1120
ANALYTICAL CHEMISTRY, VOL. 51, NO. 8, JULY 1979
Sampling of Chloroacetyl Chloride in Air on Solid Support and Determination by Ion Chromatography Phillip R. McCullough Physical Sciences Center, Corporate Research Laboratory, Monsanto Co., 800 North Lindbergh Boulevard, St. Louis, Missouri 63 166
Jimmy W. Worley" Research Department, Monsanto Agricultural Products Company, 800 North Lindbergh Boulevard, St. Louis, Missouri 63 166
A method for the determination of chloroacetyl chloride (CAC) in air is described. CAC is sampled with commercially available silica gel collection tubes, desorbed with sodium bicarbonate solution, and determined by ion chromatography. A dynamic standard generator for CAC was developed and used to validate the method in the range of 3.7-76.7 pg CAC. Average recovery was greater than 99% for both monochloroacetate and chloride. The relative standard deviation was 0.072 for monochloroacetate and 0.088 for chloride. The reliable quantitation limlt for CAC is ca. 0.01 ppm but can be improved readily if desired.
T h e determination of monochloroacetyl chloride (CAC) in air is important because of its use in the industrial workplace as a chemical intermediate and t o the finding t h a t it is a reaction product in t h e atmospheric chemistry of some chlorinated hydrocarbons ( I , 2 ) . The American Conference of Governmental Industrial Hygienists recently announced ( 3 ) its intention t o adopt a threshold limit value (TLV) for eight-hour time-weighted-average exposure t o CAC of 0.05 ppm. Consequently there is a need for a convenient method for personnel monitoring for CAC exposure. Two methods ( 2 , 4 ) have been reported previously for the determination of CAC in air. Both are based on the formation of esters of CAC in a n impinging sampling apparatus and subsequent determination of the esters by electron capture gas chromatography. T h e requirement for glass impingers and the general unsuitability for sampling periods longer than a few minutes would make these two methods unsatisfactory for personnel monitoring. We now report a convenient solid support sampling method for the determination of CAC in air by ion chromatography.
EXPERIMENTAL Reagents. Chloroacetyl chloride (99+ percent) was from Monsanto Agricultural Products Co. (St. Louis, Mo.). Sodium carbonate, sodium bicarbonate, and sulfuric acid were Mallinckrodt analytical reagent grade. Monochloroacetic acid was Fisher certified grade. Deionized water was used for all solutions. Silica gel collection tubes containing 65 mg silica gel in the front section and 35 mg in the backup section were purchased from Zink Safety Co. (St. Louis, Mo., catalog no. 226-10-100). The i.d. of these tubes is 4 mm. The two silica gel sections are ca. 14 mm and 7 mm long, respectively, and are separated by a small plug of polyurethane foam. The front section is held in place by a small section of glass wool and a metal clip. Apparatus. Portable sampling pumps were models P200 and P125 from E. I. du Pont de Nemours & Co., Inc. A Dionex Model 14 ion chromatograph, equipped with a 0.1-mL injection loop, a 3 X 150 mm anion precolumn, a 3 X 500 mm anion separator column, and a 6 X 250 mm anion suppressor column, was used. For monitoring the output of the dynamic CAC standard generator (see next section), an AID (Analytical Instrument Development, Inc., West Chester, Pa.) Model 511 flame ionization portable GC was used. The column was 6 ft X 'is in. 0.d. (stainless 0003-2700/79/0351-1120$01 OO/O
