Method for Obtaining Spectra from Sub-Milligram Quantities in Continuous Wave Nuclear Magnetic Resonance Spectrometry Johnnie L. Stewart and William L. Clapp" R. J. Reynolds Tobacco Company, Research Department, Winston-Salem, N.C. 27 102
Methods for the use of microcells in nuclear magnetic resonance spectrometry (NMR) have been reported previously by Varian Associates Applications Laboratory ( 1). These methods require transfer of the sample from a collection device to a microcell. Since the major objective of micro NMR analysis is to obtain maximum signal-to-noise from a given amount of sample, the problems of sample transfer, cleaning, filling, and positioning of microcells must be overcome if significant improvements are to be realized. This aid describes the fabrication and use of a microcell that overcomes these problems. By heat sealing a gas chromatographic collection tube and using a properly sleeved turbine, micro samples may be run in the collection tube with an increase in signal-to-noise ratio and reproducibility.
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Flgure 1. NMR spectrum of 1.3 mg of ethyl cinnamate obtained with microcell inserted in conventional 5-mm tube
EXPERIMENTAL Apparatus. A Varian A60A NMR Spectrometer, 3-mm turbine, butane-oxygen microflame torch and Scientific Products micro blood collecting tubes (150-mm X 3-mm 0.d. X 1.5-mm i.d.) were used. T h e 3-mm NMR turbine consists of a locally machined 3-mm i.d. Teflon sleeve fitted inside the stock turbine. This design permits flexibility in positioning the sample tubes, not all of which are exactly 3-mm 0.d. Procedure. Micro NMR samples submitted for analysis are trapped from a gas chromatograph into 3-mm collecting tubes. A Teflon cap is fitted over the tapered end and 30 fil of CDC13 is added. T h e tube is partially covered with dry ice, held horizontally, and the bottom heat-sealed with a butane-oxygen microflame torch. T h e tube is then positioned in the turbine a t the depth predetermined from finding the maximum effective magnetic field. T h e 3-mm tube contains sufficient tetramethylsilane to tune the instrument and to trigger the Varian C-1024 Time Averaging Computer.
Figure 2. NMR spectrum of 1.3 mg of ethyl cinnamate obtained with conventional 5-mm tube
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RESULTS AND DISCUSSION Earlier techniques required the use of a polar solvent such as methanol to transfer the sample from the gas chromatographic collection tube into a 5-mm NMR tube. The methanol was then removed, and 400 p1 of CDC13 was added. In establishing a method for the analysis of micro NMR samples, several techniques were investigated before a suitable one was found. Several types of glassware were evaluated, notably microcells of spherical, cylindrical and melting point designs. Because of the inconsistency and unpredictability of results, handling problems, and sample losses, these cells were unsatisfactory. Since total sample transfer is for all practical purposes impossible, it was decided to utilize the tube used for gas chromatographic trapping. After heat-sealing this 3-mm X 150-mm trapping tube, containing a sample of 1.3 mg of ethyl cinnamate in 30 pl of CDC13, and inserting the 3-mm tube into a 5-mm tube containing CDC13, a spectrum was obtained from a single scan as shown in Figure 1. The 3-mm tube is secured by a Teflon chuck slipped over the tapered end and inserted into the 5-mm tube. As shown in Figure 2, a 100% gain in peak height and a 50% gain in signal-to-noise were noted over those in the conventional method for placing 1.3 mg of ethyl cinnamate in a 5-mm tube containing 400 p1 of CDC13. Elimination of the outer 5-mm tube and exposure of the 3-mm tube only to the
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Figure 3. NMR spectrum of 118 p g of ethyl cinnamate obtained with microcell inserted in conventional 5-mm tube. 100 sweeps with C1024
magnetic field result in an additional 50% gain in signalto-noise. The effective magnetic field was determined with a standard solution of 43.6 mg/ml ethyl cinn'amate in CDC13 in the 3-mm tube. No signal-to-noise improvement was noticed with sample heights or volumes in excess of 15 mm or 30 pl, respectively. Since the danger of sample vortexing in this small 1.5mm i.d. tube is virtually eliminated, the tube may be spun much faster than the conventional 5-mm tube. This minimizes the spinning sidebands and consequently does not obscure the methyl region with unwanted peaks. Also, exchangeable proton peaks may be shifted by diluting the concentrated solution. ANALYTICAL CHEMISTRY, VOL. 48, NO. 3, MARCH 1976
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With this technique, the problems of sample transfer, loss, recovery, contamination, and positioning of the microcell are eliminated. The bulk of the sample is readily recovered for use in additional spectrometric analyses. Since the cost of the microcell is negligible, it may be discarded rather than cleaned. With the aid of the time averaging computer, a usable spectrum may be obtained from 100 transients on a 250-wg
sample. Also, limited information may be obtained from 1OO-wg samples as shown in Figure 3.
LITERATURE CITED (1)
Varian Instruments Application Report NMR-2.
RECEIVEDfor review August 6, 1975. Accepted October 23, 1975.
