(8)A. Arbin, P. Edlund, Acfa Pharm. Suec., 12, 119 (1975). (9)V. S . Estevez. L. F. Englert, and B. T. Ho, Res. Commun. Chem. Patho/. Pharmacol., 6 , 821 (1973). (10) Y. Grunfeld and H. Edery, Psychopharmacologia, 14, 200 (1969).
RECEIVEDfor review November 10, 1975. Accepted December 29, 1975. This work was supported by Grant DA-18 from the Medical Research Council of Canada.
Determination of Hydrogen Sulfide and Methyl Mercaptan in Mouth Air at the Parts-per-Billion Level by Gas Chromatography A. R. Blanchette" and A. D. Cooper Analytical Chemistry Department, Vick Divisions Research and Development, 1 Bradford Road, Mount Vernon, N. Y. 10553
Gas chromatographic techniques were adapted for quantifying hydrogen sulfide and methyl mercaptan at the low parts-per-billion level in mouth air samples. Major innovations were calibration of the system with hydrogen sulflde and methyl mercaptan permeation tubes, use of a variable stream splitter to produce a wide range of vapor concentrations, and adaptation of the sampling equipment to handle discrete samples of limited volume. The limits of sensltlvlty were 7 ppb for hydrogen sulfide and 15 ppb for methyl mercaptan in a 10-ml mouth air sample.
The quantification of volatile sulfur compounds in mouth air presents an interesting and unusual problem in chemical analysis, a problem which in several ways has more demanding requirements than those of air pollution studies. Interest in the clinical applications of such quantification arises from the fact that two of these compounds, hydrogen sulfide and methyl mercaptan, are important constituents of oral malodor (1, 2). Determination of the range of concentrations of these compounds in mouth air samples before and after various hygienic procedures presents the following requirements. 1) For ideal functioning the calibration system should be adapted specifically for hydrogen sulfide and methyl mercaptan. 2) Noncontinuous sampling must be used because of the limited volume of mouth air available (25 to 35 ml), instead of the continuous-flow sampling utilized for air pollution studies. 3) Measurements must be made a t the low ppb level (usually well below 1000 ppb), but in a range of concentrations due to the considerable natural variation between subjects and even in the values obtained for a single subject within a single day. 4) The method should be simple and quick, since it will be used for repeated measurements in a group of waiting subjects. T o meet these requirements, in the research described below, we made certain modifications or adaptations in existing instrumentation, as utilized for air pollution studies. Key contributions to the progress of the air pollution studies were the development of a flame photometric detector with excellent sensitivity and selectivity for sulfur-containing compounds (such as described in ( 3 ) ) ,use of permeation tubes to produce low concentrations of volatile compounds in gaseous mixtures, thereby providing a means for accurate calibration of the detector ( 4 ) ,and utilization of a gas chromatograph, equipped with such a detector calibrated with permeation tubes, for the detection and quantification of sulfur compounds in the ambient air ( 5 ) .
