Wallace, and John Sulak for excellent technical assistance. Appreciation is also expressed to Donald L. Smith, John Alexander, and Kenneth Shanklin, Wilford Hall USAF Medical Center, for providing urine specimens from postsurgery patients receiving cocaine anesthesia.
LITERATURE CITED (1) J. K. Brown, R. H. Schingler, M. G. Chaubal and M. H. Malone, J. Chromatogr., 87, 211 (1973). (2) M. L. Bastos and D. B. Hoffman, J. Chromatogr. Sci., 12, 269 (1974). (3) J. de Onis, "Coca means Cocaine. It's also a way of life", New York Times. February 9, 1973. (4) G. Volsky, "Illicit traffic of cocaine growing by leaps and bounds in Miami", New York Times, February 1, 1970. (5) H. M. Schmeck, Jr., "Cocaine is re-emerging as a major problem, while marijuana remains popular", New York Times, November 15, 1972. (6) "Cocaine found on the rise", Washington Star-News. April 10, 1974. (7) N. M. Adams, Reader's Digest, 108, 83 (1975). (8) J. E. Wallace, H. E. Hamilton, H. S. Schwertner, D.King, J. McNay. and K. Blum, J. Chromatogr.,submitted, 1975.
(9) (10) (1 1) (12) (13) (14) (15) (16) (17)
D. J. Berry and J. Grove, J. Chromatogr., 61, 11 1 (1971). F. Fish and W. D. C. Wilson, J. Chromatogr.,40, 164 (1969). H. E. Sine, N. P. Kubasik, and J. Waytash, Clin. Chem., 19, 340 (1973). J. W. Blake, R. S. Ray, J. S.Noonan, and P. W. Murdick, Anal. Chem., 48, 288 (1974). N. N. Valanju, M. M. Baden, S. N. Valanju, D. Mulligan. and S. K. Verma, J. Chromatogr., 81, 170 (1973). S. Koontz, D. Besemer, N. Mackey. and R. Phillips, J. Chromatogr., 85, 75 (1973). F. Fish and W. P. C. Wilson, J. fharm. Pharmaceut., 21, Suppl.. 135a (1969). M. L. Bastos, D. Jukofsky, and S. J. Mule, J. Chromatogr., 89, 335 ( 1974). S. J. Mule, J. Chromatogr., 55, 255 (1971).
RECEIVEDfor review May 23, 1975. Accepted September 22,1975. This research study was supported by Grants DA0059401 and DA00729-02 from the National Institute on Drug Abuse, NIH, Bethesda, Md.
Gas Chromatographic Determination of Ethylene in Large Air Volumes at the Fractional Parts-per-Billion Level J.
De Greef" and M. De Proft
Department of Biology, University of Antwerpen, Universiteitsplein, 1, 8-26 10 Wilrijk, Belgium
F.
De Winter
L 'Air Liquide Belgium, Scientific Division Laboratories, 8-262 1 Schelle, Belgium
Up to the present, quantities of ethylene as low as 1 ppb can be measured by improving the sensitivity of gas chromatographic methods. We demonstrate that ethylene can be trapped with 100% efficiency from large volumes of air on a Porapak-S precoiumn cooled at -95 O C . The precolumn is built in a gas sampling apparatus operated by a two-way six-port rotary valve. After being loaded, the coiumn is introduced into the carrier gas stream. The ethylene is totally released and swept onto the analyticai column with the carrier gas by heating the column to 100 O C with boiling water. By this method and using a standard commercial instrument, we are able to measure concentrations of ethylene as low as 0.1-0.01 ppb in a quantitative and reproducible manner.
