (Alltech Associates, Deerfield, IL) with a capillary interface was used for detection. Care was taken to isolate the high-voltage electrode from grounded surfaces. The CEC portion of the apparatus was enclosed in an acrylic plastic box fitted with an electrical interlock. The LC instrument was located outside the box. Pressure-assisted CEC operation was achieved by splitting Analytical Chemistry, Vol. 71, No. 16, August 15, 1999
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Figure 5. Isocratic CEC separation of carotene isomers in extracts of two preparations of Dunaliella algae.
the mobile phase flow through a 75 µm i.d. capillary. Pressure was adjusted by varying the length of this restrictor (ca. 80 cm) and/or varying the mobile phase flow rate (ca. 0.6-0.7 mL/min). The resulting split ratio was not explicitly determined; however, the applied pressure was typically set at 8-10 MPa. The flow was restricted at the column outlet with an 80 cm length of 50 µm i.d. capillary tubing to minimize bubble formation. Liquid Chromatography. Comparison LC separations were performed using a Varian 5000 liquid chromatograph. Detection was performed at 450 nm using a Linear 204 UV/vis detector. Columns of 250 mm × 4.6 mm dimensions were packed with the same C30 stationary phase used for CEC. Separations of R-carotene isomers were carried out with a methanol/methyl tert-butyl ether/ water (35/60/5 v/v) mobile phase composition. β-Carotene isomer separations were carried out with an acetone/water (95/5 v/v) mobile phase composition. The LC flow rate was adjusted to approximate the linear velocity for the corresponding CEC separation. The injection volume was 10 µL. RESULTS AND DISCUSSION Considerable importance has been placed on the preparation of column frits for high-efficiency packed capillaries.21,22 Approaches to frit fabrication have included creation of porous monolithic plugs by silicate-formaldehyde based chemistries22,23 and heating processes3,10,21,24,25 for substrate immobilization. Sodium or potassium silicate has also been used in combination with heating processes to “glue” silica or bonded silica particles (20) Sharpless, K. E.; Arce-Osuna, M.; Brown Thomas, J.; Gill, L. M. J. AOAC Int. 1999, 82, 288-296. (21) Boughtflower, R. J.; Underwood, T.; Paterson, C. J. Chromatographia 1995, 40, 329-335. (22) Behnke, B.; Grom, E.; Bayer, E. J. Chromatogr., A 1995, 716, 207-213. (23) Cortes, H. J.; Pfeiffer, C. D.; Richter, B. E.; Stevens, T. S. J. High Resolut. Chromatogr. Chromatogr. Commun. 1987, 10, 446. (24) Yamamoto, H.; Baumann, J.; Erni, F. J. Chromatogr. 1992, 593, 313-319. (25) Yan, C.; Schaufelberger, D.; Erni, F. J. Chromatogr., A 1994, 670, 15-23.
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together.21,26,27 Boughtflower et al. and others described the preparation of capillary end frits by point heating of the actual stationary phase in the presence of water21 or other solvents.3 The ideal process for column preparation results in an efficiently packed capillary with a stable bed structure, maintained by frits that do not adversely affect column efficiency or permeability. A very useful discussion of theoretical and practical aspects of packing capillaries has been published by Tong et al.28 The approach described in the Experimental Section is similar to published methodology, but it differs in several details. Two types of column frits can be distinguished. Initial, temporary frits are made under pressure to maintain bed integrity during depressurization. Final end frits are prepared on the resulting stabilized bed under static conditions, resulting in homogeneous, stable frits. The initial frit is created in the packing solvent (typically pentane) and is black. The possible influence of such carbonaceous frits on chromatographic performance was not investigated, but a higher failure rate due to pressurization was evident for these frits. Better pressure stability was observed for frits with an overall white appearance. The elimination of the organic solvent with water promoted the formation of such noncarbonaceous frits. During and immediately after the heating process, a slight compaction of the bed below the frit was sometimes observed. In such cases, additional frits were created at ∼1 cm intervals until further compaction did not occur. This process was intended to create a densely packed, stable bed. Because the substrate particles are rigidly held by the temporary end frits, subsequent frits can be prepared without pressurization. The resulting final inlet and outlet frits have the same apparent consistency as the column bed. It should be noted that possible chromatographic differences of “good” and “bad” frits were not evaluated; however, it