Anal. Chem. 1983, 55, 1184-1186
1184
mol 15N/mol 14N = (1122/1121)- 0.077
(3)
Using the standard procedure given in the experimental section, we obtained the results recorded in Table 11. The replicate analyses were carried out by dividing 100 cm3 of a solution containing ca. 40-80 mg of total NaN03 into three portions, which were reduced, derivatized, and analyzed separately. The mass spectrometric ratios were obtained by repetitive scanning at 20 eV; for many purposes, single scans at 70 eV would give adequate precision. Excellent agreement between molar ratios from mass measurements on NaN03 and the mass spectrometric measurements on benzamide was obtained. When the 15N was expressed as a mole fraction, the determinations appeared consistently to agree to within *0.01 mol fraction of 16N. At this level of accuracy, it is clear that the new method will not be applicable to samples whose 15N content is at or near the natural abundance. For this reason we did not correct for the natural abundance of 16Nin the authentic 14Nstandards nor did we attempt to estimate whether any isotopic fractionation occurred during the distillation of ammonia. We believe that this procedure is established as a practical routine method for determining the isotopic ratio analysis of nitrate samples. The method may be used unchanged for the corresponding determination of nitrite, and with minor modification (see Experimental Section) for ammonium salts and for mixtures of NH4+ with either NOz- or NO3-. The analysis of mixtures or nitrite and nitrate was more difficult; the reduction potentials of NO3- and NOz- are extremely similar, and we were not able to find a satisfactory method for reducing one selectively in the presence of the other. For the determination of the isotope ratio of nitrite in the presence of nitrate, recourse was made to the nitrosating capability of nitrous acid. In acidic solution, the nitrous acid was used to diazotize aniline, and the resulting diazonium cation was coupled with 2-naphthol in alkaline solution (eq 4). This procedure left NO3- unaffected. The azonaphthol
coupling product, together with unreacted aniline, was ex-
tracted into dichloromethane, and after isolation of the azonaphthol by chromatography, the mass spectrum was run at 20 eV, at which ionizing voltage the amount of fragmentation to (M - H)+was acceptably small (10% of base). We were not able to find a satisfactory method for studying nitrate in the presence of nitrite. Diazotization with aniline could not be made quantitative in the very dilute solutions used; even when a large excess of aniline and HC1 was used and the solution boiled for 1h, substantial NOz- remained unreacted. This was shown by the fact that the benzamide from 15N-richnitrate samples was contaminated by 14Nfrom added Na14N02upon subsequent reduction and acylation. Other reagents for destroying nitrite, such as urea, sulfamic acid, azide ion, cyanate ion, and ammonium ion were, for various reasons, unsatisfactory. A better solution to the problem is a chromatographic separation of the two ions prior to their determination separately by the standard method.
ACKNOWLEDGMENT We thank H. S. McKinnon for running the mass spectral analyses.
Registry No. Nitrogen-15, 14390-96-6;nitrogen, 7727-37-9; benzamide, 55-21-0;phenylazonaphthol,52882-77-6; ammonium, 14798-03-9. LITERATURE CITED Lantelme, F.; Chemla, M. Bull. SOC.Chlm. Fr. 1968, 1314. Lacoste, G.; Ollve. H.;Routle, R. Analusls 1976, 4, 172. Chem. Abstr. 1976, 85, 5 6 2 2 3 ~ . Laroche, J.; Combelles, R. "The Use of Isotopes in Soil Organic Matter Studies"; Pergamon: Oxford, 1966, p 423, and references therein. Vogel, A. I. "A Textbook of Quantltatlve Inorganic Analysis", 3rd ed.; Longmans: London, 1961; p 783. Vogel, A. I."A Textbook of Quantitative Inorganic Analysis", 3rd ed.; Longmans: London, 1961; p 255. Vogel, A. I. "A Textbook of Practical Organic Chemistry", 3rd ed.; Longmans: London, 1956; p 797. Stenhagen, E.; Abrahamsson, S.; McLafferty, F. W. "Atlas of Mass Spectral Data"; Interscience: New York, 1969; Vol. 1, p 489.