steel) and was packed with 10% DC 200 on Chromasorb W 60/80; carrier flow was 25 mL/min nitrogen. Sample injection was through a gas sampling valve (AID option 01) containing a 3.7-mL stainless steel injection loop. Initial conditioning of a column with 20 to 30 one-microliter injections of neat CAC, and continuous conditioning of the injection system with dry air containing ca. 1.0 ppm CAC, led to good, reproducible results. Dynamic CAC Standard Generator. A simple, rugged diffusion tube system (5,6 ) was designed to provide a constant source of low-concentration CAC in air. The diffusion tube was a 6 mm 0.d. x 1 mm i.d. X 120 mm length piece of glass tubing sealed a t one end. It contained CAC to a height of ca. 40 mm at the closed end. The diffusion tube was incorporated into a flow system by placing it inside an upright piece of 3/s-in. 0.d. stainless steel tubing which had been plugged at the bottom and swaged into a 3/s-in. tee at the top. The top of the diffusion tube protruded slightly into the flow path inside the tee. The diffusion tube was kept at 30.00 "C (h0.02 ") by immersing it in a constant temperature water bath. One arm of the tee was connected to the inlet of the gas sampling valve on the portable GC and the other to a cylinder of compressed, purified air. This system, when operated at 240 mL/min air flow, produced ca. 0.8 ppm concentration of CAC, established by determining weight losses from six different diffusion tubes for 4-5 days each. Air Sampling. Sampling in the laboratory was done by connecting a silica gel tube to the outlet of the gas sampling valve of the portable GC via a 1/8-in.to 1/4-in.reducer, using a 1/4-in. vespel ferrule. Field samples in production facilities were done using the portable pumps. A typical sampling rate was 65 mL/min. for 5 h (19.5 L total). Ion Chromatography. For analysis, the front section and the back-up section of each silica gel tube were transferred to separate 1-dram vials and each treated with 2.0 mL of 1.5 mM sodium bicarbonate solution. The vials then were ultrasonicated for 0.5 h or allowed to stand for at least 4 h with occasional agitation. One mL of the solution above the silica gel in the vial then was syringe-loaded into the injection port of the ion chromatograph; a Millipore 0.22-pm filter (No. SLGS 025 OS) was used during injection to remove any particulates present. Eluting solvent for the analysis of monochloroacetate and chloride was 1.5 mM sodium bicarbonate, pumped a t 40% flow. After several hours of analysis, usually at the end of the day, eluting solvent was switched to 3.0 mM sodium bicarbonate/2.4 mM sodium carbonate mixture for ca. 30 min. to allow accumulated sulfate from the silica gel (see Results and Discussion), as well as any other late-eluting species, to be expelled from the column. Regeneration of the suppressor column as necessary was done with 1.0 N sulfuric acid. Standard solutions for calibration were prepared by appropriate dilutions with 1.5 mM sodium bicarbonate of a solution prepared mol) monochloroacetic acid and 0.236 from 0.387 g (4.10 X mol) sodium chloride made up to 1000 mL. For g (4.03 x preparation of a calibration curve, 2.0 mL of each standard solution, as well as of "blank" 1.5 mM sodium bicarbonate solution, was added to a 1-dram vial and treated with the front section of an unused silica gel tube. This procedure allowed standards to be as nearly identical to samples as possible. It was essential for the chloride calibration since the silica gel itself is contaminated with low amounts of chloride (see Results and Discussion). C 1979 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 51, NO. 8, JULY 1979 Table I.
Results for Validation of CAC Method
expected CAC chloroacetate air concn, concn ppma p g/m ~b 0.83 0.77 0.42 0.39 0.17 0.15 0.083 0.077 0.042 0.039
4
e
Ib 15
I4
Time (min) Figure 1. Ion chromatogram for injection of 0.1 mL of solution containing 1.8 pg/mL monochloroacetate and 0.8 pg/mL chloride in 1.5 mM sodium bicarbonate (equivalent to a 20-L air sample containing 0.05 ppm chloroacetyl chloride). The first three peaks and part of the chloride
peak arise from t h e silica gel used Calibration solutions were prepared fresh as needed. Some deterioration with time of the low concentration standards was noted. Monochloroacetate response decreased to about 85% in three days for a 3.8 pg/mL standard (equivalent to a 20-L air sample containing 0.1 ppm CAC) and decreased to none detectable in two days for a 0.9 pg/mL standard. An ion chromatogram of a standard containing 1.8 pg/mL monochloroacetate and 0.8 pg/mL added chloride is shown in Figure 1. This represents a 20-L air sample containing 0.05 ppm CAC, the newly announced intended-TLV.