Ethylene is a powerful plant growth regulator in the successive phases of the plant life cycle ( I ) . Most plant tissues that have been studied produce between 0.5 and 5 nl/g/h ( 2 ) . Since these extremely low concentrations of ethylene drastically alter numerous physiological processes in plants, many investigators have searched after sensitive methods for its detection and determination. In reviews of methods for ethylene determination in large air volumes, we find that numerous approaches have been made ( I , 3 ) . Among these, the two following techniques are widely used. First, air samples are enriched with ethylene by accumulation of the gas produced by plant material in a closed system of constant volume for several hours. Small gas samples are then injected into a gas chromatograph with a syringe ( I ) . The other technique is a trapping method based on the isolation of ethylene by formation of a mercury complex ( 4 ) . The ethylene-mercury complex is decomposed by adding sodium chloride and the 38 * ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
volume of the released ethylene is determined manometrically. This paper reports a sensitive gas chromatographic procedure for the determination of ethylene in the ambient atmosphere of biological specimens based upon its trapping from a continuous air flow on a cooled pre-column filled with Porapak-S. The efficiency of this method is compared with that of other techniques commonly used.
EXPERIMENTAL Apparatus. The gas chromatograph used is an Intersmat Model
IGL 12DFL equipped with a dual column and a flame ionization detector. A 300-cm long, ',$-inch 0.d. stainless steel column packed with Porapak R (Waters Associates, Inc.) 50-80 mesh is employed. The flow rate of the carrier gas, Nz/02 (80:20), is 700 ml per hour, and the oven is maintained at 40 'C. A 1-mV strip chart recorder is used for measuring retention times and peak heights. At the most sensitive settings, the background noise level is 3 mm maximally. Gas samples are introduced into the gas chromatograph through a two-way six-port rotary valve manually operated. Calibration. A calibration curve of peak height vs. quantity of ethylene is made in 2 ways. ( a ) Using the same loop (constant volume), the peak heights of 2 different standard gas mixtures are measured. These mixtures contain, respectively, 102 and 1 ppm ethylene in Nz/Oz (80:20). They are products of L'Air Liquide Belgium, Scientific Division, Schelle. ( b ) A six-volume valve (L'Air Liquide Belgium Co.) is introduced into the flow system. By turning the valve counterclockwise, we can inject a definite volume of a known standard gas mixture. The volumes at the different positions of the valve are, respectively, 8, 13, 19,41,67,and 106 pl. For syringe injection gastight syringes (Hamilton Co. Inc.) are used. Trapping M e t h o d s . Mercuric Perchlorate Method. We follow the technique as proposed by Young et al. ( 4 ) . Aliquots of the ethylene-mercury complex are taken for the release and measurement of ethylene in both ways, manometrically and gas chromatographically. Manometric measurements are carried out with Warburg vessels connected with micromanometers. A special vessel is con-
Table I. Peak Height Determinations of Ethylene Quantities Injected by Syringe and by a 6-Volume Valve Syringe injection of 20 ppm C2H, in 0.5-ml air sample
6-Volume valve injection of 1 0 2 ppm C,H, in 8 pI
1 3 p1
19 p1
4 1 p1
6 7 pI
1 0 6 p1
163,(5)Q 27 (1) 42 (1) 6 1 (1) 1 3 4 (1) 225 (1) 347 (1) a peak height in mm, standard deviation between parentheses. Table 11. Ethylene Absorption in a Mercuric Perchlorate Solution as a Function of Flow Ratea Flowrate, I./h
Figure 1. Calibrated vessel for decomposing the ethylene-mercury complex and for introducing the released gas into the gas chromatograph.
0)inlet saturated (NH4)2S04solution. (0)outlet released gas
C,H, not trapped, ppm
1.8 ( 0 . 1 ) 0.32 ( O . O l ) k ' 4.7 (0.3) 0.93 (0.01) 11.6 (0.3) 1.92 (0.02) 21.2 ( 0 . 2 ) 3.71 (0.02) 25.6 (0.2) 4.76 (0.02) 27.1 ( 0 . 2 ) 5.32 (0.02) 7.17 (0.02) 33.5 (0.2) 40.1 ( 0 . 2 ) 9.73 (0.03) a The mercuric perchlorate solution was prepared according t o the method of Young et al. ( 4 ) . A standard gas mixture containing 1 0 2 ppm ethylene was used. Calibrations were carried out with two standard gas mixtures containing 1 and 1 0 2 ppm C,H,, respectively. b Between parentheses: standard deviation.
