SimuIta neous Ca rbon-Hyd rogen-Nitrogen Determination Gas Chromatography C. F. NIGHTINGALE' and J. M. WALKER Department of Chemistry, Kansas State College of Pitfsburg, Pitfsburg, Kan.
b This method uses a high frequency induction furnace for the combustion of the sample and utilizes the sensitivity and convenience of the gas chromatograph to detect combustion gases. Rapid combustion eliminates need for the liquid nitrogen trap and allows the determination of nitrogen along with carbon and hydrogen. The technique is based on the combustion and subsequent conversion of the sample to carbon dioxide, nitrogen, and acetylene in a helium atmosphere. Nitrogen, carbon dioxide, and acetylene are separated on a 5-A. Molecular Sieve column, detected b y a thermal conductivity detector, and recorded on a strip chart recorder. This method has a precision to 0.58% absolute for nitrogen, to 0.52% for carbon, and to 0.22% for hydrogen. Five- to 25-mg. samples of inorganic, aliphatic, aromatic, and heterocyclic nitrogen compounds containing oxygen, halogens, and sulfur were run.
T
wo hiETHoDs for the application of gas chromatography to microdetermination of carbon and hydrogen have appeared in the literature (1, 2 ) . Both involve combustion by modifications of the Pregl and micro-Dumas techniques and utilization of some of the advantages of the gas chromatograph as a detection system. Duswalt and Brandt ( I ) , combusted their sample in an atmosphere of oxygen; Sundberg and Maresh (6),burned their sample in a helium atmosphere in copper oxide and copper. The combustion gases were passed through calcium carbide and then frozen out in a liquid nitrogen trap. Because the combustion takes from 10 to 20 minutes, the liquid nitrogen trap must be used t o concentrate the gases for quick injection into the sample column. These methods reduce some of the disadvantages of the gravimetric method. The work described here utilizes some advantages of the gas chromatograph and speed of combustion of the high frequency induction furnace. The latter eliminates the need for a liquid nitrogen trap and permits the simultaneous determination of carbon, hydrogen, and nitrogen. Present address, The Dow Chemical Co., Midland, Mich.
The sample is placed in a quartzenclosed carbon crucible, covered with sufficient oxidizing agent, and combusted in a helium atmosphere. The combustion gases are passed through a heated combustion tube, through calcium carbide, onto a 4-foot, 5-A. Molecular Sieve column. The gases are programmed off the sample column with a linear temperature rise and detected by a thermal conductivity detector. Lack of an oxygen atmosphere has necessitated the use of an oxidizing agent. The strength of silver permanganate as an oxidizing agent and its property of losing a mole of oxygen a t a low temperature coupled with copper oxide give complete oxidation and/or pyrolysis of the sample. The combustion tube contains copper oxide and copper to ensure complete combustion and to reduce the oxides of nitrogen to elemental nitrogen. The time for loading, Combustion, separation, and recording of the sample is approximately 1 hour and 45 minutes. APPARATUS AND MATERIALS
A schematic diagram of the gas chromatograph-combustion unit is shown in Figure 1. The apparatus consists of a Leco induction furnace
a. b.
