Anal. Chem. 2000, 72, 2797-2801
Direct Temperature-Controlled Trapping System and Its Use for the Gas Chromatographic Determination of Organic Vapor Released from Human Skin Ken Naitoh, Yoshihito Inai, and Tadamichi Hirabayashi
Department of Environmental Technology, Postgraduate School of Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan Takao Tsuda*
Department of Applied Chemistry, Faculty of Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan
For controlling of trap temperature, the relationship between electric resistance of the trap tube and temperature is used. As the electric resistance of the trap tube (20 cm long stainless steel tubing) was very small, such as ca. 0.040 Ω for -70 °C and ca. 0.064 Ω for +90 °C, it was estimated by using the value of voltage output at both ends of the trap tube when a direct current (5 A) was applied for 6.5 ms at every 100 ms on the trap. By using this temperature measurement, a cycle of trapping is shortened, especially at the process of desoption, because it is possible to set a large increasing rate of temperature, such as 20 °C/s. The present trapping system has faster temperature response compared to that with a thermocouple. This system was applied for the study of the releasing of ethanol and water vapors from the human finger, which was treated as follows: dipping in 10% ethanol aqueous solution for 1 min, followed by washing with water and then drying in the air. In this case, a cycle of trapping took 53 s, and the period of total analysis was only 3 min. The present system is an efficient tool for the study of the exhalation of organic vapors from human skin. Most of the volatile organic compounds released from human skin are existent at very low concentration. Therefore it is necessary to use a sample collection method for the determination of these compounds by gas chromatography (GC). The concentration variation of these volatile solutes should be traced for a certain period, as these concentrations may depend on the health condition of each subject.1,2 The accuracy of the GC determination depends partly on the sample collection process. * Corresponding author. E-mail:
[email protected]. Phone/fax: 81-52735-5220. (1) Milton, L. L.; Frank, J. Y.; Keith, D. B. Open Tubular Column Gas Chromatography; John Wiley & Sons: New York, 1984; 210-215. (2) Walter, G. J.; Adolf, R. Sample Preparation for Gas Chromatographic Analysis; Hu ¨ thig: Heidelberg, Germany, 1983. 10.1021/ac9913309 CCC: $19.00 Published on Web 05/19/2000
© 2000 American Chemical Society
There are some studies concerning the behavior of the volatile organic compounds absorbed on the skin and/or released from it.3-7 In the collection of those volatile compounds, it is necessary to avoid both the loss of sample during the collecting process and the contamination with the substrates in air. Furthermore, as we need to analyze several to dozens of samples taken from subjects (volunteers), it is most important to employ a rapid and easy collection procedure. A noninvasive collection method is most preferable for obtaining the agreement of volunteers. The gas sample collection is mainly done on an inert material8 (e.g. glass beads) or absorbents4,7,9-12 (e.g. TENAX) packed in a cold trap. These cooling methods require a cryogen such as liquid nitrogen,8,12,17 carbon dioxide,13 or a thermoelectric device.9,14 Although the last one (Peltier’s element) makes any chemical cryogen unnecessary, it takes a rather long time for cooling and heating. (3) Javelaud, B.; Vian, L.; Molle, R.; Allain, P.; Allemand, B.; Andre´, B.; Barbier, F.; Churet, A. M.; Dupuis, J.; Galand, M.; Millet, F.; Talmon, J.; Touron, C.; Vaissiere, M.; Vechambre, D.; Vieules, M.; Viver, D. Int. Arch. Occup. Environ. Health 1998, 71, 277-83. (4) Hotz, P.; Carbonnelle, P.; Haufroid, V.; Tschopp, A.; Buchet, J. P.; Lauwerys, R. Int. Arch. Occup. Environ. Health 1997, 70, 29-40. (5) Moody, R. P.; Chu, I. Environ. Health Perspect. 1995, 103, 103-114. (6) Brown, H. S.; Bishop, D. R.; Rowan, C. A. Am. J. Pub. Health 1984, 74, 479-484. (7) Weisel, C. P.; Jo, W.-K. Environ. Health Perspect. 1996, 104, 48-51. (8) Ruther, J.; Hilker, M. J. Chem. Ecol. 1998, 24, 525-534. (9) Holdren, M.; Danhof, S.; Grassi, M.; Stets, J.; Keigley, B.; Woodruff, V.; Scrugli, A. Anal. Chem. 1998, 70, 4836-4840. (10) Nagano, H.; Miyazawa, K.; Osada, Y.; Yokoyama, T. Organohalogen Compd. 1997, 31, 150-153. (11) Peters, A. J.; Sacks, R. D. U.S. Patent 5288310, 1994. (12) Kalman, D.; Dills, R.; Peaera, C.; DeWalle, F. Anal. Chem. 1980, 52, 19931994. (13) Watanabe, K.; Seno, H.; Ishii, A.; Suzuki, O.; Kumazawa, T. Anal. Chem. 1997, 69, 5178-5181. (14) Bertman, S. B.; Buhr, M. P.; Roberts, J. M. Anal. Chem. 1993, 65, 29442946. (15) Lanning, L. A.; Sacks, R. D.; Mouradian, R. F.; Levine, S. P.; Foulke, J. A. Anal. Chem. 1988, 60, 1994-1996. (16) Ewels, B. A.; Sacks, R. D. Anal. Chem. 1985, 57, 2774-2779. (17) Kamei, T.; Tsuda, T.; Mibu, Y.; Kitagawa, S.; Wada, H.; Naitoh, K.; Nakashima, K. Anal. Chim. Acta 1998, 365, 259-266.