RECEIVED for review December 21,1982. Accepted March 14, 1983. Work supported by the Natural Sciences and Engineering Research Council of Canada. Isotopic chemicals were purchased with funds from NSERC and from the Ontario Ministry of Agriculture and Food.
Capillary Tube Sealer for Microcell Nuclear Magnetic Resonance Spectrometry Dale S. Skrabalak and Jack Henlon" New York State College of Veterinary Medicine, Cornel1 University, 925 Warren Dr., Ithaca, New York 14850
Many research and metabolism studies require NMR confirmation of compounds at microgram levels (1,2).As a result, microcell NMR and capillary sample tubes must be utilized ( 3 , 4 ) . Although the operation of microcell NMR is no more difficult than conventional NMR, sealing the associated capillary tubes can be difficult. Capillary tubes are usually heated in a flame and drawn closed. Yet this simple technique requires skill and practice (5). Described herein is a device that can be easily assembled at low cost from common laboratory equipment and used successfully with minimal skill or practice to seal capillary tubes for microcell NMR.
EXPERIMENTAL SECTION Apparatus. Figure 1shows the complete capillary tube sealing assembly. A Dremel MotoTool Model 280 (Racine, WI) is fitted with the chuck best suited to the diameter capillary tube chosen
and the MotoTool is secured in the head of the Dremel drill press, Model 210, as shown. The Dremel Tool is used because its chuck conveniently secures the capillary tube and the Model 210 drill press allows facile vertical adjustment of the capillary and water bath. Alternatively, an adjustable lab jack, ring-stand, clamps, and appropriate capillary tube holder can be used in conjunction with the heating unit described below. The heating unit (Figure 2) is situated just below the MotoTool chuck and is equipped with a removable nichrome (18 gauge) wire coil (Figure 1A and 2B). The coil (4mm i.d.) should be 5-7 mm below the chuck to prevent the latter from overheating. The supports of the heating unit are made of Bakelite (Cadillac Plastic, Buffalo, NY) and their dimensions are arbitrary once space is allotted for contacts, post connections, etc. The support which secures the heating coil is notched to provide additional room for an ice bath container during submergence of the capillary tube. Two threaded brass bars serve as contacts between electrical leads
0003-2700/83/0355-1184$01.50/00 1983 American Chemical Society
ANALYTICAL CHEMISTRY. VOL. 55. NO. 7. JUNE 1983
1185
,.
,
. . I _
.
.-
I3
~,
B
Flgure 3. Unsealed (A) and sealed ( 6 )capillary tubes.
Flgure 1. m e complete tube.seallng apparatus with heating coil (A) and affixed capillary tube (€3).
:ii Flgure 4. High tieM (300MHz) ‘H NMR spectrum of dexamethasone (20 pg) in acetone-d,. The dhmeter of the probe receiver coil was 5.5 mm and the data were acquired with 75000 pulses at a tip angle Of 70’.
Figure 2. Artist‘s conception of the “heating unn”. mumb nuts (A) are to secure heating coil (6) in place. Slots (C) are for horizontal adjustments.