Helium supply Pressure regulator E. Burrell furnace d. Magnesium perchlorate tube e. Ascarite tube f. 5 A. Molecular Sieve moisture trap 9. Differential flow regulator h. Valves 1. Leco induction furnace i. Hevi-Duty electric furnace k. Calcium carbide tube 1. 5 A. Molecular Sieve sample column m. Block n. Reference thermistor side 0. Sample thermistor side
AIodel521, a combustion tube as shown in Figure 2, and an F&M Scientific Model 202 linear programmed temperature gas chromatograph. The helium line of the gas chromatograph was opened between the moisture trap and the injection port and connected to the combustion system. An all-metal combustion tube complete with all-copper connecting tubing (Figure 2) was constructed from steel and copper. The copper tubing from the metal combustion tube was connected to the Leco quartz combustion tube (Figure 3) by Kel F tubing, a tetrafluoro hydrocarbon tubing, and wrapped with Scotch Brand tape No. 471. The quartz combustion tube was designed to eliminate the restriction a t the outlet and to provide a more laminar flow. The all-metal combustion tube was oxidized in a Temco electric muffle furnace in an air atmosphere a t 450' C. and then hydrogen-reduced with Linde hydrogen and conditioned in the system for 48 hours before using to reduce any impurities which might give an unstable base line. The combustion tube was filled one third with -Mallinckrodt wire form analytical reagent grade cupric oxide and two thirds Lvith Baker & Adamson copper metal, light turnings Code 1619, which were also oxidized and hydrogenreduced. The ends were also filled with the hydrogen-reduced copper turnings so as to prevent the loss of the cupric oxide. The combustion tube was heated with a Hevi-Duty Electric Co. Multiple Unit tube heater with the controls set on four coarse and six fine. To prevent condensation of the water of combustion in the quartz combustion tube of the Leco furnace and in the lines to the all-metal combustion tube and to the calcium carbide container, the lines were wrapped with Electrothermal KO.H T 362 heating tape. Because heating tapes could not be used on the Leco quartz combustion tube, two Sylvania Electric 150-watt projector spotlites, PAR 38 Med. Skt., were directed on the tube to raise its temperature. The F&M Scientific Gas Chromatograph wasequippedwith a5-A. Molecular Sieve column purchased from F&M Scientific complete with column heater. The following temperatures, control settings, and flow rates were used throughout the experiment: Helium flow rate, reference, 30 ml. per min.; sample, 67 ml. per min. Thermistors bridge setting, 9 ma. Column starting temperature setting, 5' C. VOL 34, NO. 11, OCTOBER 1962
m
1435
Figure 2. a.
b. c.
d.
Combustion tube
Compression valve, Bushing, 3/4 inch Bushing, 1 '/4 inches Flare nut, 3/4 inch
'/4
inch
The column was never cooled below room temperature. Control panel setting initially of 5" C. is part of over-all programming. Temperature limit set ting, 400" C. Injection port setting, room temperature. Programmed temperature rate, 6.4" C./min. Helium pressure, 14 p.s.i. Block temperature, 205' C. Bureau of Mines helium supplied by Linde Co., was used as carrier gas and purified by passing through Arthur H. Thomas Co. Ascarite (8- to 20-mesh), Mallinckrodt wire form analytical reagent grade cupric oxide heated to.800" C. with a Burrell Model T-2-9 electric furnace, anhydrous Fisher reagent chemical magnesium perchlorate, and Leco specially prepared manganese dioxide, The water of combustion was converted to acetylene by reaction with Fisher laboratory chemical calcium carbide (Electrolite 20- to 30-mesh). This allowed the determination of hydrogen as acetylene and prevented water from hydrating the Molecular Sieve sample column. The calcium carbide container was constructed of 0.25-inch copper tubing 4 inches in length, with brass compression fittings on each end. A plug of copper turnings was placed 1 inch from the top end and this section was filled with calcium carbide. The calcium carbide was changed before each run, although the need for doing so was not verified. The Leco quartz-enclosed carbon crucibles were cleaned with aqua regia and then hot chromic acid cleaning solution. The crucibles were dried on an electric hot plate and stored for use over calcium chloride. The crucibles were covered during the combustion with Leco crucible covers, No. 528-40. The silver permanganate was prepared by mixing hot saturated solutions of Mallinckrodt analytical reagent grade silver nitrate and Baker analyzed reagent grade potassium permanganate. This solution was filtered and dried of excess water using a fritted filter funnel and a suction filter flask. The precipitate was dried under the reduced pressure of a water aspirator a t a temperature of 85" C. Fisher certified reagent grade thiourea was used as a source of carbon, hydrogen, and nitrogen to determine the areas per milligram of sample element. The thiourea was washed in Mallinckrodt analytical reagent grade anhydrous ether (ether absolute), dissolved in hot distilled deionized water and recrystallized from cold water, and dried under vacuum at the temperature of 85" C. Eastman organic reagent grade 1436
0
ANALYTICAL CHEMISTRY
p-bromoacetanilide, The Matheson Co. reagent grade p-nitroaniline, Merck reagent grade benzoic acid (minimum assay of 99.5% benzoic acid), and G. Frederick Smith reagent 2,2',2"-tripyridine were other compounds used to test the method. The p-bromoacetanilide and p-nitroaniline were purified by the same method as the thiourea; the others were used without further purification. PROCEDURE
The system was connected, as in Figure 1, and the helium flow was started into the system with valves h ~ he, , hs, hd open, and hs closed. The controls on the Hevi-Duty Electric furnace, j , were set on four coarse and six fine and the system allowed to equilibrate for 4 hours. The sample column was programmed three times a t 18" C. per minute to purge the column completely. The sample was weighed on the ilinsworth Micro Balance or the Mettler Gram-Atic-Balance and placed in the bottom of the quartz enclosed carbon crucible. ii 500-mg. sample of silver permanganate was weighed on the Mettler Gram-iltic-Balance, placed in the crucible on top of the sample, and mixed by tilting the crucible to each side several times. The crucible was filled with copper turnings which had been oxidized in a flame and then covered nith a crucible cover. Valve hj was opened and valves h4
ti
2.1cm.
