Anal. Chem. 2009, 81, 7428–7435
Nanocapillaries for Open Tubular Chromatographic Separations of Proteins in Femtoliter to Picoliter Samples Xiayan Wang,† Chang Cheng,† Shili Wang,† Meiping Zhao,‡ Purnendu K. Dasgupta,§ and Shaorong Liu*,† Department of Chemistry and Biochemistry, The University of Oklahoma, Norman, Oklahoma 73019, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, P.R. China, and Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, Texas 76019 We have recently examined the potential of bare nanocapillaries for free solution DNA separations and demonstrated efficiencies exceeding 106 theoretical plates/ m. In the present work, we demonstrate the use of bare and hydroxypropylcellulose (HPC) coated open tubular nanocapillaries for protein separations. Using 1.5 µm inner diameter (i.d.) capillary columns, hydrodynamically injecting femto- to picoliter volumes of fluorescent or fluorescent dye labeled protein samples, utilizing a pneumatically pressurized chamber containing 1.0 mM sodium tetraborate solution eluent (typically 200 psi) as the pump, and performing on-column detection using a simple laser-induced fluorescence detector, we demonstrate efficiencies of close to a million theoretical plates/m while generating single digit microliter volumes of waste for a complete chromatographic run. We achieve baseline resolution for a protein mixture consisting of transferrin, r-lactalbumin, insulin, and r-2-macroglobulin. Since the pioneering work of Horvath and co-workers1,2 in liquid chromatography (LC), there has been a continuous interest in performing LC separations in ever smaller scales. Various forms of capillary LC highlight this trend.3-6 There are many benefits of downsizing LC columns: low sample requirement, negligible waste generation, improved resolution, increased separation speed, ability to use exotic eluents, and so on. There are also many hurdles: One needs injection valves capable of introducing picoliter to nanoliter sample volumes, highpressure pumps capable of generating stable flows at nanoliter/ minute to microliter/minute flow rates, picoliter to nanoliter * Corresponding author. E-mail:
[email protected]. † The University of Oklahoma. ‡ Peking University. § The University of Texas at Arlington. (1) Horvath, C. G.; Preiss, B. A.; Lipsky, S. R. Anal. Chem. 1967, 39, 1422– 1428. (2) Horvath, C. G.; Lipsky, S. R. Anal. Chem. 1969, 41, 1227–1234. (3) Ishii, D. Jasco Rep. 1974, 11, 1–7. (4) Hibi, K.; Ishii, D.; Fujishima, I.; Takeuchi, T.; Nakanishi, T. J. High Resolut. Chromatogr. Chromatogr. Commun. 1978, 1, 21–27. (5) Tsuda, T.; Novotny, M. V. Anal. Chem. 1978, 50, 632–634. (6) Dandeneau, R. D.; Zerenner, E. H. J. High Resolut. Chromatogr. Chromatogr. Commun. 1979, 2, 351–356.
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volume connectors capable of sustaining high pressures, and detectors capable of detecting picoliter to nanoliter volume eluite bands. This situation has improved in recent years. For example, injection valves with 10-20 nL injection volumes are now commercially available (see, e.g., www.vici.com), dispensing pumps based on microsyringes or a new generation of bidirectional precision pumps7 are commercially produced, picoliter-volume connectors have been microfabricated,8 and on-column laserinduced fluorescence (LIF) detectors can routinely detect fluorescent molecules just a few hundred in number.9 Issues nevertheless remain for practical capillary LC. Open tubular liquid chromatography (OTLC)10-15 utilizes open capillaries with inner diameters normally ranging from a few to tens of micrometers. In addition to the advantages common to all capillary LC formats, OTLC does not require high-pressure pumps. The theoretical limitations and characteristics of microcolumn chromatography were originally discussed in detail by Knox and Gilbert16 and Knox.17 With larger diameter open tubular columns, the mass transfer between stationary and mobile phases becomes the rate limiting