structed to decompose the ethylene-mercury complex and to introduce the released ethylene into the gas chromatograph (Figure 1).In one arm of the vessel, we bring 1 ml of the solution containing the ethylene-mercury complex; in the other, 0.6 ml 2 N HCl. Before mixing the 2 solutions, the gas space of the vessel is flushed with synthetic air, free of ethylene. After mixing both solutions, the gas phase of the vessel is displaced into the sample loop of the gas chromatograph by adding a saturated solution of (NHJzS04 into the vessel. The presence of this salt significantly reduces the solubility of ethylene ( 5 ) . To measure the amount of ethylene released in the synthetic air, 2-ml samples of the gas mixture are injected with a two-way six-port rotary valve into the gas chromatograph. Porapak-S Column Method. A copper column 10-cm long and V4-inch 0.d. is packed with 0.35-0.40 g Porapak-S 80-100 mesh. The column is welded t o stainless steel tubes of '/s inch. The tubings are connected to the apparatus with Swagelok fittings. This column replaces the sample loop. The Porapak-S column is cooled at -95 OC (mixture of liquid nitrogen in acetone) and used as a pre-column to concentrate ethylene from different gas mixtures. The ethylene is then released by quickly heating the loaded column to 100 O C . Then the column is plunged into boiling water. In this way, the ethylene is totally liberated and swept into the gas chromatograph. By this procedure, it is possible to take the trap to any location for sampling. R E S U L T S A N D DISCUSSION Table I illustrates the calibration of ethylene concentrations of known standard gas mixtures by peak heights using t h e syringe injection method on the one hand, a n d t h e method of injection with a 6-volume valve on t h e other. From t h e results, it is clear t h a t t h e peak heights measured after injection of different volumes of 102 ppm ethylene with a 6-volume valve are much more reproducible t h a n those obtained after syringe injection of 0.5 ml ethylene 20 ppm. Using this valve, t h e deviation of peak heights never exceeds t h e background noise level (max. 3 mm) at any volume measured, while t h e standard deviation is five times higher for comparable ethylene concentrations with t h e syringe injection method. Standard errors u p to 10% are frequently found in literature when t h e syringe method is
used (6). Therefore preference is given t o t h e 6-volume valve method for calibration purposes. Since we are interested in trapping small quantities of ethylene, we examine first of all t h e efficiency of t h e mercuric perchlorate method in relation to t h e gas flow passed through it. In Table 11, the results are given. A standard gas mixture containing 102 ppm ethylene is passed through t h e mercuric perchlorate solution at different flow rates and t h e ethylene not trapped is analyzed with t h e gas chromatograph directly through t h e sample loop. Flow rates are measured with a soap film flowmeter. It can be seen that there is a gradual increase in t h e amount of untrapped ethylene when t h e flow rate is increased. At flow rates of 3.71 and 9.73 l./hr, t h e amounts of untrapped ethylene are about 20 and 40%, respectively. I n t h e literature ( 4 , 7) flow rates of 18 t o 30 L/hr are used. N Butyl alcohol is not added to t h e absorbing solution t o improve the foaming action in t h e absorber ( 4 ) for two reasons. At first, we would avoid pollution of t h e analytical column by t h e evaporative loss of n-butanol. On t h e other hand, in most papers dealing with ethylene trapping in t h e ambient atmosphere of plants, mercuric perchlorate is used as a stationary phase without n-butanol being added (810). Next we study t h e quantitative release of ethylene a d sorbed into t h e mercury complex. At a flow rate of 2.79 1./ hr, a standard gas mixture of 102 ppm ethylene is passed through 35 ml mercuric perchlorate solution. Measuring directly by valve injection t h e amount of untrapped ethylene, we find t h a t 1796 p1 ethylene is adsorbed. Five-ml aliquots of this ethylene-mercury complex are treated with 2 ml of 2 N HC1 t o release t h e ethylene. Theoretically this volume contains 253 pl ethylene and we find 201 ( f 1 0 ) pl by manometric determination. By gas chromatographic analysis of 1-ml aliquots ethylene, amounts of 41 ( f 2 ) p1 are found, while 50.5 p1 was t o be expected. In both ways, 20% of t h e bound ethylene is not liberated. T o improve t h e quantification of t h e ethylene determinations, it is obvious that another method has to be found which would allow 100% trapping a n d release of small quantities of ethylene. Various Porapak types are well suited for t h e separation of volatile hydrocarbons (10, 12, 13). We select t h e Porapak-S type which is characterized by its high retention time and its large adsorption capacity for ethylene at low temperature. I n t h e first series of experiments, t h e efficiency of ethylene trapping on and release from a Porapak-S column is tested. By means of t h e 6-volume valve, 4 different volumes (8, 13, 19, and 41 p1) of a standard gas mixture containing 102 ppm ethylene are separately injected into t h e tubing ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
39
Table 111. Trapping Low Ethylene Concentrationsa on a Porapak-S Column Sample vot ume, ml
Peak height, mm
Ethylene trapped, 10- p l
92.4 13.6 (0.7)b 3.97 (0.22) 184.8 29.0 (0.6) 8.45 (0.20) 231.0 36.7 (0.6) 10.69 (0.18) 369.2 59.5 (0.4) 1 7 . 3 4 (0.12) 462.0 77.7 (1.0) 22.63 (0.30) UDifferent volumes of a gas mixture containing 4.6 ppb ethylene and a flow rate of 2.77 l./hr were employed. b Between parentheses: standard deviation.
Detection of the ethylene trapped on 2 Porapak-S pre-columns in series. For explanation, see text. Figure 2.
*r-%?w a
1 e
i
i
C
e
3 e A
Flgure 3. Columns arrangement to test the versatility of the method for trapping and releasing ethylene ( a ) Inlet carrier gas, ( b ) to analytical column, (c) outlet, (d)two-way six-port rotary valve, (e) Porapak-S pre-columns. (0 shut-off valves, (g) 6-volume valve, ( h ) inlet gas sample, ( k ) and (I), inlet and outlet of calibration gas mixture pcakheighl in m m
loo
t
/
0
0
1
2
3
4
1 6
p ~ ~ 2 ~
Flgure 4. Efficiency of the Porapak-S method in ethylene trapping on and transferring between different pre-columns
leading to 2 Porapak-S pre-columns in series. First, column 1 is cooled to -95 "C and column 2 is kept in boiling water. After the injection, the whole system is flushed with a vector gas consisting of synthetic air. Column 1is then heated up to 100 "C and the released ethylene is measured gas chromatographically. In Figure 2, the peak heights for the different volumes injected are given ( 0 ) . Second, both columns are kept a t -95 "C and different volumes of the same standard gas mixture are injected as mentioned above. While column 1 is maintained a t -95 "C, column 2 is brought to 100 "C and the released ethylene, which is eventually trapped on this column, is measured. There is no ethylene. When column 1 is also heated, we 40
ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
measure the same peak heights for the same ethylene quantities as in the first part of the experiment (Figure 2, m). All values of both experiments are within the confidence limits of 99%. Next, the influence of the flow rate of ethylene trapping on Porapak-S is examined. A standard gas mixture containing 1 ppm ethylene is passed through two cooled precolumns with different flow rates, but the second pre-column is placed in the sample loop. By passing 400 ml gas mixture through the pre-columns a t flow rates up to 24 1./ hr, all ethylene is trapped on column 1. There is no trace of 111. Maximum ethylene on column 2 larger than 3.0 X capacity of ethylene trapping is 25 ml pure ethylene per gram Porapak-S. A gas mixture containing 4.6 ppb ethylene is prepared. Its calibration is carried out with the 6-volume valve. In this way, we can work with large gas volumes of low ethylene content. Different volumes of this concentration are passed separately through a cooled Porapak-S column a t a flow rate of 2.77 1 . h . By the same procedure of cooling and heating the column, the ethylene of each volume is trapped and released, respectively, and then measured. The results are summarized in Table 111. Full linearity is obtained between the amounts of ethylene trapped and the peak heights measured. Finally, a column arrangement is set up to