barbital were virtually identical to the sensitivities of the HFID to the same compounds. Nevertheless, both detectors appeared to be about half as sensitive to thiopental as to the other three barbiturates on h weight basis. In summary, the proposed procedure was more reliable,
faster, and more precise than others previously reported in the literature, and presents possibilities of investigation with pediatric patients. Received for review August 30, 1973. Accepted January 11,1974.
Modified Electrolytic Conductivity Detector Cell for Gas Chromatography James F. Lawrence and Alan H. Moore Food Research Laboratories, Health Protection Branch, Health and Welfare Canada, Tunney's Pasture, Ottawa, Ontario K 1 A OL2
The adaption of electrolytic conductivity to the detection and quantitation of nitrogen-, chlorine- or sulfur-containing compounds in the effluents from gas chromatographs was first accomplished by Coulson ( I , 2). Patchett ( 3 ) incorporated a number of refinements into the system which increased the sensitivity of detection to 0.1 ng of organic nitrogen. Cochrane and Wilson (4, Cochrane, Wilson, and Greenhalgh ( 5 ) , Cochrane and Greenhalgh (6), and Laski and Watts ( 7 ) have examined the response of the Coulson conductivity detector (CCD) (Tracor Inc.) to a wide variety of compounds. The comparison of the CCD to E.C. (8, 9), alkali flame ( I O , I I ) , and flame photometric (sulfur mode) (6) detectors has been carried out. Although the CCD was less sensitive, its extreme selectivity allowed a much greater freedom from interferences which made it very suitable for pesticide residue analysis. The response characteristics of the CCD in the pyrolytic and oxidative modes have recently been investigated (5, 6). The detector response increased with pyrolysis furnace temperature. Oxygen flow caused an initial increase in sensitivity followed by a gradual decrease at higher flow rates. The effects of furnace temperature and hydrogen flow rates in the reductive (nitrogen) mode has recently been examined (12). They were found to be similar to that obtained in the pyrolytic and oxidative modes. The flow rate of water through the CCD system also influenced detector response ( 2 2 ) . By having the gas-water contact area flow rate equivalent to the detector cell flow rate, sensitivity was increased 2 - to %fold for the CCD (12). Equal flow rates were used by Jones and Nickless (23) who described an electrolytic conductivity detector system in which dilute HC1 was used for conductivity measurements instead of deionized water. The overall sensitivity of their system was less for nitrogen-containing compounds than that obtained with the CCD. (1) (2) (3) (4) (5)
D. M . Coulson, J. D. M Coulson. J. G . G . Patchett, J.
GasChromatogr.,3, 134 (1965). Gas Chromafogr., 5 , 285 (1966). Chromafogr.Sci., 8, 155 (1970) W . P. Cochrane and 8. P. Wiison, J. Chromatogr., 63, 364 (1971). W. P. Cochrane, B. P. Wilson, and R. Greenhalgh. J . Chromatogr.,
75, 207 (1973). (6) W. P. Cochrane and R . Greenhalgh, Intern. J . Environ. Anal. Chem., in press, 1973. (7) R. R . Laski and R. R. Watts, J. Ass. Otfic. Ana/. Chem., 56, 328 (1973). (8) D. M . Coulson, J. E. DeVries. and B. J. Walther, J. Agr. Food Chem., 8, 399 (1960). (9) R. Purkayastha and W . P. Cochrane, J. Agr. Food Chem., 21, 93 (1973). (10) R. Greenhalgh and W. P. Cochrane, J. Chromatogr.. 70, 37 (1972). (11) J. F. Palframan, J. McNab. and N . T. Crosby, J. Chromatogr., 76, 307 (1973). (12) J. F. Lawrence, J. Chromatogr., 86, 333 (1973) (13) P. Jonesand G . Nickless, J. Chromatogr., 73, 19 (1972).
The present report describes the effort to further increase sensitivity of electrolytic conductivity detection for use with gas chromatography. For this purpose, a new cell was constructed, evaluated and compared to the Coulson conductivity cell (Both cells are covered by U.S. Patent No. 3,309,845, March 21, 1967).