and the nichmme wire which is p a a d through the brasa contacts. This wire is secured with washers and thumb nuts for easy removal. The leads are secured with washers and nuts and are wired to a 6.3-V, 10-A secondary transformer (no. P6308, filament transformer, Chicago Standard Transformer COT., Chicago, IL) that is in turn plugged into a VaIiac transformer. This arrangement provides manual control of the current and heating of the nichrome wire. (NOTE For SAFETY purposes it is imperative that a secondary step-down transformer be placed in series with the Variac since no more than 3-5 V should ever be delivered to the heating coil or its exposed contacts. Also, to help prevent potential hazards, a 1-A fuse is placed in the primary Variac transformer.) An in-line ammeter may be useful in monitoring the current delivered to the beating element since optimum results are attained a t variable heating ranges. Actual beat requirements depend on such things as a capillary wall thickness, coil diameter and the number of loops in the coil, The heating unit is attached to the drill press by a post affixed to the unit and a clamp. Loosening the clamp allows the beating unit to be moved back and forth when aligning the coil and capilla~~ tube. Horizontal adjustments are made possible by slots drilled in the heating unit for its attachment to the pole’s base by thumb nuts (Figure 2 0 . ODeration. The following procedure should first be carried out with blank sample tubes while adjusting the heating current. Once the primary Variac transformer is set, the capillary tube sealing procedure is as follows: The m p l e capillary tube is faed with 30-40 pL of NMR sample solution and a clay ball weight (approximately 5 g) is attached to the bottom of the tube. The
tube (Figure 1B) is placed through the coil and gently secured in the MotoTool chuck or equivalent holding device. The lower portion of the capillary tube is then immersed in an ice water bath until its contents are completely covered. The heating coil is turned “on” and the tube is allowed to draw closed due to the added weight of the clay ball. Once closed, the bath is pulled forward (breaking the sealed tube free) and the drawn tube is trimmed with the hot coil. The drawn and trimmed tip can then be quickly flamed to assure the seal.
RESULTS AND DISCUSSION T h e apparatus and method have been extensively tested and proven routine. A sealed sample tube is shown in Figure 3 along side its empty, unsealed counterpart. A high field (Bruker, 300 MHz) ‘H micro-NMR spectrum of dexamethasone (20 )rg in 30 pL of acetone-d,) sealed within a capillary tube as described herein, is shown in Figure 4. Spectral measurements were taken 7 days after the capillary tube was sealed. Lack of solvent evaporation indicated the seal was airtight The excellent spectral resolution and high signal to noise ratio acquired from the micro sample illustrate the practicality of capillary tube micmcell NMR and therefore the usefulness of a routine capillary tube sealer. Since the apparatus is composed of common laboratory equipment, i t is an affordable alternative to expensive “specialized” e q u i p ment. The apparatus is easily used and does not require the skill and patience usually necessary to flame capillary tubes closed. Once the transformer current, coil size, and coil position are set, the heating coil can deliver relatively consistent beat while gravity supplies consistent tension. To-
1186
Anal. Chem. 1983, 55,1186-1187
gether they provide reliable routine sealing of capillary tubes.
(5) Shoolery, J. N. “Microsample Techniques in ’H and I3C NMR Spectroscopy-Study of Microgram and Submilligram Amounts of Sample with the CFT-gO”, Varlan Instrument Division: Palo Alto, CA, 1979; P5. ~
ACKNOWLEDGMENT We thank S. G. Huang of the Cornel1 University Department of Chemistry for the NMR analysis.
LITERATURE CITED (1) Melnwald, J.; Wiemer, D. F.; Eisner, T. J . Am. Chetn. SOC. 1979, 707, 3055-3060. (2) Templeton, J. F.; Jackson, C. C.; Seaman, K. L. SteroMs 1982, 39, 509-516. (3) Dewey, E. A.; Maylin, 0. A.; Ebel, J. G.; Henlon, J. D. Drug Metab. D/SPOS.1981, 9 ,30-36. (4) Skrabalak, D. S.; Maylln, (3. A. SteroMs 1982, 39, 233-244.
RECEIVED for review January 17,1983. Accepted February 28,1983. We wish to thank the Zweig Memorial Fund and the New York State Racing and Wagering Board Drug Testing and Research Program for financial support for this work. We wish to acknowledge the National Science Foundation Instrumentation Program (CHE7904825)for support of the Cornell Nuclear Magnetic Resonance Facility.