factor. Theoretically, this issue can be addressed by reducing the column diameter. The separation efficiencies (in terms of the number of theoretical plates per unit column length) are predicted to increase with decreasing column diameter; >106 plates/m are predicted to be possible with submicrometer diameter capillaries. The quest to continually improve efficiency has been unending ever since liquid phase chromatography was invented.18 The advent of uniformly packed columns was a major breakthrough;19,20 such columns provided fine and uniform pathways for the mobile (7) Global FIA Inc. Milligat Pump. http://www.globalfia.com/ItemSheets/ MG.html (accessed June 2009). (8) Liu, S. Electrophoresis 2003, 24, 3755–3761. (9) Turner, E. H.; Lauterbach, K.; Pugsley, H. R.; Palmer, V. R.; Dovichi, N. J. Anal. Chem. 2007, 79, 778–781. (10) Ishii, D.; Tsuda, T.; Takeuchi, T. J. Chromatogr. 1979, 185, 73–78. (11) Tsuda, T.; Tsuboi, K.; Nakagawa, G. J. Chromatogr. 1981, 214, 283–290. (12) Tsuda, T.; Nakagawa, G. J. Chromatogr. 1983, 268, 369–374. (13) Ishii, D.; Takeuchi, T. J. Chromatogr. Sci. 1984, 22, 400–410. (14) Jorgenson, J. W.; Guthrie, E. J. J. Chromatogr. 1983, 255, 335–348. (15) Knecht, L. A.; Guthrie, E. J.; Jorgenson, J. W. Anal. Chem. 1984, 56, 479– 482. (16) Knox, J. H.; Gilbert, M. T. J. Chromatogr. 1979, 186, 405–418. (17) Knox, J. H. J. Chromatogr. Sci. 1980, 18, 453–461. (18) Tswett, M. S. Ber. Dtsch. Bot. Ges. 1906, 234, 384–393. (19) Huber, J. F. K. J. Chromatogr. 1969, 7, 86–97. (20) Kirkland, J. J. J. Chromatogr. Sci. 1969, 7, 7–12. 10.1021/ac901265t CCC: $40.75 2009 American Chemical Society Published on Web 08/10/2009
Figure 1. Schematic diagram of the experimental setup.
phases to pass through. Each pathway can be considered equivalent to the bore of an open capillary tube. The smaller the diameter of these pathways, the shorter is the mass transport time between the mobile and stationary phases. Accordingly, smaller and smaller particles are being used to improve the efficiency of packed LC columns.21 The highest density packing possible with uniform spheres is hexagonal close packing (HCP). In HCP, both tetrahedral and octahedral voids are formed, and the largest spheres that can fit through these voids respectively have diameters of 0.225d and 0.414d where d is the diameter of the individual spheres.22 The average pathway diameters for columns packed with the present state of the art 1-2 µm diameter nonporous spherical particles range between 400-800 nm;23 this falls short of the above idealized goals. Nevertheless, the high efficiencies with these columns can be viewed as a direct consequence of the reduction of these pathway diameters. We have recently utilized submicrometer (nominally 500 nm as stated by the manufacturer, more recently measured by us by scanning electron microscopy to be 750 nm, see Figure S1 in the Supporting Information) radius open tubes, ∼0.5 m long, to separate DNA molecules; with pneumatically driven eluents at ∼100 psi, we routinely observed efficiencies >106 plates/m.24 In the present manuscript, we observe that hydrodynamically injected femtoliter to picoliter volumes of fluorescently labeled protein samples, containing a few hundred to a few thousand copies of protein molecules in the aliquot, can be separated with nearly as high efficiencies and detected in the same manner. We discuss both the potential possibilities and the paradoxes associated with this approach to protein analysis. EXPERIMENTAL SECTION Reagents and Materials. R-2-Macroglobulin (from human plasma, www.mpbio.com), Recombinant Aequorea coerulescens (21) MacNair, J. E.; Patel, K. D.; Jorgenson, J. W. Anal. Chem. 1999, 71, 700– 708. (22) Azaroff, L. V. Introduction to Solids; McGraw-Hill: New York, 1960. (23) Unger, K. K.; Skudas, R.; Schulte, M. M. J. Chromatogr., A 2008, 1184, 393–415. (24) Wang, X.; Wang, S.; Veerappan, V.; Byun, C.; Nguyen, H.; Gendhar, B.; Allen, R. D.; Liu, S. Anal. Chem. 2008, 80, 5583–5589.
