absorbance of the blank (demineralized water plus the acid) varied from 0.007 to 0.056. This corresponds to 0.02 to 0.17 pg of mercury per liter. The precision of the method was tested by replicate determinations on three natural-water samples over a period of 2 weeks. The standard deviations, based on five determinations at the 0.13-, 1.88-, and 87.8-pg/l. levels, were 10.04, j~O.21, and i ~ 7 . 1respectively. , Several natural-water samples were analyzed for mercury by the proposed method and found to contain less than 1 pg per liter. These samples were reanalyzed after adding 0.5 pg per liter of mercury to each. The recovery ranged from 80 to 128%.
Three additional samples containing 80, 184, and 228 pg of mercury per liter, as determined by a dithizone extractioncolorimetric method, were analyzed for mercury by the silver wire-atomic absorption technique. The concentration of mercury found was 88, 188, and 208 pg per liter, respectively. Extreme dilutions were used because the upper limit of the procedure is only 1.5 pg per liter. The method has proved useful for the determination of trace amounts of mercury in samples of fresh water. RECEIVED for review June 4, 1970. Accepted July 13, 1970. Publication authorized by the Director, U.S. Geological Survey.
A Method for Calibration of Nuclear Magnetic Resonance Standard Samples for Measuring Temperature Osamu Yamamoto and Masaru Yanagisawa Government Chemical Industrial Research Institute, Shibuya-ku, Honmachi, Tokyo, Japan
IN THE PRESENT DAY high resolution nuclear magnetic resonance (NMR) measurement, variation of temperature is accomplished by passing heated or cooled nitrogen gas to a cylindrical Dewar vessel in which an NMR sample tube is inserted. The cylindrical vessel also serves as a detection coil support. By controlling the gas temperature by means of a suitable device, the sample temperature may be maintained within jZ1 “C. Unfortunately, however, there seem to be only a few reliable methods for determining the sample temperature, especially the temperature of the part where the NMR signal is detected. A conventional method which has been widely employed by Varian users is that of using a supplied “standard” sample. The temperature is obtained by measuring the chemical shift of a temperature-dependent signal of a standard sample which will provide corresponding temperature in terms of the supplied calibration chart. As standard samples, acidified methanol or ethylene glycol are usually employed. But actually the supplied chart does not seem to be sufficiently accurate, and it has been indicated that the standard sample method for measuring temperature with the aid of the supplied calibration chart is not suitable for studying the system involving spin exchange, where it is frequently desirable to extract kinetic data from the spectral pattern ( I ) . In this case, temperature is one of the major spectral parameters, and it should be determined as precisely as possible. Another method for determining temperature in the NMR sample comprises the direct measurement of gas temperature by a thermocouple or a thermistor suitably placed in the NMR probe. But this method also cannot provide actual temperature, because the heat-sensor cannot be placed near the detection coil. When the heat gradient, which is always present along the elongated cylindrical Dewar, is not maintained sufficiently small, or the flow rate of the nitrogen gas is not kept constant, the determined temperature would be different from that of the sample part where the signal is detected. Thus it was hoped to determine the actual temperature of (1) A. L. Van Geet, ANAL.CHEM., 40, 2227 (1968).