EXPERIMENTAL Apparatus. An Aerograph HI-FY Model 600-C gas chromatograph fitted with a Coulson conductivity detector system (Tracor Inc., Austin, Texas) was used. The 6-ft X 6-mm 0.d. glass column was packed with 4% SE30 on 80jlOO mesh Chromosorb W/HP. Operating conditions were: column temperature, 185 "C; transfer unit temperature, 210 "C; pyrolysis furnace temperature, 780 "C; helium carrier, 60 ml/min; helium sweep, 60 ml/min; hydrogen, 50 ml/min; dc bridge potential, 30 V. A 1-cm plug of strontium hydroxide coated glass wool was placed in the end of the quartz pyrolysis tube. A 2-cm flattened nickel wire coil was inserted 2 inches from the end of the pyrolysis tube. A 1.0-mV strip-chart recorder operating a t 0.25 in./min was employed. Peak height was used to measure detector response. The herbicide, atrazine (2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine) a t a concentration of 1 pg/ml in hexane was used as the test compound. Temperature control was obtained with a constant temperature bath and circulator (Forma Scientific, Marietta, Ohio). The modified cell is depicted in Figure 1. The overall length was 5% inches. The water-jacket was constructed from 13h-inch diameter glass tubing. The dimensions of the glass capillary tubing were 0.5-mm diameter for the water entrance to the gas-water contact area and 1.0-mm diameter for the gas-water contact area and the cell arm. The electrodes consisted of platinum wire approximately 1.0 cm apart. A ball and socket joint was used to connect the effluent gas entrance to the pyrolysis tube. The pyrolysis tube, furnace, bridge circuitry, pumping system. ion exchange resin, and water were the same for both the modified cell and the Coulson cell. Tygon was used for all connective tubing. Water flow through the gas-water contact area of the modified cell was controlled by means of an adjustable screw-clamp (see Figure 1). The cell flow rate was controlled by siphon action. Both flows were equivalent so that no water escaped through the vent tube. The Coulson cell was run as outlined in the Tracor instrument manual but with the reservoir water level maintained 5'2 inch above the pump entrance. Temperature of the water was varied by inserting a 14-turn glass cooling coil, constructed from Y4-inch glass tubing, into the reservoir and connecting to the temperature bath and circulator. Sample Extraction. The extraction of atrazine spiked in potatoes (0.1 ppm) was carried out using 50 grams of sample and extracting with 250 ml benzene:acetone:lN sulfuric acid (190:10:4). The cleanup technique employed consisted of the cold bath precipitation method described by McLeod and Wales (14). The cleanedup extracts were dissolved in hexane for gas chromatography.
(14) H. A .
McLeod and P. Wales, J. Agr. FoodChem., 20, 624 (1972)
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""'"'1 GC EFFLUENT
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A
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Figure 1. Scale diagram of the modified conductivity detector and water jacket
8'C
32-C
8-C
45OC
Figure 4. Comparison of detector sensitivity of the modified cell ( A , B ) with the Coulson cell (C, D ) . Attenuation 8X. 9 . 0 ng atra-
dl
zine injected
I
I
TIME I M Z e i l
Figure 2. Effect of water flow rates on response of the modified cell A , 1.2 ml/min; E , 0.7 ml/min; C, 0 . 2 ml/min. Attenuation 8X.
Temperature 3 2 "C. 9.2 ng atrazine injected
16 .c 0
0 2 4 6 8 TIME (Minutes)
Peak size and shape for 1.0 ng atrazine at full scale sensitivity.Attenuation 1X. Temperature 3 "C
1 SPIKED SAMPLE
-
.-
-
L W
$ 8 n
z
2
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Figure 5.
e
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Figure 3. ty with A ,
I
IO
I
20 30 40 TEMPERATURE ("C)
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'
Effect of water temperature on background conductiviModified cell; E, Coulson cell. Attenuation 8X.
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Analysis of 0.1 p p m atrazine spiked in potatoes. Attenuation 4X. Temperature 18 "C
Figure 6.
RESULTS AND DISCUSSION Effect of Water Flow Rates. The effect of total flow through the modified system at 32 "C is shown in Figure 2. The peak height increased in the ratio 6:7:9 and peak area 6:8:15 for flow rates of 1.2 ml/min, 0.7 ml/min, and 0.2 ml/min, respectively. The same effect was noticed a t 1 "C. Although sensitivity increased a t slow flow rates, the base line became less stable. The fastest flow (1.2 ml/ min) gave good peak shape and thus was considered optimum. The same change in flow rate had little effect on 756
ANALYTICAL CHEMISTRY, VOL. 46, NO. 6, MAY 1 9 7 4
the peak height response of the Coulson cell although peak area increased because of peak broadening. Temperature Effect. Figure 3 illustrates the effect of temperature on background conductance of the modified and the Coulson cell. Both cells exhibited a significant increase in conductivity with a rise in temperature (a 4-fold increase from 10-50 "C). The significance of temperature became important when the 1X attenuation was used for
detection of low levels of nitrogen compounds. Also it was found that base-line noise increased greatly with a rise in water temperature making analyses at the 1X attenuation difficult at 40 "C or higher for both cells. For routine operation cold tap water (18 "C in summer and 6 "C in winter) was used for cooling the system. Water below 20 "C was satisfactory for low background conductance and a steady base line. Sensitivity. The sensitivity of the modified cell was 5 times greater than the Coulson cell under identical instrumental conditions. Figure 4 compares the response of the two cells for 9.0 ng atrazine a t 8 "C. Peak shape obtained with the modified cell was similar to that of the Coulson cell. Water temperature did not affect the sensitivity of the modified cell althcugh the response of the Coulson decreased by about 30-40% in proceeding from 8 to 45 "C. Figure 5 shows the response of 1.0 ng of atrazine on the most sensitive setting of the modified cell a t 3 "C. Halfscale deflection was obtained with about 1.3 ng atrazine. The increase in sensitivity was probably due to the modified cell constant (larger electrodes, shorter distance between electrodes) as well as the longer gas-water contact area for dissolution of ammonia. Sample Analysis. The use of the modified cell for residue analysis was examined and the results are shown in Figure 6. The injection was equivalent to 5 ng of atrazine (50 mg of sample). The vent valve was closed (effluent directed to the furnace) 1 min after injection. No interferen-
ces were encountered in the analysis of potatoes, carrots, or parsnips. Less than 0.01 ppm of the triazine could be quantitatively analyzed with the modified system. Improvements. The modified cell could be improved by using a more precise flow valve than the screw-clamp used in the present work. This would help decrease flow fluctuations and base-line noise a t more sensitive settings. An increase in the length of the gas-liquid contact area may increase sensitivity even further. The changing of the conductivity cell from a horizontal position to a vertical one (as with the Coulson) would eliminate the occasional accumulation of air bubbles near the electrodes.
