standard error of zk0.3 Hz. on the measurement of each peak frequency, a precision of &0.4' C. should be obtained on a single temperature measurement. Table I shows the standard errors in Herzogs for single determinations of proton magnetic resonance chemical shifts determined on a series of dilute aqueous solutions using the thermometer between 0' and 30' C. The first column gives the solute mole percentage; the second, the number of nieasurements of each chemical shift; the third, the standard error on a single determination of the methylene chemical shifts (which have small temperature coefficients d v ~ d t= zk0.05 Hx./O C.); and the final column, the standard error on a single determination of the water proton chemical shift (which has a large temperature coefficient d v p / d t = -0.55 Hz./"C.). All peaks were sharp
and could be readily measured. The standard error on the methylene proton determinations provides an essentially temperature-independent blank for the overall instrument-measurement accuracy, while the standard error on the water signal includes the temperature error component (dvJv/dt).u ( t ) . It is immediately apparent that the use of the thermometer permits the water signal to be measured with an error of the same magnitude as that for the temperature insensitive methylene groups. The standard error for a single water determination is 10.326 Hz. for the set of 104 determinations, while that for a single methylene group determination is 10.288 Hz. for the set of 124 determinations: the square root of the variance difference of the water and methylene signals is the temperature error component, from which it is found that the thermometer follows the
sample temperature with a standard error ~ ( t = ) hQ.28' C. Over a 9-month period, the thermometer liquid changed color from a light yellow to a dark red-brown; however, no change of the proton magnetic resonance spectrum was observed and regular temperature checks by the methanol standard showed that the thermometer calibration was accurately retained. Convenient and successful use of the thermometer in our particular study of dilute aqueous nonelectrolytes for 1000 chemical shift determinations, lead us t o believe that this proton magnetic resonance thermometric technique may have more general application to other aaueous and alcoholic solution work. LITERATURE CITED
(1) Vmian Assoc., Technical Information Publication 87-100-1 10.
Nuclear Magnetic Resonance Analysis of Carbon-1 3 Enriched Methane Ralph R. Eckstein and Albert Attalla, Monsanto Research Corp., Mound Laboratory, Miamisburg, Ohio
carbon-13 by NhIR A (nuclearofmagnetic resonance) deNALYSIS
scribed in this paper is obtained by a ratio method involving hydrogen atoms bonded to both carbon-12 and carbon-13. The most abundant isotope of carbon (98.792% carbon-12) does not exhibit nuclear magnetism ( 1 ) . Although carbon-13 (1.108% natural abundance) possesses this characteristic, its nucleus is not as sensitive to NMR absorption as the hydrogen atom and therefore gives a less intense absorption signal for equal numbers of nuclei. I n addition t o the ease of detecting the proton signal, the ratio method of determining carbon-13 enrichment obviates the necessity of preparing standard samples and calibrated working curves.
Figure 1.
NMR Dewar sample tube
Shaded portion is internally silvered
EXPERIMENTAL
The carbon-13 enriched methane was separated in thermal diffusion columns by W. >I. Rutherford of this laboratory from Matheson ultra-pure (99.96 mole %) methane gas. The enriched gas was stored in a 500-ml. round-bottom flask which was fitted with a vacuum stopcock and ball joint for transferring the sample to the Dewar system. A Varian Associates DA 60 EL (60 m.c.p.s. dual purpose instrument with external proton lock field stabilization) nuclear magnetic resonance spectrometer was used to obtain the radiofrequency absorption spectra of the liquefied gases. The spectra were produced on a flat-bed Varian recorder a t 500-C.P.S.sweep width and 100-second sweep time I
To improve the sensitivity of detection of the XhfR absorption signal and thereby increase the accuracy of the analysis, a special apparatus was designed and constructed which permits liquefaction of the methane gas (in the NMR probe), its measurement by NMR as a liquid, and, finally, recovery of practically all of the gas. The equipment as designed and built consists of a sample tube of Dewar glassware necessary for the transportation of both cooling gas and sample gas to the NMR probe. The outer and inner walls of the Dewar sample tube (Figure 1) were constructed from 15-mm. to 10-mm. borosilicate glass tubing, respectively. Both walls were continued into the 19/38
taper male joint that was evacuated from thermal insulation. The Dewar glassware used for transporting the sample and cooling gases and for supporting the sample tube in the NLIR probe is illustrated in Figure 2. The cooling gas (dry nitrogen passed through two copper heat exchangers submerged in liquid K2)enters the Dewar system a t point A and flows through the cold finger to exit a t B. After the sample chamber is evacuated, the carbon-13 enriched gas enters a t C. The cold finger, acting as a condenser, pulls the gas into the chamber where it liquefies on the cold finger and fills the sample tube. The liquid level is maintained by constant flow of cooling gas through the cold finger. Thus, the gas can be kept as a liquid in the NhIR probe for an indefinite period of time. To maintain the sample in a liquid state when not under visual observation, the proper chamber pressure was previously determined and the flow rate adjusted to maintain this pressure. No provision was made for spinning the sample tube. RESULTS AND DISCUSSION
The proton K;MR spectrum of naturally occurring methane gas consists of a large central absorption peak surrounded by two very small, approximately equally spaced, peaks on either side of the central peak. The large central peak is due to the absorption of r j energy by all protons bonded to carbon-12 atoms whereas the two small side peaks are due t o those protons bonded to carbon-13. Because the natVOL 38, NO. 13, DECEMBER 1966
1965
the area under the peaks either electronically or mechanically. The abundance of carbon-13 in a sample of highly enriched methane gas (96.2 mole %) v,-as calculated by measuring the areas of the proton NMR peaks using a planimeter and dividing the total area of all the peaks into the area of the two outer peaks. A mass spectrographic analysis of this sample agreed within experimental error ( 1 0 . 2 mole %). The per cent error in nieasuring the small central peak !vas 2% and that of the large outer peaks 0.27,. This resulted in an experimental error of &0.2% in measuring the carbon-13 abundance. ACKNOWLEDGMENT
Figure 2.