Instrument for Relative Humidlty Measurement Knut Irgum Department of Analytical Chemistry, Universlty of Ume 4, S-90 1 87 Ume A, Sweden
Relative humidity is a quantity which it is often desirable to measure in the chemical laboratory. A fairly accurate instrument to measure this quantity is the well-known hair hygrometer. Ita use, however, is often limited by voluminous physical dimensions and the fact that it has to be placed in an upright position in order to give a correct reading. Another and more serious drawback is that it has to be read visually. Another widely used method is the psychrometric measurement, which is based on the temperature decrease due to vaporization of water from a wet thermometer placed in a flow of the air to be measured. The air velocity at the probe must be greater than 3 m/s in order to obtain a correct reading. Moreover, the temperature decrease is not only a function of relative humidity but also of temperature and of barometric pressure. If an electric signal proportional to the relative humidity is required, there are instruments available which will do this, but at a price which will make the chemist think twice before investing in such an instrument. In our laboratory a dynamic gas dilution system was built to produce a controlled atmosphere for testing work environmental sampling devices. One of the variables that must be controlled is relative humidity, to within h5% a t 20 “C. Consequently, an instrument was built to monitor this.
EXPERIMENTAL SECTION A sensor for relative humidity is now available at a cost of less than $10 (Philips, Eindhoven, The Netherlands, Catalog no. 2322 691 90001; Mepco/Electra Inc., Morristown, NJ). The sensor is made from a stretched plastic film, which is gold coated on both sides, forming the dielectric and plates of a parallel plate capacitor, respectively. The dielectric constant of the plastic is changed upon the adsorbtion of water, thereby changing the overall capacitance of the sensor. The manufacturers have assigned it to consumer applications, probably due to the inherent nonlinearity which they have dealt with in their literature (1,2)without greater success. By closer examination of its response, a nearly perfect exponential relationship can be found between the capacitance and relative humidity. This means that linearization can be accomplished by charging the capacitor of a series RC circuit for a time determined by the change in capacitance of the sensor. The circuit described performs the measurement of capacitance difference and the transformation of this needed t o produce a linear voltage output. The primary task is to measure the change in capacitance when relative humidity is varied. An easy way to accomplish this is to simultaneously start two monostable multivibrators, one in 0003-2700/83/0355-1166$01.50/0
which the capacitive humidity sensor is the capacitance of the time-determining RC component. The other monostable is a reference generator with its timing components chosen to match those of the sensor monostable at 0% relative humidity. If the outputs of these two generators are exclusive-OR-wired,an output pulse which is proportional in width to the sensor capacitance change is obtained. The two monostables are triggered by the negative going edge of an astable operating at 1 kHz with 95% duty cycle. The next step to be performed is to convert the exponential pulse width response to a signal which is proportional to relative humidity. This is accomplished by charging capacitor C, with a constant voltage, E,, through a charging resistor, R, for the time which the exclusive-OR-gateoutput is high, according to
E = Eo(l- e-ton/RC)
(1)
The exponential decay of voltage increase rate across the capacitor with time matches well the relative higher increase in sensor capacitance, and thus exclusive-OR-output pulse width, at higher relative humidities. The parameters Eo and RC have then to be optimized. This is accomplished by giving C a valve of 10 nF, letting a computer step through a series of E , values, calculate the R value necessary to give 1V out at 100% relative humidity, and then compute the RMS error based on every 10% relative humidity for each Eo. The value of Eo which gives minimum error is 1.48 V with a corresponding R of 20.9 k. The capacitance vs. relative humidity data used to perform these calculations is extracted from the manufacturer’s documentation by multiplying the capacitance differences between 0 and 100% relative humidity from Table I at 1 kHz operational frequency with the percent deflection of Table I1 in the technical note (1). The circuit elements which perform this charging/decharging of the capacitor are the two pairs of paralleled 4066 CMOS transmission gates switching the capacitor C to Eo via R or to ground alternately. As the signal of interest is the voltage across the capacitor at the end of the charging pulse, some means of monitoring this voltage has to be applied. In the suggested circuit this is accomplished by a bleeding peak detector. The time constant of the hold capacitor is 1 s, assuring that ripple is kept below 1 mV but still allowing the hold voltage to decrease rapidly enough when a lowering in the relative humidity to be measured takes place. The output signal was fed directly into a CA 3162 based two-digit panel meter, thus giving percent relative humidity directly.
RESULTS AND DISCUSSION As mentioned above, it was possible to calculate the sensor capacitances from the figure given in the technical note supplied by the manufacturer. With the method described 0 1983 Amerlcan Chemical Soclety
