Anal. Chem. 2001, 73, 3687-3690
Electrophoresis in Nanometer Inner Diameter Capillaries with Electrochemical Detection Lori A. Woods, Thomas P. Roddy, Tracy L. Paxon, and Andrew G. Ewing*
Department of Chemistry, The Pennsylvania State University, 152 Davey Laboratory, University Park, Pennsylvania 16802
Separations have been achieved in 770- and 430-nminner diameter capillaries. The extremely low sample volumes involved in the study of biological microenvironments such as single cells has led to the desire to develop separation techniques in these ultrasmall capillaries. Total sample volumes as low as 12 fL have been injected using these nanometer inner diameter capillaries. Separations of several catecholamines have been accomplished in these submicrometer capillaries using both capillary zone electrophoresis and micellar electrokinetic chromatography with end-column amperometric detection. Capillary electrophoresis (CE) is a powerful separation technique that has become widely used in numerous areas of chemistry. Using high electrical potentials, CE provides fast and highly efficient separations. Separation efficiencies of 105-106 theoretical plates are typically obtained with CE.1 In addition, CE allows for the separation of extremely small volume samples ranging from nanoliters to picoliters.2,3 These characteristics make CE an excellent tool for the analysis of biological microenvironments such as single cells. CE has been utilized in the study of single cells for both whole cell and subcellular analysis. Whole cell analysis of invertebrate cells, as well as mammalian cells, has been achieved with CE.3-5 In addition, subcellular analysis of invertebrate cells has been accomplished with CE.3,6 However, subcellular investigations of mammalian cells, which are typically 3 orders of magnitude smaller in volume than invertebrate cells, have been prohibited by the volume constraints of CE.3,7 The desire to study increasingly smaller biological microenvironments such as mammalian cells has led to the need for CE techniques with even lower sample volume capabilities. Typically, CE is carried out utilizing capillaries with inner diameters (i.d.) ranging from 15 to 75 µm. The use of smaller inner diameter capillaries for CE to achieve lower sample volumes has been investigated. Electrophoretic separations have been performed in 10-,8-11 5-,7,12-14 2-,7,11,12,14,15 and 0.616,17-µm-i.d. capillaries. Separa* Corresponding author: (e-mail)
[email protected]; (fax) (814) 863-8081. (1) Gilman, S.; Ewing, A. J. Capillary Electrophor. 1995, 1, 1-13. (2) Issaq, H. Electrophoresis 2000, 21, 1921-1939. (3) Chen, J.; Ewing, A. Crit. Rev. Neurobiol. 1997, 11, 59-90. (4) Jankowski, J.; Tracht, S.; Sweedler, J. Trends. Anal. Chem. 1995, 14, 170176. (5) Yeung, E. J. Chromatogr., A 2000, 830, 243-262. (6) Chiu, D.; Lillard, S.; Scheller, R.; Zare, R. Rodriguez-Cruz, S.; Williams, E.; Orwar, O.; Sandberg, M.; Lundqvist, J. Science 1998, 279, 1190-1193. (7) Olefirowicz, T.; Ewing, A. Anal. Chem. 1990, 62, 1872-1876. 10.1021/ac010053e CCC: $20.00 Published on Web 06/19/2001
© 2001 American Chemical Society
tions of various neurotransmitters16 and indoles17 have been achieved in 0.6-µm-i.d. capillaries. Furthermore, total sample volumes as low as 180 fL have been injected using these ultrasmall capillaries.16 However, employing ultrasmall capillaries to lower the volume capabilities of CE does present some challenges including the need for sensitive and low-volume detectors. Fluorescence,8,10,11 multiphoton-excited fluorescence,14-17 and electrochemical7,9,12,13 detection have all been coupled to CE in ultrasmall capillaries with inner diameters less than or equal to 10 µm. Laser-induced fluorescence (LIF) is the most sensitive, low-volume detection method for CE.18 However, LIF detection often requires chemical derivatization.18 Furthermore, the path length dependence of LIF detection is problematic in its application to CE in ultrasmall capillaries.18,19 However, as a result of smaller laser focus, multiphoton-excited fluorescence detection provides improved mass detection limits when coupled to CE in ultrasmall capillaries.16,17 Like the LIF methods, electrochemical detection (ECD) provides extremely low limits of detection and small-volume capabilities for CE.18 ECD offers some advantages over singlephoton LIF detection for CE in ultrasmall capillaries. ECD is not dependent on path length and does not require chemical derivatization for electroactive analytes.18,19 In addition, ECD is masssensitive while LIF detection is concentration-sensitive.18,19 In this paper, electrophoretic separations in 770- and 430-nmi.d. capillaries with the use of end-column amperometric detection are