seems reasonable to avoid the production of frits with inhomogeneities and voids. A comparison of CEC and conventional LC separations is shown in Figure 3. Using the same bonding lot of polymeric C30modified silica, a 4.6 mm × 25 cm column was prepared for comparison with a 98 µm i.d. × 25 cm packed capillary. The separation of R-carotene isomers (Figure 3a) was carried out by CEC at 30 kV, using a mobile phase composition of 35/60/5 methanol/methyl tert-butyl ether/1 mmol/L borate buffer. The mobile phase linear velocity, as determined by the injection of acetone, was ∼0.7 mm/s. Using a different mobile phase system containing acetone and 1 mmol/L borate buffer, the linear velocity was ∼1.4 mm/s. This value is comparable to flow velocities reported for various C18 stationary phases. The separation was carried out by conventional LC at the same mobile phase composition and approximate linear velocity (see Figure 3b). As expected, column selectivity is equivalent for the two separations, and separation efficiency is significantly improved for CEC. Approximately 46 000 theoretical plates were generated for the CEC separation of R-carotene isomers (N/m ) 184 000; reduced plate height h ) 1.8) compared with 9100 theoretical plates for the LC separation. The lower-than-expected plate count for the (26) Zimina, T. M.; Smith, R. M.; Myers, P. J. Chromatogr., A 1997, 758, 191197. (27) Rebscher, H.; Pyell, U. Chromatographia 1994, 38, 737-743. (28) Tong, D.; Bartle, K. D.; Clifford, A. A.; Edge, A. M. J. Microcolumn Sep. 1995, 7, 265-278.
Figure 6. Gradient elution CEC separation of isomerized carotenoid standards. Conditions: 0.6 mL/min (before splitter); initial applied pressure 11.5 MPa; composition 80% acetone/buffer to 99% acetone/buffer over 18 min and hold. Buffer composition: 1 mM sodium borate.
LC separation is somewhat characteristic of polymeric C30 columns used in conventional LC. Peak shape is also improved for the CEC separation. Peak asymmetries for trans-R-carotene were 1.02 for CEC and 1.67 for the LC separation. A similar comparison was made for β-carotene isomers, separated with a polymeric C30 phase prepared with a different silica substrate. Separations by CEC and conventional LC are shown in Figure 3, parts c and d, respectively. Additional components present in Figure 3d are the result of differences in samples. High efficiency and symmetric peak shape were demonstrated for the CEC separation. Efficiencies reached up to 280 000 plates/m. The influence of injection volume on efficiency27,29 was investigated for the separation of R-carotene isomers. As expected, efficiency is improved for reduced injection volume. Injection solvent strength greatly influences sample loadability, and when feasible, samples should be dissolved in the mobile phase. This effect is quite dramatic, and injection volume should be carefully considered in method development in CEC. Because geometric isomers are often very similar in polarity, isocratic elution is appropriate for separation of such mixtures. CEC separation of iodine-isomerized mixtures of several common carotenoids is illustrated in Figure 4. These chromatograms represent the most complete separations of cis and trans caro(29) Pyell, U.; Rebscher, H.; Banholczer, A. J. Chromatogr., A 1997, 779, 155163.
tenoid isomers yet reported. Peak assignments for R- and β-carotene isomers are based on UV/vis spectroscopy and 1H NMR studies.18,30 Assignments for lutein, zeaxanthin, and lycopene isomers are based on LC NMR studies.31,32 Because iodine isomerization results in an equilibrium mixture of cis and trans isomers in constant ratios for a given carotenoid compound, it might be expected that iodine isomerization of dihydroxysubstituted carotenoids would result in similar proportions of cis and trans isomers compared with the unsubstituted parent carotenoids. Thus, the separation of isomers of lutein is similar to that for R-carotene, and likewise, the separation of zeaxanthin isomers is similar to that for β-carotene. High levels of 9-cis-β-carotene are produced by Dunaliella algae, and these algae are grown commercially as a source of this nutrient. The levels of cis isomers are known to change depending on growth conditions. Separations of extracts from different preparations of Dunaliella are shown in Figure 5. The major components of the mixture are baseline resolved, and the chromatograms represent considerable improvements over previously published separations.14 (30) Emenhiser, C.; Englert, G. E.; Sander, L. C.; Ludwig, B.; Schwartz, S. J. Chromatogr., A 1996, 719, 333-343. (31) Strohschein, S. Dissertation, Universita¨t Tu ¨ bingen, 1997. (32) Dachtler, M.; Kohler, K.; Albert, K. J. Chromatogr., B 1998, 720, 211-216.
