1460
Anal. Chem. 1986, 58, 1460-1461
Determination of Deuterium in Water by Gas-Phase Infrared Spectrophotometry Jamie J. Shakar, Charles K. Mann, and Thomas J. Vickers*
Department of Chemistry, Florida State University, Tallahassee, Florida 32306-3006
The sensltivlty of gas-phase infrared spectrophotometry for the determlnatlon of deuterium In water Is evaluated. Syringe lnjectlon of 20 pL of sample Into a heated 10-cm-path-length cell Is shown to provide a detectlon limit of about 60 ppm. The analytlcal response Is found to be linear with concentration from natural abundance (150 ppm) to at least 1.8 atom % deuterlum.
Deuterium oxide dilution has been extensively used for water volume measurements, particularly for the measurement of total body water volume in mammals. The basis of the measurement and methods employed for the determination of deuterium in aqueous media have been described in a recent report (1). Mass spectrometry, using an instrument designed specifically for 2H/1H measurements, is at this time the preferred method for total measurements. Evaluation of a commercial system for this purpose has recently been described (2). In the mass spectrometric method, a vaporized water sample is converted to hydrogen gas in a uranium furance, and the relative abundance5 of the lHZ+and 'H2H+ions are measured. Condensed-phase infrared determinations of deuterium concentration in water have been carried out using 0.2-mmpath-length cells with calcium fluoride windows ( 3 , 4 ) . Deuterium sensitivity of 1 ppm was achieved with a specially designed infrared filter photometer, but temperature regulation of the sample to d=0.005O C was required (4). In both cases a relatively large volume of sample, typically 10 mL, was required. Vapor-phase infrared measurement offers a number of advantages for deuterium measurement, if adequate sensitivity can be achieved. Both the condensed-phase infrared and mass spectrometric procedures require separation of water from the sample. This is usually achieved by vacuum sublimation. With vapor-phase infrared measurement, this separation step could be omitted. Compared to mass spectrometric measurement, the infrared method offers the advantage of employing readily available general purpose laboratory instrumentation. Compared to condensed-phase infrared measurements, the vapor-phase measurement requires only a few microliters of sample for each determination. This report describes an examination of the sensitivity of the vapor-phase method employing a general purpose dispersive infrared spectrophotometer and a 10-cm-path-length cell. It is well-known that the sensitivity of vapor-phase infrared measurements can be increased by a t least an order of magnitude by the use of commercial folded-path cells of modest internal volumes. Body water measurement on adult humans employing the deuterium dilution method typically produces changes of 30-35 ppm in deuterium abundance (1). Thus demonstration of a sensitivity of the order of 300 ppm in this study would indicate the utility of the vapor-phase infrared method for body water volume studies. MATERIALS AND METHODS Reagents. A stock solution of approximately 1% 2H20 by weight (1.6 atom % '%) was prepared by adding a weighed amount 0003-2700/86/0358-1480$01.50/0
of deuterium oxide (Aldrich Chemical, Gold Label, 99.8 atom % 2H, 15, 188-2)to a weighed amount of deionized water. The lH content of the 2H20was determined by proton NMR at the end of this study was found to be 1.2%. This variation in the deuterium content of the reagent is of no significance in the context of this study, and the 99.8% assay value was used in computing the deuterium content of standard solutions. Standard solutions were prepared by serial dilution of the stock solution with deionized water. Instrumentation. Infrared measurements were made with a Perkin-Elmer 983 spectrometer. The chopper in this instrument is placed between the source and sample, thus eliminating the effect of emission from heated samples. The sample cell was constructed from a 3-cm square block of aluminum. A 2-cm-diameter hole bored the length of the block is capped with O-ring sealed calcium fluoride (4 X 25 mm) windows to provide a cylindrical sample chamber with a volume of about 30 cm3and an absorption path of 10 cm. A port through one side wall is used to connect the cell to a mechanical vacuum pump for evacuation. A gate valve allows the cell to be isolated from the pump prior to sample introduction. A port through the top wall at the midpoint of the cell is capped with a rubber septum to permit introduction of samples by a syringe. In operation the cell is maintained at approximately 125 "C by an 80-W cartridge heater in a 1/4-in.well in the aluminum block below the midpoint of the sample chamber. A flange at one end that mates with the grooves of the Perkin-Elmer cell holder completes the assembly. Procedure. Twenty-microliter portions of sample are injected into the previously evacuated cell maintained at 125 O C , producing a pressure of about 1.2 atm in the cell. In a typical measurement, spectra are recorded at l.O-cm-' sampling interval, over a 27602670-cm-l range, requiring less than 5 min/scan. The band used for measurement is the 2720 cm-l Q branch of the 0JH stretching fundamental of lH2H0 (5). It lies in a region almost devoid of 'H20 lines, and, at the low deuterium concentrations used in this study, 2H20lines are also absent. Data sets are transferred via RS232 link to an IBM PC for processing by software that operates under the p-System furnished by Network Consulting, Inc., Burnaby, BC, Canada. All locally developed software is written in Pascal, except the Fourier transform, which is in 8086 assembly language. A novel data processing strategy, data domain averaging, was found to be superior t o peak height, area, cross-correlation or least-squares methods of data reduction (6). The sample cell vs. air measurement method employed produces a spectrum that is superimposed upon a large pedestal. Data sets are prepared by projecting a straight line between the first and last points of the region of interest. All points outside this region are nulled, and the portion of the signal below the line is . subtracted.