EXPERIMENTAL Figure 1 shows our adaptation of the equipment for analyzing
hydrogen sulfide and methyl mercaptan. Details of the system are as follows. Calibration System. Known levels in the ppb range of hydrogen sulfide and methyl mercaptan for calibration are produced as follows. The flow of nitrogen gas through the flow controller and the flowmeter (0 to 200 ml/min) is held constant a t 150 ml/min, although other flow rates can be used, depending on the desired range of concentrations. The nitrogen then passes through a copper tube (lk-in. 0.d. X 10 ft) immersed in a constant-temperature bath to equilibrate the gas a t 30 & 0.1 "C. The mixture of gases emerging after the equilibrated nitrogen has flowed through the 500-ml gas-washing bottle containing the permeation tubes is split a t the tee in a ratio adjustable by the needle valve. Unused gas is measured by a second flowmeter (0 to 200 ml/min). The smaller fraction of the split stream then passes through a glass capillary restrictor into a three-way valve where it may be diverted for measurement with a soap-film flowmeter or else may continue to the Teflon low-resistance mixing chamber (1.0-cm i.d. X 8 cm) for dilution by additional nitrogen to the desired concentration. A gas sample for calibration can then be drawn from this chamber through the transfer line, located 3.5 cm downstream from the entering mixture of diluted permeation gases. Sampling and Chromatographic Analysis. Either a calibration sample or a mouth air sample withdrawn via the disposable sampling probe (a 2- to 3-in. length of 0.12-cm i.d. Teflon tubing) is delivered through the selector valve to the Chromatronix sampling valve, from where nitrogen carrier gas (72 ml/min) sweeps it into the chromatograph for separation of the volatile sulfur compounds on the 30 ft X %-in. 0.d. FEP Teflon chromatographic column. The column is packed with 5% w/w polyphenyl ether and 0.05% w/w phosphoric acid on 30/60 mesh Teflon. The column temperature is either 58 or 72 "C. As the volatile sulfur compounds emerge from the column, they are detected by the flame photometric detector equipped with a 394-nm sulfur-specific optical filter. The detector functions a t a temperature of 120 "C, with a hydrogen flow of 100 ml/min, and with an air flow of 70 ml/min. All parts of the system in contact with the sulfur compounds were constructed of Teflon, with the exception of the capillary restrictor, the splitting tee, and the gas-washing bottle, all of which were glass. Specific modifications made to meet the requirements listed in the introduction for mouth air studies were: 1) Permeation tubes of hydrogen sulfide and methyl mercaptan were used to produce ppb levels of these two compounds, equivalent to the range expected in mouth air samples, for specific calibration of the flame photometric detector. Previous clinical workers (1, 6 ) had extrapolated from gas chromatographic responses to the concentrations produced by a sulfur dioxide permeation tube; however, we wished to bypass the problem noted by other authors ( 7 ) of differing losses, largely by adsorption, in the system for the various sulfur compounds. 2) In air pollution studies a sampling pump is commonly used in conjunction with a 6- or &port gas sampling valve to fill the sample loop. Because of the small size (10 ml) of the discrete samples of mouth air, we used a 20-ml syringe instead of the pump, after preliminary tests had shown that this modification did not adversely affect the sensitivity or reproducibility of the system. Further preliminary tests were performed t o develop a standardized procedure for the actual sampling of mouth air. These tests demonstrated that the observed sulfur levels in mouth air A N A L Y T I C A L C H E M I S T R Y , VOL. 48, NO. 4, APRIL 1976
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Calibration
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METHYL MERCAPTAN PERMEATION TUBE CONTROLLER
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Figure 1. Equipment for analyzing volatile sulfur compounds in mouth air
were not noticeably affected if the subject happened to agitate the air with his tongue or swallowed before sampling, or if the mouth air consisted of inhaled or exhaled air, and that no advantage was gained by the subject keeping his mouth closed for longer or shorter periods of time than 1 min before sampling. These results enabled us to draw up specific directions for the subjects in order to minimize interference from lung or gastrointestinal sources of sulfur compounds. Each subject was instructed to breathe in through his mouth, and then to close his mouth for 1 min while breathing normally through the nose, in order to limit air exchange in the mouth. At the end of the minute, a Teflon sampling probe connected to the analyzing equipment was inserted between closed lips, 4 cm into the mouth, avoiding contact with the tongue or mucosa. While the subject held his breath, the plunger of a syringe connected through the sampling valve to the probe was slowly withdrawn over 6 s to the 20-ml mark, thus flushing and filling the 10-ml sample loop with mouth air. To avoid carryover contamination with the volatile sulfur compounds, a fresh sampling probe was used for each determination. If saliva entered a probe during sampling, a fresh probe was inserted and the procedure repeated, since the saliva could cause partial or complete blockage. The sampling procedure took approximately 70 s, with most of this time being spent on preparing the subject for the actual sampling. For at least 1 h prior to and during the day's measurements, the subjects had refrained from actions that could affect the mouth air concentrations, such as eating, drinking, smoking, chewing gum, or brushing their teeth. Again because of the limited sample size, it was important to 730