demonstrate the versatility of this method by using parallel pre-columns in series with one other pre-column (Figure 3). Through the 6-volume valve, 4 known volumes (8, 13, 19, and 41 111) of a standard gas mixture containing 102 ppm ethylene are injected one after the other into 4 independent and cooled pre-columns (columns 1 to 4) being in series with one precolumn (column 5 ) heated to a constant 100 "C. When each of the 4 columns is loaded, column 1 is heated and the 3 other columns are kept cool and shut off by valves a t both ends. At the same time, column 5 being in series with col4 umn 1 is cooled a t -95 OC. In this way the ethylene is transferred by the vector gas from column 1 to column 5 . Afterwards, column 5 is heated and the liberated ethylene was measured. T h e same procedure is followed for all other columns loaded with ethylene. For each gas volume, the experiment is repeated 4 times. The results of these measurements are plotted in Figure 4. Peak heights are proportional to their respective ethylene quantities. I t is thus possible to transfer ethylene from one column onto another in a quantitative way. In this case, the pre-column becomes a portable extension of the analytical column proper which can be transported independently to any site of sampling. The method described is simple in its performance. I t allows ethylene determinations with a high degree of accuracy even for very low concentrations. Measurements are made in short times so that the kinetics of physiological processes involving ethylene production or consumption can easily be studied.
ACKNOWLEDGMENT T h e first two authors are indebted to the Scientific Division Laboratories of L'Air Liquide Belgium, Schelle, for procuring all facilities to make this scientific cooperation possible. All three of us thank G. Martin, Head of the Department. T h e technical assistance of w. Van Dongen is also acknowledged.
LITERATURE CITED (1) F. B. Abeles, "Ethylene in Plant Biology," Academic Press, New York, 1973. (2) A. C. Leopoid. "Hormonal Regulation in Piant Growth and Development," H. Kaidewey and Y. Vardor. Ed., Veriag Chemie, Weinhelm. 1972, p 245. (3) H. P. Burchfieid and E. E. Storss, "Biochemical Applications of Gas Chromatography,'' 4th ed., Academic Press, New York, 1970, p 206.
(4) R. E. Young, H. K. Pratt, and J. B. Biale, Anal. Chem., 24, 551 (1952). (5) E. M. Beyer Jr., and W. P.Morgan, Plant Physiol., 46, 352 (1970). (6) J. D. Goeschl, H. K. Pratt, and B. A. Bonner, Pl8nt Physioi., 42, 1077 (1967). (7) R. E. Holm and J. L. Key, Plant Physiol., 44, 1295 (1969). (8) B. G. Kang and P. M. Ray, Planta, 87, 206 (1969). (9) A. L. Abeles and F. B. Abeles. Plant Physiol., 50, 496 (1972). (10) A. E. Linkins, L. N. Lewis, and R. L. Palmer, Plant Physiol., 52, 554 (1973). (11) 0. L. Hollis, "Advances in Gas Chromatography-1965,'' A. Zlatkis and S. Ettre, Ed., Preston Technical Abstracts Co.. Evanston. Ill., 1966, p 56. (12) R. M. Muir and E. W. Richter, "Piant Growth Substances-1970," D. J. Carr Ed., Springer-Verlag, Berlin, Heidelberg, New York. (13) E. F. Elstner and J. R. Konze, FEBS Lett., 45, 18 (1974).
RECEIVEDfor review August 21, 1975. Accepted October 14, 1975. This work was financially supported by the Belgian "Fonds voor Kollektief Fundamenteel Onderzock" (grant'no. 2.9009.75).
Gas Chromatographic Determination of Free Mono-, Di-, and Trimethylamines in Biological Fluids Stephen
R. Dunn,*
Mlchael L. Simenhoff, and Laurence G. Wesson, Jr.
Division of Nephrology, Department of Medicine, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pa. 19 107
Short chain aiiphatlc amines are thought to be toxic at low concentrations in certain pathological states. in order to assay those substances in biological fluids, a method was developed to separate free dimethylamine (DMA) from trimethylamine (TMA) by gas chromatography with over 85 % resolution and very littie tailing. Using a flame ionization detector and an ammonia column conditioner, both TMA and DMA were detectable at a concentration of 0.470 ng/pi and reproducible to 4 ~ 4 . 8 %maximum. A second column was used to confirm the identity of DMA and to separate monomethylamine. An ultrafiltrate of the biological fluid sample can be run every 10 minutes.