RESULTS AND DISCUSSION For t h e validation of this method, three samples a t each of 10 different CAC-loadings, equivalent to 20-L air samples containing 0.04-0.83 ppm CAC, were generated using CAC diffusion tubes. This was accomplished by collecting CAC with a silica gel tube attached to the exit of the gas sampling valve on the portable GC, through which was flowing 0.83 or 0.77 ppm CAC in air a t 240 mL/min (0.92 or 0.85 pg CAC/ min). Collection for 4.25 to 85 min gave the desired incremental loadings, with the 85-min samples being full 20-L air samples. T h e front section of each silica gel sample and t h e back-up section of one 85-min sample were desorbed and analyzed as described in the Experimental section. Quantitation was by peak height, using calibration curves generated from standards containing ca. 0, 2, 5, 10, 25, and 50 pg/mL each of monochloroacetate and added chloride. T h e back-up section analyzed showed no detectable monochloroacetate (minimum detectable, ca. 0.4 pg/mL). The results for the front sections are shown in Table I. T h e average recovery of both monochloroacetate and chloride is 99+ percent. T h e pooled coefficients of variation or relative standard deviations, are 0.072 for monochloroacetate and 0.088 for chloride. The
(m),
1121
31.6 29.5 15.8 14.8 6.3 5.9 3.2 3.0 1.6 1.5
found chloroacetate concn, p g/ m ~ c
, d
30.0 (0.013) 29.2 (0.029) 16.1 (0.016) 15.2 (0.033) 6.3 (0.063) 6.4 (0.039) 3.1 (0.032) 3.1 (0.032) 1.6 (0.156) 1.5 (0.133)
expected chloride concn, p g/m ~b 12.3 11.5 6.2 5.8
2.5 2.3 1.2 1.2 0.6 0.6
found chloride concii, p g im L C ~
~ J
10.3 ( 0 , 0 9 7 ) 9.7 (0.036) 5.6 (0.056) 5.3 (0.019) 2.8 (0.089) 2.6 (0.019) 2.0 ( 0 . 0 ) 2.0 (0.050) 1.7 (0.059) 1.8 (0.222)
a Based on a 20-L air sample. Based on weight losses from CAC diffusion tubes used. Results presented as average of three samples, followed by the relative standard Pooling of the individudeviation ( R S D ) in parentheses. al RSDs gave the result of 0.072. Bartlett’s test (Ref. 7 ) for homogeneity of variance gave a x i value of 1 9 . 8 9 . This is less than 21.67 ( x ’ for 9 degrees of freedom at the 0.01 level) and indicates that the hypothesis of equal varChloride iance can be accepted with 99% confidence. results corrected for “average” background chloride (intercept of least squares calibration curve). f Pooling of the individual RSDs gave the result of 0.088. Bartlett’s test gave a x 2 value of 6.53 (see f-o o t~ n o t e-d ) ._ _ ____-
-
CV for each should be better if the precision for the lowest concentration of each were improved; this probably could be done using electronic integration rather than peak height quantitation. The “average” recovery of chloride is misleading since individual recoveries are low a t the higher concentrations and high at the lower concentrations. The reason for this is not known, but may have to do with inadequate correction for the background chloride found on the silica gel. The background chloride may be variable among silica gel tubes used. One “blank” ion chromatography standard showed 1.9 pg/mL background chloride from the silica gel, along with 1.0 pg/mL fluoride and 255 pg/mL sulfate; identities of these species are based on retention times. T h e fluoride, and two other unknown peaks, elute early and do not interfere with the monochloroacetate and chloride analyses. Sulfate does not interfere directly; it does not even elute from t h e separator column with 1.5 m M sodium bicarbonate. Apparently, however, buildup of sulfate and possibly other late-eluters on the column as many samples are run changes the column characteristics such t h a t monochloroacetate and chloride retention times decrease. Brief switchover to the stronger eluting solvent 3.0 mhl sodium bicarbonate/2.4 m M sodium carbonate causes t h e accumulated sulfate and any other species to be expelled. Use of a different solid support or cleanup of the silica gel presently used might improve the procedure, particularly for chloride determination. However, the hydrophilic nature of silica gel probably is advantageous for t h e retention and hydrolysis of CAC, and the commercial “as is” availability of these silica gel tubes has strong appeal when considering implementation of routine monitoring a t a large industrial plant. The performance of this method under field conditions was evaluated in four separate tests totaling over 100 samples. No detectable breakthrough of CAC into the back-up section was observed; t h e largest loading was 92 pg monochloroacetate, obtained over a period of 4.2 h a t 90 mL/min. Therefore, humidity or other agents do not pose a problem for retention of CAC a t the levels of interest. Additionally, no interfering
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 8, JULY 1979
peaks have been observed in the ion chromatograph analysis of the field samples. A potential drawback to this method is that it may not be specific for chloroacetyl chloride. Other potential sources of monochloroacetate, such as monochloroacetic or chloroacetic anhydride, may be positive interferences if present. This disadvmitage is partially offset by the ability to determine both monochloroacetate and chloride in the same analysis. Insufficient chloride in a sample may be taken as evidence that the observed monochloroacetate did not come entirely from CAC. The reversed situation of too much chloride, from other sources, is not expected to be a significant problem in the industrial or testing atmospheres for which this method is primarily intended. For reliable detection of low levels of CAC, samples should be analyzed promptly to guard against the deterioration that is noted with low level standards. The useful detection limit for monochloroacetate is ca. 0.4 pg/mL, corresponding to 0.01 ppm CAC in air for a 20-L air sample and use of 2.0 mL desorbing solution. Sensitivity probably can be increased readily simply by increasing the injection volume from the
current 0.1 mL and/or increasing the size of air sample.