Sample and cooling gas transport‘ chamber
The authors thank Robert C.Glover for his assistance in preparation of the glass Dewar system and Walter J. Haubach for his helpful suggestions on equipment design. LITERATURE CITED
ural relative abundance of the two carbon isotopes is in the ratio of 98.9 to 1.1, the relative intensities of the peaks will follow in the order 0.55:98.9:0.55. The small doublet arises from spin-spin coupling (e) between the hydrogen and carbon-12 atoms. This does not occur between hydrogen and carbon-12,
as the latter is not magnetically active. The determination of the relative abundance of the two carbon isotopes in carbon-13 enriched methane gas is obtained simply from the ratio of the intensities of the two sets of proton absorption peaks. These intensities can be obtained directly by integrating
(1) Pople,
V. A.. Schneidcr, IT. G.,
Bernsteia, H. J., “High-Resolution Nuclear Magnetic Resonance,” p. 307, l\lcGraw-Hill, New York, 1959.
( 2 ) Zbid., p. 91. Mound Laboratory is operated by &Ionsaiito Research Corp. for the Atomic Energy Commission under Contract KO,
AT-33-1-GEX-53.
Multicell Diffwsion Trays for Determining Inorganic Fluoride in Physiological Materials Clyde
R. Nicholson,
Davies, Rose-Hoyt Pharmaceutical Division, The Kendall Co., Kendall Research Center, Barrington, 111.
for an efficient and Idetermining reliable analytical procedure for fluoride in physiological N
BEARCHISO
materials, the microdiffusion method of Wharton (9) was found to be the most appropriate. Reliability in determining fluoride in tooth enamel was good, and as many as 20 analyses could be completed in a day. Fluoride determinations of urine samples were acceptable when run in new polypropylene Conway diffusion cells, but variations increased beyond acceptable limits with continued reuse, even though they were carefully cleaned each time. I n attempting to solve this problem, polyethylene diffusion cells were vacuum formed using a polypropylene cell as a mold. Results with both tooth enamel and urine samples mere excellent, Reproducibility mas better than that obtained in new Conway units, possibly due t o the abiIity t o maintain a more positive seal during the diffusion cycle. The cover of these disposable units was a circle of Mylar or polyethylene film sealed to the middle annular ring with silicone grease. This improvement in reliability led to the production of a compact niulticell disposable tray, the cells of which ivere 1966
ANALYTICAL CHEMISTRY
sealed during the diffusion cycle with a single sheet of polyethylene film. The use of these trays of diffusion cells, coupled with some innomtions in procedure technique, resulted in improyed reliability and greatly increased efficiency in deterining 0.0 pg. F to 4.0 pg. F in physiologic$ materials. EXPERIMENTAL
Reagents and Apparatus vere as outlined by Wliarton ( 2 ) for 5-em. spectrophotometric cells, except for the following innovations. Diluted Spectrophotometric Reagent. To 167 ml. of the single spectrophotometric reagent was added 42 ml. of 70% HC104 and the final volume was brought to 1000 ml. with redistilled deionized water. This reagent is stable indefinitely. 10” X 12”, 30 cell
laboratory production froni Comet Industries, Inc., Bensonville, Ill., of 30-mil low density polyethylene. The master mold of hard maple was designed t o pioduce units with a single annular conipartnient 40 min. in
diameter and 10 mni. deep, with a total capacity of about 5 nil. The center well with 2.5 nim. walls is 14 mm. in diameter and 5 nim. deep. Covers for Diffusion Cell Trays are 11” X 13”, 3-mil polyethylene sheets, which are used with D o v Corning high vacuum silicone grease to seal the cells during the diffusion cycle. Repeating Pipet. The 10-ml. Repipet by Labindustries, 1740 University Avenue, Berkeley 3. Calif., is used to dispense 6.0 nil. of the diluted Zr SPdDNS to each 25-m1. sample flask. Procedure. The diffusion cell tray is prepared for use by applying a liberal coating of high density silicone grease around each cell before placing the KaOH in the center wells. Samples for analysis, containing 0.0 t o 4.0 pg. F, are then placed in the single annular chamber. One i d . or more of liquids such as urine or blood is used: smaller aliquots may be diluted to approsimately 1.0 ml. with deionized water. Dry samples, as tooth enamel, are wet with 1.0 ml. of deionized water before placing 0.5 nil. of concentrated HC104 in the annular chamber, away from the sample. (The smooth surface of the polyethylene permits complete separation of sample and HClOd until