The possible existence of toxic effects on the part of low molecular weight aliphatic amines in uremia has gendered a need for their assay with reasonable accuracy a t the low concentrations in which they exist in biological fluids (1-3). Previously, paper chromatography was used to determine primary and secondary amines by converting them to the 2,4-dinitrophenyl derivatives ( 4 ) . Blau ( 5 ) combined ion exchange and descending paper chromatography to determine a wide variety of biologically occurring amines including mono-, di-, and trimethylamine as hydrochloride salts. Thin layer chromatography of 5-dimethylamino-1-naphthalenesulfonamides (DANS-amides) (6) of the amines was performed after separation from the tissue proteins (7) with subsequent spectrophotofluorometric quantitation. These methods were for the most part laborious and often required desalting, extractions, or ion exchange methods either to remove interfering substances or to convert the amines into a species suitable for chromatographic separation. James e t al. (8) separated the amines in question on a four-foot column using hexadecanol-15% liquid paraffin as the liquid phase. However, the lower limit that could be detected with their technique was 4 pg for the methylamines. Sze, Borke, and Ottenstein (9) successfully separated the lower aliphatic amines on a nine-foot column using 15% diglycerol +5% tetraethylenepentamine ( T E P ) on Chromo-
sorb W. Umbreit et al. (10) determined amines a t low levels by injecting the aqueous hydrochloride salts with their conversion on column to the free amines via a caustic loaded precolumn. Separation of mono-, di-, and trimethylamine from extracts of fish tissue was achieved by Gruger (11) using Chromosorb 103 porous polymer. Andre and Mosier (12) combined an Ascarite precolumn with a Chromosorb 103 column to separate methylamine from dimethylamine a t a concentration of 5 ng/pl. Miller e t al. (13)separated dimethylamine from trimethylamine a t the pg/ml level using Graphon and 2% T E P . DiCorcia e t al. (14) using different amounts of Graphon and TEP obtained excellent separation of all three amines in water. T h e column was good for analytical standards but unsatisfactory in separating traces of amines in aqueous biological samples. Recently the same author (15) using 4% Carbowax (PEG) 20 M 0.8% KOH on Carbopack B separated the methylamines in water a t a concentration of 9 ng in a 4.5 p1 injection. Since these methods proved unsatisfactory for repeated sampling of biological samples with low amine concentrations, a modified method was developed.
+
EXPERIMENTAL Apparatus. A Perkin-Elmer 990 gas chromatograph with a flame ionization detector was used for all analyses. The primary column was a 0.25-inch o.d., 2-mm i.d., 6-foot coiled glass column packed with 10% amine 220 and 10% KOH on SO/lOO mesh acidwashed Chromosorb W (Supelco Co., Bellefonte, Pa). Conditioning of the column was achieved by heating to 130 "C overnight with approximately 5 ml/min flow of nitrogen. The column was operated at 60 f 1 "C isothermally using nitrogen as carrier at 17 ml/min. The hydrogen flame was optimized for this particular carrier flow by installing a Nupro fine-metering valve (Penn Valve Co., Willow Grove, Pa.) between the hydrogen gas regulator and the detector. Other operating parameters were injector port temperature, 190 i: 5 OC; manifold temperature, 220 f 5 O C ; air flow, 400 ml/min. A 6-foot coiled glass column, 0.25 inch o.d., 2-mm i.d. packed with 28% Pennwalt 223 4% KOH on SO/lOO Gas-Chrom R (Applied Science Labs, State College, Pa.) was conditioned 24 hr at 150 O C with low flow and used to confirm the absence of methylamine in the sera. Operating parameters for this column were column temperature, 60 O C isothermal; injector
+
ANALYTICAL CHEMISTRY, VOL. 48, NO. 1. JANUARY 1976
41
