Accelerated Art icles Anal. Chem. 1994,66, 3849-3851
IH-NMR Spectroscopy on the Nanoliter Scale for Static and On-Line Measurements Nian Wu, Timothy L. Peck, Andrew G. Webb, Richard L. Magin, and Jonathan V. Sweedler' Beckman Institute for Advanced Science and Technology, University of Illinois at Champaign-Urbana, Urbana, Illinois 6 180 1
NMR spectroscopy is one of the most powerful methods available for structural elucidation and for determination of the physical environment of an analyte. However, there has been limited success in scaling NMR to work with smaller volume samples. We report the developmentof nanoliter-scale NMR detection cells, formed by wrapping a radio frequency microcoil directly around a fused silica capillary. This creates 5-200-nL volume NMR detection cells from an 1mm length of 75-530-pm4.d. fused silica capillary. The design and construction of optimized coils are described in detail. Using such NMR detection cells in a static mode, 5 TIS). As the entire capillary is contained in the bore of the magnet, this is not a concern in the present work. On-line electrophoreticseparations are inherently different from previous LC-NMR; in the E-NMR experiments, each (24) Curran, S. A.; Williams, D. E. Appl. Specfrosc. 1987.8, 1450.
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analyte is moving past the detector at a different rate25and hence has a different residence time, 7 , in the detector cell. For an electropherogram measured between 3 and 30 min, there is a 1 order of magnitude difference in residence times. Because NMR is a nondestructive detection technique, a variable acquisition time approach can be used where longer observation times are used for later eluting bands so that either more scans or a longer relaxation time between each scan can be used to increase the S N R of the later eluting bands. Similar variable detector acquisition approaches have been used in fluorescence detection to improve The electric field and electrophoretic current can have a profound effect on the observed intensities of the individual analyte NMR bands. Although complicating factors, these effects are not necessarily a disadvantage as the dependence can yield molecular information difficult to obtain using other analytical methods.17J* In the first E-NMR experiments by Packer,28several possible complications are described, including the fact that the orbits of thechargecarriers areinfluenced by the main NMR magnetic field, Bo, and that the electrophoretic current produces a magnetic field gradient in the sample. The first effect is small for any orientation of the current and magnetic field as each molecular collision randomizes thedirection and magnitudes of thevelocities (with the exception of a small polarization of the molecules from the applied electric field).18 In most previous E-NMR, the main magnetic field, Bo, is parallel to the direction of current flow so that the current-induced magnetic field has no component in the Bo d i r e c t i ~ n . ' ~In, ~our ~ arrangement (see Figure l),the electrophoretic current-induced magnetic field introduces a perturbation to the main magnetic field. However, the induced field gradient is linear and external compensation can be used.30v31 In previous work, resistive heating has been described as one of the most serious problems with E-NMR.I7-I9 The temperature rise degrades the separation efficiency and directly affects the electrophoretic mobilities and diffusion rates. For aqueous samples, a >2% change in diffusion coefficient per degree temperature rise is expected; in many CE applications, the temperature rise in the capillary can be greater than 10 0C,32-34One of the advantages of CE compared to previous large-scale electrophoretic systems is that the high surfaceto-volume ratio minimizes the temperature gradient across the small-diameter capillary. However, these thermal effects are the reason that the capillary diameters cannot be increased to > 100 pm without degrading the separation efficiency. It is important to note that all of the factors affecting the NMR band intensities are experimentally interrelated; eq 1 predicts an improved SNR by increasing the solution flow rate because of the introduction of new spins. However, if the flow rate ( 2 5 ) Huang, X.;Coleman, W. F.; Zare, R. N. J. Chromatogr. 1989, 480, 95. (26) Sweedler, J. 4' .; Shear, J. B.; Fishman, H. A,; Zare R. N.; Scheller, R. Anal. Chem. 1991, 63, 496. (27) Shear, J. B.; Dadoo R.; Fishman, H. A,; Scheller, R. H.; Zare, R. N. Anal. Chem. 1993, 65, 2977. (28) Packer, J.; Rees, C.; Tomlinson, D. J. Ado. Mol. Relaxation Processes 1972, 3, 119. (29) Holz, H., Muller, C. J. Magn. Reson. 1980, 40, 595. (30) Holz, M.; Muller. C. Ber. Bunsenges. Phys. Chem. 1982, 86, 141. (31) Holz, M.; Lucas, 0.;Muller, C . J. Magn. Reson. 1984, 58, 294. (32) Terabe, S.;Otsuka, K.; Ando, T. Anal. Chem. 1985.57, 834. ( 3 3 ) Nelson, R.; Paulus, A,; Cohen, A,; Guttman, A,; Karger, B. J. Chromatogr. 1990, 480, 11 1, (34) Bello, M.; Besi, P., Righetti, P. G. J. Chromatogr. 1993, 652, 317.