the sample readily and with reasonable accuracy, particularly for kinetic studies. Recently, some attempts have been made to solve the problem. Van Geet ( I ) proposed a method of measuring temperature of the sample in the NMR probe with a thermistor directly inserted in a spinning sample tube. This elaborate device comprises a thermistor probe covered by a tubing of polytetrafluoroethylene in the lower portion and a support which permits the thermistor to stay at rest around the spinning sample tube without touching its wall. The direct temperature reading is provided by a resistance bridge and meter placed as far from the probe as desired. He also built a spinning thermistor probe which spins along with the sample tube remaining closed in a pressure cap (2). He observed by this technique that the chemical shift between the CH2 and OH groups of ethylene glycol fits a straight line, while that of methanol does not, and should be approximated by a quadratic equation ( I ) . Forstn and his coworker (3) employed a method of calibrating temperature, which consists of making a capillary containing methanol-ethylene glycol mixture, and inserting it in a NMR sample tube together with a liquid sample of which NMR spectrum will be measured. Temperature is obtained from the shift of the OH proton of the mixture before and after an actual NMR measurement. The relation between the OH proton shift and temperature is previously determined by inserting the capillary into a xylene solution in the NMR sample tube, and measuring its shift values at various temperatures, which in turn are calibrated by a thermocouple inserted in the sample tube. Neuman and Jonas also reported another calibration method with a thermocouple in a spinning sample tube (4). Although the above methods succeeded in giving accurate measurements of the sample temperature in the NMR probe, they seem to put the operator to some bother in measuring temperature because of delicacy of the device or the trouble of (2) A. L. Van Geet, Rea. Sei. Instrum., 40, 177 (1969). (3) K.-I. Dahlqvist and S. ForsCn, J . Phys. Chem., 73, 4124 (1969). (4) R. C. Neuman and V. Jonas, J. Amer. Chem. SOC.,90, 1970 (1968).
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Figure 1. Schematic representation of dual-tube assembly (in mm). For clearer understanding of details, the figure is not drawn in the exact proportion A. Pure material used as a temperature standard B. Liquid giving a lock signal
inserting a calibrated capillary into a sample tube at each time measurement, particularly when the sample is sensitive to air or moisture so that the tube should be sealed off. On the other hand, the conventional method with “standard” sample, if the temperature-dependent shift of the latter is properly calibrated, is considered still to provide one of the
1’=-0.9832+187.1 ( 2 0.8’C) I
I
,
,
I
120 150 180 210 Methanol O H shift in Hz at 100M H r
Figure 3. Calibration chart for methanol standard sample, similar to Figure 2 Temperature standards used are (a) p-dichlorobenzene, (b) cinnamyl alcohol, (c) water, (d) rerr-butylbromide, and (e) rrans-1,2-dichloroethylene most convenient methods for measuring temperature with reasonable accuracy. In this work, as an effort to overcome the difficulties mentioned above, a novel and fairly simple method is given for calibrating the shift-temperature relation of the standard sample with good accuracy. This method is based on the fact that in a high resolution NMR spectrum the solid material does not give any signal, but does show a sharp signal when it melts. If a pure material
il
( b ) 102.6Hz
40
70
100
130
240
160
Ethylene glycol OH shift in Hz at 1OOM Hz
Figure 2. Calibration chart for ethylene glycol standard sample. The experimental formula giving temperature value (“C) was obtained by regression analysis, in which 6 is the shift difference between OH and CH2 protons expressed in Hz a t 100 MHz Materials indicated in the figure were used as temperature standards: ( a ) 1,3,5-tribromobenzene, (b) benzoic acid, (c) p-dibromobenzene, (d) 1,3,5trichlorobenzene, (e) p-dichlorobenzene, and (f) cinnamylalcohol 1464
( a ) 102.9Hz
(c)
102.OHz
Figure 4. NMR signal of pdibromobenzene placed in the part (A) in Figure 1, below melting point (a), a t about melting point (b),and above melting point (c). Figures in Hz represent the shift difference (at 100 MHz) between the CHs and OH protons measured by replacing the dual-assembly by the standard sample to be calibrated
ANALYTICAL CHEMISTRY, VOL. 42, NO. 12, OCTOBER 1970
with a definite melting point is placed in the NMR sample tube, and measured in the NMR probe with increasing temperature, the signal will appear as soon as the material melts. If, in this instance, the standard sample is inserted into the NMR probe without any change of other measuring conditions, and its temperature-dependent chemical shift is determined, then the shift value can be calibrated by use of the melting point of the pure material. EXPERIMENTAL