presented. Separations of several catecholamines have been accomplished in these submicrometer capillaries using capillary zone electrophoresis (CZE). In addition, the first separations using micellar electrokinetic chromatography (MEKC) in ultrasmall capillaries have been obtained. Total sample volumes as low as 12 fL have been injected using these nanometer inner diameter capillaries. (8) Hogan, B.; Yeung, E. Anal. Chem. 1992, 64, 2841-2845. (9) Berquist, J. Tarkowski, A.; Ekman, R.; Ewing, A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 12912-12916. (10) Gilman, S.; Pietron, J.; Ewing, A. J. Microcolumn Sep. 1994, 6, 373-383. (11) Lindberg, P.; Stjernstrom, M.; Roeraade, J. Electrophoresis 1997, 18, 19731979. (12) Sloss, S.; Ewing, A. Anal. Chem. 1993, 65, 577-581. (13) Ewing, A. J. Neurosci. Methods 1993, 48, 215-224. (14) Gostkowski, M.; McDoniel, J.; Wei, J.; Curey, T.; Shear, J. J. Am. Chem. Soc. 1998, 120, 18-22. (15) Gostkowski, M.; Shear, J. J. Am. Chem. Soc. 1998, 120, 12966-12967. (16) Wei, J.; Gostkowski, M.; Gordon, M.; Shear, J. Anal. Chem. 1998, 70, 34703475. (17) Gostkowski, M.; Wei, J.; Shear, J. Anal. Biochem. 1998, 260, 244-250. (18) Swinney, K.; Bornhop, D. Crit. Rev. Anal. Chem. 2000, 30, 1-30. (19) Timperman, A.; Sweedler, J. Analyst 1996, 121, 45R-52R.
Analytical Chemistry, Vol. 73, No. 15, August 1, 2001 3687
EXPERIMENTAL SECTION Reagents. Dopamine (DA), catechol (CAT), isoproternol (ISO), dihydroxyphenylacetic acid (DOPAC), norepinephrine (NE), epinephrine (E), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), and 2-(N-moropholino)ethanesulfonic acid (MES) were obtained from Sigma (St. Louis, MO). 4-Methylcatechol (4-MC) was obtained from TCI America (Portland, OR). Sodium dodecyl sulfate (SDS) and a 48% aqueous solution of hydrofluoric acid were obtained from Aldrich (Milwaukee, WI). All chemicals were used as received. The separation buffers used were 100 mM MES containing 2% 1-propanol adjusted to pH 5.5 with sodium hydroxide and 10 mM TES containing 20 mM SDS adjusted to pH 7.1 with sodium hydroxide. All standards were prepared as 100 mM stock solutions in 0.1 M perchloric acid and were diluted to the desired concentration with the appropriate buffer. Instrument. A CE system with end-column amperometric detection was utilized. This system has been previously described.12,20 Briefly, 50-60 cm of fused-silica capillary with an outer diameter of 150 µm and inner diameter of either 770 ( 40 or 430 ( 40 nm (Polymicro Technologies, Phoenix, AZ) was employed. The inner diameter of the capillaries was confirmed using scanning electron microscopy (SEM) and is reported as the average and standard deviation of four measurements. Amperometric detection was performed using a two-electrode configuration. A carbon fiber with a 5-µm diameter (Amoco Performance Products, Greenville, SC) and a 500-700-µm electroactive area was employed as a working electrode. The carbon fiber microelectrode was positioned just inside the end of the capillary.12 Electrochemical detection was performed at 0.7 mV versus a Ag/AgCl reference electrode. The detection system was enclosed in a copper mesh Faraday cage to minimize external noise. Injections were performed electrokinetically, and injection volumes were calculated based on electroosmotic flow measured with a neutral marker. Procedures. The ultrasmall capillaries were filled with the use of a liquid chromatography pump (Scientific Systems, State College, PA). The output from the pump was directed through an ODS Hypersil column (Keystone Scientific, State College, PA). The ultrasmall capillary was connected perpendicular to the ODS Hypersil column with the use of a cross-connector (Keystone Scientific). The flow from the pump was directed through the ODS Hypersil column to alleviate back pressure caused by filling the small inner diameter of the capillary. The capillaries were filled with a mixture of methanol and doubly distilled water at pressures of 4000-4500 psi. Approximately 2 mm of the polyimide coating was removed from the capillary to expose the fused silica. The exposed portion of the capillary was placed in HF for 15 min. The inner diameter of the capillary was etched open to accommodate the carbon fiber microelectrode. After the etching was complete, the exposed portion of the capillary was placed in a sodium carbonate solution to neutralize the acid and then washed with water. Safety Considerations. A safety interlock box was used to protect the user from high voltage. Hydrofluoric acid can cause severe burns and must be used with extreme care. It should be neutralized with sodium carbonate prior to disposal. (20) Huang, X.; Zare, R.; Sloss, S.; Ewing, A. Anal. Chem. 1991, 63, 189-192.