Optically Transparent Thin Layer Electrode for Anaerobic Measurements on Redox Enzymes Barbara J. Norris, Marilyn L. Meckstroth, and William R. Heineman* Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 4522 7
A spectropotentiostatic method using an optically transparent thin layer electrode, OTTLE, for measuring E"' and n values of redox enzymes was recently reported (1). The OTTLE was of the minigrid-microscope slide design with the bottom edge immersed in a small cup of solution (2). I t was pointed out that measurements could be made on solution volumes of less than 1 ml with a cell design which eliminated the cup. This is an important consideration since many enzymes are available only in minute amounts. We describe here an OTTLE with which spectropotentiostatic measurements can be made on a total solution volume of down to 500 ~ 1An . equally important feature of this cell is the ability to remove dissolved oxygen from solution by a vacuum degassing technique. Electrochemical measurements on the many redox enzymes which are oxidizable by molecular oxygen must be performed under such anaerobic conditions.
EXPERIMENTAL Apparatus. The optically transparent thin layer cell is shown in Figure 1. It is basically of the minigrid-microscope slide variety of OTTLE ( 2 ) . The solution cup was eliminated and the auxiliary electrode was moved into the thin layer with the reference electrode probe located between the working and auxiliary minigrid electrodes. The cell was adapted to oxygen removal with vacuum/ nitrogen cycling by using a previously reported vacuum. degassing bulb and special Ag/AgCl reference electrode (3). The front face of the cell consisted of clear lucite, 1 X 1% X % inches. Two blocks of '/* X '/4 X '14 inch lucite were attached to the front face with 1,2-dichloroethane. Holes were drilled through the blocks to accommodate valves (No. 1MM1, Hamilton Co., Reno, Nev.). The back of the cell was a microscope slide cut to the dimensions of the front. Three layers of 2-mil pressuressensitive Fluorofilm DF-1200 Teflon tape (Dilectrix Corp., Farmingdale, N.Y.) cut in 2-mm strips, were placed around the edges of the glass for spacers. The working and auxiliary electrodes, 1 X 3.5 cm pieces of gold minigrid (500 wires per inch, 60% transmittance, Buckbee Mears Co., St. Paul, Minn.), were placed 5 mm apart and were sandwiched between the faces of the cell. The minigrids extended outside of the cell for electrical contact. The cell was sealed around the edges with epoxy. A silver/silver chloride, 1.0 M KC1 reference electrode contacted the cell through valve V2 located between the minigrids. Placement of the reference probe between the auxiliary and working minigrids was critical for obtaining best potential control of the OTTLE minigrid electrode. A vacuum degassing bulb was attached to the other valve, V1; this bulb allowed the cell to be connected to a vacuum/nitrogen train. Details of the reference electrode, vacuum degassing bulb, and vacuum/nitrogen train have been described (3). No problems were encountered in preparing vacuum-tight cells. Careful application of epoxy to completely cover the edges consistently resulted in good cells. Cell lifetime was usually limited by fraying of the fragile minigrid contacts rather than by the development of leaks. 630
ANALYTICAL CHEMISTRY, VOL. 48, NO. 3, MARCH 1976
A Princeton Applied Research Model 173 potentiostat in conjunction with a Model 175 Universal Programmer was used for applying potentials t o the cell. Potentials applied for the spectropotentiostatic experiments were measured with a Digitec 261C digital voltmeter. Optical measurements were made with a Harrick Rapid Scan Spectrometer, Model RSS-B. Reagents. Solutions were prepared in pH 7.00,O.l M phosphate buffer (Buffer Titrisol, E M Laboratories, Elmsford, N.Y.) and 0.1 M NaCl (Suprapur, EM Laboratories). Horse heart cytochrome c (Type VI, 95-100% pure, Sigma Chemical Co., St. Louis, Mo.) and 2,6-dichlorophenolindophenol(99% pure, Fluka, Columbia Organic Chemicals, Columbia, S.C.) were used without further purification. Procedure. The following procedure was used for oxygen removal from the enzyme solution and for filling the OTTLE. Close adherence to this procedure was necessary for optimum results. The thin layer cell was first evacuated by attaching it to the vacuum/nitrogen train by means of the degassing bulb and applying a vacuum. The stopcock on the bulb and the two valves were opened, and three cycles of vacuum-nitrogen were applied, leaving the cell under nitrogen pressure. The valves and stopcock were closed and the cell was removed from the train. A solution of previously degassed 1.0 M KC1 was added by syringe to the reference tube, and the Ag/AgCl wire was reinserted. The cell was again attached to the train, the stopcock was opened, and two cycles of nitrogen-vacuum were applied to the bulb. The valve V1 to the cell body was opened and the vacuum-nitrogen cycle was repeated three more times, leaving the cell under nitrogen pressure. The reference valve V2 was then opened and the vacuum carefully applied until the KC1 solution rose t o the top of the reference tube, a t
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Figure 1. Optically transparent thin layer cell ( A )Top view, (6) Side view
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