Anal. Chem. 2003, 75, 3929-3933
Nanoinjector for Capillary Electrophoresis and Capillary Electrochromatography Eiji Iizuka and Takao Tsuda*
Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan Motonori Munesue and Shinichi Samizo
Chemco Scientific Co., Ltd., 2-8-14, Nakazaki, Kita-ku, Osaka 530-0016, Japan
A rotary valve nanoinjector was devised for use in capillary electrophoresis (CE) and capillary electrochromatography (CEC). A fused-silica capillary tip was inserted in a small through-hole in the rotor. The narrow and short capillary tip, with an inner volume of 6-24 nL, was embedded in the hole using epoxy resin. The injection volume was confirmed chromatographically by comparing the peak areas obtained with the nanoinjector to those of a conventional injector. In addition, both the rotor and stator of the injector were made of a nonconducting material, polyimide resin, to be utilized for CE and CEC. The application of the nanoinjector for CE was demonstrated. The rotary valve injector has already been generally utilized as a sample injection method for high-performance liquid chromatography (HPLC). The injector used in capillary electrophoresis (CE) and capillary electrochromatography (CEC) should have a sample volume on the order of nanoliters. For example, if the injector volume is 2/100 of the column volume, the sample volume of the injector for CE is approximately 6-24 nL when the inner diameter of the capillary column used is 50-100 µm. Since the inner volume of conventional injectors is now on the order of a micro- or submicroliter, it is not possible to use them directly for CE and CEC. The study of microinjectors for CE was initially introduced by Tsuda et al.1 The thickness of the rotor used was 5 mm, and the inner diameter of a drilled hole used for the sample chamber was 0.3 mm. As the rotor and stator of this injector were made of ceramic and poly(tetrafluoroethylene), respectively, it was possible to use it under an applied high voltage. Takeuchi and Ishii studied a 20-nL injector for use in micro HPLC.2 A very thin rotor, 0.7 mm, made of stainless steel was used. The use of a very thin rotor (0.7 mm) has two drawbacks, First, extremely sophisticated technology is needed in the production process, Second, a thin rotor is unstable. A slide-type nanoinjector using a ruby rotor for CE was introduced by Hanai and Tsuruta.3 The ruby rotor sample cham-
ber size was 50 µm in inner diameter and 1 mm in length. Owing to the failure to make a connection with a very small dead volume, the electropherogram demonstrated by the apparatus lost efficiency. As the conventional injector was not applicable directly for CE due to its large injection volume, on the order of microliters, split injection devices have been adopted. A split part followed by an autorotary injector has been demonstrated under continuously applied voltage.4 An in-line six-way injector with a 0.38-µL sample loop was operated electronically to control the injection period.5-7 Sample introduction was achieved by opening the sample flow channel at a certain period under electrically control.8 In CE, electrokinetic injection and hydrodynamic injection have been generally used.9-15 In the case of microchip CE, the injection is performed by controlling both the applied voltage and the time period of sampling.16-18 In the above injections, sample volumes are defined only by injection time and applied voltage or pressure. Micromechanical valve injection might be preferable, which allows volume-defined injection for fused-silica capillary-based CE. For on-line use of an injector in CE, it is necessary to make a nanovolume sample chamber. Therefore, one must keep the inner (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16)
* Corresponding author. Phone/Fax: 81-52-735-5220. E-mail: takao@ ach.nitech.ac.jp. (1) Tsuda, T.; Mizuno, T.; Akiyama, J. Anal. Chem. 1987, 59, 799-800. (2) Takeuchi, T.; Ishii, D. HRC & CC, J. High Resolut. Chromatogr. Chromatogr. Commun. 1981, 4, 469-470. 10.1021/ac020778y CCC: $25.00 Published on Web 07/02/2003
© 2003 American Chemical Society
(17) (18)
Hanai, T.; Tsuruta, H. Instrum. Sci. Technol. 1994, 22 (2), 151-155. Tsuda, T.; Zare, R. N. J. Chromatogr. 1991, 559, 103-110. Deml, M.; Foret, F.; Bocek, P. J. Chromatogr. 1985, 320, 159-165. Verheggen, Th, P. E. M.; Beckers, J. L.; Everaerts, F. M. J. Chromatogr. 1988, 452, 615-622. Evans, C. E. Anal. Chem. 1997, 69, 2952-2954. Evans, C. E.; Ponton, L. M. Anal. Chem. 2001, 73, 1974-1978. Jorgenson, J. M.; Lukacs, K. D. HRC & CC, J. High Resolut. Chromatogr. Chromatogr. Commun. 1981, 4, 230-231. Jorgenson, J. M.; Lukacs, K. D. Anal. Chem. 1981, 53, 1298-1302. Tsuda, T.; Nomura, K.; Nakagawa, G. J. Chromatogr. 1982, 248, 241247. Tsuda, T.; Nomura, K.; Nakagawa, G. J. Chromatogr. 1983, 264, 385-392. Burton, D. E.; Sepaniak, M. J.; Maskarinec, M. P. Chromatographia 1986, 21, 583-586. Smith, R. D.; Udseth, R. H.; Loo, J. A.; Wright, B. W.; Ross, G. A. Talanta 1989, 36, 161-169. Fishman, H, A.; Amudl, N. M.; Lee, T. T.; Scheller, R. H.; Zare, R. N. Anal. Chem. 1994, 66, 2318-2329. Manz, A.; Effenhauser, C. S.; Burggraf, N.; Harrison, D, J.; Seiler, K.; Fluri, K. J. Micromech. Microeng. 1994, 4, 257-265. Seiler, K.; Fan, Z. H.; Fluri, K.; Harrison, D. J. Anal. Chem., 1994, 66, 3485-3491. Rosaria, J. R.; Ferrigno, R.; Girault, H. H. J. Electroanal. Chem. 2000, 492, 1-22.