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After sample collection, the sample should be desorbed from the trap by rapid heating and then led to GC for analysis. Electrical heating of the capillary trap must provide a versatile and efficient sample desorption method.15,16 Generally, a Nichrome ribbon or other electrical resistance elements are used as a heater. In other words, they are kinds of indirect heating. Most of the trap methods have a temperature detection device for controlling to cool the absorbent lower than ambient temperature and to heat it to allow thermal desorption to take place. The trap temperature is generally measured by a sensor such as thermocouple or a thermistor. It is difficult to fix these sensors on cold trap tube very tightly to obtain a real value of the cold trap temperature and also to estimate an average temperature of the whole cold trap from a local value. Therefore, the trap tube temperature measured by using these sensors is not always equal to the real value, because of the geometrical and/or mechanical incompleteness of the arrangement of these sensors. In this report, we propose a new direct method for measurement of the trap temperature. The new method utilizes the temperature coefficient of the electric resistance of the trap tube made of metal alloy. When a certain direct current is applied to the metal tube, the voltage generated between both sides of the trap tube relates directly to the electric resistance of the trap tube. Thus the measurement of trap tube voltage informs us of its temperature. In this study, we use a trap tube made of stainless steel. By the trap system using the new temperature detection, the period of the thermal desorption process can be reduced to one-third compared to the homemade trap system with a conventional thermocouple. Thus we have succeeded also in suppressing the overheating of the trap. There are few studies for direct sampling of volatile compounds from the human skin in short periods (less than several minutes). For example, we have already proposed the instrumentation for the determination of ethanol concentrations in human perspiration by using GC.17 The alcohol is included in sweat secreted after its intake. The system can measure concurrently both the ethanol amount in sweat secreted and the absolute amount of sweat carried with an air stream. The latter can be determined by means of a hygrosensor. Anyway, it is possible to estimate every 5 min the ethanol concentration by using both water and ethanol absolute amounts. This sampling system is noninvasive and very easy to handle. The purpose of this study is to find a more efficient collecting system. Aiming at wide applications, we have improved the direct online sampling system of organic vapors releasing from human skin. First, we devise an automatic collection system, which allows one to trap the organic vapors included in relatively large amounts of gas carrier, for example a volume of 150 mL in 30 s. Second, we employed the direct detection system for the temperature of the trap tube. With our new instrumentation, it is possible to measure the sample amount every 3 min. Our proposed system is applied for the study of the releases of ethanol and water vapors from the thumb after dipping it for a very short period in an aqueous solution including ethanol. EXPERIMENTAL SECTION Instrumentation for Organic Vapor Released from Human Skin. A schematic diagram of a direct online sampling system is shown in Figure 1. The direct online sampling system consisted 2798 Analytical Chemistry, Vol. 72, No. 13, July 1, 2000
Figure 1. Schematic diagram of direct online sampling system. The temperature of the trap tube was measured by thermocouple or estimated by the value of its electric resistance. For operation of the a-f lines of the six-way valve, see the text.