3688 Analytical Chemistry, Vol. 73, No. 15, August 1, 2001
Figure 1. Comparison of the electropherograms obtained using a 1 mM solution of DA and CAT, a 10 s at 10 kV injection, a 25-kV separation potential, and a 91 mM MES with 2% 1-propanol (pH 5.5) buffer in a (a) 770-nm-i.d. capillary and (b) 12-µm-i.d. capillary.
RESULTS AND DISCUSSION Electrophoresis in 770-nm-i.d. Capillaries. In this initial application of CE-ECD in submicrometer inner diameter capillaries, the performance of the capillaries was investigated. Separations have been achieved in 770-nm-i.d. capillaries using CZE. A separation of DA and CAT in a 770-nm-i.d. capillary is shown in Figure 1a. As expected, the cationic DA elutes first followed by the neutral CAT. These peaks are well resolved and elute in approximately the same time as in an identical separation in a 12-µm-i.d. capillary (Figure 1b). While the elution times for these two separations are similar, their peak efficiencies are quite different. The peak efficiencies for the separation in the 770-nm-i.d. capillary have been calculated to be 1400 for DA and 5200 for CAT. These are much smaller than the peak efficiencies for the identical separation in the 12-µm-i.d. capillary. These efficiencies were calculated to be 19 000 for DA and 84 000 for CAT. For capillaries with inner diameters larger than 40 µm, it has been shown that utilizing smaller inner diameter capillaries results in higher peak efficiencies.21 However, the extremely small internal volume of the submicrometer inner diameter capillary might result in some additional phenomena such as increased wall interactions that decrease peak efficiencies. The total sample volumes have been calculated to be 8 pL for the separation in the 770-nm-i.d. capillary and 1.2 nL for the separation in the 12-µm capillary. Another concern with the utilization of submicrometer inner diameter capillaries for CE-ECD was the reproducibility in their use for obtaining quantitative information. A calibration of a 770nm-i.d. capillary has been obtained using separations of 5-pL (21) Lukacs, K.; Jorgenson, J. J. High Resolut. Chromatogr. 1985, 8, 407-411.
Table 1. Calibration of a 770-nm-i.d. Capillary Using DA and CATa DA
CAT
concn (mM)
amt (amol)
peak area
concn (mM)
amt (amol)
peak area
5.0 1.0 0.5 0.1
15000 2800 1500 270
5.2 ( 0.3 1.3 ( 0.2 0.56 ( 0.04 0.10 ( 0.02
5.0 1.0 0.5 0.1
7400 1400 820 130
3.75 ( 0.09 0.74 ( 0.08 0.40 (0.08 0.08 ( 0.02
a Amounts of catecholamines and the corresponding peak areas obtained from separations of DA and CAT at four different concentrations. Separations at each of the four concentrations were repeated three times. Peak areas given are the mean and standard deviation from three separations. The separations were obtained in a 770-nmi.d. capillary using 10 s at 10 kV injections, a 25-kV separation potential, and a 91 mM MES with 2% 1-propanol (pH 5.5) buffer.
Figure 2. Comparison of the electropherograms obtained in a (a) 770-nm-i.d. capillary with 90 mM MES with 2% 1-propanol (pH 5.5) buffer with a solution of 5 mM DA, 5 mM ISO, 5 mM 4-MC and CAT, and 25 mM DOPAC and (b) a 14-µm-i.d. capillary with a 90 mM MES with 2% 1-propanol (pH 5.5) buffer with a solution of 30 µM DA, ISO, 4-MC, and CAT. A 10 s at 10 kV injection and a 25-kV separation potential were used.
samples of DA and CAT at four different concentrations. The concentration, amount, and corresponding peak area for each analyte is shown in Table 1. The correlation coefficients for amount and peak area have been calculated to be 0.997 for DA and 0.999 for CAT. This linear relationship suggests that CE-ECD in submicrometer inner diameter capillaries can be used reproducibly to obtain quantitative information. After obtaining the calibration of the submicrometer inner diameter capillary, a multicomponent separation was accomplished in a 770-nm-i.d. capillary (Figure 2a). The cationic species, DA and ISO, elute first and are resolved in less than 7 min. They are followed by the coelution of the two neutrals, CAT and 4MC. The anionic analyte, DOPAC, elutes last. This peak exhibits tailing and some additional band broadening. This occurrence might be the
Figure 3. MEKC separation obtained in a 770-nm-i.d. capillary using a 5 s at 5 kV injection, a 25-kV separation potential, and a 10 mM TES and 20 mM SDS (pH 7.1) buffer with a 200 µM solution of CAT, 4-MC, NE, and E.