Figure 1 shows the sample tube used in this work, which was made by Shigemi Standard Co. Ltd., Shinjuku-ku, Tokyo, Japan. The inner wall of the outer tube and the outer surface of the inner tube are ground to fit so that the extension of the inner tube with smaller diameter is kept vertical in a fixed position. A small amount of pure material with known melting point is placed in the bottom of the extension of the inner tube ( A ) . The space between the inner and outer tubes in the bottom portion ( B ) is filled with suitable liquid, one of whose signal is conveniently used as a lock signal, The inner tube is capped or, preferably, sealed off to avoid absorption of moisture by the material, especially at low temperature. The whole assembly is placed in the NMR probe in the usual manner and the NMR measurement is made with sample spinning. The materials used were purified by conventional methods in combination with the preparative gas-chromatography or zone-melting technique. They are indicated in Figures 2 and 3. These materials are selected from easily available compounds with suitable purity giving a rather sharp signal which does not overlap the signals of the liquid contained in part ( B ) . This is also true for the selection of the liquid giving a lock signal, provided that it remains as liquid at any measuring temperature. Of course, if the spectrometer used is of external lock type, the liquid in part ( B ) is unnecessary, or it may be a liquid compound giving no NMR signal. It is essential that the amount of the pure material in part ( A ) be small enough that in the whole volume of the material the temperature gradient may be negligible within the desired accuracy. This is also important in order to reach thermal equilibrium as fast as possible over the volume of the material after insertion of the assembly. At first, the temperature setting in the probe is slightly below the melting point of the material, and the temperaturedependent shift of the standard sample is determined. Then the dual-tube assembly is inserted in the probe with the precaution not to change any other measuring conditions and the signal is located after sufficient time interval to reach thermal equilibrium in the sample tube. This requires more than 1 hour. If the signal is not observed, the temperature setting is increased by about 0.5 "C, and the above procedure is repeated until a sharp signal of the material is obtained. As an example, the obtained result for p-dibromobenzene as a temperature standard is shown in Figures 4 and 5 . Figure 5 shows that the change of the signal height is sufficiently sharp that it is possible to correlate the obtained shift value of the standard sample with the melting point of the material within 1 0 . 5 "C. The NMR measurements were carried out at 100 MHz by a Varian HA-100 spectrometer with a V-4343 VariableTemperature Accessory. The shift difference between OH and CH2or C H a protons of the standard samples is expressed in Hz measured at 100 MHz. RESULTS AND DISCUSSION
Figures 2 and 3 show the obtained results for supplied standard samples of ethylene glycol and methanol, respec-
I
100
105 Shift ( Hz )
110
Figure 5. Signal height of p-dibromobenzene plotted against shift difference in ethylene glycol at the same temperature setting
tively. The regression analysis leads to the linear equations described in the figures, which provide temperature values in terms of the O H proton shifts (in Hz at 100 MHz). In contrast with Van Geet's observation, the methanol shift is linear over the temperature range measured. From our experience, it seems that the OH proton shift is slightly different depending upon preparation conditions of the sample, probably due t o the different amounts of added acid, water, and other substances, if any. The errors given by the regression analysis are well within jZl.0 "C but the precision of the temperature controller used is f1.O "C, so that the over-all accuracy of the temperature should be considered as large as + 1.0 "C. The main feature of the method herein described resides in that in this procedure one can calibrate the standard sample without special device and in the condition which is almost the same as in the actual measurement. It is unnecessary either to stop the sample spinning or to remove the pressure cap from the sample housing. Thus all environmental and flow rate conditions are the same as the situation of the actual measurement. An additional merit of this procedure is that, once a standard sample is calibrated by this method, other standard samples can be calibrated with the first, and a great saving both in time and in labor may be obtained. It is interesting to note that the difference between the value obtained after this calibration and the value given by the supplied calibration chart is often as large as 5 "C or more on the one side, while it is almost zero on the other side of the measurable temperature range. This means that great care must be taken when the Arrhenius parameters are calculated from the kinetic data in the NMR method, as suggested by Van Geet (1). ACKNOWLEDGMENT
The authors express their gratitude to Hiroshi Tomita and Yasuko Nakano of our institute for the purification of the materials used as temperature standards by the preparative gas-chromatography and zone-melting technique, respectively. RECEIVED for review May 5 , 1970. Accepted July 7, 1970.
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