Analytical Chemistry, Vol. 75, No. 15, August 1, 2003 3929
Figure 1. Schematic diagram of proposed nanoinjector for CE. (a) Knob screws; (b-1) inlet stator (PI); (b-2) outlet stator (PI); (c) 5/16-in. union nut; (d) fused-silica capillary tubing (7 cm × 100 µm) for sample inlet (d-1) and vent (d-2); (e) rotor (PI); (f) central pin; (g-1) inlet capillary tubing; (g-2) capillary column; (h) sample chamber.
diameter of the sample chamber small enough for handling a sample volume on the order of nanoliters. Since it is technically difficult to make a very narrow sample chamber using a mechanical technique, we embedded a fused-silica capillary tubing of 50100-µm inner diameter in a rotor made of resin. In this way, a volume-defined injection on the order of nanoliters is achieved. Thus, a new nanoinjector of 6-24 nL has been successfully made using a nonconducting material. EXPERIMENTAL SECTION Structure of the Rotary Valve Nanoinjector. A schematic diagram of the rotary valve nanoinjector is shown in Figure 1. The injector consists of one rotor, two stators, and one central pin. The rotor and stator were made of polyimide (PI) resin (Vespel S, SP-1, Du Pont Kabushiki Kaisha, Tokyo, Japan). Nanovolume sample chambers ranging from 6 to 24 nL were set in the rotor. All the parts were specially selected for use at high voltage. The channel for liquid flow was designed to be straight and short. Sample was introduced to a sample chamber through a fused-silica capillary tubing (100-µm i.d., 7-cm length), d-1 in Figure 1. After filling a microsyringe (10 µL, Ito Co., Tokyo, Japan) with sample, its needle was connected with the fused-silica capillary of d-1 by using a small tip of polytetrafluoro tubing (0.25mm i.d., 15-mm length). The sample was introduced into a sample chamber in a rotor. Then the rotor position was rotated to the position for a separation column. Sample Chamber. The sample chamber was constructed by inserting a fused-silica capillary tip (outer diameter 0.375 mm, supplied from G-L Science, Tokyo, Japan) into a hole (∼0.4-mm i.d.), which was drilled in the rotor. The rotor thickness is 3 mm. The hole for inserting the fused-silica capillary tip was precisely made using equipment consisting of a compact drilling machine (Product No.: K-15, Hozan Tool Industrial Co., Ltd., Osaka, Japan) and an optical stage (XYZ axis steel extended contact-bearing stages, type TSD-605C, Sigma Koki Co., Saitama, Japan). Parts of the injector were set on the optical stage for drilling. After inserting 3930 Analytical Chemistry, Vol. 75, No. 15, August 1, 2003
the fused-silica capillary tip into the hole, it was fixed with epoxy glue. Then the rotor surface was polished using a fine grinding stone. A rotor with a different injection volume was produced by changing the inner diameter of the fused-silica capillary tubing inserted in the rotor. Thus, the injection volume becomes 6 and 24 nL for fused-silica capillary tubings of 50- and 100-µm inner diameters, respectively. Stator. The internal structure of the stator is shown in Figure 2a. The end of the capillary column was devised to contact the rotor directly. This device is shown precisely in Figure 2b (expanded view of the right side of Figure 2a). The sample leading tip made of poly(tetrafluoroethylene) (1.5-mm o.d., 4-mm length) is compressed constantly by a small compression spring (1.59mm o.d., 0.29 mm wire size, 3.5-mm length; Marumiya Spring Co., Ltd., Tokyo, Japan). The compression spring is made of piano wire. The spring holder and a union nut, shown in Figure 2a, are arranged to hold the compression spring. These devices allow the sample leading tip to very delicately contact the rotor. Estimation of Inner Volume of Sample Chamber. The