result of adsorption effects that are amplified by the extremely large surface area-to-volume ratio of the capillary or the relatively long residence time of DOPAC in the capillary. A similar separation in a 14-µm-i.d. capillary is shown in Figure 2b. These separations demonstrate that fairly complex analyses can be carried out in ultrasmall capillaries. MEKC in 770-nm-i.d. Capillaries. As shown in Figure 2, neutral analytes such as CAT and 4-MC coelute in CZE. However, MEKC, another mode of CE, can be used to separate neutral substances.22,23 In MEKC, neutrals are separated on the basis of hydrophobicity.22 Hydrophobic species are retained in the capillary for a longer time because they interact more strongly with the micelles than hydrophilic species.22 In addition to providing a method for the separation of neutrals, MEKC can also provide improved selectivity in the separation of ionic substances.22,23 Utilizing MEKC, a separation of four analytes including two neutrals has been achieved in a 770-nm-i.d. capillary (Figure 3). The two neutrals, CAT and 4-MC, are separated on the basis of their hydrophobicity with the more hydrophilic CAT eluting first. As expected, they are followed by the two cationic analytes, NE and then E. In MEKC, cationic species are retained in the capillary longer than neutrals because they interact with the negatively charged Stern layer of the micelles, as well as ion pair with a free SDS monomer.22,23 The NE and E peaks in this separation exhibit additional band broadening that might be the result of increased interactions with the walls of the capillary or the micelles. In fact, more band broadening is observed in MEKC separations in ultrasmall capillaries than in similar separations in larger capillaries.22,23 This occurrence might be the result of adsorption effects that are amplified by the extremely large surface area-to-volume ratio of the capillary. The origin of the small peak that elutes at ∼3 min is unknown. It was also seen in an injection of only the separation buffer. Electrophoresis in 430-nm-i.d. Capillaries. The desire to lower the volume capabilities of CE even further than allowed by the use of 770-nm-i.d. capillaries has led to investigation of the use of 430-nm-i.d. capillaries for CE-ECD. To confirm the inner diameter of the capillary, a SEM image of a cross section of the 430-nm-i.d. capillary was obtained. A SEM image of the inner bore (22) Ewing, A.; Wallingford, R.; Olefirowicz, T. Anal. Chem. 1989, 61, 292A303A. (23) Wallingford, R. Ewing, A. Anal. Chem. 1988, 60, 258-263.
Analytical Chemistry, Vol. 73, No. 15, August 1, 2001
3689
Figure 6. Electropherogram obtained in a 430-nm-i.d. capillary using a 1 mM solution of DA and CAT, a 1 s at 1 kV injection, a 25-kV separation potential, and a 98 mM MES with 2% 1-propanol (pH 5.5) buffer.
accomplished (Figure 6). The peak efficiencies for this separation have been calculated to be 460 for DA and 2700 for CAT. Figure 4. SEM image of a cross section of a 430-µm-i.d. capillary showing the size and integrity of the inner bore. This capillary is not etched. Scale bar represents 0.5 µm.
Figure 5. Electropherogram obtained in a 430-nm-i.d. capillary using a 1 mM solution of DA and CAT, a 10 s at 10 kV injection, a 25-kV separation potential, and a 98 mM MES with 2% 1-propanol (pH 5.5) buffer.
of a 430-nm-i.d. capillary and a portion of the fused-silica capillary wall at a magnification of 50 000 is shown in Figure 4. Separations have been achieved in 430-nm-i.d. capillaries using CZE (Figure 5). The total sample volume for this separation has been calculated to be 1.2 pL. However, 430-nm-i.d. capillaries have been used to separate much smaller samples. Using a 1-s injection at 1 kV, an electrophoretic separation of a 12-fL sample of DA and CAT was (24) Chien, J.; Wallingford, R. Ewing, A. J. Neurosci. Methods 1990, 34, 1-15.
3690 Analytical Chemistry, Vol. 73, No. 15, August 1, 2001
CONCLUSION Electrophoresis in 770- and 430-nm-i.d. capillaries with endcolumn amperometric detection has been investigated. It has been shown to be both reproducible and quantitative. Total sample volumes as low as 12 fL have been injected using these nanometer inner diameter capillaries. The extremely small sample volumes involved in single cell analysis have led to the desire to develop this technique as a method of lowering the volume capabilities of CE. While application of the small-volume capabilities provided by the use of submicrometer inner diameter capillaries are not limited to single cell analysis, CE in ultrasmall capillaries will allow for many new and exciting investigations in this field. It could be used to sample directly from the inside of a single mammalian cell to determine neurotransmitter levels in the cytoplasm and possibly to directly sample individual vesicles from the cell. This knowledge would provide insight into neurotransmitter storage within a cell and could be used to develop models of neurotransmitter transport, a process that is not well understood.24 In addition, it could also be used for investigation of the effects of drug treatment on neurotransmitter storage and transport. ACKNOWLEDGMENT This work was supported by the National Science Foundation. Received for review January 12, 2001. Accepted May 18, 2001. AC010053E