green fluorescent protein (rAcGFP1, www.clontech.com), human transferrin (www.lifetech.com), and ATTO-TAG FQ amine derivatization kit (www.invitrogen.com) were purchased as indicated. All other proteins, sodium dodecyl sulfate (SDS) and hydroxypropylcellulose (HPC) were from www.sial.com. Sodium hydroxide, methanol, and sodium tetraborate decahydrate (Na2B4O7) were from www.fishersci.com; all fused-silica capillaries were from www.polymicro.com; nanocapillaries (360 µm o.d., 1.5 µm i.d.) were custom-made. All solutions were prepared using ultrapure water (UPW, Barnstead Nanopure, www.thermo.com) and filtered through a 0.22 µm filter (P/N 100370-922, www.vwrsp. com) and degassed by vacuum before use. Apparatus. The experimental setup is schematically shown in Figure 1. The helium-pressurized chamber, consisting of a cap and a base (7.8 mm i.d., 29.5 mm deep, accommodates a 600 µL microcentrifuge tube to serve as a sample/eluent vial) was machined in-house from poly(methylmethacrylate). A septum was put between the base and the cap to ensure a seal. A high-pressure regulator was used to control the applied pressure ( R-lactalbumin> insulin> R-2macroglobulin. These differing slopes result in the retention values that approach each other at high concentrations and as a result, despite improved efficiencies, selectivity and hence resolution, are lost. This was presumably due to the protein conformation changes with the concentrations. The conformations of these proteins likely differ to a greater extent at low concentrations while at high ionic strengths they all assume the most tightly packed conformation where the exterior exposed surface area is minimized. Similar results, notably that resolutions deteriorate while separation efficiencies improve with increasing eluent concentration, were also observed with NaOH as the eluent (detailed data are shown in Figure S3 in the Supporting Information). SDS was also tested as an additive in the eluents, but there was no beneficial effect on the separation (Figure 5). The concentrations used here are below the critical micelle concentration of SDS. Any adsorption of the surfactant, either on the wall (unlikely, as the wall is already negatively charged) or association with the analyte (unlikely, because above the pI, the analyte is also negatively charged), is expected to reduce the retention. Paradoxically, as the log tR log [SDS] plot in the inset indicates, the retention increases on SDS addition. The log tR - log [SDS] plots are almost linear with positive slopes but the slopes differ among the analytes in a manner that much like increasing eluent concentration, addition of SDS results in loss of selectivity. Further work was carried out solely with 1.0 mM Na2B4O7 as the eluent, without additives. Effect of Capillary Inner Diameter. Capillary diameter is a key parameter that affects the separation efficiency. We have tested capillaries with three different diameters (1.5, 2.4, and 4.2
Figure 5. Effect of SDS as an additive on the separation of the four proteins transferrin (T), R-lactalbumin (L), insulin (I), and R-2macroglobulin (M) in a 1.5 µm i.d., 70 cm long (65 cm effective) capillary at 300 psi. Samples were injected for 8 s at 100 psi. The inset shows log tR - log [SDS] plots.
µm). Figure 6 presents the separation results when all other experimental conditions were maintained the same (including column lengths: 50 cm total and 45 cm effective and eluent pressure 150 psi). With the 4.2 µm capillary, the analytes eluted fast and without resolution. With the 2.4 µm capillary, the analytes are nearly resolved; predictably, even better resolution is obtained with the even smaller 1.5 µm capillary. It is reasonable to conclude that efficiency and resolution will continue to improve with narrower capillaries. Presently, extruded capillaries are not available in diameters that are still smaller but we hope to report on results of similar separations using nanofabricated channels in the near future. Meanwhile it is interesting to note that based on the diameters and the applied pressure, the void retention times are 9.65, 29.5, and 75.7 min, respectively for the 4.2, 2.4, and 1.5 µm i.d. capillaries; all of the traditionally computed k′ from the largest to the smallest capillary are negative. Table S1 in the Supporting Information lists the values; these do not scale with capillary bore in any consistent manner. The results of a different experiment, where at a constant applied pressure the length of the larger capillaries were adjusted until almost the same retention time was obtained for the group of analytes is shown in Figure 7. Detailed numerical data are shown in Table S2 in the Supporting Information for the two smaller bore capillaries, and it is interesting that although the resolution is better for the smallest bore capillary, the efficiency in terms of the number of plates (for all but transferrin, also plates/ m) is the best for the intermediate bore capillary. Effect of Flow Rate (Elution Pressure). The effect of elution rate (or elution pressure) on both resolution and separation efficiency was systematically examined for a 70 cm long, 1.5 µm i.d. capillary. We measured the resolution between the transferrin and the lactalbumin peaks as well as the respective plate heights. As shown in Figure 8, both the optimum resolution and efficiency was obtained at a pressure of ∼200 psi; low micrometer plate Analytical Chemistry, Vol. 81, No. 17, September 1, 2009
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Figure 6. All separation capillaries had the same length (50 cm total and 45 cm effective). Sample injection duration and pressure were adjusted to ensure injections of sample plugs of nearly equivalent length with 4 s at 15 psi for a 4.2 µm capillary, 5 s at 40 psi for a 2.4 µm capillary, and 8 s at 100 psi for a 1.5 µm capillary. The elution pressure was 150 psi.