7 3 . 4C Figure 3.
F
ELUTION TIME IMINUTES)
Figure 4. Typical chromatogram for carbon-hydrogen-nitrogen determination
and hl were closed. The sample Ivas placed in the Leco induction furnace. The calcium carbide container, k , was removed, filled with fresh calcium carbide, and replaced. The calcium carbide was always replaced before each run. Valves h, and h4 n ere opened, and hs was closed and the system allowed t o purge. To speed the process of purging, the column was heated a t a programmed temperature rate of 35" C.per minute and held a t the maximum temperature until the process was complete. The remainder of the procedure begins when the bloryer is turned on to cool the column. The controls t o the chromatograph are adjusted to their proper settings and the projector spotlites are turned on at the end of 1-1 minutes. At the end of 15 minutes, the timing is interrupted and the Leco induction furnace is energized. -it the end of the 30-second time delay, the furnace fires and the clock. recorder, and programmer are turned on and the combustion is allowed t o proceed for 30 seconds and then the Leco induction furnace is turned off. The column blower is turned off at the end of I8 minutes. The areas of the peaks n-ere integrated with a Keuffel 8: Esser Co. No. 4212 planimeter and the area per milligram was calculated for the thiourea. A mean value of the area per milligram was taken and used to calculate the per cent compositionof the other compounds. RESULTS
LC
Quartz combustion tube
A series of runs was made using carbon dioxide, sodium carbonate decahydrate, and thiourea to establish the location of the peaks and the retention times of the nitrogen, carbon dioxide, and acetylene on the Molecular Sieve column under these operating conditions. Thiourea was used as a standard to obtain the areas per milligram for nitrogen, carbon dioxide, and acetylene. A series of combustions was run, and the results are tabulated in Table I. The mean areas per milligram were used to calculate the per cent composition of the other compounds as well as thiourea. Benzoic acid was used in this project to test the ability of this method t o oxidize a n aromatic ring compound. The per cent composition was calculated from the mean area per milligram obtained from thiourea and recorded in Table 11.
DISCUSSION
A typical chromatogram is shown in Figure 4. Results of the nitrogencarbon-hydrogen determinations by the gas chromatographic method, Table 111, show the variety of compounds which were analyzed by this method and the over-all absolute precision obtained. The compounds were chosen to provide various structures as aell as interfering elements. As stated by Dusivalt and Brandt ( I ) , the combustion products of the sulfur and halogen components are removed by a section of silver metal in the combustion tube. This was unnecessary in our case because we used silver permanganate as a n oxidizing agent. The analyses of thiourea, benzoic acid, p-bromoacetanilide, and 2,2’,2“ -tripgridine show excellent agreement Ivith the theoretical values. The results of the combustion of ammonium nitrate show a low value for nitrogen and a high value for hydrogen. This could indicate a hydrated sample but drying the ammonium nitrate a t 105’ C. for 4 hours did not change the values. p-Kitroaniline s h o w all lon. values but shows a much lower value for carbon than for other elements and no explanation is readily available. Results indicate a need for further study including reduction of sample size and the changing of instrumental parameters. Under these conditions one might utilize rapid programming followed later by a reduction in rate t o differentiate between the C 0 2 and CzHz peaks. The use of the above parameter changes could possibly eliminate
Table 1.
Results of Nitrogen-Carbon-Hydrogen Determinations b y Combusting Thiourea
Theory, 70 C H 36.80 15.78 5.30
N 37.95 37.23 37.83 35.69 36.44
N
Table II.
Found, yo C H 15.26 5.54 15.07 5.49 16.25 5.15 16.27 5.63 16.07 4.69 Mean dev.