volume of the sample chamber was estimated from the peak area of the standard sample obtained by liquid chromatography (LC). Laboratory-constructed LC equipment, a capillary column (100µm i.d. and 20 cm long) and a UV detector, 254 nm (type HC2001, Asahi Techneion, Inc., Tokyo, Japan) were used. A rotary valve injector (inner volume 0.2 µL, model 7520, Rheodyne, L. P.) were used as a standard. The peak area obtained by the nanoinjector was compared to the peak area obtained with a 0.2µL injector, using 0.3 mM thiourea in 50% ethanol aqueous solution. Reproducibility of Injection Volume under CE Conditions. The reproducibility of the nanoinjector was evaluated by electropherogram. The measurements of peak area were repeated 5 times under the following experimental conditions. Fused-silica capillary tubing (50-µm i.d., 29.4-cm length) was used as a column, 0.01 M phosphoric acid buffer (containing 0.1% ethylene glycol)
Figure 2. (a) Inside expanded view of newly devised stator. (b) Expanded view of shaded area in (a).
was used as the background electrolyte, and aqueous 1.1 mM thiourea was used as the standard sample. A little ethylene glycol was added to the background electrolyte to smooth the contact of background electrolyte to the inner wall. In other words, the contact angle was lowered. The applied voltage was -15 kV (21 µA). A pressure of ∼2.5 × 105 Pa was added at both ends of the capillary column. A UV detector, 254 nm, was used. The background electrolyte in the two reservoirs at both ends of the capillary column was compressed with a 10-mL syringe under pressurized helium gas, as shown in Figure 3. Thus, after introducing helium gas into the syringe, the gas was compressed in the cylinder. Operation of Capillary Electrophoresis with a Nanoinjector. The newly designed nanoinjector was applied to capillary electrophoresis. The arrangement in capillary electrophoresis with the nanoinjector is shown in Figure 3. A given gas pressure was added at both ends of the capillary column in order to prevent bubble formation in the column. The applied voltage was -18 kV, and the pressure at both ends was ∼2.2 × 105 Pa. For safe operation, the system was surrounded by Pyrex glass walls to avoid direct contact except for the nanoinjector knob, which was grounded electrically. A nonconductive resin bar (∼20 cm long)
was attached to the handling part of the knob. Operation of the nanoinjector was done by handling this bar. A UV detector, 254 nm, was used, and 0.01 M phosphate buffer (pH 7.0) containing 0.1% ethylene glycol was used as background electrolyte. The sample was a mixture containing pyridoxamine, thiourea, guanosine 5′-monophospate disodium salt (GMP), uridine 5′-monophosphate disodium salt (UMP), and adenosine 5′-diphosphate monosodium salt (ADP). The concentration of each component in the sample solution was ∼1 mM. RESULTS AND DISCUSSION Designs of Rotor and Stator. Since CE and CEC operate under high voltage, the injector parts directly in contact with a liquid should be made of engineering resin. As the rotor and stator of the proposed injector are made of polyimide resin, it can be operated at less than 18 kV without any problem. The key to the device is the construction of the nanovolume sample chamber. The sample chamber was devised by embedding a fused-silica capillary tip into a small hole in the rotor. The especially arranged machine for drilling this hole proved very accurate. By using this mechanical arrangement, a small hole (0.4mm i.d., 3-mm length) can be precisely drilled with good Analytical Chemistry, Vol. 75, No. 15, August 1, 2003
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Figure 3. Schematic diagram of end-pressurized CE system using nanoinjector.