Figure 7. The chromatograms were, respectively, obtained with a 155 cm long (150 cm effective) × 4.2 µm i.d., a 70 cm long (65 cm effective) × 2.4 µm i.d., and a 50 cm long (45 cm effective) × 1.5 µm i.d. capillary. All sample injections were 8 s at 100 psi, and all separations were performed at 150 psi with 1.0 mM Na2B4O7.
heights were routinely obtained. We expect that the optimum pressure will change with the proteins selected and other experimental parameters such as eluent composition and capillary dimensions. An elution pressure higher than that optimum for maximum efficiency may often be desirable to increase separation speed. Effect of Column Length. The effect of capillary length on separation performance was tested using 1.5 µm i.d. capillaries 50-110 cm in length using a constant pressure gradient; the results are shown in Figure 9. As with conventional columns, the resolution values between the analytes increase with increasing capillary length. For hard to resolve molecules, increasing the column length, as practiced in conventional separations, will also apparently be a straightforward solution. However, this is carried out at the expense of reduced separation speed. Table S3 in the 7434
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Figure 8. Resolution between transferrin and R-lactalbumin as a function of linear velocity (separation pressure) on a 70 cm long (65 cm effective) × 1.5 µm i.d. capillary. Sample injections 8 s at 100 psi; 1.0 mM Na2B4O7 eluent.
Supporting Information shows detailed performance data for the different column lengths. It will be noted which specific column generates the maximum plate counts, plates/m or plates per unit time varies from one analyte to another. Capillary Surface Coating. The surface charge has been considered to have a great impact on separations of charged molecules.22,24,45,46 When we coated the nanocapillary with HPC (essentially eliminating the ζ potential) and used it for the same separations, no significant difference could be observed albeit the HPC-coated capillary may have provided a marginally more (45) Pennathur, S.; Santiago, J. G. Anal. Chem. 2005, 77, 6772–6781. (46) Pennathur, S.; Santiago, J. G. Anal. Chem. 2005, 77, 6782–6789.
roles in the separations. We continue to try to unveil the mechanism for separation of biomolecules in nanocapillaries and hope to communicate the findings in the near future. We have demonstrated protein separations in open tubular nanocapillaries, with high chromatographic efficiencies. With a very simple LIF detector, we can attain limits of detection of a few thousand molecules. With single molecule detection strategies that are presently common48,49 and seemingly straightforward to adapt to the present nanocapillary, the detection limits can be vastly improved. Using pressure/hydrodynamic injection, we need only femtoliters to picoliters of sample for each separation. Most cells can provide this volume of sample; this separation technique, combined with micro/nanofluidic manipulations,50,51 can thus be a powerful tool for single cell analysis.
Figure 9. Resolution as a function of capillary length. All separations were carried out under a constant pressure gradient (4.3 psi/cm column length) using 1.5 µm i.d. capillaries of (A) 110, (B) 90, (C) 70, and (D) 50 cm lengths; the effective lengths in each case were 5 cm smaller. The eluent was 1.0 mM Na2B4O7.
efficient separation (see Figure S4 and Table S4 in the Supporting Information). This implies that electrostatic interaction is not the prevailing mechanism for the retention of the proteins or their separations. However, this does not preclude surface charge on the capillary walls affecting the conformation of a protein analyte molecule. The precise conformations of the protein molecules in a capillary of radial dimensions comparable to that of the analytes themselves has never been elucidated, and the detailed separation mechanism is not fully understood. It is possible that hydrodynamic separation28 and radial migration47 both play significant
ACKNOWLEDGMENT This work is partially supported by NIH (Grant 1R21EB00851201A1), NSF (Grant CHE-0514706), and OCAST (Grant AR082-051). SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review June 10, 2009. Accepted July 25, 2009. AC901265T (47) Zheng, J.; Yeung, E. S. Anal. Chem. 2002, 74, 4536–4547. (48) Lee, J.-Y.; Li, J.; Yeung, E. S. Anal. Chem. 2007, 79, 8083–8089. (49) Widengren, J.; Kudryavtsev, V.; Antonik, A.; Berger, S.; Gerken, M.; Seidel, C. A. M. Anal. Chem. 2006, 78, 2039–2050. (50) Huang, B.; Wu, H.; Bhaya, D.; Grossman, A.; Granier, S.; Kobilka, B. K.; Zare, R. N. Science 2007, 315, 81–84. (51) Byun, C. K.; Wang, X.; Pu, Q.; Liu, S. Anal. Chem. 2007, 79, 3862–3866.
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