of the following parts: (1) A sampling probe was attached to the skin surface directly, which was part of an apparatus for the measurement of sweating rate (sampling area of skin: 1 cm2; Kenz Perspiro OSS-100, Suzuken, Nagoya, Japan).17-20 The absolute amount of water due to perspiration and exhalation from skin was estimated by a hygrosensor and a thermister, both of which were installed inside the probe. (2) A stainless steel open tubular of 200 mm long and 0.8 mm inner diameter was used as a trap tube. The inner wall of the tubing was covered with chemically bonded poly(ethylene glycol) 20M (type UACW, kindly donated from Frontier Laboratory, Ltd., Koriyama, Japan). The open tubular trap was coiled with 8.5 mm in outer diameter and coiled six times, and its inner volume was 100 µL. (3) Liquid carbon dioxide was used as coolant for the trap. An electrical solenoid valve (type 8264D9, Asco-Japan, Nishinomiya, Japan) was set between a spraying nozzle and a cylinder of liquid carbon dioxide, which was used for automatic regulation of cold trap temperature. The spraying nozzle was made by stainless steel tubing (1/16′ o.d. and 0.6 mm i.d.), and the mouth was enlarged by using a small drill to be cone shaped. The mouth was set just in front of the coiled trap tube. When the electrical solenoid valve opened, the cold trap could reach to an appropriate low temperature rapidly and keep it constant by automatic on-off regulation of liquid carbon dioxide spraying. (4) An electric direct current source supplied a number of dc pulses (5 A; duration period 6.5 ms) to the trap tube itself for heating it to an appropriate temperature for the thermal desorption of organic substrates. (5) The pneumatic switching valve (type 5011, 6 port, Rheodyne, Cotati, CA) was used for control sampling period. In both processes of thermal desorption and preprocess, lines a-f, b-c, and d-e in Figure 1 were (18) Kamei, T.; Naitoh, K.; Nakashima, K.; Ohhashi, T.; Kitagawa, S.; Tsuda, T. J. Pharm. Biomed. Anal. 1998, 15, 1563-1569. (19) Kamei, T.; Naitoh, K.; Nakashima, K.; Ohhashi, T.; Kitagawa, S.; Tsuda, T. Instrum. Sci. Technol. 1997, 25, 1, 39-53. (20) Kamei, T.; Tsuda, T.; Kitagawa, S.; Naitoh, K.; Nakashima, K.; Ohhashi, T. Anal. Chim. Acta 1998, 365, 319-326.
connected. In the trapping process, the lines of a-b, c-d, and e-f were connected. Four on-off solenoid valves (Takasago Electric, Inc., Nagoya, Japan) controlled the valve operation by aid of gas pressure. (6) Two different temperature detectors for the trap tube were used. At first experiment, a wire of AlumelChromel thermocouple (outer diameter 0.32 mm) was used for temperature measuring. It was placed on the outside of the trap coil by using a thermal conductive tape, and its junction point was 0.6 mm long. For the second experiment, temperature was newly estimated from the value of the electric resistance of the trap tube. As the electric resistance of the trap tube (20 cm long) was very small, such as ca. 0.040 Ω for -70 °C and ca. 0.064 Ω for +90 °C, the electric resistance was estimated by using Ohm’s law from the value of voltage when direct current (5 A) was applied for 6.5 ms at every 100 ms on the trap. The voltage was measured between both ends of the cold trap, for example, the value of voltage at -70 °C being 200 mV. The linear relationship between electric resistance and temperature was established by using baths kept at several different temperatures, in which the cold trap was immersed. A thermocouple attached on the cold trap was used for calibration. There was a good linear relationship between the temperature of the cold trap and its electric resistance. For avoiding any leakage of electric current, polytetrafluoroethlene tubings (0.9 mm inner diameter) were used for the connection with both ends of the stainless steel trap tube. (7) A homemade timing and temperature controller was employed. This system could control timing for the period of switching valve rotation, the period of sampling, the period of injection, and the temperature of the trap tube. Feedback from the temperature sensor regulates the number of the pulse of the current of 5 A, which duration period is 6.5 ms. When there is a high demand of heating, 5 A current was continuously applied. A dc pulse was used both for the heating of the trap and the measurement of its temperature. (8) A gas chromatograph (GC-7A, Shimadzu, Kyoto, Japan) equipped with flame ionization detector (FID) was used. Trapping Procedures. The procedure for trapping was controlled by automatic homemade controller. It involved two processes: one was a “preprocess” and the other was the “net process”. In the preprocess, the trap was set at an appropriate temperature for cleaning. During this preprocess, nitrogen carrier gas was passed through the trap tube and led into the GC, and another gas stream was also kept through the sampling probe followed to an exhaust port of the switching valve. The above preprocess