ANALYTICAL CHEMISTRY, VOL. 48, NO. 4 , APRIL 1976
minimize s mpling losses by dsorption. We found that this could be achieved ifthesample remained in the loop for less than 5 s before being swept onto the chromatographic column. Calibration samples were likewise withdrawn with the syringe. This procedure yielded a theoretical sampling rate of approximately 200 ml/min, which is half the slowest flow of calibration gases in the mixing chamber. This ensured against attempted withdrawal of a calibration sample faster than it was produced, which would have caused gas t o be drawn back from the waste line of the mixing chamber. 3) To obtain a broad range of concentrations a t the low ppb level of interest to us, and do so with low gas flows as well as rapid equilibration between concentration changes, we incorporated a variable stream splitter between the gas-washing bottle, which serves as a permeation-tube chamber, and the dilution chamber. The nitrogen gas flow over the permeation tubes was held at about 150 ml/min, and the gaseous mixture emerging from the gas-washing bottle containing these tubes was split a t the tee in a ratio of 4:l or greater, with this ratio being adjustable by a needle valve. Ratios lower than 4:l with a gas flow over the permeation tubes of 150 ml/min require excessive pressure in the gas-washing bottle to force the resulting stream through the capillary restrictor. Consequently, to obtain high concentrations of calibration gases, it is better to remove the splitting tee and to direct the total flow from the gas-washing bottle to the dilution chamber instead of lowering the split ratio. The larger fraction of the split stream was vented while the smaller fraction was passed on through the capillary restrictor. By turning the three-way valve, the split ratio of the s h a m was accu-
rately measured by the soap-film technique. Thus, only the smaller portion of the initial calibration stream had, upon arrival in the mixing chamber, to be diluted with nitrogen to the desired low concentrations. To achieve good mixing, the diluent stream of nitrogen was aimed directly at the entering stream of calibration gases. To accommodate the flows of diluent necessary to obtain low ppb levels but without inducing pressures that would affect either the gas flow from the permeation tubes or the split ratio of calibration gases, it was critical to use a mixing chamber of low resistance and to vent waste from this chamber. Because of the high reactivity of sulfur-containing molecules, we used Teflon beyond the capillary restrictor and up to the detector so that all samples contacted relatively nonreactive surfaces. The entire system was tested in a clinical investigation, involving 21 subjects studied for up to 10 days, to determine the range of concentrations for hydrogen sulfide and methyl mercaptan, and from this to define the extent of variability among subjects. This system was further utilized in these subjects to define the different patterns in the levels of these volatile sulfur compounds over a 3-h period following two different rinsing procedures. 4) To facilitate the speed and simplicity desirable for repeated measurements in clinical studies, we used an arrangement similar to that of Solis-Gaffar et al. ( 6 ) in which, following turning of the sampling valve, nitrogen carrier gas swept a mouth air or calibration sample into the chromatograph. The rapid sampling made it possible, for example, to run a crossover study with rigid timing schedules and in which approximately 60 samples were obtained during each 4-h daily test interval.
RESULTS AND DISCUSSION Calibration. A broad range of concentrations of the calibration samples was produced by varying the split ratio, or the flow of nitrogen diluent, or both. For example, with a gas flow of 150 ml/min over a hydrogen sulfide permeation tube having an output of 700 ng/min, a concentration range of 1.2 to 230 ppb was obtained with appropriate split ratios of from 49:l t o 4:l and with dilution flows of from 0.4 t o 8.0 l./min. This range can be extended to 1150 ppb by directing the total flow from the gas-washing bottle directly to the mixing chamber. The variable split allowed a broad range of the lower concentrations with low gas flows since it was necessary to select only a small portion of the calibration gas from the gas-washing bottle for dilution. Previous workers had obtained a broad range by adjusting the gas flow over the permeation tube (5, 7) a procedure