reproducibility. A fused-silica capillary of 50-µm inner diameter was used as the insertion tip in the hole. The geometrical toughness of the tip is quite high and does not deform under moderate pressure. The end edge of the fused-silica capillary would damage the surface of the rotor if allowed to directly contact the stator. Therefore, it is necessary to avoid direct contact of both the inserted fused-silica capillary tip end and the capillary column ends on the surface of the stator and rotor, respectively, because they are harder than the resin. A proposed device of the rotor and stator are shown in Figure 2b. It has a sample leading tip made of poly(tetrafluoroethylene), a compression spring, and a spring holder. The sample leading tip made of poly(tetrafluoroethylene) shows excellent low friction and flexibility. The compression spring presses the sample leading tip locally with a constant fixed force. By using this device, it is possible to press the sample leading tip softly to the rotor without damage to the surface. There is a small space at both ends of the sample chamber, shown in Figure 2b. At this stage of the experiment, dead space is less than 100 nL. There is no leakage of liquid from the contact surface of the rotor and stator, when this device is used. Estimation of Inner Volume of the Sample Chamber. Estimation of the sample chamber volume is very important because this volume is the basis for the actual nanoinjector volume. It is also allows one to confirm that no unnecessary volume results in the rotor manufacturing process. The estimate of the sample chamber volume was performed using the same nanoinjector housing for nanorotors and conventional rotors. The chromatograms obtained using these two rotors are shown in Figure 4. Five runs in a series were tested. The relative standard deviations of the peak area for nanorotors and conventional rotors are 0.699 and 2.19%, respectively. The volume of the nanorotor has been estimated to be 21.2 nL. This estimated volume is less than the value calculated from the sample chamber composed with a tip of fused-silica capillary (100-µm i.d.,3-mm length), 23.6 nL. Although the origin of this difference is not clear, one comes 3932 Analytical Chemistry, Vol. 75, No. 15, August 1, 2003
Figure 4. Chromatogram using nanoinjectors and standard injectors (a) 23.6 nL and (b) 0.2 µL. Flow: constant-pressure mode of 1.3 kgf/ cm2. Eluent: mixture of water and ethanol (50/50). Column: capillary column (100-µm i.d., 20-cm length). Standard sample: 0.3 mM thiourea in 50% ethanol aqueous solution.
from overestimation of the inner diameter of the fused-silica capillary. Reproducibility and Operation of Nanoinjection under CE Conditions. The 6-nL sample chamber was made using a fusedsilica capillary tubing of 50-µm inner diameter and 3-mm length. The reproducibility of the CE operation using the nanoinjector is demonstrated, as shown in Figure 5. The electropherogram for the mixture of five components is shown in Figure 6. The volume of the relative standard deviation for the thiourea peak area is 3.14%. The thiourea peak, which elution time was 2 min, is very sharp and the performance of the nanoinjector is quite good. The
Figure 5. Reproducibility of newly constructed 6-nL injector in CE mode. Applied voltage: -15 kV (21 µA). Background electrolyte: 0.01 M phosphate buffer containing 0.1% ethylene glycol. Column: fused-silica capillary (29.4 cm × 50 µm). Sample volume: 6 nL. Sample: thiourea (1.1 mM). The ends of the separation column were pressurized at ∼2.5 × 105 Pa.
Figure 6. Separation using a 6-nL nanoinjector in CE mode. Applied voltage: -18 kV (28-29 µA). Pressurized CE: ∼2.2 × 105 Pa. Background electrolyte: 0.01 M phosphate buffer, containing 0.1% ethylene glycol. Column: fused-silica capillary (29.4 cm × 50 µm). Sample: (1) pyridoxamine (1 mM), (2) thiourea (1.1 mM), (3) GMP (1 mM), (4) UMP (0.99 mM), and (5) ADP (0.98 mM).
reproducibility of injection with the proposed injector is as good or better than the accuracy obtained with electrokinetic or hydrodynamic injection using the ordinary manual operation. The dead volume other than the sample chamber was kept small (less than 100 nL), and the device can be operated under high voltage.
CONCLUSION Although many CE instruments are available, an injection device still has not been developed that is comparable with the system of injection in liquid chromatography in terms of reproducibility of injection. Therefore, there is a great need to develop a small injector for CE. A nanoinjector has been presented, in which a sample chamber is made with a piece of fused-silica capillary. Since a fused-silica capillary is itself geometrically very rigid, the sample volume of the nanoinjector can be kept constant for a long period. Mounting the sample chamber vertically in the rotor ensures more stability than with groove mounting on the rotor surface, because the wear from friction between rotor and stator is reduced. Longer life is thus guaranteed. The present injector has been constructed with good reproducibility. The skills required for the construction of the nanoinjector are precise positioning of the hole in the rotor and straight drilling of the hole. Excluding these requirements, the other construction processes are relatively easy. The 6-nL sample chamber is the second-smallest injector reported to date.1-3 However, a 3-mm rotor is employed, so it is more robust than the world’s smallest type. We are now undertaking the development of an automatic nanoinjector. ACKNOWLEDGMENT This research was partially supported by a Grant-in-Aid for Scientific Research (B), 12440209, from the Ministry of Education, Culture, Sports, Science and Technology. We thank Takasago Electric, Inc. for the supply of rotors and very helpful discussions. Received for review December 20, 2002. Accepted May 8, 2003. AC020778Y
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