was maintained except during the periods of the trapping process. The net trapping process consisted of the following steps: (a) A finger was pressed on the sampling probe for sampling. Water and organic substances released from the skin (finger) were carried first to the exhaust port of the switching valve (a-f line in Figure 1). The content of water in the nitrogen stream was measured by the hygrosensor in the sampling probe. After the rate of water released from the skin became constant (generally it took 15 s), the precooling step began, in which the trap temperature was cooled to the suitable temperature for trapping. (b) Then the switching valve was rotated to the trapping position. Water and volatile organic substances involved in the nitrogen gas stream were led into the trap tube and they were trapped in it. The total amount of water vapor led into the cold trap was
recorded. (c) Next, the switching valve was rotated to the thermal desorption position, and the trap tube was heated directly by an electrical current for the thermal desorption of the sample. Water and organic compounds stored in the trap tube were immediately vaporized by the application of electric current and led into a capillary column in the GC. Analytical Conditions. The experimental conditions for the determination of ethanol by GC were as follows: a capillary column [large bore capillary glass column of 40 m long, 1.2 mm inner diameter, and 1.6 mm outer diameter, coated with G-205 (5% phenyl methyl silicone; thickness of stationary phase 2 µm), kindly donated by Chemical Evaluation and Research Institute of Japan, Tokyo]; column temperature, 63 °C; carrier gas, nitrogen gas, 20 mL/min; sampling gas stream, nitrogen gas, 300 mL/min; an integrator (Chromato-Integrator D-2500, Hitachi, Tokyo). Calibration for Ethanol. The calibration for ethanol was performed as follows. Standard ethanol aqueous solutions (0.01, 0.02, 0.04, 0.06, and 0.10 v/v%) were led into the probe via a narrow tubing at constant flow rate of 1 µL/min for 30 s by a pump. The amount of the standard solution injected, 0.5 µL, was also measured by the hygrosensor. The injection of each standard sample solution was repeated 10 times with a 3 min interval. The accuracy of the hygrosensor was reported in our previous studies.18,19 The minimum detectable amount of water by KenzPerspiro is 0.002 mg/min. Water and Ethanol Absorbing Procedure in/on Skin. Ethanol aqueous solution was absorbed in/on skin when a thumb was dipped in it. For reliable and reproducible results, the contacting process of the skin with the solution was specified as follows: (a) A 100 mL volume of 10% ethanol aqueous solution was kept in a 100 mL glass vial. The thumb was dipped into the vial and vibrated gently with a pattern of drawing a circle for 1 min. (b) After the thumb was pulled out, it was washed with a stream of tap water for 15 s, and then it was kept in the air for natural drying for 90 s. (c) Next, the thumb was attached on the sampling probe in order to measure the amount of water and ethanol being released. RESULT AND DISCUSSION Direct Temperature Control of Cold Trap. Temperature variations of the a cold trap measured by using a thermocouple and a new electric resistance system are shown as curves of (a) and (b) in Figure 2, respectively. The temperature of the trap tube was controlled at 90 ( 5 °C during the preprocess of trapping. In the net trapping process, the trap tube was cooled to -70 ( 3 °C by spraying liquid carbon dioxide, shown as the step of I in Figure 2. The period required for this cooling process, from 90 to -70 °C, was within 15 s. Then the temperature of the trap tube was kept constant at -70 ( 3 °C for 30 s, which is denoted as II in Figure 2. After the sample trapping, the trap tube was directly heated by applying electric direct current. Time periods are required for the sample desorption process, shown as the step of III in Figure 2. These periods of (a) and (b) in Figure 2 are 30 and 8 s, respectively. The new apparatus controlled with electric resistance required less than one-third of the period compared to that with the homemade thermocouple device. The temperature increase rate of 20 °C/s attained by the present system is fairly rapid compared to other experiments, such as 5-7,8 16.5,9 and 4 °C.14 After attainment of the temperature for the desorption cycle, Analytical Chemistry, Vol. 72, No. 13, July 1, 2000
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Figure 2. Effect of the temperature detection difference between using the thermocouple and electric resistance. The temperature of the trap tube was measured by the thermocouple (a) and estimated by the value of its electric resistance (b). I: precooling of the trap tube, 15 s. II: trapping process, 30 s. III: desorption process, 30 s in (a), 8 s in (b).
Figure 3. Calibration curve for ethanol. The correlation coefficient between the peak area and amount of ethanol injected was 0.999.