Diflerence, 7c N C H $1.15 -0.52 +0.24 $0.43 -0.71 +0.19 +1.03 $0.47 -0.15 -1.11 $0.49 $0.33 -0.36 +0.29 -0.61 10.82 iO.50 10.30
Results of the Carbon-Hydrogen Determinations Obtained from the Combustion of Benzoic Acid“
- Difference, H C 5 15 + O 66 68 84 4 93 -0 01 68 94 5 03 +0 09 Mean dev. 1 0 25 Sample neight corrected for minimum assay of benzoic acid
Theory, C 68 85
Table 111.
5%
Found, %
H 4 95
C G9 51
E+0 20 -0 02 $0 08 1 0 10
Summary of Results of the Combustions of Various Compounds
Compound N Thiourea 37.03 Benzoic acid Ammonium nitrate 32.98 ~~~.~~~~ p-Bromoacetanilide 6.55 2,2’,2”-Tripyridine 18.22 p-Nitroaniline 19.84 ~
Found, 70 C H 15.78 5.30 69.07 5.04 5.85 44.96 3.97 77.57 4.64 50.22 4.26 Mean dev.
the need for purging between runs and reduce the total time for each analysis. LITERATURE CITED
(1) Duswalt, A. A., Brandt, IT. W., AKAL.CHEM.32,273 (1960). ( 2 ) Sundberg, 0. E., hlaresh, C., Ibid., p. 274.
Difference, % C H 1-0.23 0.00 0.00 +0.22 $0.09 -2.02 +o.x1 $0.01 +0.07 iO.20 +0.20 4-0.34 -0.11 -0.44 -1.95 -0.12 10.58 10.52 10.22 N
Other elements S 0
0 0, Br
0
RECEIVED for review April 16, 1962. Accepted August 13, 1962. Division of Analytical Chemistry, 142nd Meeting, ACS, Atlantic City, N. J., September 1962. Abstracted from a thesis by C. F. Nightingale submitted in partial fulfillment for the degree of Master of Science a t Kansas State College of Pittsburg, Pittshurg, Kan.
Electrochemical Determination of Organophosphorous Compounds G. G. GUILBAULT, D. N. KRAMER, and P. L. CANNON, Jr. Protective Development Division,
U. S.
Army Chemical Research and Development laboratories, Army Chemical Center, Md.
b An electrochemical method i s described for the determination of anticholinesterase organophosphorous compounds, such as 0,O diethyl 0 [2 (ethylthio)ethyl] phosphorothionate (Systox), isopropyl methylphosphonofluoridate (Sarin), diethyl-pnitrophenyl monothiophosphate (parathion), and s-(1,2-dicarbethoxyethyl)0,O-dimethyldithiophosphate (malathion). A constant current of 2 5 pa. i s applied across two platinum electrodes, and the change in potential upon enzymatic hydrolysis of butyrylthiocholine iodide b y cholinesterase is recorded vs. time. Anticholinesterase compounds inhibit the hydrolysis, caus-
-
-
-
ing a corresponding decrease in the slopes of the depolarization rates, AE/Af. This decrease i s a direct measure of the concentration of organophosphorous material. Without pre-incubation of enzyme, 0.03 to 0.5 pg. per ml. of Sarin, and 0.3 to 9.2 pg. per ml. of Systox may be determined with an accuracy of about Three to ten minutes incuba1 tion permits the determination of 2 X 10-4 t o 3 X 10-3 pg. per ml. of Sarin, 0.01 to 0.20 pg. per ml. of Systox, 0.18 to 1.8 pg. per ml. of parathion, and 1.8 to 18 pg. per ml. of malathion, with accuracies of from 0.9 to 3.2%.
yo.
M
ORQAKOPHOSPHOROUS compounds are highly toxic anticholinesterase agents which are of increasing importance because of their daily use as insecticides, plasticizers, petroleum additives, and polymers. Some of the more important of these materials are Sarin, Gystox, parathion, and malathion. Kramer and Gamson (6) have recently given a comprehensive review of current analytical methods for organophosphorous compounds. At present, the most sensitive methods involve either a colorimetric determination by the amine-peroxide reaction (7) or an enzymatic method, following the AKY
VOL. 34, NO. 11, O C T O B E R 1962
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