RESULTS AND DISCUSSION Spectra. Figure 1illustrates the appearance of the spectra over the concentration range examined. The lowest concentration run in each set corresponds to natural abundance, 0.015% 'H. The highest concentration on the analytical curve is approximately 0.1%. The reference spectrum used in quantitation is obtained with a solution having a deuterium concentration of l.6-1.8%. The highest spectrum in Figure 1 is a reference spectrum that has been divided by a factor of 10 to allow presentation on the same scale as the lowconcentration spectra. The spectra in Figure 1are each the ensemble average of five individual runs. There are weaker 0 1986 American Chemical Soclety
ANALYTICAL CHEMISTRY, VOL. 58, NO. 7, JUNE 1986
1461
Table I. Analytical Curve Data no.
set dataa REP
PTS
slope
intercept
coeff
corr
std deviation of slope
std deviation of intercept
limit of detection, %
1 2 3 4
2 10 5 5
5
0.576 0.577 0.576 0.611
0.0132 0.0111 0.0133 0.0148
0.999 98 0.999 99 0.999 97 0.999 99
0.0010 0.0014 0.0025 0.0017
0.0010 0.0011 0.0019 0.0012
0.0054 0.0056 0.0099 0.0060
4 4 4
“REP is number of replicate measurements at each concentration, and PTS is number of concentration points in set.
w 6o
0
40
a 0 20
co
m
4 0 2670
2690
2710
2730
2750
WAVENUMBER, cm-‘ Figure 1. ‘H’HO spectra for various concentrations of deuterium in water. The lowest spectrum corresponds to natural abundance. For clarity, an offset has been added to each of the other spectra. Concentrations, in % ‘H, are 0.015, 0.031, 0.055, 0.095, and 1.61. The highest concentration spectrum has been divided by 10. Absorbance values have been multiplied by 10 000.
spectral features for lH2H0 on both sides of the 2720 cm-l feature, but increasing the spectral window to include these features in the data domain averaging process degrades the limit of detection and increases the time required for a scan. Slit Width Selection. A slit width of 0.303 mm was found to produce the best results with the spectrophotometer used. With a slit width of 0.864 mm the absorption line was so broad that it became difficult to discriminate against the background at low concentration, and the limit of detection was degraded. With a slit width of 0.185 mm the absorption line was sharper, but the decreased light throughput of the system increased the noise associated with the measurement sufficiently that the limit of detection was again degraded. A different optimum slit width might be found with a different spectrophotometer. Analytical Curve Results. Table I summarizes the analytical curve results for four independent calibration runs carried out over a period of several weeks. Set 4 corresponds to the spectra shown in Figure 1. Data domain averaging (6) was employed with sets 1,3, and 4. The scans in set 2 covered only 31 cm-l and did not provide enough points to permit useful data domain averaging. The results reported for this set were obtained by correlation (7). Each set includes four or five calibration points in the 0.015-0.1% 2H concentration range in addition to the sample used to obtain the reference spectrum. Set 1consisted of duplicate scans for each sample and five scans of the reference spectrum. Ten 31-point scans were averaged for each sample in set 2. In sets 3 and 4 results are for the average of five scans at each concentration. The limit of detection is defined as 3 times the standard deviation of the intercept divided by the slope. These results indicate that changes in deuterium concentration of the order of 60 ppm can be detected at the 99% confidence level with a 10-cm-path-length gas cell and syringe introduction of 20-bL samples.
Comparison of scatter for multiple scans of a single sample injection with the scatter for multiple injections makes it evident that the limit of detection is to some extent affected by the uncertainty associated with syringe delivery of the sample. Thus some improvement in the limit of detection can be anticipated by use of a more elaborate sample delivery device, such as the sampling loop devices used in chromatographic measurements. Of greater consequence for the sensitivity of the vapor-phase method for deuterium measurement is the possibility of increasing the absorption path length. By use of a multipassing cell the sensitivity can be increased by at least an order of magnitude. Commercial devices are available that provide a path length of 1m for a total volume within the sample cell of less than 50 cm3 (e.g., the Specac GC/TR interface marketed in the US by Aries, Inc., Acton, MA). This suggests that the gas-phase infrared method provides the ability to detect changes of a few parts per million in deuterium concentration. The analytical curve data indicate good linearity in response to at least 1.8% 2H. Thus the method is useful for a wide range of deuterium concentrations and should prove applicable to a variety of problems requiring deuterium determination, including the measurement of total body water volume. Serum Samples. To provide a preliminary assessment of the likely utility of the method for deuterium determinations without prior isolation of the water fraction, spiked serum samples were injected directly into the heated cell. Spectra apparently identical with those in Figure 1were obtained, with no notable change with time or number of injections. When the sample cell was inspected a t the end of this process, the only evidence of serum injection was a collection of small flakes of dried material in the bottom of the cell. It is evident from these tests that there are no spectroscopic or instrumental problems associated with the direct injection of serum samples.
ACKNOWLEDGMENT We acknowledge with thanks the contribution of Rick Flurer, who participated in the initial design and testing of the vapor-phase cell and preliminary infrared measurements on water vapor. LITERATURE CITED (1) Halliday, D.; Mliler, A. 0. Biorned. Mass Spectrom. 1977, 4 , 82-87. (2) Wong, W. W.; Cabrera, M. P.; Kleln, P. D. Anal. Chern. 1984, 56, 1852-1868. (3) Stansell, M. J.; MoJlca,L., Jr. Clin. Chern. (Winston-Salem, N . C . ) lS88, 74, 1112-1124. (4) Byers, F. M. Anal. Biochem. 1979, 98, 208-213. (5) Benedict, W. S.; Gailar, N.; Plyler, E. K. J . Chern. Phys. 1958, 2 4 , 1139-1165. (6) Mann, C. K.; Vickers, T. J. Appl. Spectrosc., in press. (7) Mann, C. K.; Goleniewski, J. R.; Slsmanidls, C. A. Appl. Spectrosc. 1982, 36, 223-227.
RECEIVED for review November 8, 1985. Accepted February 3, 1986.