overheating and wave fluctuation of the temperature appeared in the profile of curve a but not curve b in Figure 2. The former phenomenon might be derived by a time delay in the response of the thermocouple. This defect can be reduced by the proposed new cold trap temperature regulation system. It is clearly shown in curve b in Figure 2. The reliability of the new temperature-controlled system is very good. It could operate without any problem for 1 month. The recovery of ethanol is 98.8% with standard deviation 6.7% in the test standard mixtures ranging from 0.01 to 0.1% ethanol aqueous solution. The sample volume used in the new system was 150 mL/30 s. This volume is fairly large compared to other experiments, such as 150,8 20,9 and 25 mL/min.10 Thus one cycle for sample collection can be completed within 53 s in the present system. A good first-order linear relationship between the peak area and amount of ethanol injected is shown in Figure 3. The correlation coefficient is 0.999. The new system can work well for the determination of ethanol under conditions of water vapor, even though its rate is on the order of 1.0 mg/min. Measurement of the Amount of Ethanol Released from Human Skin after Its Absorbtion. We applied the present system for the determination of the ethanol vapor released from the human skin after dipping the skin in the ethanol aqueous solution for 1 min. Ethanol and water might be adsorbed in the skin during the action of dipping. As this absorbed ethanol is 2800 Analytical Chemistry, Vol. 72, No. 13, July 1, 2000
Figure 4. Gas chromatogram of a series of successive samplings on a thumb after dipping it into 10% ethanol aqueous solution for a period of 1 min. (a)-(f) are ethanol peaks. (1)-(6) are injections being performed every 3 min.
Figure 5. Relationship between the amount of ethanol and water vapor released and the elapsed period. Experimental conditions: The skin was dipped with 10% ethanol aqueous solution for periods of 60 s and then washed and dried. The symbols of O and 0 correspond to the amounts of ethanol and water released, respectively.
released from the skin gradually, samplings have been performed continuously. A series of 6 successive samplings was adopted with a 3 min interval between each sampling. A series of 6 gas chromatograms obtained in a run is shown in Figure 4. Arrows (v) indicate each injection, and ethanol peaks a-f correspond to the injection of 1-6, respectively. The retention time of ethanol is 2.97 ( 0.02 min under the given conditions. The theoretical plate numbers (N) of peaks c, d, and e are 7544, 7613, and 7050, respectively. These values are almost 2 times larger than the same experiment by using the apparatus with a thermocouple (N ) 3300). Ethanol peak heights of (a)-(f) in Figure 4 are decreasing sharply in order. The decreasing rate of the amount of ethanol released from the skin would correlate with the mechanism of the skin exhalation scheme. Exhalation Rate of Ethanol and Water Vapor Absorbed. Amounts of ethanol and water vapor released from the thumb after contact with 10% ethanol aqueous solution are shown in Figure 5. Logarithmic values of ethanol amount released from the thumb had a negative linear relationship with logarithmic values of elapsed time (min) after its dipping, shown as open circles in Figure 5. A similar tendency can be observed in the amount of water, shown as open squares in Figure 5. The amount of ethanol
released decreased proportionally to one-tenth of the initial value at 17 min after the dipping. However, the decreasing rate of released water was one-sixth of the initial value. Therefore water is not released as fast as ethanol from the skin. By using the presented system, we can study the releasing patterns of ethanol and water in a relatively short period (10-20 min). In a skin absorption mechanism, hydration at the shallow layer of the skin might be an important factor. Several reviews have discussed skin hydration as well as organic absorption.5,6 Our new system can obtain direct information concerning the rates of water vapor and volatile organic compounds released from the human skin simultaneously. This proposed system can be widely applicable to various investigations for the interaction between the skin of a human being and volatile organic compounds, as well as water. CONCLUSION An instrumentation for a direct online sampling system of organic vapor released from human skin is demonstrated. The proposed system has the following characteristic points: direct sampling from human skin; very rapid analysis with relatively large volume of sample gas stream; successive analyzing with 3 min intervals. Kinetic studies on the releasing manners of vaporizable materials and water, which are absorbed on/in skin, become possible by using our new GC system. Hence, logarithmic values of ethanol and water amounts are proportionally decreasing as the increase of the logarithmic value of elapsed time. The amount of released ethanol and water at 17 min after skin contacting with
ethanol aqueous solution is decreased to 1/10 and 1/6 of the initial stage, respectively. The new system is fully automatic and easy to handle. One of the most important characteristics of the proposed method is that sampling can be performed in a noninvasive manner. Therefore, increased participation of volunteers looks promising. Furthermore the new system proposed in this paper may be useful for various applications, for example, studying amounts of residual cosmetics and also the rate of release from skin exposed to chemically harmful environments. Our proposed new cold trap system uses a unique sensing method for the measurement of cold trap temperature. By using a feedback operation with this sensor, rapid cooling and heating control becomes possible. This device may be a good tool for study in the environmental field and GC/MS. ACKNOWLEDGMENT This research was partly supported by the aid of the Ministry of Education, Science, and Culture (B11559008). The authors are thankful for the technical support of Mr. H. Samejima and Mr. T. Akahoshi, Chemicals Evaluation and Research Institute, Mr. T. Maeda, DKK, Dr. C. Watanabe, Frontier Laboratory, and Takasago Electric, Inc.
Received for review November 17, 1999. Accepted March 29, 2000. AC9913309
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