ACKNOWLEDGMENT The aut,hors are grateful to R. A. Frame for much of the work on the diffusion calibration system for CAC, and to L. A. Peterfreund and D. L. Sheriden for analyses of some of the field samples. Helpful discussions with W. E. Dahl, E. E. Debus, and M. L. Rueppel also are appreciated. LITERATURE CITED (1) R. Butler, A. Snelson, and I. J. Solomon, Abstracts of the Division of Environmental Chemistry, 174th NafbnalMeeting of the American Chemical Society, Chicago, Ill.,Sept. 1977, p 59. (2) J. A. Dahlberg and I. B. Kihlman, Acta Chem. Scand., 24, 644 (1970). (3) J. D. Stewart, Ed., Occup. Safety Health Reporter, 8, 24 (1978). (4) P. W. Langvardt T. J. Nestrick, E. A. Hermann, and W . H. Braun, J . Chromatogr.. 153, 443 (1978). (5) A. H. Miguel and D. F. S. Natusch, Anal. Chem., 47, 1705 (1975). (6) A. P. Altshuller and I. R. Cohen. Anal. Chem., 32, 802 (1960). (7) D. G. Taylor, R. E. Kupel, and J. M. Bryant, "Documentation of the NIOSH Validation Tests", DHEW (NIOSH) PublicationNo. 77-185, Cincinnati, Ohio, 1977.
RECEIVED for review December 26, 1978. Accepted April 2 , 1979.
Bacterial Membrane Electrode for the Determination of Nitrate R. K. Kobos," D. J. Rice, and D. S. Flournoy Department of Chemistry, Virginia Commonwealth University, Richmond, Virginia 23284
A novel potentiometric sensor has been devised for nitrate by coupling the bacterium Azofobacfervinelandiiwith an ammonia gas-sensing electrode. Nitrate is reduced to ammonia by a two-step process involving the enzymes nitrate and nitrite reductases contained in the bacterial cells. The response of the bacterial sensor is linear over the concentration range of 1 X to 8 X M with a slope between 45 to 50 mV/decade. The electrode is useable for a period up to two weeks. Nitrate-containing samples were analyzed with an accuracy and precision between 3 to 4 % . The advantages of using intact bacterial cells instead of isolated enzymes to carry out the multistep reduction are discussed.
A novel approach to the development of bioselective sensors has been the use of intact bacterial cells in place of isolated enzymes at the surface of a. membrane electrode (1-5). This approach offers several economic and analytical advantages over conventional enzyme electrodes. These advantages include: an increased electrode lifetime, regeneration of electrode response (31,and the elimination of tedious, time consuming enzyme isolation and purification steps. The use of bacteria would be particularly superior for systems in which more than one enzymatic step along with cofactors are required to produce the measured product. Previously reported bacterial electrodes (1-4) utilized the bacterial cells to mediate a single enzymatic process. A hybrid electrode for nicotinamide adenine dinucleotide (NAD) has also been reported (5) in which a combination of an enzyme and bacterial cells are employed to carry out a two-step process. In this study, the use of bacterial cells to mediate a multistep enzymatic process to produce a bacterial sensor is investigated. 0003-2700/79/0351-1122$01.00/0
The system chosen for study involves the bacterial reduction of nitrate to ammonia by the following enzymatic sequence:
NO3- + NADH NO;
+ 3NADH
nitrate reductase
nitrite reductase
+
3"
NO2- + NAD+ + H20 (1)
+ 3NAD'
f 2H20
(2)
The ammonia produced can be detected with an ammonia gas-sensing electrode resulting in a bacterial sensor responsive to nitrate ions. This assimilatory enzyme system is found in several strains of bacteria and fungi (6). The advantages of using the bacterial cells can clearly be seen by comparison to similar systems in which isolated enzymes were used (7,s). An early attempt to prepare an enzyme electrode for nitrate met with limited success because of difficulties in isolating the nitrite reductase enzyme in sufficient quantity ( 7 ) . An electrochemical based enzymatic method for nitrate has recently been reported in which a different source of this enzyme was used (8). For this enzymatic determination, the two enzymes, nitrate and nitrite reductases, have to be isolated, purified, and then immobilized in a reactor column. Furthermore, the necessary cofactors must be included in the buffer solution. Although this system works well, it is somewhat complex. An alternate approach is to use bacterial cells which contain the necessary enzymes and cofactors as well as a means of cofactor regeneration. In this study a bacterial electrode for nitrate is reported in which a strain of Azotobacter, which possesses the desired enzymes (6, 9, I O ) , is coupled to an ammonia gas-sensing electrode. Because of its importance, many methods exist for the determination of nitrate. However, most of these methods are subject to interferences from common ions, e.g., chloride. perchlorate, chlorate, iron, sulfate, carbonate, and oxalate. 6 1979 American Chemical Society