which requires high flows; or by relying upon adjustment of the flow of diluent gas into the mixing chamber ( 8 ) , a procedure which also requires high flows since the entering permeation stream has not been split; or else by using an exponential dilution flask (9, 10). This last method can be utilized with low gas flows and is a good technique for air pollution studies. However, it is not rapid enough for clinical work because of the time required for the flask t o dilute to the desired concentration. In addition, the detector has to be used to monitor the flask and thus is not available for sample measurement a t those times. Our studies with a variety of concentrations in the frequent mouth air samples required that measurements of the latter be interspersed with measurements of the calibration samples. Use of the split ratio permitted quick selection of the desired concentration by adjustment of the needle valve, with rapid equilibration between concentration changes. With use of a low-resistance mixing chamber vented to the atmosphere through nonrestricting lines, nitrogen diluent flows of up to 8 l./min caused a pressure increase of less than 0.2 psig in the upstream section of the calibration system, when the flow through the gas-washing bottle was 150 ml/min or less. Because of this small back pressure, changes in diluent flow did not affect gas flow through the permeation chamber or the split ratio of the gas stream a t the splitting tee. Consequently, no pressure corrections
were necessary for the flowmeter calibration curves. T o ensure accurate detector readings, several calibration samples covering the range of concentrations of interest were run on each test day, since preliminary checks had indicated that one-point verification of the calibration curve was not valid. Room air samples were also run periodically throughout a test period to confirm the continued absence of interference from ambient air. We verified that no detectable carryover existed between samples by running a room air sample free of the volatile sulfur compounds immediately after mouth air samples with high levels of both compounds. Measurement of Mouth Air Concentrations. Threshold sensitivity of the system remained constant for several weeks a t a time. Based upon a minimum signal equal t o twice that of background noise, the consistent limit of sensitivity was 7 ppb for hydrogen sulfide and 15 ppb for methyl mercaptan, when contained in a 10-ml sample of mouth air. The lower mean levels we report were determined by reading any signal below this consistent sensitivity limit as zero. From the values derived from 18 to 30 samples obtained from each of 10 subjects over 6 to 10 days, we determined that the mean concentrations of hydrogen sulfide in different subjects ranged from approximately 2 to 10 times greater than those of methyl mercaptan, with an average of about 4 times greater for the group. The mean values per subject over that period ranged from 65 to 698 ppb of hydrogen sulfide and from 10 to 188 ppb of methyl mercaptan. The equipment thus quantified these compounds a t and below the very low levels (about 118 ppb and 25 ppb, respectively, calculated from the ng/lO ml values given by Tonzetich (2)) a t which they become objectionable to the observer when present in mouth air. In the subsequent sections of the clinical investigation, the equipment proved reliable and sensitive enough to delineate even subtle differences in the time pattern of mouth air concentrations after oral rinsing, as well as to differentiate with statistically significant results between the effects of a test rinse and a control rinse. These clinical results will be described in a separate publication. Besides being used for comparative evaluations, the system described above could be applied to investigate the effect of variables such as oral disease upon compounds present in mouth air. The above system of design and construction with standard components is one that can be readily adapted to a variety of clinical studies.
ACKNOWLEDGMENT The authors express their appreciation to Henry McQuiston for his editorial assistance in the preparation of this manuscript. LITERATURE CITED (1) J. Tonzetich. Arch. OralBiol., 16, 587 (1971). (2) J. Tonzetich, J. Dent. Res., 5 4 (Abst. No. 71), 62, Special Issue A (1975). (3) S. S.Brody and J. E. Chaney. J. Gas Chromatogr., 4, 42 (1966). (4) A. E. O'Keeffe and G. C. Ortman, Anal. Chem., 36, 760 (1966). (5) R. K. Stevens, J. D. Mulik, A. E. O'Keeffe, and K. J. Krost, Anal. Chem., 43, 827 (1971). (6) M. C. Solis-Gaffar, H. P. Niles, W. C. Rainieri, and R. C. Kestenbaum, J. Dent. Res., 54, 351 (1975). (7) R. K. Stevens, A . E. O'Keefe. and G. C. Ortman, Enwiron. Sci. Techno/., 3, 652 (1969). (8) F. P . Scaringelii, A E. O'Keeffe. E. Rosenberg, and J. P. Bell, Anal. Chem., 42, 871 (1970). (9) F. Bruner, P. Ciccioli, and F. Di Nardo, Anal. Chem., 47, 141 (1975). (10) F. Bruner, C. Canulii. and M. Possanzini, Anal. Chem., 45, 1790 (1973).
RECEIVEDfor review October 24, 1975. Accepted January 5. 1976. ANALYTICAL CHEMISTRY, VOL. 48, NO. 4, APRIL 1976
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