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is increased by increasing the applied voltage (and hence electrophoretic current), the increased temperature gradient across the capillary can offset the SNR gain resulting in an overall degradation of performance.
EXPERIMENTAL SECTION MicrocoilFabrication. The experimental setup for the online 1H-NMR detection in CE is shown in Figure 1. The microcoil is wound directly onto fused silica capillaries (751 355 pm (i.d.1o.d.); 2501355-, 5301695-, and 75/135-pm capillaries; Polymicro, Phoenix, AZ) with 42-gauge (63.1 pm diameter) varnished copper magnet wire using a pin vice and micromanipulator under a stereomicroscope. We typically use 14-17 turns of wire for a total detection length of between 0.9 and 1.1 mm. In general, a bifilar winding technique is used where the diameter of the secondary wire is chosen to provide an adequate interturn spacing for the solenoid. A uniformly wound solenoid with a constant value of interturn spacing is achieved after removal of the second wire. In our present application, wechose to use a single-wire,tightly wound solenoid to minimize susceptibility-induced static magnetic field distortions as described above. Variations of this geometry include the use of a secondary wire that remains open circuit on the coil form or that is driven in parallel with the primary wire. To begin the winding process, one end of the wire is secured to the capillary using a small droplet of epoxy cement and then weighted to maintain constant tension. The wire is slowly wound onto the capillary by gently rotating the pin vice. When the appropriate number of turns has been wound onto the capillary, the wire is secured using a second droplet of epoxy. Although the presence of epoxy does lead to susceptibility-induced line broadening of the NMR spectrum, this effect is minimized by restricting the use of epoxy to the ends of the coils only. Upon the disconnection of the weights, the completed coil is electrically connected to a semirigid coaxial cable (UT85SS, Roseberger Micro-coax, Collegeville, PA) by the application of solder,conductive epoxy, or silver paint. The impedance-matching nonmagnetic capacitors that match the coil to the 5 0 4 transmission line generally have a nonzero value of susceptibility and can lead to substantial line broadening of the N M R spectrum. Therefore, the impedance matching capacitors are placed at the opposite ends of the 3-cm section of coaxial cable. The degradation in the electrical performance of the circuit that results from separation of the tank circuit elements (Le., coil and impedance-matching capacitors) is offset by the narrower line widths in the NMR spectrum. Electrical Characterization and NMR. Each microcoil is tested using a HP8751A network analyzer and HP87511A S-parameter test set (Hewlett-Packard, Palo Alto, CA) by measuring the input reflection coefficient (SI 1) to accurately impedance match the circuit to 50 Q at 300 MHz. The microcoil assembly is then mounted in an in-house modified NMR probe in which capillary and coil are perpendicular to the static magnetic field over the detection region.16 In order to ensure against damaging the rf amplifier by the high voltages used in CE, the separation voltageis kept below thecalculated breakdown voltage of the silica capillary walls at the position of the microcoil. For example, a maximum separation voltage of 12.5 kV is used with a 48-cm-long capillary (an electric
field of 260 V/cm) with an inner diameter of 75 pm and an outer diameter of 355 pm (140-pm walls) and where the coil is positioned 8 cm from the grounded end. In this manner, the potential at the coil never exceeds 2 kV, and the intensity of the electric field across the capillary wall (1 50 kV/cm) remains below the 250-400 kV/cm dielectric breakdown specification of the capillary. An additional test of capillary fidelity and breakdown potential is performed by application of 12.5 kV to the capillary with a buffer solution in a darkened room both for visual inspection of arcing and to monitor the CE current just prior to insertion into the magnet. We find this to be important following repeated experiments, as a minute amount of buffer solution can leak onto the surface of the dielectric separating the anode and cathode in the probe assembly, potentially leading to arcing in the probe. Furthermore, any abnormal electrophoretic leakage current is readily measured at the high-voltage power supply and may indicate that the capillary had been damaged during the coil construction or capillary mounting/positioning. NMR spectroscopy experiments ('H) are performed using a GN-300 (7.05 T)/89 mm NMR spectrometer. The NMR software installed on the GN-300 is MacNMR (Tecmag Corp., Houston, TX), available for the Apple Macintosh. The maximum sensitivity, i.e., signal-to-noise per square root of total data acquisition time, of the NMR experiment is obtained using an excitation pulse and interpulse repetition delay that both approach zero.35 However, this leads to distortions of the line shapes in the NMR spectra. Provided that the repetition delay is optimized for the particular choice of tip angle of the excitation pulse, there is negligible degradation in the sensitivity (Le.,