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Figure 7. Gradient elution CEC separation of a composite food extract. Conditions: 0.6 mL/min (before splitter); initial applied pressure 9 MPa; composition 80% acetone/buffer to 99% acetone/buffer over 17 min and hold. Buffer composition: 1 mM sodium borate.
The separation of mixtures containing both polar and nonpolar compounds is usually accomplished by gradient elution. Gradient elution presents a special challenge with CEC. Although commercial instrumentation is not yet available, gradient elution CEC has been demonstrated with custom apparatus. Two approaches have been used. Huber et al.33 and Taylor et al.34,35 modified CE apparatus to accept gradient generation from a liquid chromatograph. In addition to this approach, Yan et al. has also described gradient generation by the use of dual, programmed power supplies.36 Solvent composition is varied by controlling the potential applied to solvent reservoirs joined at a “T” union. Regardless of the approach, changes in mobile phase composition inevitably alter current and flow rate. Because of these variations in flow, gradient elution methods from conventional LC and CEC may not be directly comparable. CEC separations were carried out on samples containing both xanthophylls (polar carotenoids) and hydrocarbon carotenoids. Because of the wide range in polarity of the analytes, isocratic separations are not practical, and pressure-assisted gradient elution CEC separations were performed. The column was pressurized at 8-10 MPa to increase system reliability and stability, and CEC current was observed to increase with applied pressure. Operation was also possible in a pressure-driven mode, without applied potential. The increases in separation efficiency realized by pressure-assisted CEC compared with pressure-driven LC were (33) Huber, C. G.; Choudhary, G.; Horvath, C. Anal. Chem. 1997, 69, 44294436. (34) Taylor, M. R.; Teale, P.; Westwood, S. A.; Perrett, D. Anal. Chem. 1997, 69, 2554-2558. (35) Taylor, M. R.; Teale, P. J. Chromatogr., A 1997, 768, 89-95. (36) Yan, C.; Dadoo, R.; Zare, R. N.; Rakestraw, D. J.; Anex, D. S. Anal. Chem. 1996, 68, 2726-2730.
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relatively small, especially compared with the dramatic improvements observed between isocratic CEC and LC separations (i.e., Figure 3). Separation of carotenoid standards is shown in Figure 6. The sample was prepared by mixing isomerized solutions of polar (lutein, zeaxanthin, echinenone) and nonpolar (R- and β-carotene) carotenoid standards. Excellent separations of the polar and nonpolar carotenoid isomers were achieved in less than 20 min. Most of the important cis and trans isomers are baseline resolved; only the 9-cis- and trans-R-carotene partially coelute. A CEC separation of a food extract is illustrated in Figure 7. The sample is a saponified extract of a composite food sample (baby food; see Experimental Section). Because of the presence of the very nonpolar hydrocarbon lycopene, the separation required slightly longer than 30 min under the given conditions. Although peak area reproducibility was not assessed, retention time reproducibility (typically 5% RSD) was poorer than for conventional LC. Separation efficiency for the lycopene isomers is lower compared to that for the other carotenoid isomers, probably because of reduced electroosmotic flow under the given mobile phase composition (acetone/buffer, 99/1 v/v). Very narrow peak bandwidths were observed for lutein and other polar carotenoids in the extracted food sample (Figure 7). These peaks also exhibit pronounced tailing and are much wider at the base than are later eluting peaks. This apparent focusing effect was not observed for the same components in solution standards. The identity of the affected components was verified by spiking the extracts with xanthophyll standards. In addition to the reduction in peak bandwidth, retention was also reduced compared to solution standard samples. The source of this anomaly has not been identified and is under investigation.
CONCLUSIONS Polymeric C30 stationary phases are suitable for use in capillary electrochromatography and generate flow velocities similar to those of C18 stationary phases. Although such phases exhibit relatively low efficiencies in pressure-driven liquid chromatography, column efficiencies for the polymeric C30 phase operated in the CEC mode were comparable to those obtained with C18 phases. The combination of high efficiency and high selectivity permits better separations of carotenoid mixtures than have previously been demonstrated.
ACKNOWLEDGMENT We thank Bischoff Chromatography (Leonberg, Germany) for providing ProntoSIL. M.P. wishes to thank the Alexander von Humboldt Foundation for financial support. The authors further thank Chao Yan (Unimicro Technologies, Pleasanton, CA) and John Dorsey (Florida State University, Tallahassee, FL) for helpful discussions. Received for review March 23, 1999. Accepted May 23, 1999. AC990311W
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