CONCLUSIONS The modified cell has several advantages over the commercially available Coulson cell. The new apparatus is much more compact, has a water-jacket for temperature control, and is about 5 times more sensitive. The new cell also has an adjustable control valve to obtain optimum flow conditions for analysis. Temperature and water flow rates have significant effects on the response of the modified cell. Flow rates of about 1.2 ml/min and a water temperature of less than 20 "C proved most satisfactory for analysis. Received for review October 2, 1973. Accepted December 17, 1973.
Estimation of Choline and Acetylcholine in Tissue by Pyrolysis Gas Chromatography William 8 . Stavinoha and Susan T. Weintraub Department of Pharmacoiogy. The Un/vers/tyof Texas Heaith Science Center a t San Antonio. San Antonio. Texas 78284
Measurement of brain acetylcholine and choline is critical in the study of neurochemical processes. Consequently, a large number of analytical techniques, which employ bioassay, enzyme assay, chemical procedures or instrumental methods for the estimation of these biogenic amines, have been described (1-3). In general, reported acetylcholine values are comparable when one accounts for differences in animal sacrifice ( 4 ) . However, a wide variation in the reported values for brain choline concentration can be seen in recent literature ( 5 , 6). Because of this wide range, methods of sacrifice may not fully explain the divergence in reported data. Recently, a method for the simultaneous measurement of choline and acetylcholine has been published which utD . J Jenden and L. R . Campbell, in "Analysis of Biogenic Amines and Their Related Enzymes,'' Vol. 19, D. Glick, Ed., Interscience, New York, N.Y., 1971 I . Hanin. in "Advances in Biochemical Psychopharmacology." Vol. 1, E. Costa and P. Greengard, Ed., Raven Press, New York. N.Y., 1969 P I . A Szilagyi, D. E. Schmidt, and J. P Green, A n a / . Chem . 40, 2009 (1968) W 5.Stavinoha, S. T Weintraub, and A T Modak, J Neurocnem. 20, 361 (1973) J . Schuberth. B. Sparf. and A . Sundwall, in "Drug and Cholinergic Mechanisms in the CNS," E. Heilbronn and A. Winter, Ed., Research Institute for National Defense, Stockhoim, 1970. Isabel Eade, Catherine Hebb, and S. P. Mann, J. Neurochem., 20, 1499 (1973).
ilizes a chemical demethylation procedure to produce volatile derivatives ( 7 ) . This method is quite sensitive, and the use of GC-MS provides positive identification of the compounds in question. However, the methodology is complex and time-consuming, and the chemical demethylation requires meticulous anhydrous conditions. Pyrolysis has been widely applied to the gas chromatographic separation of nonvolatile compounds (8, 9 ) , and this method offers an alternate means to demethylate quaternary halides. While the measurement of acetylcholine by pyrolysis gas chromatography has previously been reported, the application of this technique to the simultaneous assay of choline and acetylcholine has been unsuccessful (3, 10). Commonly used isolation methods for quaternary compounds-precipitation by ammonium reinickinadequate for a k , tetraphenylboron, or periodide-are preparing choline for measurement by pyrolysis gas chromatography (11). (7) D. J Jenden. R. A. Booth, and Margaret Roch, Ana/. Chem., 44, 1879 (1972). (8) R . L. Levy, in "Chromatographic Reviews," Vol. 8 . M Lederer. Ed , Elsevier, New York, N . Y . , 1966. (9) "Gas Chromatography, 1968," C. L. A. Harbourn, Ed.. Elsevier, Amsterdam, 1969. pp 385-416. (10) P. I . A. Szilagyi, J P. Green, 0 Monroe Brown, and S. Margolis, J . Neurochem.. 19, 2555 (1972) (11) W B. Stavinoha and S T. Weintraub, unpublished work, 1973. A N A L Y T I C A L C H E M I S T R Y , VOL. 46, NO. 6 , M A Y 1 9 7 4
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